International Buffalo Information Centre
BUFFALO BULLETIN
ISSN : 0125-6726
(IBIC)
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IBIC is a specialized information center on
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being the buffalo information center of buffalo
research community through out the world.
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any aspect of research or development, progress
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S. Sophon
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International Buffalo Information Centre,
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Buffalo Bulletin (December 2013) Vol.32 No.4
CONTENTS
Page
Case Report
Recto-cervico-vaginal prolapse and its clinical management in a Mehsana buffalo
C.F. Chaudhari and V.S. Dabas........................................................................................................239
Delivery of a dicephalus sternopagus tetrabrachius tetrapus dicaudatus monster
in a Murrah buffalo by caesarean section
Gyan Singh, A. K. Pandey, Ravi Dutt, Shyam Sunder, Sandeep Kumar
and Suresh Chander..........................................................................................................................242
Cerebral babesiosis in a riverine buffalo (Bubalus bubalis) and
its successful therapeutic management
Vikrant Sudan, R.L. Sharma, M.K. Borah and R. Yadav...................................................................245
Original Article
Single strand conformation polymorphism within butyrophilin gene and
its relationship with milk yield in Indian riverine buffaloes
D.S. Kale, B.R. Yadav, Anupama Mukherjee and Jagdish Prasad...................................................253
Oxidized low density lipoprotein receptor 1 (OLR 1) gene polymorphism in
Mehsana buffaloes (Bubalus bubalis)
Manisha Deshpande, D.N. Rank, P.H. Vataliya and C.G. Joshi......................................................260
Evaluation of follicular atresia and electrophoretic pattern of follicular fluid proteins
in acyclic buffalo (Bubalus bubalis)
F.A. Khan, G.K. Das, Megha Pande, Rajendra Singh and S.K. Ghosh............................................265
Reproductive cycle stage bias in physiological and immune responses to
endotoxin challenge in Murrah buffaloes (Bubalus bubalis)
Z.A. Pampori and S. Pandita.........................................................................................................270
Buffalo Bulletin (December 2013) Vol.32 No.4
CONTENTS
Page
Original Article
Performance of Murrah buffaloes fed sunflower heads based complete diets in terms
of nutrient utilization and rumen fermentation pattern
D. Nagalakshmi, D. Narsimha Reddy and M. Rajendra Prasad...................................................283
Prevalence of sub-clinical mastitis in lactating buffaloes detected by comparative evaluation
of indirect tests and bacteriological methods with antibiotic sensitivity profiles in Bangladesh
Ch. Venkata Seshaiah, S. Jagadeeswara Rao, Y. Ramana Reddy,
J.J. Kisku and M.A. Samad............................................................................................................293
Prevalence and antibacterial susceptibility in mastitis in buffalo and cow in
district Lahore-Pakistan
Yasser Saleem Mustafa, Farhat Nazir Awan and Tooba Zaman....................................................307
Laparoscopic biopsy technique of liver and spleen in buffalo calves
K. Srinivasa Rao, Makkena Sreenu, K.B.P. Raghavender and P.V.S. Kishore...............................315
Impact on hematological parameters in young and adult Murrah buffaloes exposed to
acute heat stress
N. Haque, A. Ludri, S.A. Hossain and M. Ashutosh.......................................................................321
Comparative evaluation of different surgical approaches of caesarean sections in
buffaloes under field conditions
G.G. Chandore, S.P. Meshare and M.V. Ingawale.........................................................................327
Case Report
Buffalo Bulletin (December 2013) Vol.32 No.4
RECTO-CERVICO-VAGINAL PROLAPSE AND ITS CLINICAL MANAGEMENT
IN A MEHSANA BUFFALO
C.F. Chaudhari and V.S. Dabas*
CASE HISTORY AND CLINICAL
OBSERVATIONS
ABSTRACT
The present communication places on
record the clinical management of recto-cervicovaginal prolapse in a Mehsana buffalo.
A five-year-old Mehsana buffalo was
presented with the history of severe straining for
the previous two days. Further, it was reported to
have repeated the same at 15 day intervals on three
prior occasions. Moreover, the animal was reported
to have parturated normally three months before,
and the afterbirths had been expelled within six
hours postpartum. There was no breeding history
and the animal was apparently healthy. Clinically, a
baseball-sized soiled, oedematous vaginal part and
a coconut-sized rectal mass (Figure 1) was found to
be prolapsed. The animal was straining so severely
that each time, the perineum touched the ground
with expulsion of a small quantity of faeces and
muco-purulant discharge. Accordingly, the case
was diagnosed to be severe endometritis leading to
recto-cervico-vaginal prolapse, and it was decided
to treat medicinally.
Keywords: buffalo, Bubalus bubalis, rectocervico-vaginal prolapse, rope truss
INTRODUCTION
Prolapse of genital organs is a common
reproductive problem which adversely affects
overall performance of the affected animal. Samad
et al. (1987) reported the incidence of genital
prolapse as 42.9% among various obstetrical
problems in buffaloes. Although, the prolapse of
various elements viz. vaginal, cervico-vaginal,
uterus and rectum has been reported in buffaloes
(Sah and Nakao, 2003; Singh et al., 2011;
Kumbhar et al., 2009 and Patil et al., 2011), the
cervico-vaginal prolapse together with prolapsed
rectum is a rare disorder. Therefore, the present
communication reports a rare case of recto-cervicovaginal prolapses in a Mehsana buffalo with its
successful clinical management.
TREATMENTS AND DISCUSSION
The animal was secured in the travis to
achieve caudal epidural analgesia using 5 ml 2%
lignocaine hydrochloride solution. Following
washing of all the integuments with a mild
Veterinary Clinical Research and Experiential Learning Complex, College of Veterinary Science
and Animal Husbandry, Navsari Agricultural University, Navsari - 396 450, Gujarat, India,
*E-mail: vsdabas@yahoo.co.in
239
Buffalo Bulletin (December 2013) Vol.32 No.4
antiseptic solution and application of liquid paraffin,
the prolapsed masses were replaced manually.
Thereafter, the vaginal douching was performed
with mild potassium permanganate solution and the
rope truss was applied (Figure 2) for retention and to
prevent recurrence. Parenterally, Inj. Intacef 3 gm,
Inj. Meloxicam 20 ml and Inj. Chlorphemaramine
maleate 10 ml were given besides intra-uterine
infusion of Inj. Oxytetracycline 20 ml diluted in 20
et al., 1982). The main goal in the treatment of
uterine prolapse is the replacement of the organ at
its original place followed by a method to keep it in
the retained position and to clear the basic etiology
of the condition. In the present case, no difficulty
was encountered for replacement of either of the
prolapsed masses, and caudal epidural analgesia
using 5.0 ml 2% lignocaine hydrochloride solution
was found enough to decrease straining and
desensitize the perineum. Likewise, application
of the rope truss to exert pressure on the sides of
the vulva and simultaneous use of parenteral and
intra-uterine antibiotic therapy respectively helped
in retention and removal of possible infection of
the prolapsed elements. Kumbhar et al. (2009),
Mudasir et al. (2009) and Dharani et al. (2010)
also successfully managed the genital prolapses in
buffaloes by antibiotic therapy and application of
rope truss.
ml of distilled water and it was recommended that
this continue for the next five days. The rope truss
was removed after three days. The animal had an
uneventful recovery, and no further recurrence was
reported.
Cervico-vaginal prolapse is a most common
reproductive disorder of ruminants, usually in the
late gestation period, and can be recognized by
the protrusion of varying parts of the vaginal wall
and cervix through the vulva (Arthur et al., 2001).
It isconsidered to be the major problem causing
heavy economic losses to the farmers (Khan et al.,
1984). Around 65% of Nepali buffaloes expressed
vaginal prolapse at the last trimester (Sah and
Nakao, 2003). However, postpartum prolapse of
genital organs accounts for about 22 percent of the
total reproductive disorders in buffaloes (Pandit
REFERENCES
Arthur, G. H., D. E. Noakes, T. J. Parkinson
and G. C. W. England. 2001. Veterinary
Reproduction and Obstetric, 8th ed. Harcourt
Figure 1. Prolapsed recto-cervico-vaginal mass.
Figure 2. Retention of prolapse by rope truss.
240
Buffalo Bulletin (December 2013) Vol.32 No.4
(India) Private Limited.
Dharani, S., G. Suman Kumar, K. Sambasivarao
and K. Moulikrishna. 2010. Management
of a severe post-partum vagino-cervical
prolapse in a graded Murrah buffalo with
Renault’s truss and antibiotic therapy.
Buffalo Bull., 29(4): 311-314.
Khan, M.Z., S.K. Verma and S.K. Khar. 1984.
Studies on antepartum prolapse of vagina
in buffaloes. Haryana Agri. Univ. J. Res.,
14(3): 282-285.
Kumbhar, U. B., A. A. Suryawanshi, J. B. Mulani
and D. S. Raghuwanshi. 2009. Clinical
management of post-partum eversion of
uterus in Marathwadi buffalo. Veterinary
World, 2(5): 202.
Mudasir, Q., S. P. Shukla and S. P. Nema. 2009.
Haemato-biochemical changes during
prepartum cervicovaginal prolapse in a she
buffalo. Buffalo Bull., 28(3): 148-150.
Pandit, R. K., S. K. Gupta and S. R. Pattabiraman.
1982. A clinical study of vagina and uterus
in buffaloes. Indian Vet. J., 59: 975-980.
Patil, A. D., U. B. Kumbhar and K. Thorat. 2011.
Pre-partum rectal prolapse in a buffalo.
Intas Polivet, 12(1): 46-47.
Sah, S. K. and T. Nakao. 2003. Some characteristics
of vaginal prolapse in Nepali buffaloes. J.
Vet. Med. Sci., 65(11): 1213-1215.
Samad, H.A., C.S. Ali, N.U. Rehman, A. Ahmad
and N. Ahmad. 1987. Clinical incidence of
reproductive disorders in buffaloes. Pak.
Vet. J., 7(1): 16-19.
Singh, B., K. P. Singh, S. V. Singh, J. P. Singh and
H. N. Singh. 2011. Post-partum cervicovaginal prolapse in a buffalo. Intas Polivet,
12(1): 32-33.
241
Case Report
Buffalo Bulletin (December 2013) Vol.32 No.4
DELIVERY OF A DICEPHALUS STERNOPAGUS TETRABRACHIUS TETRAPUS
DICAUDATUS MONSTER IN A MURRAH BUFFALO BY CAESAREAN SECTION
Gyan Singh*, A. K. Pandey, Ravi Dutt, Shyam Sunder, Sandeep Kumar and Suresh Chander
fusion or non-separation, the types of the twin may
differ viz. thoracopagus (40%), omphalopagus
(33%), pyopagus (18%), cephalopagus (2%) and
ischiopagus (2%; Fernando, 1993). These might
arise due to genetic and environmental factors.
They are rare in other species, and reports in
buffaloes are meager.
ABSTRACT
Congenital bovine fetal anomalies can
be divided into heritable, toxic, nutritional, and
infectious categories. Although uncommon in most
herds, inherited congenital anomalies are probably
present in all breeds of cattle, and but reports in
buffaloes are meager. In some herds, the occurrence
of inherited anomalies has become frequent and
economically important. A rare case of dystocia due
to a dicephalic sternopagus tetrabrachius tetrapus
dicaudatus monster was relieved by caesarean
section.
Keywords: buffalo, caesarean,
monster, conjoined, Bubalus bubalis
CASE HISTORY AND OBSERVATIONS
A full-term Murrah buffalo about six and
half years old in her second parity with dystocia
was brought to the Teaching Veterinary Clinical
Complex. It had a history of straining for the
previous 8 - 10 h but had been unable to delivered
the fetus. The gestation period was over and water
bags had ruptured. Both hind limbs along with
one pelvis were hanging outside through vulva.
Per vaginal examination with proper lubrication
after epidural analgesia revealed two tails in the
birth canal and fetuses that were joined at the
sternal region. The hind limbs of the other fetus
present in flexed position breech presentation (i.e.
a dicephalic-sternopagus tetrabrachius tetrapus
dicaudatus monster).
Forced extraction was attempted by a
local veterinarian but did not succeed. The animal
was recumbent with severe tympany. The owner
was advised for caesarean section. The caesarean
dicephalus,
INTRODUCTION
A monster is an individual having multiple
anomalies involving many organs and systems of
the body. Fetal anomalies and monstrosities are
the most common cause of dystocia in bovines.
Conjoined twins arise from a single ovum and are
monozygotic. Monsters are mostly encountered in
cattle with an overall incidence of one in 100,000
bovine births (Roberts, 1971). Conjoined twins
develop after the development of embryonic plate
(Whitlock et al., 2008). Depending upon the site of
Teaching Veterinary Clinical Complex, College of Veterinary Sciences, Lala Lajpat Rai University of
Veterinary and Animal Sciences, Hisar 125004 (Haryana), India, *E-mail: vetgyan@rediffmail.com
242
Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 1. The dicephalussternopagus tetrabrachius tetrapus dicaudatus monster.
Figure 2. Photograph showing a pair of hearts.
Figure 3. Photograph showing two livers and gall bladders.
243
Buffalo Bulletin (December 2013) Vol.32 No.4
REFERENCES
section was performed as per routine surgical
method (paramedian, lateral to milk vein) and
a dicephalic-sternopagus tetrabrachius tetrapus
dicaudatus monster was delivered. External genitalia
indicated the sex of both fetuses as female. The
dam was administered systemic antibiotics, antiinflammatories, ecbolics, calcium boro-gluconate
and multivitamins. The monster was presented for
post mortem examination.
Fernando, A. 1993. Practical Guide to High Risk
Pregnancy and Delivery, 2nd ed. Baltimore,
Mosby Year Book. pp. 50-68.
Kumar, S., S. Prasad, U. Sharma, R. Sharda, Y.P.S.
Dabas and S.N. Maurya. 1999. Dicephalus
tetrapus tetrabrachius monster in Murrah
graded buffalo. Indian J. Anim. Reprod.,
20(2): 171.
Prasad, J.K., S. Prasad, A. Kumar and G.K. Singh.
2006. Thoraco-sternopagus monster: A rare
case of fetal dystocia in buffalo. Indian J.
Anim. Reprod., 27(2): 122-123.
Roberts, S.J. 1971. Veterinary Obstetrics and
Genital Disease (Theriogenology). CBS
Publisher and Distributor, India. p. 73.
Sahu, S.B. and R.K. Pandit. 1999. Dicephalus
thoraco-sternopagus Siamese monster in
a buffalo- a case report. Indian Vet. J., 76:
745-746.
Selvaraju, M., D. Kathiresan and C. Veerapandian.
2002. Dystocia due to conjoint twin monster
in a buffalo a case report. Indian Vet. J., 79:
721-722.
Whitlock, B.K., L. Kaiser and H.S. Maxwell.
2008. Heritable bovine fetal anomalies.
Theriogenology, 70(3): 535-549.
DESCRIPTION OF MONSTER
Each conjoined fetus had a separate
abdominal cavity; pair of fore and hind limbs,
ovaries, kidneys, one each of head, vertebral
column, tail, complete gastrointestinal tract, and
spleen. But both the livers were joined with each
other. The right gall bladder was three times larger
in size than left. The genital organs (oviducts,
uterus, cervix and vagina) ill developed with
external genitalia. The anal opening in each of
the conjoined fetus was patent. Thoracic cavities
having a heart and lungs were separate, externally
conjoined at the sternum. A similar type of monster
was reported by Kumar et al. (1999) having
duplication of all body parts except heart and lungs.
Dystocia due to a dicephalus thoraco-sternopagus
Siamese monster (Sahu and Pandit, 1999) and a
conjoined twin monster (Selvaraju et al., 2002)
have been reported as rare cases in buffaloes. A
thoraco-sternopagus twin arises due to embryonic
duplication of a germinal area whose body structure
are partially but not completely duplicated (Robert,
1971). Normal per-vaginal delivery of such types
of conjoint twins is difficult due to their enlarged
and abnormal size resulting in dystocia. The present
case study suggested that caesarean section may be
the treatment of choice in fetal monstrosities.
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Case Report
Buffalo Bulletin (December 2013) Vol.32 No.4
CEREBRAL BABESIOSIS IN A RIVERINE BUFFALO (Bubalus bubalis) AND ITS
SUCCESSFUL THERAPEUTIC MANAGEMENT
Vikrant Sudan1, R.L. Sharma2,*, M.K. Borah2 and R. Yadav
ABSTRACT
babesiosis” vis-à-vis bovine diseases exhibiting
analogous neurological signs has been discussed.
This seems to be the pioneer documentation of
“cerebral babesiois” in a buffalo from the semi-arid
enzootic areas of the Indian subcontinent.
Babesiosis has long been recognized as
persistent major constraint restricting the cross
border movement and export of highyielding
buffalo breeds from their native homelands to the
advanced countries. The disease accounts for high
mortality, poor growth and lower productivity of
the bubaline host. A she Murrah buffalo, aged 2
years and weighing approximately 300 kg, owned
by a person from a weaker section of the society,
suffered from critically high temperature (110oF),
Keywords: Murrah buffaloes, cerebral babesiosis,
Babesia bovis, nervous signs, diminazine
aceturate
INTRODUCTION
ruminal hypotonocity (1/3 minutes), anorexia,
aggressiveness, grinding of teeth, corneal opacity,
cessation of defaecation, abducted hind limbs,
icterus, and mild anaemia. She was observed
repeatedly making to and fro movements. Clinical
and haematological findings coupled with the
presence of Babesia bovis piroplasms in the
circulating erythrocytes made us infer that the
animal was suffering from the cerebral form of
babesiosis. The buffalo promptly responded the
specific therapy and successfully restored normal
haematological indices and erythrocytes free from
the piroplasms. Differential diagnosis of “cerebral
The buffalo -the incredible Asian dairy
animal, popularly known as the “Black Diamond”
-plays a versatile role in the socio-economic
uplift of its owners from the rural agricultural
communities. It is the largest highenergy milk and
lean meat producer in India (Gupta and Singh,
2002). Amongst a few parasitic diseases affecting
growth, development and productivity of the dairy
animal, bovine babesiosis caused by six species of
the pathogen, has since long being recognized as
an economically important disease of the wild as
well as the domesticated buffalo population in the
Department of Parasitology, College of Veterinary Sciences and Animal Husbandry, U.P. Pandit Deen Dayal
Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura281001, India
2
Department of Veterinary Parasitology, Apollo College of Veterinary Medicine, Agra Road, Jaipur-302031,
India, *E-mail: rlsharma2008@gmail.com
3
Department of Veterinary Medicine, Apollo College of Veterinary Medicine, Agra Road, Jaipur-302031,
India
1
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Buffalo Bulletin (December 2013) Vol.32 No.4
the buffalo shed, housing, sanitation, etc. were not
in absolute conformity with scientific standards.
These were reported to be of typical traditional
type, suited for small animal holdings of three to
five animals.
A closer look at the animal revealed severe
dyspnoea, frothy nasal discharge, lacrymation,
grinding of teeth, and abducting hind limbs, and it
was standing in a typical posture with arched back
and head extended in the forward direction. There
was no haemoglobinurea. The eyes were slightly
opaque suggestive of progressively developing
corneal opacity. Ophthalmic examinations of
the eyes in respect of symmetry, confirmation
were carried out from 2-3 feet distance with the
least head restraint. In absence of tonometric and
ophthalmic equipmental facilities, ocular pressure
was roughly assessed by application of gentle
pressure on the orbits. The ruminal motility of the
animal was assessed by pressing the fist in the left
paralumbar fossa. Peripheral blood (5 ml aliquot in
EDTA) was aseptically collected and the body hair
coat was carefully searched for acarine parasites.
Rectal coprological samples were also collected.
The samples were brought to the laboratory for
identification of the pathogen(s) and its vectors
using standard keys/ techniques (Bowmann et al.,
2003; Urquhart et al., 2003; Soulsby, 2005; Hendrix
and Robinson, 2006 and Taylor et al., 2007).
Overall clinical assessment of the
patient was suggestive of grave prognosis as the
temperature was critically high. The owner was
suitably informed. In order to overcome serious
neurological plight, the animal was forthwith
managed by symptomatic fluid and supportive
therapy, comprising of intravenous drip of 4 liters
of normal saline solution (NSS), intramuscular
injection of meloxicam 0.5 mg/ kg body weight,
Based on the identification of the pathogen, the
tropics and subtropics (Ristic, 1988; Roberts et al.,
1998; Aiello and Mays, 1998 and Lefevre et al.,
2010). The disease is characterized by exceptionally
high pyrexia, extensive erythrolysis leading to fastdeveloping anaemia, icterus, haemoglobinurea and
eventual death of the host (Wright, 1972; Urquhart
et al., 2003; Soulsby, 2005; Taylor et al., 2007 and
Lefevre et al., 2010). The prevailing epizootiological
determinants on the Indian subcontinent offer
the most favored and optimum opportunity for
faster propagation of the acarine intermediate host
(Boophilus microplus) and in situ development of
the pathogen- an apicomplexan parasite restricting
the cross border movement of buffaloes and export
of high yielding buffalo breeds from native home
land to the advanced countries. The authors have
come across an interesting and unusual observation
about the pathogen associated with the cerebral
form of the disease involving central nervous
system (CNS), “cerebral babesiosis”, in buffalo.
This is described herein.
MATERIALS AND METHODS
A she Murrah buffalo, aged 2 years and
weighing approximately 300 kg, was presented
on the 8th of July, 2011, before the clinicians at
the Teaching Veterinary Clinical Complex, Apollo
College of Veterinary Medicine, Jaipur. The cow
was reported having suffered from high temperature
and subsequent anorexia for the previous 10 days.
Further enquiry revealed that the animal had been
administered systemic antibiotics, antipyretics and
appetizers by a field veterinarian but to no eventual
success for previous 7 days. The owner belonged to
a socio-economically weaker section of the Indian
society, with meager family income from sale of
milk and agricultural labour. The management of
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Buffalo Bulletin (December 2013) Vol.32 No.4
transient blindness (Figure 1). Neither of the eyes
was completely blind. Ruminal hypotonocity was
quite evident (1 per 3 minutes).
Laboratory investigation of the thin and
watery peripheral blood revealed suppressed
heamatological indices [Haemoglobin- 11.0 g/dl,
PCV- 34%, TLC- 4.0x103/ μL] and altered differential
cell counts [Neutrophils 18%, Lymphocytes 78%,
Monocytes 2% and Eosinophils 2%], these data
were suggestive of the animal suffering from
a milder form of anaemia, severe leucopenia,
lymphocytosis and moderate eosinophilia.
Microscopic examination of the Leishman
stained thin smear evidenced characteristic intraerythrocytic piroplasms identified as Babesia
bovis (Figure 2) whereas, copro samples did not
demonstrate parasitic ova and/or cysts. Blood of
the animal re-examined on Day-21 post therapy
revealed that the altered haematological indices
had returned to normal levels and the erythrocytes
were completely free from the pathogen.
animal was given specific therapy consisting of
diminazine aceturate 5.0 mg/kg body weight. The
animal was re-examined on Day-21 post therapy
as evidenced by progressive restoration of body
temperature to normal by Day-3 post therapy. The
blood of animal was reexamined on Day-21 post
therapy.
RESULTS
Clinical examination of the buffalo
revealed lusterless dull hair coat infested with
the developmental instars, including adults of
Boophilus microplus over dewlap, axilla, ventral
abdomen, udder and the peri-anal region. The
animal was weak, uneasy and frequently kicking
down. The conjunctival mucous membrane was
congested and palid progressively turning to icteric.
The muzzle was dry with frothy nasal discharge
from both the nostrils. There was excessive
drooling salivation, rapid shallow respiration
(32 /minutes) besides, accelerated pulse (80 /
minutes) and almost dry and hard rectal faeces. The
superficial lymph nodes were neither swollen nor
oedematous. The rectal temperature was critical,
reaching 110.0oF. Signs of respiratory distress
DISCUSSION
On the Indian subcontinent, in the enzootic
semi-arid desert, bubaline babesiosis is mainly
caused by B. bovis and B. bigemina (buffalo
strain). The one host tick Boophilus microplus
is the vector of the disease in India, transmitting
both transstadially and transovarianly bebesiosis
in the susceptible exotics and / or their cross-bred
progenies in buffaloes (Urquhart et al., 2003;
Soulsby, 2005; Taylor et al., 2007 and Lefevre et
al., 2010). Though Babesia spp. are host and vector
specific, yet the pathogens have been documented
intertransmissible from buffalo to cattle and vice
versa (Callow et al., 1976). The nymph and adult
instars of the tick are widely distributed in the
were quite evident and the animal was breathing
in an arched back position with extended head and
abducted limbs. Auscultation sounds revealed harsh
sounds from the lungs and audible heart beat from
distance. The animal was seen uneasy, frequently
exhibiting to and fro movements. Ophthalmic
examination of the eyes in respect of symmetry
and confirmation did not reveal any deformity and
/ or blindness. The eyeballs were devoid of intraocular lesions suggestive of trauma, insect bite and
/ or inherited genetic defects. However, the animal
was progressively developing corneal opacity and
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Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 1. Corneal opacity in the affected animal.
Figure 2. Blood smear showing intra erythrocytic piroplasms.
248
Buffalo Bulletin (December 2013) Vol.32 No.4
semi-desert environment, prevailing in a majority
of the Middle Eastern and South Asian countries,
including India (Radostits et al., 1994; Aiello
and Mays, 1998 and Taylor et al., 2007). Once
the adult female tick is infected, it can transmit
the infection for 32 generations (Markov and
Abramov, 1966). Epizootilogically, vector borne
diseases are regulated by four component systems,
wherein susceptible hosts, optimum environments,
the parasite and its vector play an integral role
and account for the high occurrence of the disease
especially during summer months (May to June)
in the semi-desert ecosystem of Jaipur. Extremely
high temperature, high milk yield and heat stress
during summer months are recognized major
predisposing factors.
The pathogen is widespread in buffalo
native to Africa, Asia, and Australia. Young animals
are reasonably resistant to the pathogen and do not
cause clinical disease. In older animals, clinical
signs can be very severe; however, differences in
pathogenicity may occur with various Babesia spp.
isolates associated with different geographic areas.
The anemia may occur very rapidly, with 75% or
more of the erythrocytes being destroyed in just a
few days. After the onset of hemoglobinuria, the
prognosis is guarded. Among fully susceptible
older animals, the mortality may reach 70% without
treatment. Anaemia, anorexia and / or anoxia are
contributory factors to the weakness and loss of
condition seen in cattle that survive the acute phase
of the disease (Purnell, 1981; Ristic, 1988 and
Aryeetey and Jimenez-Lucho, 2002).
Interestingly, to our knowledge, this seems
to be the first ever documentation of “cerebral
babesiosis” associated with B. bovis infection in the
buffaloes from the sub-tropical, semi-arid, enzootic
region of the Indian subcontinent. The disease
occurs sporadically especially in adult bovines
and generally terminates fatally. Infections with B.
bovis have been incriminated as the main causal
agent of the “cerebral syndrome”, characterized
by paddling of limbs, ataxia, and mania followed
by death in the majority of cases. Pathogenesis
of the syndrome has been debatable. Possibilities
of crossing the CNS barriers by the parasite and
causing auto immune disorders, characterized by
intra-vascular agglutination of the erythrocytes in
the cerebral capillaries with consequential embolism
/ thrombosis could not be ruled out (Soulsby, 2005;
Taylor et al., 2007 and Lefevre et al., 2010). The
diary animals, especially exotics and / or their
cross-bred progenies, have been documented to
be more susceptible to the disease than the native
breeds of buffaloes in the enzootic areas (Taylor et
al., 2007 and Lefevre et al., 2010).
The characteristic clinical signs in buffalo,
reported here-in, were consistent with the critically
“nervous signs” encountered in cattle [marked
fall in general condition, caesation and / or
ruminal hypotonocity, elevated body temperature,
accelerated pulse and respiratory rate suggestive of
dyspnoea and trachycardia, drooling saliva, frothy
nasal discharge, constipated rectal faeces, etc.],
coupled with sudden development of pathognomic
neurological signs (aggressiveness, incoordinated
gait and convulsions, paddling of limbs, persistent
abduction of hind limbs, ataxia, mania) and
demonstration of the pathogen in circulation were
suggestive of acute “Cerebral Babesiosis” with
grave prognosis. Further, depressed haematological
indices confirming anaemia, severe leucopenia,
lymphocytosis and moderate eosinophilia and
above all, successful response to single specific
anti-bebesial therapy and complete elimination
of the pathogen from the circulation on Day-21
post therapy, made authors to strongly speculate
and believe that the buffalo suffered from cerebral
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Buffalo Bulletin (December 2013) Vol.32 No.4
form of Babesiosis. The nervous signs appeared
due to blood stasis incidental to clogging of brain
capillaries by accumulation and/ or agglutination of
parasitized erythrocytes (Purnell, 1981; Aiello and
Mays, 1998; Taylor et al., 2007 and Lefevre et al.,
2010). The buffalo, while undergoing critical phase
of the disease, exceptionally high pyrexia and high
parasitemia, might have adversely affected the in
situ physiology of pituitary and adrenal glands
resulting in poor feed intake, nutrient utilization
and rise in body temperature to critical levels and
consequently, faster deterioration of the general
health of the animal (Wright and Goodger, 1977;
Radostits et al., 1994; Soulsby, 2005; Taylor et al.,
2007 and Lefevre et al., 2010).
Activation and spontaneous release
of kallireins and kininins (vasoactive amines),
consequential to host defence-parasite interaction
might have played a significant role in the
pathobiology and development of “cerebral
babesiosis”. The cytokines have been incriminated
in vasodilatation and increased permeability of
the affected fine blood vessels supplying tissues.
Synchronously, the activity was coupled with
concentration and sequestration of the parasitized
erythrocytes, obstructing the free blood flow (Wright
and Goodger, 1977; Mahoney and Seal, 1977;
Purnell, 1981; Aryeetey and Jimenez-Lucho, 2002;
neurological signs viz. aggressiveness, absence of
haemoglobinuria, circling, loss of herding instinct,
paresis, muscular tremors, stiffness of hind legs and
incoordinated gait seems logical and imperative.
In listeric encephalitis, the animal suffers from
unilateral facial nerve paralysis causing drooping of
the eyelids and ears, dilated nostrils, and drooling
of saliva, incidental to pharyngeal nerve partial
paralysis. The duration of the disease is invariably
longer (2-6 weeks). The lesions are mainly
localized in the brain stem and the clinical signs
indicate dysfunction of the third to seventh cranial
nerve (Radostits et al., 1994 and Lefevre et al.,
2010).Whereas the rabies affected animal makes
unprovocated attacks and is unable to swallow
and / or drink. It is characterized by early paralysis
of the throat and masseter muscles. The animal
often exhibits changed behavior, stops eating and
drinking, frequently passes urine, seeks solitude,
produces characteristic loud bellowing sounds,
and its lower jaw drops (Radostits et al., 1994
and Lefevre et al., 2010). Polioencephalomalacia
(PEM) is primarily a non-infectious neurological
disease of the young and intensively reared cattle
and buffaloes. It is incidental to the depressed
activity of the tissue thiamine and related enzymes.
The disease is characterized by amaurosis “glass
eye” and strabismus followed by neurological signs
Allred, 2003 and Lefevre et al., 2010). Eventually,
during the acute phase of the disease, the buffalo
seems to suffer the coagulopathy syndrome of the
disease resulting in blood stasis, tissue anoxia and
specific neurological signs originating from the
brain (Wright, 1972; Wright and Goodger, 1977
and Lefevre et al., 2010).
The differential diagnosis of the cerebral form
of the babesiosis vis-à-vis other disease conditions,
(listeriosis,
rabies,
polioencephalomalacia,
cerebral theileriosis), exhibiting almost analogous
and recumbancy. The disease is not prevalent on
the Indian subcontinent (Radostits et al., 1994 and
Lefevre et al., 2010). The cerebral form “turning
sickness” is characterized by a marked fall in general
condition and lactation, caesation of rumination
and digestive disturbances, pre mortal moderately
elevated body temperature, accelerated pulse
and respiratory rate, drooling saliva, frothy nasal
discharge and respiratory distress, coupled with
sudden development of pathognomic neurological
signs (occasional circling, head pressing and
250
Buffalo Bulletin (December 2013) Vol.32 No.4
ACVM, Jaipur, India for facilities provided.
persistent abduction of hind limbs, animal falling
in lateral recumbancy and finally transient posterior
paresis) and demonstration of the Theileria
piroplasms in erythrocytes and Koch blue bodies
in the affected lymphoid tissues (Radostits et al.,
1994; Lefevre et al., 2010 and Sudan et al., 2012).
However, the bubaline cerebral form of babesiosis
is devoid of circling signs, haemoglobinuria,
paresis, muscular tremors, stiffness of hind legs,
gastrointestinal disorders, etc.
It would be interesting to precisely
investigate through well-planned experimental
studies in buffaloes to elucidate (a) comparative
efficacy of anti-babesial drugs ensuring complete
elimination of the pathogen from the cerebral
circulation; (b) prevalence of the disease,
epizootiological determinants and reasons for
its underreporting by the 0field veterinarians in
the enzootic areas; (c) pathophysiological impact
of the disease on endocrine glands especially
the pituitary and adrenal glands, feed intake and
nutrient utilization, spontaneous marked fall in
lactation and deterioration of the general health
of the affected animal besides, onset of shock and
failure of thermoregulation, reaching critical levels
in buffaloes; (d) mechanism of in vivo migration
and access of the pathogen to cerebral capillaries
and / or tissues and multiplication of the pathogen
therein and intravascular sequestration of the
parasitized erythrocytes; and (e) immunological
response of the host, prior to sudden onset of
sequential events discussed above and development
of specific neurological signs.
REFERENCES
Aiello, S.E. and A. Mays. 1998. The Merck
Veterinary Manual, 8th ed. Merck & Co. Inc.
New Jursey, USA. 2305p.
Allred, D.R. 2003. Babesiosis: Persistence in the
face of adversity. Trends Parasitol., 19: 5155.
Aryeetey, R. and V. Jimenez-Lucho. 2002.
Babesiosis: Current treatment options.
Infect. Dis. 4: 319-326.
Bowman, D.D., R.C. Lynn, M.L. Eberhard and A.
Alcaraz. 2003. Georgis Parasitology for
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of Elsevier, USA.): 422p.
Callow, L.L., R.J. Parker, B.J. Rodwell and M.L.
Ottley. 1976. Piroplasmosis in buffaloes
and its serological diagnosis based on a
homology between buffalo and bovine
immunoglobulins. Aust. Vet. J., 52: 40-41.
Gupta, S.C. and B.P. Singh. 2002. Fasciolosis
in cattle and buffaloes in India. J. Vet.
Parasitol., 16: 139-145.
Hendrix, C.M. and Ed Robinson. 2006. Diagnostic
Tarasitology for Teterinary Technicians,
3rd ed. Mosby IMC (Elsevier) St Louis
Missouri, USA. 304p.
Lefevre, P.S., J. Blancou, R. Chermette and G.
Uilenberg. 2010. Infectious and Parasitic
Diseases of Livestock. Lavoisier Tec & Doc,
France. 1985p.
Mahoney, D.F. and J.R. Seal. 1961. Bovine
babesiosis; Thick blood films for the
detection of parasitaemia. Aust. Vet. J., 37:
44-47.
Markov, A.A. and LV. Abramov. 1966. Reciprocal
ACKNOWLEDGEMENT
The authors express their deep sense
of gratitude and sincere thanks to the Dean,
251
Buffalo Bulletin (December 2013) Vol.32 No.4
adaptations of certain blood parasites and
their hosts, p. 268-269. In Proceedings of
1st International Congress of Parasitology,
Italy. I: 268
Purnell, R.E. 1981. Babesiosis in various hosts,
p. 25-63. In Ristic, M. and J. Kreier (eds.)
Babasiosis. Acedemic Press, New York,
USA.
Radostits, O.M., D.C. Blood and C.C. Gay. 1994.
Veterinary Medicine: A tTextbook of the
Diseases of Cattle, Sheep, Pigs, Goats and
Horses, 8th ed. Bailliere Tindall, London,
UK. 1763p.
Ristic, M. 1988. Babesiosis of Domestic Animals
and Man. CRC Press, Boca Raton, Florida,
USA. 264p.
Soulsby, E.J.L. 2005. Helminths, Arthropods and
Protozoa of Domesticated Animals, 7th ed.
Bailliere Tindal, London, UK.
Sudan, V., R.L. Sharma, R. Yadav and M.K. Borah.
2012. Turning sickness in a cross bred cow
naturally infected with Theileria annulata.
J. Parasit. Dis., 36(2): 226-229.
Taylor, M.A., R.L. Coop and R.L. Wall. 2007.
Veterinary Parasitology, 3rd ed. Edition
Blackwell Publishing LTD, UK. 874p.
Urquhart, G.M., J. Armour, J.L. Duncan, A.M.
Dunn and F.W. Jenningsis. 2003. Babesiosis,
pp. 242-246. In Veterinary Parasitology.
Blackwell Science LTD, UK.
Wright, I.G. 1972. Studies on the pathogenesis of B.
argentina and Babesia bigemina infections
in splenomectized calves. Z. Parasitenkd.,
39: 85-102.
Wright, I.G. and B.V. Goodger. 1977. Acute Babesia
bigemina infection: Changes in coagulation
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Original Article
SINGLE STRAND CONFORMATION POLYMORPHISM WITHIN BUTYROPHILIN GENE
AND ITS RELATIONSHIP WITH MILK YIELD IN INDIAN RIVERINE BUFFALOES
D.S. Kale1, B.R. Yadav2, Anupama Mukherjee3 and Jagdish Prasad3
ABSTRACT
linear model procedure (SYSTAT) for association
study indicated BTI1 SSCP was significantly
associated (P≤0.05) with 305-day lactation
milk yield of Murrah buffaloes. The Murrah
buffaloes with BTI1BB genotypes had 683.93 kg
and 320.48 kg higher milk yield as compared to
Butyrophilin has been shown to be a
mammary-specific gene and used as a genetic
marker to investigate allelic substitution effects
on milk production traits. The aim of the present
study was to study genetic polymorphism of the
butyrophilin gene using SSCP in the Murrah,
Surti and Bhadawari breeds of riverine buffaloes
(Bubalus bubalis). The association study was
carried out to find possible relationships (if any)
with milk production traits in Murrah buffalo. The
SSCP analysis of the intron region of butyrophilin
revealed polymorphic BTI1 SSCP in buffaloes.
DNA sequencing of three polymorphic SSCPs
within the Murrah buffalo butyrophilin gene
revealed three variants.viz, A, B and C. The
frequencies of identified buffalo butyrophilin
variants were: A = 0.6, B = 0.31 and C = 0.09 in
55 Murrah buffaloes; in Surti A = 0.5, B = 0.3 and
C = 0.2 while in Bhadawari A= 0.4, 0.5 and 0.1.
The Murrah buffalo butyrophilin variant sequence
(EU194868) was found in 96 % pairwise percent
similarity with Bos taurus nucleotide sequence
(AF005497). The statistical analysis using general
BTI1AA and BTI1CC genotypes, respectively.
The association of BTI1 SSCP polymorphism with
milk production traits will be useful for genetic
improvement of buffaloes for milk production traits.
Keywords: riverine buffaloes, Bubalus bubalis,
butyrophilin, DNA polymorphism, riverine PCRSSCP, milk yield
INTRODUCTION
The buffalo is the most important farm
animal species in Asia, especially in India, where it
is extensively used for milk, meat, fuel and fertilizer
(from manure) production , as well as for draught
power (Borghese, 2005). India possesses more than
half of the world buffalo population and many of
the fine breeds are found in its different parts of
Department of Animal Genetics and Breeding, Nagpur Veterinary College, Maharashtra Animal and
Fishery Sciences University (MAFSU), Seminary Hills, Nagpur-440006, Maharashtra, India, E-mail:
deepakkaleccmb@gmail.com
2
Livestock Genome Analysis Laboratory, National Dairy Research Institute (NDRI), Karnal-132001, Haryana
State, India
3
Department of Animal Genetics and Breeding, Faculty of Veterinary Science and Animal Husbandry,
Allahabad Agricultural Institute Deemed University (AAIDU), Allahabad-211007, Utter Pradesh, India
1
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Buffalo Bulletin (December 2013) Vol.32 No.4
the country. Indian buffaloes are hardy, thrive well
on poor quality nutrition and are good converter
of inputs into milk as compared to cattle (Biswas
et al., 2003). However, their inherent potential
for growth and production has not been exploited
due to inadequate information about the genetic
basis of production traits and breeding strategies.
The riverine buffalo contributes significantly to
the Indian agriculture system and to the economy.
out to analyse butyrophilin gene polymorphism
using PCR-SSCP in Murrah, Surti and Bhadawari
breeds of riverine buffaloes (Bubalus bubalis).
DNA sequencing was done for polymorphic
SSCP variants obtained in Murrah buffaloes. The
association study was carried out to find possible
relationships (if any) with milk production traits in
Murrah buffalo.
Nearly 55% of milk production in India comes
from buffaloes (FAO, 2007). There is therefore a
MATERIALS AND METHODS
needed to analyze and establish molecular markers
for production traits in buffaloes. Currently,
genetic marker research applied to animal breeding
and production is focussed mainly on analysing
mutations located within candidate genes and
their association with economically important
production traits (Kale and Yadav, 2011).
The Polymerase Chain reaction – single
strand conformation polymorphism (PCR-SSCP)
(Orita et al., 1989) method is a powerful method
for identifying sequence variation in amplified
DNA fragments. Compared with the PCR-RFLP
technique, PCR-SSCP has the advantage of being
able to detect single base substitutions in other
locations besides enzymatic restriction sites.
Butyrophilin is the major glycoprotein of the bovine
milk fat globule membrane (MFGM), accounting
for over 40 and 50% of the total protein on weight
and molar bases, respectively (Jack and Mather,
1990). Butyrophilin has been proved as a good
candidate to serve as genetic marker because of its
exclusivity to the mammary gland and its purported
role in milk fat secretion (Mather, 2000). Recently
the association of butyrophilin gene polymorphism
with milk yield, protein yield and SNF yield has
been reported in Korean dairy proven and young
bulls (Jang et al., 2005).
Therefore the present work has been carried
The study was conducted on representatives
of three important breeds of riverine buffaloes
i.e. 55 Murrah, 10 Surti and 10 Bhadawari breed
animals from Haryana and Punjab, Gujarat and
Utter Pradesh and Madhya Pradesh states of India
respectively. Blood samples (10 ml) were collected
by jugular veinipuncture using vacuum tubes
containing acid citrate dextrose solution (ACD)
as an anticoagulant. Genomic DNA isolation was
performed from blood using the phenol chloroform
extraction protocol (Clamp et al., 1993) with some
modifications. The PCR primers (Table1) for PCRSSCP analysis were designed using PRIMER3
software for the butyrophilin gene in buffalo on the
basis of the cattle GenBank sequence.
The polymerase chain reaction (PCR)
was carried out on about 100 ng/ μl of genomic
DNA in a 25 μl reaction volume. The reaction
mixture consisted of 2.5 μl of 10x PCR assay
buffer containing 1.5 mmol MgCl2, 200 μmol each
of dNTPs, 0.75 U Taq DNA polymerase and 10
pmole of each primer. Amplification was carried
out in a Biometra thermal cycler using PCR cycling
conditions as (95°C for 5 minutes) and 34 cycles of
45 seconds at 95°C, annealing temperature (TA°C)
and 72°C consecutively, followed by a five minutes
final extension at 72°C. In the process of PCR
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Buffalo Bulletin (December 2013) Vol.32 No.4
amplification the annealing temperature (TA) was
optimised for each primer sequence (Table 1). The
PCR amplification was verified by electrophoresis
of the PCR products with loading dye (95%
formamide, 0.25% bromophenol blue and 0.25%
xylene cyanol) on 2% (w/v) agarose gel in 0.5 x
TBE buffer using a 100 bp ladder as a marker for
confirmation of the length of the PCR products. The
agarose gels were stained with ethidium bromide
(0.5 μg/ml). The amplified products (5 μl) were
detected on 2% agarose gel using 1 μl of loading
dye as a stop dye, electrophoresed and visualized
using UV light after ethidium bromide staining.
The butyrophilin gene PCR products were
resolved by SSCP analysis. The various factors
were tested for each fragment and optimized.
viz, amount of PCR product acrylamidebisacrylamide concentration, presence of glycerol,
voltage, electrophoresis run time and at room
temperature using ice chilled circulating water
to electrophoresis assembly. The amplified PCR
products were diluted in denaturing solution (95%
formamide, 10 mmol NaOH, 0.05% xylene cyanol
and 0.05% bromophenol blue, 20 mmol EDTA)
and heat denatured at 95°C for ten minutes. After
denaturation PCR products were immediately
transferred to chilled ice pack and kept in -20°C for
10 minutes. The PCR products were resolved on
a non-denaturing 12% acry1amide: bis-acrylamide
(49:l) gel. The vertical gel electrophoresis was
carried out in a Bio-Rad Protean® II Xi Cell
service and analysed using various analytical tools
for polymorphism detection. The DNA sequence
polymorphism observed were used to genotype the
Murrah, Surti and Bhadawari buffalo populations.
The frequency of polymorphic alleles, genotypes
and their accordance with Hardy-Weinberg law
was assessed by POPGENE 1.31 software (http://
www.ualberta.ca/~fyeh). The association between
polymorphic allelic variants of butyrophilin genes
and milk production traits were analysed using
GLM procedure (SYSTAT). The following model
was used,
Yijkl = μ + gi+ si+ pj + hk + eijkl
Yij : observation on jth animal ith genotype
μ : population mean
: effect of ith genotype (i=1, 2)
gi
si : effect of i season
pj : effect of j parity
hk : effect of k year
eijkl : random error
RESULTS AND DISCUSSION
The butyrophilin gene was screened for
SSCPs using primers BTI1, BTI3 and BTI4 (Table
1). The SSCP patterns for all the three primers were
best resolved in 12% acrylamide-bisacrylamide with
the same SSCP conditions viz. voltage (200 volts),
temperature (25°C), time (12 h) with 10% glycerol.
The butyrophilin gene fragments amplified using
BTI1 yielded three polymorphic SSCP patterns viz.
A, B and C in 12% non-denaturing PAGE (Figure
1) while fragments amplified using BTI3 and
BTI4 primers did not exhibit polymorphic SSCP
fragments. The frequencies of polymorphic SSCP
patterns in BTI1 amplified fragments of buffalo
were: A = 0.6, B = 0.31 and CC = 0.09 in 55
Murrah buffaloes; in Surti A = 0.5, B = 0.3 and C
electrophoreses unit using 1X TBE buffer at 1012.5 volts/cm for 12 h at room temperature. Gels
were silver-stained (Sambrook and Russell, 2001)
and photographed using a digital camera for
butyrophilin SSCP pattern analysis.
The PCR products representing different
SSCP patterns in Murrah buffalo were directly
sequenced using automated DNA sequencing
255
Buffalo Bulletin (December 2013) Vol.32 No.4
= 0.2 while in Bhadawari A= 0.4, 0.5 and 0.1. This
study revealed polymorphic SSCP in the intron 1
region of the butyrophilin gene of Murrah, Surti
and Bhadawari breeds of buffaloes.
The PCR products generated using BTI1
primer representing three SSCP patterns (A, B
and C) in Murrah buffalo were sequenced using
automated DNA sequencing services (Bangalore
Genei). The nucleotide sequences arising from this
study were submitted in GenBank (EU194868,
EU1997977 and EU199798). The polymorphic
Murrah buffalo butyrophilin variant A sequence
(EU194868) identified using BTI1 primer was
compared with Bos taurus reference sequence
(AF005497) using alignment tool (Geneious
Software) which revealed eleven computational
mutations. The Murrah buffalo butyrophilin
variant sequence (EU194868) was found to have
96% pairwise percent similarity with Bos taurus
nucleotide sequence (AF005497). The BTI1 SSCP
polymorphism was used to genotype Murrah, Surti
and Bhadawari populations of buffaloes in which
BTI1 SSCP genotypes were in Hardy-Weinberg
proportions (Table 2).
Association of butyrophilin allelic
variants with milk production traits in Murrah
buffaloes: The variance analysis indicated BTI1
SSCP was significantly associated (P≤0.05) with
yield, fat and SNF percentage in Murrah buffaloes
with different genotypes are given in Table 3.
Presently with improvements in selection
and breeding technologies, selection pressure
has been steadily growing. Among the tools at
the disposal of modern animal breeders, marker
assisted selection conceivably seems to be the most
direct control, for it can hasten the rate of genetic
gain of desirable traits in farm animals. Hence,
in the present study, the sequence variations in
buffalo butyrophilin gene were investigated by
SSCP analysis of three amplified fragments. Three
SSCP variants were detected in the BTI1 amplified
fragment. The SSCP polymorphism was designated
as BTI1 SSCP. The variance analysis indicated
BTI1 SSCP was significantly associated (P≤0.05)
with 305-day lactation milk yield of Murrah
buffaloes. The Murrah buffaloes with BTI1BB
genotypes had 683.93 kg and 320.48 kg higher
milk yield as compared to BTI1AA and BTI1CC
genotypes respectively. The statistical association
of butyrophilin gene polymorphism revealed using
SSCP followed by DNA sequencing is one of the first
reports in Murrah buffaloes Similarly in previous
studies, Jang et al. (2005) studied association of
butyrophilin candidate genes with production
traits in Korean dairy proven and young bulls and
reported that BTN3 was associated with 305-day
305-day lactation milk yield of Murrah buffaloes.
The Murrah buffaloes with BTI1BB genotypes
had 683.93 kg and 320.48 kg higher milk yield
as compared to BTI1AA and BTI1CC genotypes,
respectively. The positive association of BTI1
SSCP polymorphism with milk production traits
may be useful for improving milk performance
in dairy buffaloes. However, it was observed that,
BTI1 polymorphism genotypes did not significantly
differ with fat and SNF percentage trait values of
Murrah buffaloes. The least squares means for milk
production traits (p<0.05). Bhattacharya et al.
(2006) reported HaeIII PCR-RFLP polymorphism
in crossbred cattle and identified AA, BB and AB
genotypes having significant effects (p≤0.05) on
total milk solid, fat and SNF percentages.
CONCLUSION
In the present study, PCR-SSCP after
direct sequencing was found to be feasible
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Buffalo Bulletin (December 2013) Vol.32 No.4
Table 1. Details of primer sequences used for buffalo butyrophilin DNA polymorphism analysis using PCRSSCP.
Name of
Primer
BTI1
BTI3
BTI4
TA / RE/
Gene region
Primer Sequence
FP
RP
FP
RP
FP
RP
5′-CCTGCTTATTTCCCTAGTCTC-3′
5′-CCACCCTAAGGTTAGTCAATC-3′
5′-AACTGGCTATAAAGCCCTAGA-3′
5′-ACTACACAAGGGAACTGAGGT-3′
5′-AGATCTCACAGACATTCCAGA-3′
5′-TGCTGAACCAGAGGTAGAGTA-3′
Table 2. The frequency of BTI1 SSCP genotypes and variants in Murrah,
buffaloes.
Number/ Frequency of genotypes
Buffalo
Breed
N
AA
BA
BB
CA CB
CC
Murrah
55 33/0.6 0/0
17/0.31
0/0
0/0
5/0.09
Surti
10
5/0.5
0/0
3/0.3
0/0
0/0
2/0.2
Bhadawari
10
4/0.4
0/0
5/0.5
0/0
0/0
1/0.1
57°C/ intron1
57°C/ intron3
62°C/ intron4
Surti and Bhadawari breeds of
Variant Frequency
A
B
C
0.6 0.31
0.09
0.5 0.30
0.2
0.4
0.5
0.1
Table 3. Means and standard error (SE) of studied traits in reference to BTI1 polymorphism in Murrah
buffalo.
Genotype
n
Milk Yield ± SE
FAT*±SE
SNF*±SE
BTI1 AA
33
2204.56 ± 223.75
0.297NS±0.004
0.315NS±0.001
BTI1 BB
17
2888.49S± 295.83
0.296NS±0.006
0.315NS±0.002
BTI1 CC
05
2568.01± 415.78
0.309NS±0.008
0.318NS ±0.002
*are scale-transformed values; Within columns, means marked by the same superscriptS did differ
significantly at P≤0.05.
Figure 1. Single strand conformation polymorphism (BTI1) within butyrophilin gene in riverine
buffaloes where,
A, B and C = SSCP patterns
04-08 = Animal numbers
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Buffalo Bulletin (December 2013) Vol.32 No.4
technique to reveal nucleotide sequence variation.
The variance analysis revealed that the BTI1
SSCP variant BTI1BB genotype was differing
significantly with 305-day lactation milk yield
of Murrah buffaloes. Validation of identified
polymorphism with a larger dataset might be good
candidates for marker assisted breeding for better
milk production in dairy buffaloes. The observed
nucleotide sequence variation in the butyrophilin
gene and positive association with production traits
can be useful information resource for buffalo
genetic improvement, conservation, management
and breeding decisions.
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candidate genes with production traits in
Korean dairy proven and young bulls. Asian
Austral. J. Anim., 18: 165-169.
Kale, D.S. and B.R. Yadav. 2011. Polymorphism of
leptin gene in Murrah buffalo. Indian Vet.
J., 88(8): 55-57.
Mather, I.H .2000. A review and proposed
nomenclature for major proteins of the
milk-fat globule membrane. J. Dairy Sci.,
83: 203-247.
Orita, M., Y. Suzuki, T. Sekiya and K. Hayashi.
1989. Rapid and sensitive detection of point
mutation and DNA polymorphisms using
polymerase chain reaction. Genomics, 5:
874- 879.
POPGENE 1.31 software, (http://www.ualberta.
ca/~fyeh).
Rozen, S. and H.J. Skaletsky. 1998. Primer3. Code
ACKNOWLEDGMENTS
The financial grant as National Fellowship
to Dr. B.R. Yadav from the Indian Council of
Agricultural Research (ICAR) and assistance in the
laboratory by Mr. R.K. Tonk, Mr. Naresh Kumar
and Mr. Nankoo Singh are acknowledged.
REFERENCES
Bhattacharya, T.K., S.S. Misra, D.S. Feroz, S.
Shukla, P. Kumar and A. Sharma. 2006.
Effect of butyrophilin gene polymorphism
on milk quality traits in crossbred cattle.
Asian Austral. J. Anim., 19: 922-926.
Biswas, T.K., T.K. Bhattacharya, A.D. Narayan,
S. Badola, K. Pushpendra and A. Sharma.
2003. Growth hormone gene polymorphism
and its effect on birth weight in cattle and
buffalo. Asian Austral. J. Anim.., 16: 494497.
Borghese, A.M.M. 2005. Buffalo population
and strategies in the world. In Buffalo
258
Buffalo Bulletin (December 2013) Vol.32 No.4
available at http://wwwgenome.wi.mit.edu/
genome_software/other/primer3.htm
Sambrook, J. and D.W. Russel. 2001. Molecular
Cloning: A Laboratory Manual, 3rd ed. Cold
Spring Harbour, New York.
259
Original Article
Buffalo Bulletin (December 2013) Vol.32 No.4
OXIDIZED LOW DENSITY LIPOPROTEIN RECEPTOR 1 (OLR 1) GENE POLYMORPHISM
IN MEHSANA BUFFALOES (Bubalus bubalis)
Manisha Deshpande*, D.N. Rank, P.H. Vataliya and C.G. Joshi
ABSTRACT
INTRODUCTION
Oxidized low density lipoprotein receptor 1
(OLR1) is the major protein that binds, internalizes
and degrades oxidized low-density lipoprotein. It
is reported that SNP 8,232 in the 3′-UTR in OLR1
was associated with milk fat yield and percentage.
This study was aimed to reveal the PCR-RFLP
pattern of OLR1 (3’UTR) in the Mehsana breed
of buffaloes. A fragment of OLR1-288 bp was
amplified by PCR, and subsequently, RFLP study
was carried out to identify genotypes of the
animals with PstI restriction enzyme. It revealed
monomorphic patterns.
Further representative samples were
cloned and in vector and after sequencing sequence
variation in nucleotide sequences of OLR1was
analysed. On comparison with published cattle
sequence the nucleotide sequence variation between
cattle and buffalo was present at nine nucleotide
positions i.e. 85, 91, 116, 129, 151, 168, 171, 217,
240.
The use of polymorphic markers in
breeding programmes could make selection more
accurate and efficient. The studies which are
attempted to detect genes affecting quantitative
traits via linkage to genetic markers can be divided
into two categories: analysis of candidate genes
and genome scans based on within-family genetic
linkage. The candidate gene approach consists of
the study of different genes potentially involved
in the physiological process (e.g., milk proteins
synthesis, milk fat synthesis) and identification
for each gene of the allele responsible for desired
phenotype. OLR1 is identified as a candidate gene
for milk production traits (Khatib et al., 2007).
Khatib et al. (2006) reported that SNP 8,232 in
the 3′-UTR was associated with milk fat yield and
percentage in Italian Brown Swiss.
OLR1 is the major protein that binds,
internalizes and degrades oxidized low-density
lipoprotein. The oxidized form of the low-density
lipoprotein (oxLDL) is involved in endothelial cell
injury, dysfunction, and activation, all of which are
implicated in the development of atherosclerosis
(Mehta and Li, 1998). It has been shown that oxLDL
and its lipid constituents have numerous damaging
effects on secretary activities of the endothelium,
including induction of apoptosis (Imanishi et al.,
2002). OLR1 was initially identified in bovine
Keywords: Mehsana buffaloes, Bubalus bubalis PCRRFLP, OLR1 gene, polymorphism
Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Anand
Agricultural University, Anand 388001, India, *E-mail: manisha2878@yahoo.com
260
Buffalo Bulletin (December 2013) Vol.32 No.4
followed by 35 cycles at 94°C for 1 minute, 54°C
for 45 seconds and 72°C for 1 minute with a final
extension at 72°C for 10 minutes.
For the PCR-RFLP analysis, 10 μl of
each PCR amplified product was digested with
5 units of the PstI (CTGCA↓G) in a 30μl total
reaction and incubated in a water bath at 37°C for
16 h. The digestion products were separated by
electrophoresis on a 2% agarose gel in 0.5% TBE
buffer.
aortic endothelial cells by Sawamura et al. (1997).
In addition to binding oxLDL, OLR1 removes
aged and apoptotic cells from blood circulation
(Oka et al., 1998). Information on OLR1 gene
polymorphism in Mehsana buffalo is very scanty;
hence, the present study was undertaken to reveal
the PCR-RFLP pattern of the OLR1 locus in the
Mehasana breed of buffaloes.
MATERIALS AND METHODS
Cloning and sequencing
PCR products from a representative 288
bp OLR1 3’UTR sample was purified and cloned
in pTZ57R/T vector (InsT/Aclone™ kit). Ligated
recombinant vector was transformed in competent
E. coli (DH5-α) cells. Recombinant plasmids
were extracted and used for cycle sequencing
and subjected to automated DNA sequencing on
ABI PRISM® 310 Genetic Analyzer (Applied
Biosystems, USA). Sequencing was carried out
using BigDye® Terminator v3.1 Cycle sequencing
kit (Applied Biosystems, USA). Sequence data
obtained was analyzed in silico by employing
software tools viz. NCBI BLAST, SeqScape and
ClustalW to access the genetic variation.
Buffalo population, sampling and DNA
extraction
To analyze the status of OLR1/ Pst1
polymorphism, blood samples were collected
randomly from 60 unrelated buffaloes of the
Mehsana breed registered under the progeny
testing programme of the Dudhsagar Research
and Development Association, Mehsana, Gujarat
state. DNA was extracted using standard protocol
by the phenol: chloroform extraction procedure
(Sambrook et al., 1989).
Molecular genotyping
Primers reported by Khatib et al. (2006)
for Bos taurus could not amplify the OLR1 3’ UTR
region in the Mehsana buffalo. Hence, new primer
pairs were designed (F: 5’CTG G AGGAAAA
GAAGGAAACC3’
R:
5’TGCTGTGA
CCTTGAGTTAGGC3’) using Bioinformatics
tools, Primer 3.0 and Primer Express softwares
(http://www.genome.wi.mit.edu/cgi_bin/primer/
primer3) on the basis of gene sequences available
in the data base NW_174132.2 to amplify the
desired fragment.
PCR was carried out in a final reaction
volume of 25 μl. Amplification cycling conditions
involved initial denaturation 94°C for 10 minutes,
RESULTS AND DISCUSSION
A 288 bp fragment of OLR1 gene loci was
amplified by PCR, using the designed primers. PCR
amplicons were digested with restriction enzyme
PstI. It was expected OLR1 had a PstI restriction
site at 215 bp and would produce two fragments
of 215 and 73 bp. On screening the OLR1/Pst1 in
the 60 Mahsana buffaloes, all the samples showed
an identical restriction pattern with the absence of
restriction site producing 288 bp fragment only
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Buffalo Bulletin (December 2013) Vol.32 No.4
(Figure 1). All the animals revealed only one
genotype (BB) and a monomorphic pattern with B
allele fixed in the Mehsana buffalo.
The molecular basis of OLR1/Pst1
polymorphism is mutation C→A in recognition
site of Pst1 restriction enzyme.
After sequencing and assembling the
sequences it was observed that the sequence has a
mutation at the site of Pst1i.e at nucleotide position
215. The recognition site of PstI 5’-C T G C A^G-3’
is mutated, T is deleted, and A added. Hence the
sequence observed is 5’–C- G A C A^G-3’(5’-C
T G C A^G-3’ →5’-C T G C A^G-3’) hence, the
The results of the present study, i.e. the
monomorphic pattern in the buffalo, are not in
accordance with Khatib et al. (2006).
A consensus sequence of 288 bp OLR1
UTR 3’ was obtained by assembling the forward
and reverse compliment sequence using the
Seq Scape software programme. The consensus
sequences were then aligned with known sequences
for OLR1 UTR 3’in GenBank using NCBI BLAST
and ClustalW programme. The nucleotide sequence
alignment of OLR1 with published sequences
revealed 96% to 99% homology (Table 1).
The nucleotide sequence variation
absence of the site in the fragment.
between cattle and buffalo for OLR1 (3’UTR)
Figure 1. OLR/Pst1 PCR-RFLP of OLR 1 gene 3’ UTR 288bp OLR 1 3’ UTR gene PCR
fragment in Mehsana buffalo digested by pstI.
Lane 1, 2 : OLR/Pst1 digest single fragment of 288 bp showing absence of site in the fragment
Lane 3 : 100 bp Ladder
Lane 5 : PCR product of OLR1 of 288 bp
Table 1. Bubalus bubalis OLR 1 - Blastn in GenBank + EMBL + DDBJ.
Sr.
No
Accession
1
BT029784.1
2
D89049.1
Location/
Source
Max
Indent
Bos taurus oxidised low density lipoprotein
(lectin-like) receptor 1 (OLR1), mRNA,
complete cds
(1-288)
862-1149
96 %
Bos taurus mRNA for lectin-like oxidized LDL
receptor, complete cds
(1-288)
858-1145
96 %
Description
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Buffalo Bulletin (December 2013) Vol.32 No.4
Plate 1. Clustal W results of OLR1 3’UTR.
263
Buffalo Bulletin (December 2013) Vol.32 No.4
was observed at nine nucleotide positions i.e. 85,
91,116,129,151,168,171,217,240 (Plate 1).
No buffalo sequences were available in
the GenBank database for OLR1 3’UTR before
preparing this manuscript. One genomic nucleotide
sequence of 288 bp consisting of OLR1 3’UTR was
submitted to Genbank of NCBI database accession
number GQ385226.
Natl. Acad. Sci. USA, 95: 9535-9540.
Sambrook, J. and D.W. Russell. 2001. Molecular
Cloning: A Laboratory Manual, 3rd ed. Cold
Spring Harbor Laboratory, Cold Spring
Harbor, New York.
Sawamura, T., N. Kume, T. Aoyama, H. Moriwaki,
H. Hoshikawa, Y. Aiba, T. Tanaka, S. Miwa,
Y. Katsura, T. Kita and T. Masaki. 1997.
An endothelial receptor for oxidized lowdensity lipoprotein. Nature, 386: 73-77.
REFERENCES
Imanishi, T., T. Hano, T. Sawamura, S. Takarada
and I. Nishio. 2002. Oxidized low density
lipoprotein potentiation of Fas-induced
apoptosis through lectin-like oxidized-low
density lipoprotein receptor-1 in human
umbilical vascular endothelial cells. Circ.
J., 66: 1060-1064.
Khatib, H., S.D. Leonard, V. Schutzkus, W. Luo
and Y.M. Chang. 2006. Association of
the OLR1 gene with milk composition in
Holstein dairy cattle. J. Dairy Sci., 89(5):
1753-1760.
Khatib, H., K. Rosa, F. Weigal, F. Schiavini, E.
Snatus and A. Bagnato. 2007. Additional
support for an association between OLR1
and milk fat traits in cattle. Anim. Genet.,
38: 308-310.
Mehta, J.L., and D.Y. Li. 1998. Identification and
autoregulation of receptor for OX-LDL in
cultured human coronary artery endothelial
cells. Biochem. Bioph. Res. Co., 248: 511514.
Oka, K., T. Sawamura, K. Kikuta, S. Itokawa, N.
Kume, T. Kita and T. Masaki. 1998. Lectinlike oxidized low-density lipoprotein
receptor 1 mediates phagocytosis of aged/
apoptotic cells in endothelial cells. Proc.
264
Buffalo Bulletin (December 2013) Vol.32 No.4
Original Article
EVALUATION OF FOLLICULAR ATRESIA AND ELECTROPHORETIC PATTERN OF
FOLLICULAR FLUID PROTEINS IN ACYCLIC BUFFALO (Bubalus bubalis)
F.A. Khan1,*, G.K. Das1, Megha Pande1, Rajendra Singh2 and S.K. Ghosh3
ABSTRACT
follicle, atresia, follicular fluid
Histological examination of H&E stained
sections of ovaries collected from cyclic and
acyclicbuffaloes (n=6/group) was done in order
to evaluate the degree of follicular atresia. The
percentages of healthy and atretic follicles were
different (P<0.05) between cyclic (53.1% and
46.9%, respectively) and acyclic (10.7% and 89.3%,
respectively) buffaloes. Electrophoretic patterns of
follicular fluid proteins, studied by SDS-PAGE of
follicular fluid samples aspirated from small (5.06.9 mm), medium (7.0-9.9 mm) and large (≥ 10
mm) follicles of cyclic and acyclic buffaloes (n=
6/group), did not reveal any apparent differences
between the groups. DNA fragmentation patterns
evaluated by using DNA isolated from the cell
pellets obtained after centrifugation of the follicular
fluid samples from small-, medium- and largesized follicles of cyclic and acyclic buffaloes (n=5/
group) showed a typical apoptotic oligonucleosome
ladder pattern in the acyclic group. In conclusion,
these results indicate an increased rate of follicular
atresia without any qualitative changes in the
follicular fluid proteins during ovarian acyclicity in
buffalo.
INTRODUCTION
Follicular development in buffalo is a
continuous process with concurrent growth and
regression of ovarian follicles occurring during
the reproductive cycle. Similar to those in cattle,
follicular dynamics in buffalo follows a wave
pattern with predominance of two-wave cycles
(Baruselli et al., 1997). During each follicular wave,
there is recruitment of a group of follicles (cohort)
out of which usually one is selected for progressive
growth to become the dominant follicle while the
others undergo regression. Dominant follicles can
have variable fates-ovulation, persistence or atresia,
depending upon several factors like physiological
state, stage of the cycle, pathological conditions
or hormonal treatments etc. An increased rate
of follicular atresia has been implicated as one
of the major factors for reproductive failure in
buffalo (Rajesha et al., 2001). Atresia of ovarian
follicles is associated with various morphological,
biochemical and histological changes (Kaipia and
Hsueh, 1997). A recent study on the follicular
characteristics during ovarian acyclicity in buffalo
showed that all the large-sized (≥ 10 mm) follicles
Keywords: buffaloes, Bubalus bubalis, ovary,
Animal Reproduction Division, Department of Pathobiological Sciences, 1656 Linden Drive University of
Wisconsin-Madison, Madison, WI 53706, *E-mail: fakhan3@wisc.edu
2
Centre for Animal Disease Research and Diagnosis
3
Germplasm Centre, Indian Veterinary Research Institute, Izatnagar 243122, India
1
265
Buffalo Bulletin (December 2013) Vol.32 No.4
characteristics of the ovarian follicles, especially
the granulosa and theca cell layers. Classification
of follicles into healthy and atretic was done as
described previously (Guraya, 1979).
Follicular fluid was aspirated from small,
medium-sized and large follicles of cyclic and
acyclic buffaloes (n=6/group), centrifuged at 1000
g and 4°C for 10 minutes and the supernatant
was collected for analysis. The electrophoretic
pattern of follicular fluid proteins was studied by
a standard protocol (Laemmli, 1970) using 10%
polyacrylamide gel in a vertical slab gel apparatus
(Bangalore GENEI, India).
For studying the apoptotic oligonucleosome
pattern, the cell pellets obtained after centrifugation
of the follicular fluid samples from small-,
medium- and large-sized follicles of cyclic and
acyclic buffaloes (n=5/group) were used. DNA
was isolated using a DNeasy Kit as per the protocol
recommended by the manufacturer (Quiagens
Ltd.). For visualizing the probable presence of the
oligonucleosome ladder pattern characteristically
shown by apoptotic DNA fragments (Hermmann
et al., 1994), 10 μl of DNA sample obtained was
electrophoresed in 2% agarose gel containing 1 μg/
ml of ethidium bromide and visualized in a UV-Gel
Documentation system (Syngene, UK).
Comparison of percentage normal and
atretic follicles between cyclic and acyclic groups
was done by Chi-square test using STATSTM Version
1.1, Decision Analyst Inc., USA.
are estrogenically inactive, which indicates a
derangement in the normal follicular development
culminating in follicular atresia rather than ovulation
(Khan and Das, in press). Studies on biochemical
composition of the follicular fluid demonstrated
alterations in certain components like nitric oxide,
ascorbic acid, glucose, cholesterol and alkaline
phosphatase during acyclicity in buffalo leading
to the conclusion that oxidative stress caused by
the imbalance of nitric oxide and ascorbic acid
possibly results in follicular atresia manifested
as inactive estrogen status and increased alkaline
phosphatase levels in the follicular fluid (Khan and
Das, in press; Khan et al., 2011). Available evidence
suggests that follicular fluid protein concentrations
are not affected by the reproductive state of the
animal (Khan et al., 2011). However, the effect of
acyclicity on the electrophoretic pattern of follicular
fluid proteins remains unknown. The objectives of
the present study were to evaluate follicular atresia
in ovarian follicles of acyclic buffaloes using
ovarian histology and DNA fragmentation patterns
(oligonucleosome ladder pattern) characteristic
of apoptosis, and to examine the changes in the
electrophoretic pattern of follicular fluid proteins
during acyclicity in buffalo.
MATERIALS AND METHODS
Ovaries were collected from cyclic and
acyclic buffaloes (n=6/group) at a local abattoir
and transported to the laboratory on ice within 30
minutes. The ovaries were fixed in 10 % formalin,
embedded in paraffin wax, cut into thin sections of
5-7 μm thickness and stained with haematoxylin
and eosin (H&E) using routine histological staining
procedure (Luna, 1968). The stained slides were
examined by light microscopy for histological
REFERENCES AND DISCUSSION
The percentages of healthy and atretic
follicles were different (P<0.05) between cyclic
(53.1% and 46.9%, respectively) and acyclic (10.7%
and 89.3%, respectively) buffaloes. The concomitant
266
Buffalo Bulletin (December 2013) Vol.32 No.4
it can be concluded that there are no qualitative or
quantitative alterations in the protein component of
the follicular fluid during acyclicity in buffalo.
In summary, results of the present study
indicate an increased rate of follicular atresia
characterized by histological alterations in the
follicle and fragmentation of granulosa cell
DNA without any characteristic change in the
electrophoretic pattern of follicular fluid proteins
during ovarian acyclicity in buffalo.
presence of both healthy and atretic follicles in
cyclic buffalo ovaries indicates that growth and
regression of follicles occur concurrently further
confirming the previous observations that follicular
development is a continuous process in buffalo
(Baruselli et al., 1997). Based on the presence of
an almost equal proportion of healthy and atretic
follicles in cyclic buffaloes, it seems apparent that
growth and regression of follicles occur at a similar
rate during the estrous cycle. The presence of both
healthy and atretic follicles in acyclic buffaloes
provides support to our earlier notion that follicular
development does continue during acyclicity as
well (Khan and Das, in press). The predominance
of atresia in acyclic buffalo ovaries is in line with
observations during acyclicity based on estrogenic
status (Khan and Das, in press) and the findings of
Guraya (1979) who reported an increased incidence
of atresia in buffalo ovaries during anestrus.
A prominent apoptotic DNA fragmentation
(oligonucleosome ladder) pattern was observed in
DNA isolated from the granulosa cells and oocytes
of all the three follicle size categories (Figure 1;
lanes 1, 2 and 3) in acyclic buffaloes. In contrast,
no such pattern was evident in the cyclic group
(Figure 1; lanes 4, 5 and 6). The higher percentage
of atresia indicated by the above results compared
to the observations on histology can be inferred to
be a reflection of changes occurring in the DNA
earlier than the manifestation of the morphological
alterations during follicular atresia.
ACKNOWLEDGMENTS
The authors thank Drs. Prafull Singh, M.Y.
Wani and S.R. Bisla for their help during DNA
extraction and SDS-PAGE and the staff of the
Division of Pathology, Indian Veterinary Research
Institute for their assistance with the ovarian
histology.
REFERENCES
Baruselli, P.S., R.G. Mucciolo, J.A. Visintin, W.G.
Viana, R.P. Arruda, E.H. Madureira, C.A.
Oliveira and J.R. Molero-Filho. 1997.
Ovarian follicular dynamics during the
estrous cycle in buffalo (Bubalus bubalis).
Theriogenology, 47: 1531-1547.
Guraya, S.S. 1979. Morphological and
histochemical observations on buffalo
ovaries during anoestrus. Indian J. Anim.
Sci., 49: 423-432.
Herrmann, M., H.M. Lorenz, R. Voll, M. Grunke,
W. Woith and J.R. Kalden. 1994. A rapid and
simple method for the isolation of apoptotic
DNA fragments. Nucleic Acids Res., 22:
5506-5507.
SDS-PAGE analysis of follicular fluid
proteins did not reveal any apparent difference in the
overall electrophoretic pattern between acyclic and
cyclic groups (Figure 2). Pertinently, in an earlier
study on the biochemical composition of follicular
fluid (Khan et al., 2011), no difference in the total
protein concentration was recorded between cyclic
and acyclic buffaloes. Based on these observations,
267
Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 1. Electrophoretic pattern of follicular fluid proteins (Lane M-Marker; Lanes 1-3: small, mediumsized and large follicles of the acyclic group, respectively; Lanes 4-6: small, medium-sized and
large follicles of the cyclic group, respectively).
Figure 2. Granulosa cell DNA fragmentation pattern (Lanes 1-3: small, medium-sized and large follicles
of the acyclic group, respectively; Lanes 4-6: small, medium-sized and large follicles of the
cyclic group, respectively).
268
Buffalo Bulletin (December 2013) Vol.32 No.4
Kaipia, A. and A.W. Hsueh. 1997. Regulation of
ovarian follicle atresia. Annu. Rev. Physiol.,
59: 349-363.
Khan, F.A. and G.K. Das. 2012. Follicular
characteristics
and
intrafollicular
concentrations of nitric oxide and ascorbic
acid during ovarian acyclicity in water
buffalo (Bubalus bubalis). Trop. Anim.
Health Pro., 44(1): 125-131.
Khan, F.A., G.K. Das, Megha Pande, R.A. Mir
and Uma Shankar. 2011. Changes in
biochemical composition of follicular fluid
during reproductive acyclicity in water
buffalo (Bubalus bubalis). Anim. Reprod.
Sci., 127: 38-42.
Laemmli, U.K. 1970. Cleavage of structural
proteins during the assembly of the heat of
bacteriophage T4. Nature, 227: 680-685.
Luna, L.G. 1968. Manual of Histological Staining
Methods of the Armed Forces Institute of
Pathology, 3rd ed. McGraw-Hill Book Co.,
New York, USA.
Rajesha, D., J.P. Ravindra and M. Narayanaswamy.
2001. Ovarian antral follicular activity
and serum estradiol-17β concentrations in
buffaloes during different periods of the
year. Indian J. Anim. Sci., 71: 641-643.
269
Original Article
Buffalo Bulletin (December 2013) Vol.32 No.4
REPRODUCTIVE CYCLE STAGE BIAS IN PHYSIOLOGICAL AND IMMUNE RESPONSES
TO ENDOTOXIN CHALLENGE IN MURRAH BUFFALOES (Bubalus bubalis)
Z.A. Pampori1 and S. Pandita2
hyperglycemia was registered which culminated
in hypoglycaemia. Total leukocytes were higher in
the estrous than the diestrous stage but endotoxin
challenge did not affect the total counts. However,
there was neutrophilia after endotoxin challenge
in both the stages of the reproductive cycle in
ABSTRACT
Reproductive cycle in large domestic
animals has a distinctly different hormonal milieu
with estrus dominated by estrogen and diestrous
by progesterone. Since the sex steroids are caught
up in disease severity, present study investigated
variability in immune responses upon endotoxin
challenge at day 0 (estrous) and day 10 (diestrous)
of the reproductive cycle in Murrah buffaloes.
Physiological responses like rectal temperature,
heart rate, pulse rate and immune responses like
plasma TNFα, nitric oxide, xanthine oxidase,
cortisol and glucose were evaluated before and
after LPS challenge (E. coli 055:B5 0.6 μg/kg
body weight). Physiological as well as immune
responses were heightened during estrous as
compared to the diestrous stage of the cycle after
endotoxin challenge. The area under the curve
(AUC) for rectal temperature and heart rate was
significantly (P<0.05) higher at estrous as compared
to diestrous. Integrated responses of TNFα, nitric
oxide and xanthine oxidaes to LPS challenge
calculated as AUC were significantly (P<0.05)
higher at estrous as compared to the diestrous stage
of the estrous cycle. AUC for plasma cortisol, an
anti-inflammatory mediator, was significantly
(P<0.001) higher at the diestrous than the estrous
stages of cycle. During first 2 h of endotoxin insult,
buffaloes. The results indicate that the underlying
physiological attributes of stage of reproductive
cycle represents a source of variability in immune
competence when challenged.
Keywords: Murrah buffaloes, Bubalus bubalis,
Reproductive cycle, LPS, TNFα, Nitric oxide,
Cortisol
INTRODUCTION
The buffalo is an economically important
livestock species in Asian and Mediterranean
countries with India having 56% of total world
buffalo population which hold good potential for
production. Several lines of investigation have
reported that sex steroids influence the immune
response to the antigenic challenges and outcome
of the insult (Olsen and Kovacs 1996; Jorg et
al., 1998; Giltay et al., 2000; Losonczya et al.,
2000; Marco et al., 2009). Many workers have
demonstrated that the hormone environment
Division of Veterinary Physiology, Sher-e-Kashmir University of Agricultural Sciences and Technology of
Kashmir (SKUAST-K), J&K State 190006, India, E-mail: drzap64@gmail.com
2
Dairy Cattle Physiology Division, National Dairy Research Institute (ICAR), Karnal 132001, India
1
270
Buffalo Bulletin (December 2013) Vol.32 No.4
at the time of infection has a profound effect on
the outcome of microbial infection in the female
reproductive tract (Ramadan et al., 1997; Kaushic
et al., 2000; Brabin, 2002). Studies indicate that
uterine immune function is enhanced during the
follicular phase and estrogen treatment enhanced
uterine immune function in ovariectomized ewes,
mares and cows (Washburn et al., 1982; Roth et
al., 1983; Carson et al., 1988; Lander et al., 1990).
Wulster et al. (2003) reported in gilts, enhanced
susceptibility to uterine infections after intrauterine challenge with E. coli and Arcanobacterium
pyogenes during diestrus. Jason and Joseph (2008)
reported that proestrous mice are protected from
cardiovascular and immunological dysfunction
following trauma-haemorrhage insult. Tibbetts et al.
(1999) reported estrogen to have proinflammatory
effects on neutrophil and macrophage infiltration in
the mouse uterus during the estrous cycle. Several
lines of investigation in humans suggest that
disease expression is affected by the reproductive
status and diseases like multiple sclerosis, asthma
or systemic lupus erythematosis are exacerbated
during specific periods of the menstrual cycle or
pregnancy (Skobeloff et al., 1996; Case and Reid,
1998; Whitacre, 2002). Sartin et al. (2003) have
shown that estrogen plus progesterone implants
favourably alter the time course or disease severity
of many of clinical manifestations associated with
coccidiosis and endotoxemia in calves. Ansar et
al. (1985) and Cutolo et al. (1995) maintained
that androgens and progesterone exert suppressive
effects on both humoral and cellular immune
responses and seem to represent natural antiinflammatory hormones whereas estrogens exert
immune-enhancing activities.
Cytokine responses to provocative
stress challenges modelled by endotoxin (LPS)
administration, as well as active infection has
received great attention as markers and mediators of
both homeostatic and pathophysiological processes
in vivo. Realising the probable influence of sex
steroids in immune responses, the present study
was planned to investigate variability in immune
responses in vivo in cyclic buffaloes at estrus
and diestrous stages of cycle in which distinctly
different sex steroid milieu are maintained.
MATERIALS AND MERHODS
Ten apparently healthy female cyclic
Murrah buffaloes of 1st parity were selected from
the cattle research station of the institute and
grouped into two E (estrous) and D (diestrous)
with five buffaloes in each group. Group E had
cyclic buffaloes at day 0 of estrous cycle whereas
Group D at day 10 of estrous cycle. Stage of cycle
in the buffaloes was confirmed through rectal
examination by the concerned and well experienced
veterinarian at the farm. All these Murrah buffaloes
were maintained under routine management and
nutritional practices as followed in the herd at the
institute. Four animals from each group were given
a single intravenous bolus of LPS (E. coli 055:B5
from Sigma Chemical Co., St. Louis, Missouri,
USA.) 0.6 μg/kg body weight, in 10 ml sterile normal
saline in the jugular vein. One animal serving as
control from each group was administered 10 ml of
sterile normal saline intravenously as placebo. The
experiment was conducted in the morning in the
month of September, with the average maximum
temperature 30.5°C and the minimum 23.3°C.
Before LPS challenge physiological parameters
like rectal temperature, heart rate and respiration
rate were recorded, and 6 ml of blood was drawn
in heparinised vacutainers (Becton-Dickinson
and Company, USA.) from the jugular vein in all
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Buffalo Bulletin (December 2013) Vol.32 No.4
1.56 μM. Inter assay and intra assay coefficients of
variance were 3.4% and 4.62% respectively.
Plasma tumor necrosis factor α was
evaluated by using a bovine tumor necrosis factor-α
ELISA kit from Cusabio Biotech Co., Ltd. The
sensitivity of assay was 0.05 ng/ml and inter assay
and intra assay coefficient of variance was 6.25 and
5.4% respectively.
Plasma xanthine oxidase was estimated by
using xanthine oxidase assay kit from Bio Vision
Research Products, USA. The detection limit was
1 mU/100ml of reaction volume. Inter assay and
intra assay coefficients of variance were 7.5 and
6.75% respectively.
Plasma cortisol was determined by using
a cortisol EIA kit from Cayman. The sensitivity of
the assay kit was 6.6 pg/ml. The inter assay and
intra assay coefficients of variance were 6.7 and
6.25% respectively.
Sex steroids estrogen and progesterone
were estimated by RIA using 3H tracers. Antiserum
for estradiol was procured from Sigma Chemical
Co., St. Louis, Missouri, USA. whereas that
for progesterone was a gift of Dr. B.S.Prakash.
Progesterone in plasma was estimated by a direct
RIA technique developed by Kamboj and Prakash
(1993). Estrogen was extracted in benzene and
counting of β-radiation was performed in a
Beckman β counter, USA. Recoveries of estradiol
and progesterone were 86% and 95%, respectively.
Inter-assay and intra-assay coefficients of variance
were 13.3% and 10.8% in estradiol and 9.4% and
10.25% in progesterone.
The data analysis was performed using
a Systat 12 software package (Systat Software
Inc 1735 Technology Dr., Ste.430, San Jose, CA
95110, USA). Analysis of variance of the data was
performed using two way ANOVA with variables,
group and time, included in the model as fixed
animals after taking all necessary aseptic measures.
After LPS challenge, physiological parameters
were recorded and 6 ml blood drawn in heparinised
vacutainers, at 1, 2, 4, 8 and 24 h from all animals
including control. During the whole experiment,
the animals had free access to drinking water and
fodder. The blood samples were transported to the
laboratory on ice within 30 minutes. Total leukocyte
count (TLC) and differential leukocyte count (DLC)
were determined immediately after collection as
described by Schalm et al. (1975), using Field stain
for DLC. Plasma was separated by centrifugation
and was stored at -20°C in aliquots of 0.5 ml in 1.5
ml micro-centrifuge tubes till analysis. The present
experimentation was conducted in buffaloes after
taking proper permission from the ethics committee
of the Institute vide IAEC No. 23/09-21/11/2009.
Plasma nitric oxide was estimated as total
nitrite (NOx) using a modified Griess reaction
as described by Miranda et al. (2001). The test
involved preparation of Griess-I (2% sulfanialamide w/v in 5% HCL), Griess-II (0.1% N-Inaphthyl ethylendiamine dihydrochloride w/v in
Milli Q water) and vanadium chloride -III (VCl3,
8 mg per ml of 1 M HCL). The chemicals were
purchased from Sigma Chemical Co., St. Louis,
Missouri, USA. Deproteinization of plasma was
achieved by acetonitrile as described by Ghasemi
et al. (2007). 100μl of each deproteinized sample
and standard (sodium nitrite) was pipetted out in
duplicates in a 96 well microtitre plate. 100 μl of
VCl3 reagent was added to each well, followed by
100μl of Griess reagent (Griess-I + Griess-II in
1:1 ratio) immediately. Incubation at 37°C for 30
minutes was carried out before absorbance was read
at 540 nm wavelength in an ELISA plate reader
(Microscan MS-5608A). The concentration was
determined from the standard curve using a linear
regression equation. Detection limit of NO was
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Buffalo Bulletin (December 2013) Vol.32 No.4
significantly different from the 54.41±0.51 beats/
min in Group D at LPS challenge. However, the
heart rate varied significantly (P<0.001) between
the time intervals; it peaked at 1 h and started
declining thereafter to baseline values (Figure 1b).
Integrated heart rate responses to LPS challenge
calculated as AUC with baseline control subtracted
for the first 8 h was significantly (P<0.01) higher in
Group D than Group E buffaloes (Table 1.1).
Group E buffaloes registered a significantly
(P<0.001) higher 24 h mean respiration rate
(26.45±0.34
breath/min) than Group D
(24.41±0.34) at LPS challenge. Respiration rate
varied significantly (P<0.001) between the time
intervals; it started increasing soon after LPS
challenge, reached its peak at 1 h (28.87±0.59),
remained higher than baseline even at 8 h post
challenge and reached normal baseline beyond 8
h (Figure 1c). Respiration rate responses to LPS
challenge calculated as AUC with baseline control
subtracted for first 8 h was not significantly different
in Group E and Group D females (Table 1.1).
With respect to the immune mediators,
Group E buffaloes registered a significantly
(P<0.001) higher mean of 24 h plasma TNFα
(2.079±0.07 ng/ml) than Group D (0.523±0.07 ng/
ml) at LPS challenge. TNFα levels increased shortly
after challenge, peaked at 2 h but then receded
to 0 h level at 8 h post challenge in Group D but
remained elevated beyond 8 h in Group E (Figure
2a). Integrated TNFα responses to LPS challenge
determined as area under time x concentration
curve (AUC) with baseline control subtracted for
first 8 h was significantly (P<0.01) higher in Group
E than in Group D buffaloes (Table 1.1).
Group E buffaloes registered significantly
(P<0.001) higher 24 h mean plasma NO (39.20
±0.61 μM) than Group D buffaloes (26.53±0.61
μM) at LPS challenge. Plasma NO levels increased
effects and Tukey’s Honestly-significant difference
test was employed. Values are presented as mean ±
S.E. Graph and charts were prepared in Microsoft
Excel 2007. Area under concentration x time curve
(AUC) was calculated by commonly approached
numerical approximation method called the
trapezoidal rule.
RESULTS
In Group E buffaloes, the average estradiol
levels were 10.41±0.61 pg/ml which differed
significantly (P<0.01) from the 7.16±0.70 pg/
ml registered in Group D buffaloes. The average
plasma levels of progesterone in Group E and Group
D buffaloes were 0.28±0.05 ng/ml and 2.21±0.13
ng/ml, respectively; the difference between the two
groups was significant (P<0.01).
The important physiological parameter
rectal temperature was recorded before LPS
challenge (0 h) and at 1 h, 2 h, 4 h, 8 h and 24 h post
challenge. Group E (estrous) buffaloes registered
an average of 24 h rectal temperature significantly
(P<0.01) higher (101.804±0.17°F) than Group
D (diestrous) buffaloes (100.963±0.17°F) at LPS
challenge. Temperature variation between the time
intervals was statistically significant and peaked
between 2 to 4 h after LPS challenge in both the
groups. Subsequently rectal temperature declined
to the normal 0 h levels beyond 8 h (Figure 1a).
Integrated rectal temperature responses to LPS
challenge calculated as AUC (area under curve)
with baseline control subtracted for first 8 h
was significantly (P<0.05) higher in Group E as
compared to Group D Murrah buffaloes (Table
1.1).
Group E buffaloes registered 24 h mean
heart rate 55.37±0.57 beats/min; this was not
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Buffalo Bulletin (December 2013) Vol.32 No.4
significantly (P<0.001) during the course of LPS
challenge with levels reaching their peaks at 8 h
post challenge (Figure 2b). Integrated NO response
to LPS insult calculated as AUC with baseline
control subtracted for 24 h was significantly
(P<0.01) higher in Group E than Group D cyclic
buffaloes (Table 1.1).
The mean plasma level of xanthine
oxidase was not different between Groups E and
D buffaloes amounting to 5.78±0.10 and 5.62±
mU/ml, respectively, as an average of 24 h at LPS
challenge. However, plasma XO levels varied
significantly (P<0.001) between time intervals and
registered peaks at 8 h after LPS challenge. Levels
remained higher than 0 h even after 24 h of challenge
(Figure 2c). Integrated XO responses to endotoxin
challenge determined as AUC with baseline control
subtracted for 24 h was significantly (P<0.001)
higher in Group E than in Group D buffaloes (Table
1.1).
Group E buffaloes registered average 24
h plasma cortisol significantly (P<0.001) lower
(1.30±0.02 ng/ml) than Group D (1.75±0.02 ng/
ml) at LPS challenge. Plasma cortisol levels
varied significantly (P<0.001) between the time
intervals, registered peaks at 8 h, and then dropped
to baselevel by 24 h post challenge. In Group D
cortisol was significantly higher than in Group
E from h 2 post challenge and remained high for
24 h (Figure 2d). Integrated cortisol responses
to challenge calculated as AUC for first 8 h were
significantly (P<0.001) higher in Group D than in
Group E buffaloes (Table 1.1).
Buffaloes in Group E registered a 24 h
mean plasma glucose level significantly (P<0.001)
higher (58.80±0.80 mg/dl) than Group D females
(53.33±0.80 mg/dl) at LPS challenge. Plasma
glucose levels varied significantly (P<0.001)
between the time intervals and registered
hyperglycemia with peaks at 1 h after LPS
challenge. Thereafter, the glucose levels receded
below base level only at 2 h post challenge in Group
D but not in Group E whereas buffaloes of both
the groups registered hypoglycaemia at 4 h to 8 h
post challenge; levels returned to normal baseline
by 24 h (Figure 2e). Plasma glucose response to
Table 1. Integrated responses of various immune mediators calculated as area under time x concentration
curve (AUC) after LPS challenge at different stages of reproductive cycle in Murrah buffaloes.
Parameters
Rectal temperature 0-8 h (°F x h)
Heart rate 0-8 h (beats/min x h)
Respiration rate 0-8 h (breath/min x h)
TNFα for 0-8 h (ng/ml x h)
Nitric oxide for 0-24 h (μM/L x h)
XO for 0- 24 h (mU/ml x h)
Cortisol 0- 8 h (ng/ml x h)
Glucose 0-2 h (mg/dl x h) [Hyperglycaemia]
Glucose 2-8 h (mg/dl x h)
[Hypoglycaemia]
Estrous (d 0)
16.43 a ± 3.81
20.625 a ± 2.136
20.500 ± 1.323a
11.297a ± 1.34
358.217a ± 25.07
61.210a ± 4.17
10.413b ± 0.32
22.091a ± 1.46
Diestrous (d 10)
4.13 b ± 2.01
12.875 ± 2.230b
17.250 ± 2.016a
3.636b ± 0.28
224.962b ± 14.80
32.360b ± 1.37
14.862a ± 0.35
11.334b ± 0.90
-25.707a ± 2.49
-43.345a ± 5.04
Values in the same row with different superscripts differ significantly (P<0.05).
274
Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 1a.
Figure 1b.
Figure 1c.
Figure 1a-c. Temporal changes in physiological parameters after LPS challenge in cyclic Murrah
buffaloes. Data represents means ± SE (n= 10), *P< 0.05 at same point of time.
275
Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 2a.
Figure 2b.
Figure 2c.
Figure 2d.
Figure 2e.
Figures 2a-e. Temporal changes in plasma TNFα (ng/ml), NOx (μM/L), XO (mU/ml), cortisol
(ng/ml) and glucose (mg/dl) after LPS challenge in cyclic Murrah buffaloes. Data represent
means ± SE (n= 10), *P< 0.05 at the same point of time.
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Buffalo Bulletin (December 2013) Vol.32 No.4
DISCUSSION
LPS determined as AUC for 0 to 2 h and 2 -8 h
of LPS challenge revealed hyperglycemia during
the first 2 h with AUC significantly (P<0.001)
higher in Group E buffaloes as compared to Group
D buffaloes and hypoglycaemia from 2-8 h post
challenge with AUC significantly (P<0.001) lower
in Group D than Group E buffaloes (Table 1.1).
Buffaloes in Group E had a significantly
(P<0.01) higher mean total leukocyte count
(12.25±0.31x103) than Group D (10.90±0.31x103)
at LPS challenge. However, there was no
significant change in total leukocyte count between
0 h (11.30±0.31) and 24 h (11.85±0.31) after LPS
challenge (Figure 3). Lymphocyte percentage
did not significantly differ between Group E and
D buffaloes. Group E and D buffaloes registered
neutrophilia after LPS challenge with average
neutrophil percentages of 22.250±0.80 and
25.00±0.08 at 0 h increasing significantly (P<0.01)
to 29±0.80% and 28±0.80%, respectively, at 24 h
after LPS challenge (Figure 3).
During the last two decades, sex hormones
have become recognized as integral signalling
modulators of the mammalian immune system.
Endotoxin challenge in animal models to profess
cytokine responses has received great attention as
indicators for and mediators of both homeostatic
and pathophysiological processes in vivo.
Variability in the levels of immune mediators like
TNFα, xanthine oxidase or nitric oxide between the
stages of the reproductive cycle strongly suggest
the role of sex hormones in immune-modulation,
which is supported by Bouman et al. (2005).
Immunological evidence suggests that female sex
hormones play a role in the aetiology and course of
chronic inflammatory diseases (Cutolo and Wilder,
2000).
The plasma estradiol levels were
reasonably high in Group E and perceptible in
Group D whereas plasma progesterone was far
Figure 3. Changes in total leukocyte count (TLC) & differential leukocyte count (DLC) before and after
24 h of LPS challenge in cyclic Murrah buffaloes. Data represent means ± SE (n=10), superscript
A & B or a & b differ significantly (P<0.05).
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Buffalo Bulletin (December 2013) Vol.32 No.4
less in Group E than Group D buffaloes. Present
findings were similar to the report of Shafie et al.
(1982) who recorded peak levels of estradiol (20
pg/ml) during follicular phase and 16.7 pg/ml on
day 11of estrous cycle. Since blood sampling was
done after the onset of estrus , the levels of estrogen
observed in present investigation could not have
reflected peaks. However, the sex steroid levels
observed in present study were comparable to the
levels reported by Sartori et al. (2004) in Holstein
Frisian heifers, Dhali et al. (2006) in mithun and
Kanai and Shimizu (1984) in swamp buffaloes.
The progesterone levels in Group D and Group
E buffaloes were lower than the levels reported
by Shafie et al. (1982) in buffaloes but closely
approximated the levels reported by Rao and
Pandey (1982) in buffaloes, Dhali et al. (2006) in
mithun and Mondal and Prakash (2003) in Sahiwal
cows. The present study demonstrated that Group E
and Group D buffaloes had distinctively different
sex steroid hormone backgrounds.
Group E buffaloes reported higher
values for physiological parameters at endotoxin
challenge as compared to Group D indicating
dimorphism in responses to endotoxin in cyclic
females during estrus and diestrous characterised
by different sex steroid milieu. The Group E
buffaloes were, therefore, moderate responders
in agreement with the findings of Kahl et al. (2009)
in cattle, Horadagoda et al. (2002) in buffaloes,
Michie et al. (1988) in humans, and Schlafer et al.
(1994) in sheep. Due to variations in sex steroid
levels with the stage of estrous cycle, estrogen and
progesterone might be candidates for hormones
regulating immune responses to LPS challenge.
Plasma TNFα levels were perceptibly low at all time
points in Group D buffaloes as compared to Group
E; however, the TNFα levels elevated immediately
after LPS challenge and were at peak after only 1
h in both the groups of animals, but TNFα levels
remained significantly higher than 0 h level for a
longer duration in Group E buffaloes as compared
to Group D. TNFα, a primary inflammatory
cytokine produced by immune cells, governs the
secretion and cascade of other cytokines involved
in immune reactions to antigenic challenge, and the
outcome of disease is, therefore, largely monitored
by this cytokine. Buffaloes at estrus with high
TNFα may counter the challenge effectively by
involving other immune mediators or cytokines
but at the same time animals might get exposed to
pathophysiological risks also. Similarly, NO was
higher in Group E buffaloes at every point of time
than Group D and peaked at 8 h post challenge in
both the groups. Whereas XO peaked at 8 h post
challenge and was significantly different in Group E
and Group D low responders. Since the estrogen is
believed to enhance the immune reactions (Jason
and Joseph, 2008; Tibbetts et al., 1999), Group E
animals had estrogen as a predominant sex steroid
present in circulation, and this could probably have
been responsible for heightened physiological
responses to endotoxin challenge as compared to
Group D animals with progesterone as a major
circulatory sex steroid. In the present study, Group
E females registered higher levels of immune
response mediators than Group D females,which is
than in Group D buffaloes. LPS challenge resulted
in increased circulatory levels of XO as well as
NO, which both play an important role in immune
defence and homeostasis. XO participates in the
activation of systemic inflammatory cells such as
increased adherence and/or rolling of neutrophils
to the endothelium, which, if not balanced, leads
to lung injury with poor prognosis. However, at the
same time, elevated nitric oxide counteracts XO
functions by reducing P-selectins, and thus helps
in the maintenance of homeostasis (Lance et al.,
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Buffalo Bulletin (December 2013) Vol.32 No.4
1997).
and Druckman, 2005). The overall frail responses
of immune mediators to endotoxin challenge
in Group D buffaloes could probably be the
handiwork of progesterone, which predominates
during diestrous.
White blood cells are indispensible cellular
components of two important arms of the immune
system, the cell mediated and humoral immunity.
In the present study, the shifts in the counts of two
major cells with neutrophilia was evident after LPS
challenge in both the groups of buffaloes. Any type
of stress is recognised by neutrophilia (Pampori et
al., 2010), and in the present study, neutrophilia
suggested that the buffaloes were under stress due
to LPS challenge.
The present study revealed that the
variability in immune response mediators were
present as a function of endocrinological character
of the two stages (estrous and diestrous) of the
estrous cycle. Therefore, sex hormone balance
remains a crucial factor in the regulation of immune
and inflammatory responses and the therapeutical
modulation of this balance should represent a
part of advanced biological treatments for many
diseases. Further, the variability in immune
responses inherent in female subjects at different
stages of estrous cycle could affect the outcome of
interventions.
A good quantity of investigation supports
the view that progesterone suppresses immunity
and increases susceptibility to infections as reported
by Ramadan i. (1997) in sheep, White et al. (1997)
in humans , Kaushic et al. (2000) in mice, WulsterRadcliffe et al. (2003) in gilts and Kahl et al. (2009)
in cattle. Progesterone, referred to commonly as
immunosuppressive, was supported by the present
study and probably may be a requirement for
reception and attachment of an embryo in the uterus
and or a protection from its rejection. The exact
mechanism by which the progesterone suppresses
immunity is not well understood; however,
recently Li et al. (2009) reported that progesterone
inhibited immune response to lipopolysaccharide
by modulating Toll-like receptor (TLR) signalling
and inhibited TLR4 and TLR9-triggered IL-6
and nitric oxide production in macrophages,
significantly inhibited LPS-induced nitric oxide
synthase (iNOS), and up-regulated expression of
suppressor of cytokine signalling (SOCS1) protein.
Reports suggest that anti-inflammatory properties
of progesterone in rodents and humans are mainly
mediated through inhibition of production and
release of a number of proinflammatory cytokines
and inhibition of NO production (Miller and Hunt,
1996). The anti-inflammatory steroid cortisol was,
however, reported high and sustained in Group
D buffaloes as compared to Group E and these
findings are comparable to the findings of Schlafer
et al. (1994), Kahl et al. (2009). Probably estrogen,
a predominant sex steroid in Group E buffaloes
favoured production of proinflammatory cytokines
whereas progesterone, predominant in Group D
buffaloes, disapproved it but favoured production
of the anti-inflammatory hormone cortisol. This
anti-inflammatory property of progesterone is of
special importance during pregnancy (Druckman
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Original Article
PERFORMANCE OF MURRAH BUFFALOES FED SUNFLOWER HEADS BASED
COMPLETE DIETS IN TERMS OF NUTRIENT UTILIZATION AND RUMEN
FERMENTATION PATTERN
D. Nagalakshmi*, D. Narsimha Reddy and M. Rajendra Prasad
ABSTRACT
TCA precipitable nitrogen was higher (P<0.01)
with the SFH diets than the conventional diet. The
ammonia nitrogen was higher in the 40% SFH diet
fed animals compared to the other two diets. The
results of this study indicated that SFH could form
a roughage source for ruminants by incorporating
An experiment was conducted to study
the effect of feeding expander extruder processed
sunflower head (SFH) based complete diets
incorporated at 40 and 50% and compared with
conventional ration (concentrate mixture to meet
protein requirements for maintenance and ad lib
sorghum straw). These three rations were fed to
fistulated buffaloes in a 3 x 3 Latin square design.
A 6d metabolic trial was conducted each time
after a preliminary feeding period of 21 days and
rumen liquor was collected after each trial for 3
consecutive days, 5 times a day, before feeding (0 h)
and at 2 h intervals after feeding (2, 4, 6 and 8 h) to
assess nutrient utilization and rumen fermentation
pattern. The organic matter, crude protein, ether
extract, crude fibre, acid detergent fibre and
cellulose (P<0.05) digestibilities were higher
(P<0.01) in animals fed either of the complete diets
compared to conventional feeding. The buffaloes
fed the 40% SFH diet digested higher (P<0.01) dry
matter, energy and neutral detergent fibre (P<0.05)
compared to the conventional group. The calcium
balance was comparable, while phosphorus
(P<0.01) and nitrogen (P<0.05) balances were
higher on the 40% SFH diet compared to the
conventional diet and intermediate on the 50%
SFH diet. The ruminal pH was lower (P<0.01),
while total volatile fatty acids, total nitrogen and
in an expander extruder processed complete diet
at either the 40 or 50% level. Out of these two
complete diets, the expander extruder processed
complete diet containing 40% SFH proved better in
terms of nutrient utilization and rumen fermentation
pattern.
Keywords: Murrah buffaloes, Bubalus bubalis,
sunflower heads, nutrient utilization, rumen
fermentation, buffaloes, expander extruder
INTRODUCTION
In India, a huge gap exists between demand
and supply of feed resources for livestock feeding,
which is to the tune of 10% for dry fodder, 35% for
concentrates and 33% for green fodder and could
further increase to 11, 45 and 35%, respectively
by the year 2020 (Ramachandra et al., 2007).The
efficient use of available feed resources (crop
residues and agro-industrial by-products, grains
and oil seed meals) along with employing suitable
feed processing techniques could greatly help to
Department of Animal Nutrition, College of Veterinary Science, Sri Venkateswara Veterinary University,
Rajendranagar, Hyderabad 500 030, Andhra Pradesh, India, *E-mail: dnlakshmi@rediffmail.com
283
Buffalo Bulletin (December 2013) Vol.32 No.4
horizontal mixer. The mineral mixture and vitamin
supplements were prepared in a premix and added
directly into the mixer. The preheated molasses was
added in the mixer while mixing and then mixed for
10 minutes to obtain complete feed in mash form.
The complete feed containing approximately 13.0%
moisture was processed in an expander-extruder by
the procedure standardized by Nagalakshmi et al.
(2007). The mash feed was conditioned with steam
pressure of 0.6-1.0 kg/cm2 for the 40% SFH diet
and 1.0-1.2 kg/cm2 for the 50% SFH diet, then
passed through a single continuous barrel of the
expander-extruder, where the feed was pushed
forward and extruded through a die hole of 16mm
diameter fitter at other end of the barrel. At the last
section of the barrel, the temperature of 85-90oC
bridge the gap between nutrient availability and
nutrient requirements. One such crop residue is
deseeded sunflower heads (SFH); this byproduct of
the sunflower crop is available in huge quantities
after deseeding of sunflowers. About 765 x 103
metric tons of SFH is estimated to be available
in India for livestock feeding.SFH contains about
7.43% crude protein (CP) and 63.67% total
digestible nutrients (TDN) (Madan Mohan et al.,
1997). In spite of higher nutritive value compared
to conventionally used straws, presently most of
the SFH is either used as manure or burnt in fields
(Nagalakshmi et al., 2003). In situ and in-vitro
studies revealed that SFH could be incorporated in
complete diets either as the sole roughage source at
40% level or at the 50% level in combination with
10% sorghum straw (Nagalakshmi et al., 2005).
Further, expander extruder processing of crop
residues based complete diets increased nutrient
utilization, palatability and reduced cost of feeding
in ruminants (Nagalakshmi et al., 2010). Thus the
present study was conducted to evaluate complete
diets containing 40 or 50% SFH selected from
in-vitro studies and processed with an expanderextruder in terms of nutrient utilization and rumen
fermentation pattern in buffaloes.
was achieved and here the feed was subjected
heating for about 30 seconds before extruding out.
The hot pellets were cooled in a batch cooler and
stored in gunny bags.
Experimental diets
The deseeded SFH procured from nearby
sunflower fields was sun dried and used as the
main roughage source in the complete diets. The
experimental diets were 1. Expander-extruder
processed complete diet containing 40% SFH,
2. Expander-extruder processed 50% SFH along
with 10% sorghum straw and 3. Conventional diet
comprising concentrate mixture offered to meet
the protein requirements for maintenance and
sorghum straw available ad libitum. The ingredient
composition of all three diets is given in Table 1.
MATERIALS AND METHODS
Processing of experimental diets
Two complete diets were formulated with
40 and 50% SFH in a roughage concentrate ratio of
40:60 and 60:40, respectively (Table 1). The SFH
and concentrate ingredients except for molasses
and micro ingredients were first proportioned
and batched for 100 kg as per the formula. They
were then ground in a hammer mill with an 8mm
sieve. The ground material was conveyed to a
Animals, feeding regime and housing
management
Three adult male graded Murrah buffaloes
(328.5 + 19.58 kg) having permanent rumen fistula
were randomly allotted to three diets in 3x3 Latin
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Buffalo Bulletin (December 2013) Vol.32 No.4
square design. The experimental animals were
housed in well ventilated stalls and offered feed
3 times a day to meet nutrient needs as per Kearl
(1982) requirements. Clean fresh water was made
available throughout the experimental period. In
each trial the buffaloes were fed the respective diets
for 30 days (preliminary period) and then followed
by a metabolic trial of 7 days duration. There was a
switch over period of 10 days between each trial.
Van Soest et al. (1991) and Talapatra et al. (1940),
respectively. The gross energy was estimated
as per the procedure described in the manual
of the Gallemkemp Automatic Ballistic Bomb
Calorimeter. The SRL samples were analysed for
total N, TCA-insoluble N (Cline et al., 1958),
residual N and food and protozoal N (Singh et
al., 1968), ammonia N (Schwartz and Schoeman,
1964) and TVFA (Barnett and Reid, 1956).
Rumen studies
At the end of each metabolic trial, rumen
liquor was collected for 2 consecutive days, 5 times
a day, once before feeding designated as 0 h and
then at 2 h intervals after feeding (2, 4, 6 and 8 h)
to assess the rumen fermentation pattern. On days
of rumen liquor collection, feed was offered at 7
am (before 0 h collection) and at 4 pm (after 8 h
collection) to avoid the effect of continuous feeding
on concentration of rumen metabolites. The pH
of rumen fluid was estimated immediately after
collection with help of digital pH meter. The rumen
liquor was then strained through four layer muslin
cloth. About 5 ml of strained rumen liquor (SRL)
of each animal from every collection was preserved
by adding 2 drops of saturated solution of mercuric
chloride in plastic vials for estimation of total
volatile fatty acids (TVFA). The ammonia nitrogen
(NH3-N) concentration in SRL was estimated
immediately after collection. The remaining SRL
was deep frozen in plastic vials after adding two
drops of 1:4 sulphuric acid for estimation of total
nitrogen and other nitrogen (N) fractions.
Statistical analysis
The data was subjected to analysis of
variance (Snedecor and Cochran, 1980) and the
means were tested for significance by Duncan’s
multiple range test (Duncan, 1955).
RESULTS AND DISCUSSION
The chemical composition of concentrate
mixture, sorghum stover and two expander extruder
pelleted complete diets is given in Table 2. The
crude protein (CP) content of SFH was about two
and a half times higher and the neutral detergent
fibre was lower than conventionally used sorghum
stover.
The nutrient digestibilities and nutrient
balances in buffaloes fed SFH based complete
diet is presented in Table 3. The digestibilities of
dry matter (DM), organic matter (OM), CP, ether
extract (EE) and CF was significantly (P<0.01)
higher in the SFH based complete diets compared
to the conventional diet, which might be attributed
to uniform grinding and blending of concentrate
and roughage in the former rather than the separate
feeding of the roughage and concentrate in the latter.
Further, EEP might have resulted in the binding
of fat and protein molecules with each other or
with other components of the feed, thus protecting
Analytical procedure of feeds, faeces and urine
The feed samples were analysed for
proximate constituents and phosphorus as per the
procedure of AOAC (1997). The fibre fractions and
calcium were determined as per the procedure of
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Buffalo Bulletin (December 2013) Vol.32 No.4
them from rumen microbes and exposing them for
efficient digestion in small intestine (Broderick et
al., 1991; Hauck et al., 1994) resulting in higher
fat and protein digestibilities. Such beneficial
effects on nutrient digestibility with expanderextruder processing of various crop residues based
complete diets was reported in Ongole bull calves
(Reddy and Reddy, 1999; Reddy et al., 2002),
buffalo bulls (Nagalakshmi and Reddy, 2010b) and
sheep (Thirumalesh et al., 2003) fed maize cobs,
sugarcane bagasse, cotton stalks and bajra straw
based complete diets, respectively. Reddy and
Reddy (1998) reported higher DM, OM, CP and CF
digestibilities in Ongole calves fed EEP processed
complete diets containing 28.5% SFH as the sole
roughage source compared to a conventional diet
(concentrate and chopped sorghum straw). In the
present study, among the complete diets the DM, CF
and nitrogen free extract (NFE) digestibilities was
higher (P<0.01) in the 40% SFH based complete
diet compared to the 50% SFH diet, which might
be due to the higher SFH proportion or an effect
of the higher roughage concentrate ratio in latter
complete diet. Gelatinization of starch components
of feed and loosening of bonds between lignin and
soluble carbohydrates (hemicelluloses, xylose, etc)
during expander extruder processing resulted in
buffaloes digesting more (P<0.01) energy when
fed the SFH based complete diets compared to
the conventional diet. Similarly, higher energy
digestibility was observed in calves fed EEP
processed diet containing cotton stalks compared
to a conventional diet (Kirubanath et al., 2003).
No effect on ADF and cellulose digestibility
was observed with inclusion of sugarcane bagasse
(Reddy et al., 2001), maize cobs (Reddy and Reddy,
2000) or cotton stalks (Nagalakshmi and Reddy,
2010b) at the 40% level in expander extruder
processed complete diets. But in the present study,
a higher digestibility of ADF and cellulose was
observed when incorporated at the 40 or the 50%
levels, which might be due to differences in the
variation in ADF and cellulose content in the above
crop residues.
All the animals were on positive nitrogen
(N) and mineral balances, indicating that these
diets could supply these nutrients in the required
proportion (Table 3). The N balance was higher
(P<0.05) in the 40% SFH diet followed by the
50% SFH diet and was lowest on the conventional
diet. Nitrogen retention depends upon factors like
N intake and energy availability. Higher dietary
energy level and greater protein intake increases
the N retention (Baruah, 1983). Higher digestible
crude protein (DCP) and DE intakes by the
animals fed 40% SFH diet, followed by 50% SFH
diet compared to conventional ration resulted in
similar trend for N balance. Similar to the present
findings, Thirumalesh et al. (2003) observed
higher N balance in lambs fed a 40% bajra straw
based EEP diet compared to a conventional diet.
No significant effect on calcium and phosphorous
retentions was observed in calves fed a 40%
sorghum straw (Reddy and Reddy, 1999a) or a
sugarcane bagasse (Reddy et al., 2002) based EEP
diets compared to conventional rations. While
Kishan Kumar et al. (2010) observed higher
calcium and phosphorous balances in calves fed a
palm press fibre based complete diet compared to
a conventional diet. In the present study, no effect
of complete diet, expander extruder processing and
SFH inclusion was observed on calcium balance,
while the phosphorous balance was higher on the
40% SFH diet compared to the conventional diet
and the balance in buffaloes fed the 50% SFH diet
was intermediate between the other two diets.
The nutritive value of diets and plane of
nutrition is presented in Table 3. The DCP, total
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Buffalo Bulletin (December 2013) Vol.32 No.4
that the pH of SRL was dictated by the TVFA
concentration. The pH concentration was lower
(P<0.01) while the TVFA concentration was higher
(P<0.01) in the buffaloes fed the complete diets
in comparison to those fed the conventional diet.
The increased TVFA concentration in the SRL
of the complete diet fed animals might be due to
increased availability of fermentable energy in the
complete diets. Similarly, Nagalakshmi and Reddy
(2010b) and Reddy et al. (2001) observed higher
TVFA concentration in buffalo bulls fed expander
extruder processed complete diets containing
40% of either cotton stalks or sugarcane bagasse,
respectively. The TVFA concentration in general
attained peak 2 h after feeding, reduced (P<0.01)
by 4 h after feeding, and gradually fell up to 8h
after feeding. The total nitrogen concentration
peaked (P<0.01) at 2 h after feeding in all the groups
and the peak was maintained till 6 h of feeding.
Similarly, the peak levels for ammonia nitrogen
and TCA precipitable nitrogen was observed 2
h after feeding and the levels were maintained
even up to 8 h after feeding. The peak of total
nitrogen, ammonia nitrogen and TCA precipitable
N observed at 2 h post feeding might be due to
active degradation of protein and hydrolysis of non
protein nitrogen substances for microbial protein
synthesis. The higher N intake by buffaloes fed the
40% SFH based complete diet could have resulted
in a higher ammonia nitrogen concentration in
the SRL. The higher TCA precipitable nitrogen
concentration observed in the complete diets
containing either 40 or 50% SFH might be due to
efficient utilization of NH3 by rumen microbes with
simultaneous availability of carbohydrates and
higher organic matter digestibility (Table 3). The
residual-N in SRL gradually increased and peaked at
4 h post feeding when SFH complete diets were fed
while the animals fed conventional diets, the peak
digestible nutrients (TDN) and digestible energy
(DE) content of the diet was highest (P<0.01) in the
40% SFH containing complete diet compared to the
other two diets, which was due to higher nutrient
digestibilities and balances recorded when fed this
diet (Table 4). The daily DM intake in all the groups
met the standard requirements recommended
by Kearl (1982) for 325 kg body weight (6.0 kg
DM) indicating that the diets containing SFH were
palatable to buffaloes. The DM intake was lower
(P<0.05) when fed the 50% SFH complete diet
compared to the conventional diet, while the intake
on the 40% SFH diet was comparable (Table 3).
The water consumption was higher (P<0.01) in
buffaloes fed the 50% SFH diet followed by the
40% SFH diet compared to the conventional diet.
Increase of water intake on SFH based diets was
reported by previous workers in calves (Reddy
and Reddy, 1998) and sheep (Reddy et al., 2004).
Nagalakshmi and Reddy (2010a) reported higher
water intake in buffaloes fed a sugarcane bagasse
based expander extruder processed diet. The higher
water intake in the 50% SFH diet than the 40% SFH
diet was due to the higher roughage component
(60%) in this diet. The TDN intake was comparable
among all the groups but the DCP and DE intake
per kg W0.75 was higher when fed the 40% SFH
diet in comparison to the conventional diet and the
50% SFH based complete diet. The DCP and TDN
intake per kg metabolic body weight by all groups
of buffaloes was higher than the standard intakes
of Kearl (1982) (2.54 g DCP, 34.49 g TDN per kg
W0.75 for 325 kg body weight for maintenance).
Both the hour of sampling and diet
influenced the pH, TVFA and various nitrogen
fractions but no significant interaction of these
factors was observed on above rumen parameters
(Table 5). The pH and TVFA concentration
were inversely related to each other, indicating
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Buffalo Bulletin (December 2013) Vol.32 No.4
Table 1. Ingredient composition (kg/100kg) of SFH based complete diets.
Ingredient
Sunflower heads
Sorghum straw
Maize
Groundnut cake
Cottonseed cake
Wheat bran
Deoiled rice bran
Molasses
Urea
Salt
Mineral mixture
Vitamin mixture (g/qt)
Concentrate
mixture
EEP- 40% SFH
EEP- 50% SFH
--30.0
16.0
11.0
-39.0
-1.0
1.0
2.0
20.0
40.0
-20.0
9.0
6.0
6.5
5.0
10.0
0.5
1.0
2.0
10.0
50.0
10.0
10.0
15.0
5.0
--7.0
-1.0
2.0
10.0
Table 2. Chemical composition (% DM basis) of experimental diets.
Constituent
Organic matter
Crude protein
Ether extract
Crude fibre
Nitrogen free extract
Neutral detergent fibre
Acid detergent fibre
Hemicellulose
Cellulose
Calcium
Phosphorus
Concentrate
mixture
85.02
18.16
2.38
31.04
33.44
70.87
50.33
20.54
30.42
2.45
0.73
EEP- 40%
SFH
85.92
13.82
2.10
29.09
40.91
57.27
38.84
18.43
27.93
2.16
0.73
288
EEP- 50%
SFH
84.07
13.29
2.45
32.37
35.97
56.10
45.73
10.37
31.53
1.67
0.72
Sorghum
stover
87.83
2.45
1.30
34.45
49.64
75.57
71.01
5.23
57.64
1.31
0.72
Sunflower
heads
84.56
7.82
2.86
34.34
39.37
55.66
46.27
16.10
30.17
2.51
0.45
Buffalo Bulletin (December 2013) Vol.32 No.4
Table 3. Nutrient digestibility (%) and balances (g) in buffaloes fed SFH based complete diets.
Nutrient
Nutrient digestibility
Dry matter
Organic matter
Crude protein
Ether extract
Crude fibre
Nitrogen free extract
Cell contents
Neutral detergent fibre
Acid detergent fibre
Hemicellulose
Cellulose
Energy
Nutrient balance
Calcium
Phosphorus
Nitrogen
Conventional
diet
EEP-40% SFH
EEP- 50%
SFH
SEM
50.66c
54.34b
47.88b
50.62b
31.68c
76.26ab
64.56
46.29b
43.73b
61.53b
51.40b
46.82b
67.00a
69.64a
62.23a
64.18a
61.65a
80.36a
71.43
63.22a
53.25a
79.89a
63.80a
66.78a
59.80b
62.30a
50.07a
63.17a
55.06b
70.98b
66.21
54.89ab
52.75a
67.30ab
59.20a
55.42ab
2.385**
2.336**
2.333**
2.414**
4.592**
1.475**
1.312
2.814*
1.573**
3.306*
2.136*
3.128**
77.45
11.23b
25.62b
85.02
20.97a
46.74a
90.35
16.25ab
36.34ab
2.682
1.529**
3.640*
Means with different superscripts in a row differ significantly: *P<0.05; **P<0.01.
abc
Table 4. Nutritive value and plane of nutrition of buffaloes fed sunflower heads based diets.
Nutrient
Nutritive value
Crude protein %
Digestible crude protein %
Total digestible nutrients %
DE (Mcal/kg)
Nutrient intake
DMI/kgW0.75 (g/d)
DCP intake/kg W0.75 (g/d)
TDN intake W0.75 (kcal/d)
DE intake/kg W0.75 (kcal/d)
Water intake/kg DMI (L)
Conventional
diet
EEP-40% SFH
EEP- 50%
SFH
SEM
8.87c
4.35c
51.41b
2.53b
13.80a
10.03a
63.69a
4.36a
12.42b
7.08b
56.04b
2.86b
0.744
0.841
1.863
0.318
83.61a
3.70b
43.92
216.9b
4.40b
77.50ab
7.80a
49.85
342.6a
5.96ab
71.30b
5.05b
40.00
204.4b
6.83a
2.172*
0.621**
2.215
26.96*
0.392**
Means with different superscripts in a row differ significantly: *P<0.05; **P<0.01.
abc
289
Buffalo Bulletin (December 2013) Vol.32 No.4
Table 5. Rumen fermentation pattern in buffaloes fed sunflower heads based complete diets.
Diet
Conventional diet
EEP-40% SFH
EEP-50% SFH
Period
0h
2h
4h
6h
8h
SEM
pH
TVFA
(meq/
dl)
NH3-N
(mg/dl)
Total-N
(mg/dl)
TCA-ppt-N
(mg/dl)
Residual N
(mg/dl)
7.07a
6.29b
6.47b
**
60.34b
79.07a
83.80a
**
12.52b
18.88a
13.58b
**
84.70b
111.93a
115.6a
**
25.75b
34.92ab
42.93a
**
35.63
43.37
39.24
NS
6.68a
6.56bc
6.44c
6.42c
6.75ab
**
0.050
68.00bc
90.81a
78.43b
71.95bc
62.82c
**
1.958
9.62b
20.00a
17.31a
14.39ab
13.65ab
**
0.893
77.28c
125.56a
118.78ab
109.47abc
89.31bc
**
4.386
24.81b
44.03a
42.17ab
34.19ab
27.47ab
*
2.217
31.01c
45.16ab
46.25a
40.65abc
33.99bc
*
1.841
Means with different superscripts in a sub-column differ significantly: *P<0.05; **P<0.01; SEM: Standard
error mean.
abc
level was observed at 2 h and thereafter the levels
fell drastically. Expander-extruder processing of
complete diets containing various crop residues as
the sole roughage source, viz., maize cobs (Reddy
and Reddy, 2000), sugarcane bagasse (Reddy et
al., 2001), cotton stalks (Nagalakshmi and Reddy,
2010) increased the concentration of total nitrogen
and TCA precipitable nitrogen in rumen liquor of
buffalo bulls compared to conventional diets.
The results of this study indicated that
sunflower heads can form a roughage source for
ruminants by incorporating at 40-50% level. Out
of these two complete diets, the expander extruder
processed complete diet containing 40% SFH
proved better in terms of nutrient utilization and
rumen fermentation pattern.
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Original Article
PREVALENCE OF SUB-CLINICAL MASTITIS IN LACTATING BUFFALOES DETECTED
BY COMPARATIVE EVALUATION OF INDIRECT TESTS AND BACTERIOLOGICAL
METHODS WITH ANTIBIOTIC SENSITIVITY PROFILES IN BANGLADESH
J.J. Kisku and M.A. Samad*
ABSTRACT
was SFMT (50.00 and 26.66%), respectively. The
highest prevalence of SCM was recorded at > 9
to 12 years of age (23.33%), 4th parity (16.67%)
Buffaloes, like cattle and goats, play
a major part in the subsistance economy of rural
people in Bangladesh. These livestock species are
prone to the intramammary infections (IMI), which
are associated with a lot of economic impact to the
farmers. The prevalence and importance of clinical
and sub-clinical mastitis (SCM) have been reported
in cows and goats from Bangladesh and this study
was undertaken to evaluate the indirect tests and
bacteriological methods for the prevalence of SCM
associated with host risk factors and antibiotic
sensitivity profiles of bacterial isolates recovered
from milk samples of apparently healthy mammary
quarters of lactating buffaloes of an organized
farm in Bangladesh during the period from June to
November 2010. A total of 120 quarters milk samples
from 30 available apparently healthy lactating
cross-bred (Nili-Ravi × Murrah and Nili-Ravi ×
local) buffaloes were subjected to the Whiteside
test (WST), the surf field mastitis test (SFMT) and
the California mastitis test (CMT); those positive
by the WST, SFMT and CMT were 35 (29.16%), 32
(26.66%) and 39 (32.50%) with an overall 56.66%
prevalence of SCM in lactating buffaloes. The test
with the highest diagnostic performance, for both
animal-wise and quarter-wise prevalence of SCM
was the CMT (56.66% and 32.50%), followed by
the WST (53.30% and 29.16%), and the lowest
and late lactation (30.0%). The daily average milk
production was insignificantly (p > 0.05) decreased
in buffaloes (4.5 ± 0.72 liter / day) that had IMI
(SCM) in comparison to buffaloes (4.8 ± 0.88
liter / day) without IMI. The CMT positive milk
samples (n = 39) were subjected to bacterial culture
isolation (the gold standard test for comparison of
indirect mastitis test). Among the bacterial isolates
of IMI, Staphylococcus spp. (30.77%) showed
the highest frequency, followed by Streptococcus
spp. (20.51%), Bacillus spp. (15.39%) and
Escherichia coli (12.82%) as a single infection,
and also recorded as mixed infection (12.82%)
and 7.69% remained as unclassified bacterial
growth. Moderate to high antibiotic sensitivity of
Staphylococcus spp., Streptococcus spp., Bacillus
spp. and E. coli was obtained with gentamicin,
ciprofloxacin, enrofloxacin and chloramphenicol,
but these organisms were found mostly resistant
or less sensitive to ampicillin, amoxicillin and
streptomycin. It may be concluded from these
results that there is a high prevalence (56.66%)
of SCM in buffaloes in Bangladesh and that the
associated pathogens have already developed
resistance due to indiscriminate use of antibiotics
and accordingly, there is a need for proper attention
to control of mastitis in buffaloes.
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh2202, Bangladesh *E-mail: masamad88bau@yahoo.com
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Buffalo Bulletin (December 2013) Vol.32 No.4
(Samad, 2000; Islam et al., 2004). Among the
many diseases that occur in buffaloes, mastitis is a
frustrating, costly and extremely complex disease
that results in a marked reduction in quality and
quantity of milk (Harmon, 1994). The prevalence of
mastitis has been reported to be lower in buffaloes
(SCM 27% & CM 4%) as compared to cows
(SCM 36% & CM 5.5%). This lower prevalence
might be attributed to the tighter teat sphincter of
buffaloes as compared to that of cows (Uppal et
al., 1994). However, bubaline mastitis has been
recognized as an economically serious disease as
it is in cows in India (Pal et al., 1989; Kumar and
Thakur, 2001; Sharma et al., 2007; Kavitha et al.,
2009). and Pakistan (Hussain et al., 1984; Khan et
al., 2004; Khan and Muhammad, 2005; Bachaya
et al., 2005; Sharif and Ahmad, 2007; Muhammad
et al., 2010; Sharma et al., 2010). but such a status
has not been evaluated in buffaloes in Bangladesh
(Samad, 2000). Generally mastitis occurs in two
forms, clinical (overt) and sub-clinical (hidden).
Clinical mastitis (CM) can be characterized by five
cardinal signs of udder inflammation (redness, heat,
swelling, pain and loss of milk production), while
the SCM is bereft of any obvious manifestation of
inflammation. SCM is 3 to 40 times more common
than the CM and causes the greatest overall losses
in most dairy herds (Schultz et al., 1978). Annual
losses in the dairy industry due to mastitis was
approximately 2 billion dollars in the USA and 526
million dollars in India, in which SCM is responsible
for approximately 60 to 70% of these dollar losses
(Merril and Galton, 1989; Varshney and Naresh,
2004). Lactating animals with SCM are those with
no visible changes in the appearance of the milked
/or the udder, but milk production decreases by 10
to 20% with undesirable effect on its constituents
and nutritional value, rendering it of low quality
and unfit for processing (Khan et al., 2004). Apart
Keywords: buffaloes, Bubalus bubalis, sub-clinical
mastitis, lactating buffaloes, indirect tests, bacterial
pathogens, antibiotic sensitivity
INTRODUCTION
Buffaloes occupy a prominent place
in the social, economic and cultural life of rural
communities in most Asian countries. There are 170
million buffaloes in the world, 97% (164.9 million)
in Asia, 2% (3.4 million) in Africa mainly in Egypt,
and 0.2% (0.34 million) in Europe, mainly in Italy
(FAO, 2004; Singh and Barwal, 2010). India has
56% (95.2 million), Pakistan 14% (23.8 million),
China 13% (22.1 million) and Bangladesh 0.76%
(1.3 million) of world buffalo population nearly
98% (166.6 million) of water buffaloes in Asia.
The buffalo contributes 72 million tons of milk and
three million tones of meat annually to world food,
much of it in areas that are prone to nutritional
imbalances. In addition, buffaloes are the source of
20 to 30% of the draught power in Southeast Asia,
which is why the buffalo has been called the ‘living
tractor’ of the east (Asia). They are also considered
a ‘walking fertilizer factory,’ producing about 6.8
kg of dung daily (Cockrill, 1974). The 1.3 million
buffaloes of Bangladesh produce about 96,000 MT
of milk and 16,000 MT of meat annually (DLS,
2005). These buffaloes are mainly reared under
rural conditions scattered throughout Bangladesh,
although there is an organized Government Buffalo
Breeding and Development Farm , which was
established in 1985 and is situated in Bagerhat
district of Bangladesh. Recently, the Bangladesh
Government has undertaken a project to establish
buffalo farms in 12 districts of Bangladesh (Anon,
2011). Research reports on buffalo health and
production are very limited from Bangladesh
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Buffalo Bulletin (December 2013) Vol.32 No.4
house. All the available lactating buffaloes were
subjected to clinical and physical examination
with special interest towards the udders and teats.
A total of 30 lactating buffaloes (120 mammary
quarters) which had normal udders, quarters and
teats, were included for this study. The age (3 to
12 years), parities (1 to 6) and stage of lactation
(early, mid and late) were recorded for each of the
randomly selected 30 buffaloes. The average milk
yield per day varied from 3.0 to 6.5 liters (Table 4).
Milk samples were collected after proper
disinfection of teat surface with 70% ethanol. Then
20 ml of milk sample was collected aseptically
from each quarter in separate sterile screw-capped
vials after squiring few streams (Buswell, 1995).
All indirect tests were conducted at the spot before
milk sample collection for culture. The milk
samples of all the 120 quarters were subjected to
the White Side Test (WST), the Surf Field Mastitis
Test (SFMT) and the California Mastitis Test
(CMT).
from causing colossal economic losses, this disease
also poses the risk for the transmission of zoonotic
diseases like tuberculosis, brucellosis, leptospirosis
and streptococcal sore throat to human beings
(Sharma et al., 2005; Samad, 2008). The invisible
changes in SCM can be recognized indirectly by
several diagnostic methods including CMT, WST,
SCC, pH, chloride and catalase tests. Direct isolation
and identification of intra-mammary infections
(IMI) may have significant benefit as preventing
CM and may lead to further understanding of
the dynamics of the disease (Lam et al., 1996).
In addition, the assessment of SCM etiological
pathogens aids to classify the healthy sound milk
samples from those of pH hazards. Considering
these factors, the present study aimed to elucidate
the prevalence of SCM in lactating buffaloes by
comparative evaluation of three indirect tests, to
isolate and identify the major bacterial pathogens
causing IMI with their antibiotic sensitivity patterns
and to investigate the association between host
determinants and prevalence of SCM in lactating
buffaloes.
White side test (WST)
The WST was performed with a prepared
WST reagent (4% sodium hydrochloride) as per
the procedure described by Kumar and Thakur
(2001) and Sharma (2008). In brief, each milk
sample was thoroughly mixed carefully to avoid
violent shaking. The sample was sufficiently mixed
to ensure an even distribute the sample. Then 50
μl of milk were placed on a glass slide with a
dark background by micropipette. Subsequently
20 μl of WST reagent (4% sodium hydroxide)
were added to the milk sample and the mixture
was stirred rapidly with a toothpick for 20 to 25
seconds. A breaking up of milk in flakes, shreds
and viscid mass was indicative of a positive
reaction, while milky and opaque and entirely free
of precipitant was indicative of a negative reaction.
MATERIALS AND METHODS
This study on SCM in lactating buffaloes
was carried out on an organized Government Buffalo
Breeding and Development Farm, situated in the
district of Bagerhat, Bangladesh during the period
from June to November 2010. Buffaloes were well
managed under a semi-intensive husbandry system
with raised floor. They were often provided with
some green grass in addition to natural pasture
and concentrate diet and were kept together in a
common shed, but at advanced stage of pregnancy
and lactation stage, they are maintained in separate
sheds at a short distance from each other in a
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Buffalo Bulletin (December 2013) Vol.32 No.4
Surf field mastitis test (SFMT)
The SFMT was performed and scored
following the method described by Muhammad et
al. (2010) In brief, 2.0 ml milk sample was drawn
from a bottle into a cup and an estimated 2.0 ml
reagent (Surf Excel® , Uniliver Bangladesh, 3.0%
Antibiotic sensitivity tests
Antibiotic sensitivity test of 20 bacterial
isolates of single infection was performed to seven
different antibiotics by the disc diffusion method
(Oxioid Ltd., UK) as described by Bauer (1966)
and Ellner (1978). The disc concentration of
antibiotics included ampicillin (10 μg), amoxicillin
(10 μg), ciprofloxacin (5 μg), chloramphenicol (30
μg), enrofloxacin (5 μg), gentamicin (30 μg) and
streptomycin (10 μg). The procedure in brief was
nutrient and blood agar cultures (24 h at 37°C)
for each isolated organism were evenly spread
over plates. About 2.5 ml of different bacterial
suspensions were poured over the plates. Then the
plates were tipped to one side and the surplus fluids
were removed by suction. Cultures were allowed
to dry for one hour at 37°C after which different
antibiotics discs were carefully placed over the
surface of the plate with the help of alcohol-flamed,
fine pointed forceps. The discs were so placed
that there was enough space around each disc for
diffusion of the antibiotic. Plates were incubated for
48 h at 37°C and the zone of inhibition around each
disc was measured. The inhibition of the growth
was demonstrated by a clear zone of growth around
the discs due to the result of two processes viz. (a)
diffusion of the antibiotics and (b) growth of the
bacteria. Sensitivity was expressed as ‘3+’, ‘2+’,
‘1+’ and ‘–’, expressing high, moderate, sensitive
and resistant levels of susceptibility, respectively.
solution) was squirted from a polyethylene bottle.
Mixing was accomplished by gentle circular motion
of the paddle in a horizontal plane for a few seconds.
The reaction developed almost immediately with
milk containing a high concentration of somatic
cells. The peak of reaction was obtained within 30
seconds and immediately scored as 1+, 2+ and 3+
and score ≥ 1+ considered positive for SCM.
California mastitis test (CMT)
The CMT was performed by using CMT
kit (Leucocytest®, Synbiotics Corporation-2,
France) as per the kit manufacturer’s instructions
as described by Rabbani and Samad (2010). In
brief, 2.0 ml of milk sample was taken in the CMT
paddle and equal volume (2.0 ml) of CMT reagent
was added in each cup, rotated for few seconds and
then the result was recorded within 30 seconds as 0
(negative) and T (trace) were considered negative
or normal, while CMT scores of 1+ (weak positive),
2+ (distinct positive) and 3+ (strong positive) were
taken as indicators of sub-clinical mastitis (SCM).
Bacteriological studies
The CMT positive milk samples (n =
39) were subjected to bacteriological culture as
per method described by Quinn et al. (1994) Each
bacterial colony was examined macroscopically
(colony morphology) and microscopically (Gram’s
stain) as described by Merchant and Packer (1967).
Identification of all isolates was performed by using
standard biochemical tests (Buchnan and Gibbon,
1984).
Statistical analysis
Results were analyzed by Chi-square
test to observe the significant influence of age,
parity, lactation stage and milk yield on SCM
of lactating buffalo cows and Cochran’s test for
sensitivity and specificity of different diagnostic
tests using Statistical Package for Social Science
(SPSS) Version 13.0 (Coakes et al., 2006).
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Buffalo Bulletin (December 2013) Vol.32 No.4
RESULTS AND DISCUSSION
Comparative evaluation of indirect tests
The comparative evaluation of indirect
tests (WST, SFMT, CMT) for the detection of
SCM in lactating buffaloes is presented in Tables 1
and 2. The positive reaction of these indirect tests
seem to depend on the presence of somatic cells
(leukocytes) in the milk (Sharma et al., 2008). and
the number of the somatic cells is mainly associated
with the severity of intra-mammary infections
(IMI). The principle of these indirect tests is that the
reagents (detergents) dissolve or disrupt the outer
cell wall and the nuclear cell wall of leukocytes
(somatic cells), which are primarily fat (detergent
dissolves fat). DNA is released from the nuclei
of somatic cells and it strings or gels together to
form a stringy mass. As the number of leukocytes
in a quarter increases due to IMI, the amount of
formed-gel increases parallel linearly (Sharma et
al., 2010). In addition, the accuracy, sensitivity
and specificity of these indirect tests are varied
due to different chemicals the detergents contain.
Of the 120 milk samples, 32 (26.70%)
were positive and 81 (67.5%) were negative for
SCM by all the three tests (Table 2). Three (2.5%)
samples were positive by WST and CMT but not
by SFMT. Four (3.3%) samples were positive by
CMT alone. The Qa for Table 2 was 10.57, which
exceeds the critical value and indicates that the tests
differ significantly from each other in the diagnosis
of SCM as positive or negative. The results of the
comparative evaluation of these three indirect tests
showed highest diagnostic value of CMT in both the
animal-wise (56.66%) and quarter-wise (32.50%)
in comparison to WST (50.0% & 26.66%) and
SFMT (50.0% & 26.66). It appears that CMT had
significantly higher diagnostic value in comparison
to WST and SFMT. These findings support Sharma
et al. (2007) who reported 66.0%, 68.60% and 72.0%
prevalence of SCM in buffaloes using Modified
The comparative diagnostic values of
indirect tests and bacteriological methods were
evaluated on 120 mammary quarter milk samples
of 30 apparently healthy lactating cross-bred (NiliRavi × Murrah and Nili-Ravi × local) buffalo cows
in an organized Government Buffalo Breeding
and Development Farm, Bagerhat, Bangladesh.
Animal and quarter-wise prevalence of SCM
Animal-wise prevalence of SCM was
53.30%, 50.0% and 56.66%, while quarterwise prevalence of SCM was 29.16%, 26.66%
and 32.50% by using WST, SFMT and CMT,
respectively (Table 1). These results support the
findings of Sharma et al. (2007) who reported
66.00%, 68.60% and 72.0% animal-wise, and
38.99%, 42.0% and 45.0% quarter-wise prevalence
of SCM by using modified WST, modified CMT
and somatic cell count (SCC). In addition, Said
and Abd-el-Mlik (1968) reported 38.07% SCM
in buffaloes using WST and CMT. Anwar and
Chaudhary (1983) reported an overall 47.5%
prevalence of SCM in buffaloes using the Strip
Cup test, pH test and WST. Rehman et al. (1983)
reported prevalence of SCM 59.2% in cows and
36.8% in buffaloes using direct and indirect tests.
Hussain et al. (1984) reported SCM 33% in cows
and 8% in buffaloes using WST. Bachaya et al.
(2005) reported 77.98% animal-wise and 58.75%
quarter-wise prevalence of SCM in buffaloes using
SFMT. Sharif and Ahmad (2007)
reported an overall 51.0% animal-wise and 37.75%
quarter-wise prevalence of SCM by using SFMT in
buffaloes. These differences in the prevalence of
SCM might be due to differences in management
practices, methods of detection, breeds of animals,
immune response of animals and climatic conditions.
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Buffalo Bulletin (December 2013) Vol.32 No.4
Prevalence of SCM associated with host risk
factors
It appears from Table 5 that the highest
prevalence of SCM in buffaloes was recorded in the
> 9 to 12 year (23.33%) age group in comparison to
the > 6 to 9 year (20.00%), the > 12 year (06.67%)
and the 3 to 6 years (06.67%) groups. Kumar and
Sharma (2002) reported the highest prevalence
of SCM in buffaloes between 5 and 7 years of
WST, Modified CMT and SCC, respectively.
Quarter side-wise prevalence of SCM
The respective quarter side-wise
prevalence of SCM in LF (left front), LH (left
hind), RF (right front) and RH (right hind) using
WST (30.0%, 26.66%, 20.0% & 40.0%), SFMT
(23.33%, 30.0%, 20.0% & 33.33%) and CMT
(33.33%, 30.0%, 23.33% & 43.33%) are presented
in Table 3. It appears that the highest prevalence
of SCM was recorded in the RH quarter by all the
tests: WST (40.0%), SFMT (33.33%) and CMT
(43.33%), whereas lowest prevalence was in the
RF quarter represented as 20%, 20% and 23.33%
by WST, SFMT and CMT, respectively. These
observations support the report of Sharif and
Ahmad (2007) who reported highest prevalence of
SCM in RH quarter (30.46%) in comparison to LF
(24.51%), LH (21.19%) and RF (23.84%) quarters.
However, Khan and Muhammad (2005) reported
a high prevalence of SCM in LH quarter (37%) in
comparison to LF (18.5%), RF (14.8%) and RH
(29.6%) quarters. Saini et al. (1994) also reported
higher prevalence of SCM in hind quarters in
comparison to front quarters but the highest in left
hind quarters. Dhakal (2006) reported insignificant
(p > 0.05) differences in the prevalence of SCM
among LF (8.0%), LH (6.0%), RF (10.0%) and
age. Sharma et al. (2007) reported the highest
prevalence of SCM in animals 5 to 9 years of age.
The maximum prevalence of SCM
in buffaloes was recorded during the 4th parity
(16.67%), followed by 1st (13.33%), 2nd and
3rd (10.0%) and the lowest during the 5th and 6th
(03.33%) parity (Table 5). These results support
the findings Sharma et al. (2007) who reported
maximum prevalence of SCM during the 3rd and
4th parity. Kumar and Sharma (2002) also recorded
majority of SCM cases during the 3rd parity.
Kavitha et al. (2009) reported increased prevalence
of mastitis with increase of parity in buffaloes.
An obvious trend of increasing prevalence
of SCM was observed with the increased of stage
of lactation. The highest prevalence of SCM was
recorded in late lactation (30.0%) in comparison to
early (20.00%) and mid (13.33%) lactation (Table
5). These observations are in conformity with the
findings of Sharma et al. (2007) who reported
maximum prevalence of SCM in the late lactation
followed by early and mid lactation. Patil et al.
(1995) also reported the highest prevalence of SCM
during late lactation period as compared to early and
mid lactation. Higher prevalence of SCM during late
lactation might be due to fact that during this period
buffaloes are more vulnerable to usher infection.
RH (8.0%) quarters. Kavitha et al. (2009) also
did not find any significant (p > 0.05) difference
on the prevalence of SCM in buffaloes among the
LF (7.03%), RF (10.1%), LH (10.93%) and RH
(10.93%) quarters. It may be concluded from these
reports that the prevalence of SCM was higher in
hind-quarters than fore-quarters, which may be
due to the greater chances of hind-quarters being
soiled with urine or contaminated from the tail.
298
Buffalo Bulletin (December 2013) Vol.32 No.4
process (Harmon, 1993). Therefore, hygiene at
Effects of SCM on milk production
The daily average milk production in
lactating buffaloes that had intramammary infection
(IMI) showed an insignificant (p > 0.05) decreased
(4.5 ± 0.72 liter / day) in comparison to buffaloes
(4.8 ± 0.88 liter / day) that had no IMI (Table 4).
This finding supports the observation of Moroni et
al. (2006) who reported no drastic decrease in milk
yield among the SCM affected buffaloes compared
to healthy contemporaries. However, Dua (2001)
has estimated Rs 17,233.2 million due to SCM in
buffaloes as compared to Rs 6,962.9 million due
to clinical mastitis in India. Munro et al. (1984)
reported effects of mastitis on milk production,
milk composition and quality of milk products.
milking is of paramount important in the control
of IMI in lactating animals. Streptococci and
E. coli are environmental pathogens, and their
occurrences in mastitis are mainly associated with
type of bedding and wallowing habits of buffaloes.
Antibiotic sensitivity
The emergence of drug resistant organisms
causing mastitis due to indiscriminate use of
antibiotics is well established in bovine mastitis in
Bangladesh (Kader et al., 2002). Moreover due to
lack of prophylactic agents, chemotherapy continues
to play a major role in the therapeutic management
of mastitis. For the success of the treatment,
sensitivity testing plays a pivotal role. Recently
newer antibiotics have been introduced for the
treatment of both SCM and clinical mastitis. Thus,
it has become imperative to control this dreaded
disease with most effective antibiotic therapy.
Hence, the present study was also designed to probe
into in vitro sensitivity of isolated bacterial species
from cases of SCM against a range of traditional
as well as newly introduced antibiotics potentially
useful in mastitis treatment and control programs.
The antibiotic sensitivity of randomly
selected 20 different single culture isolates of
Staphylococcus spp. (5 isolates), Streptococcus
spp. (5 isolates), Bacillus spp. (5 isolates) and E.
coli (5 isolates) were tested with seven different
antibiotics (Table 7). It appears from the results of the
antibiotic sensitivity profiles that all the tested four
isolates of staphylococci, streptococci, bacilli and
E. coli were found moderately (2+ / 20%) to highly
(3+ / 80%) sensitive to gentamicin, ciprofloxacin,
endrofloxacin and chloramphenicol, whereas all
the bacterial isolates showed resistance (- / 0%)
to less sensitivity (1+ / 20%) against ampicillin,
amoxycillin and streptomycin (Table 7). These
Bacterial pathogens
The major agents involved in bacterial
intramammary infection (IMI) isolated from milk
samples were Staphylococcus spp. (30.77%),
followed by Streptococcus spp. (20.51%),
Bacillus spp. (15.39%), E. coli (12.82%) and
mixed (12.82%) species (Table 6). These results
support the report of Khan and Muhammad (2005)
who reported Staphylococcus aureus was found
with the highest frequency (45%), followed by
Streptococcus spp. (23%), E. coli (18%) and
Bacillus spp. (14%) in buffaloes. Similar results
have also been observed by Memon et al. (1999)
who reported Staph. aureus as the major pathogen
(38%), followed by Str. uberis (13%), E. coli (11%)
and Klebsiella pneumoniae (11%). Bhalerao et al.
(2000) also reported Staph. aureus as the major
pathogen (54.55%), followed by streptococci
(36.36%), E. coli (4.55%) and Klebsiella (2.27%).
Khan et al. (2004) reported Staph. aureus (45%)
as the major pathogen, followed by Streptococcus
spp. (23%), E. coli (18%) and Bacillus spp. (14%).
Staphyococci are usually spread during the milking
299
Buffalo Bulletin (December 2013) Vol.32 No.4
Table 1. Prevalence (animal-wise and quarter-wise) and severity of sub-clinical mastitis in lactating
buffaloes.
S/N
1
2
3
Test used
Types
White Side
Test
Surf Field
Mastitis Test
California
Mastitis Test
Animal-wise
Quarter-wise
Animal-wise
Quarter-wise
Animal-wise
Quarter-wise
Total
number
tested
30
120
30
120
30
120
Number positive (%)
1+
2+
3+
Total
No. (%)
09 (30.00)
23 (19.16)
08 (26.60)
20 (16.66)
09 (30.00)
25 (20.83)
04 (13.33)
07 (05.83)
05 (16.66)
08 (06.66)
05 (16.66)
09 (07.50)
03 (10.00)
05 (04.16)
02 (06.66)
04 (03.33)
03 (10.00)
05 (04.16)
16 (53.30)
35 (29.16)
15 (50.00)
32 (26.66)
17 (56.66)
39 (32.50)
Table 2. Comparison of three indirect tests to detect sub-clinical mastitis in lactating buffaloes.
White Side
Test (WST)
+
+
Surf Field Mastitis
Test (SFMT)
+
-
California Mastitis
Test (CMT)
+
+
+
Samples
No. (%)
32 (26.70)
81 (67.50)
04 (03.30)
03 (02.50)
Test of
Significance
Cochran’s Q
value (Qa)
was 10.57 for
2 df at p = 0.00
Table 3. Quarter-side-wise prevalence of sub-clinical mastitis in lactating buffaloes.
S/N
Test used
1
White Side
Test (WST)
2
Surf Field
Mastitis Test
(SFMT)
3
California
Mastitis Test
(CMT)
Quarter
side
LF
LH
RF
RH
Total
LF
LH
RF
RH
Total
LF
LH
RF
RH
Total
No. of
samples
tested
30
30
30
30
120
30
30
30
30
120
30
30
30
30
120
Number positive (%)
1+
07 (23.33)
05 (16.66)
03 (10.00)
08 (26.66)
23 (19.16)
05 (16.66)
05 (16.66)
03 (10.00)
07 (23.33)
20 (16.66)
06 (20.00)
06 (20.00)
05 (16.66)
08 (26.66)
25 (20.83)
2+
01 (03.33)
02 (06.66)
02 (06.66)
02 (06.66)
07 (05.83)
01 (03.33)
03 (10.00)
02 (06.66)
02 (06.66)
08 (06.66)
03 (10.00)
02 (06.66)
01 (03.33)
03 (10.00)
09 (07.50)
3+
01 (03.33)
01 (03.33)
01 (03.33)
02 (06.66)
05 (04.16)
01 (03.33)
01 (03.33)
01 (03.33)
01 (03.33)
04 (03.33)
01 (03.33)
01 (03.33)
01 (03.33)
02 (06.66)
05 (04.16)
Total
No. (%)
09 (30.00)
08 (26.66)
06 (20.00)
12 (40.00)*
35 (29.16)
07 (23.33)
09 (30.00)
06 (20.00)
10 (33.33)*
32 (26.66)
10 (33.33)
09 (30.00)
07 (23.33)
13 (43.33)*
39 (32.50)
LF = Left front quarter, LH = Left hind quarter, RF = Right front quarter, RH = Right hind quarter,
*Insignificantly (p > 0.05) higher prevalence.
300
Buffalo Bulletin (December 2013) Vol.32 No.4
Table 4. Comparison of milk production between sub-clinical mastitis negative (n = 13) and positive (n = 17)
lactating buffaloes.
Animal
No.
1
2
3
4
5
6
7
Milk production
(l/d)
Normal
SCM
6.0
5.5
4.0
4.0
5.0
5.0
5.5
4.0
5.5
4.5
4.0
4.0
6.5
5.0
SCM = Sub-clinical mastitis
Animal
No.
8
9
10
11
12
13
14
Milk production
(l/d)
Normal
SCM
4.0
3.0
4.0
3.5
5.0
4.0
4.0
4.5
4.0
5.0
4.5
5.5
4.5
l/d = Liter / day
Animal
No.
15
16
17
Total
Mean
± SD
Milk production
(l/d)
Normal
SCM
5.0
4.5
5.5
62
4.8
± 0.88
77
4.5*
± 0.88
n= No. of animals *Decreased insignificantly (p > 0.5)
Table 5. Host risk factors associated with sub-clinical mastitis in lactating buffaloes detected by California
Mastitis Test (CMT).
S/N
1
2
3
Risk factors
Age (years)
3 to 6
> 6 to 9
> 9 to 12
> 12
Total
Parity
1st
2nd
3rd
4th
5th
6th
Total
Lactation period
Early (0 to 10 weeks)
Mid (>10 to 20 weeks)
Late (>20 to 24 weeks)
Total
No. of
Buffaloes
tested
Positive
Nagative
No. (%)
No. (%)
05
11
10
04
30
02 (06.67)
06 (20.00)
07 (23.33)*
02 (06.67)
17 (56.67)
03 (10.00)
05 (16.67)
03 (10.00)
02 (06.67)
13 (43.33)
08
05
05
07
03
02
30
04 (13.33)
03 (10.00)
03 (10.00)
05 (16.67)*
01 (03.33)
01 (03.33)
17 (56.67)
04 (13.33)
02 (06.67)
02 (06.67)
02 (06.67)
02 (06.67)
01 (03.33)
13 (43.33)
11
07
12
30
05 (16.67)
03 (10.00)
09 (30.00)*
17 (56.67)
06 (20.00)
04 (13.33)
03 (10.00)
13 (43.33)
*Highest insignificant (p > 0.05) values.
301
Buffalo Bulletin (December 2013) Vol.32 No.4
Table 6. Frequency distribution of bacteria isolated from milk samples (n = 39) of lactating buffaloes.
S/N
Bacterial species
+ ve, No. (%)
S/N
Bacterial species
+ve, No. (%)
1
Staphylococcus spp.
12 (30.77)
5
Staphylococcus spp.+Streptococcus spp.
02 (05.13)
2
Streptococcus spp.
08 (20.51)
6
Bacillus spp. + Staphylococcus spp.
02 (05.13)
3
Bacillus spp.
06 (15.39)
7
Escherichia coli + Streptococcus spp.
01 (02.56)
4
Escherichia coli
05 (12.82)
Mixed infection
05 (12.82)
Single infection
31 (79.49)
Unclassified bacterial growth
03 (07.69)
8
n = 39 CMT positive mammary quarter milk samples
Table 7. Percentage in vitro sensitivity of different bacterial isolates to different antibiotics.
S/N
1
2
3
4
5
6
7
Antibiotics
Gentamicin
Ciprofloxacin
Enrofloxacin
Chloramphenicol
Amoxicillin
Ampicillin
Streptomycin
R = Resistance
Status
Staphalococcus spp.
Streptococcus spp.
Bacillus spp.
E. coli
R
00.00
00.00
00.00
00.00
LS
00.00
00.00
00.00
00.00
MS
20.00
20.00
20.00
40.80
HS
80.00
80.00
80.00
60.00
R
00.00
00.00
00.00
00.00
LS
00.00
00.00
00.00
00.00
MS
40.00
20.00
20.00
20.00
HS
60.00
80.00
80.00
80.00
R
00.00
00.00
00.00
00.00
LS
00.00
00.00
00.00
00.00
MS
40.00
40.00
20.00
60.00
HS
60.00
60.00
80.00
40.00
R
00.00
00.00
00.00
00.00
LS
00.00
00.00
00.00
00.00
MS
60.00
40.00
20.00
80.00
HS
40.00
60.00
80.00
20.00
R
60.00
60.00
60.00
20.00
LS
40.00
40.00
40.00
80.00
MS
00.00
00.00
00.00
00.00
HS
00.00
00.00
00.00
00.00
R
40.00
40.00
60.00
40.00
LS
60.00
60.00
40.00
60.00
MS
00.00
00.00
00.00
00.00
HS
00.00
00.00
00.00
00.00
R
80.00
60.00
80.00
40.00
LS
20.00
40.00
20.00
60.00
MS
00.00
00.00
00.00
00.00
HS
00.00
00.00
00.00
00.00
LS = Less sensitive
MS = Moderately sensitive
302
HS = Highly sensitive
Buffalo Bulletin (December 2013) Vol.32 No.4
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antibiotics in the treatment of mastitis in Bangladesh.
The high (56.66%) prevalence of SCM in
the lactating buffaloes of the Government Buffalo
Breeding and Development Farm - ultimately
indicates the bad quality of milk available to the
consumers. This is mainly due to the non-use of
regular screening tests for SCM and the unhygienic
management of animals and milking system. The
CMT was found more sensitive than WST and
SFMT, thus it could be used for regular screening
of SCM in lactating buffaloes; however, SFMT
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regular screening of SCM by using any available
indirect test and the tested effective antibiotics
be evaluated in vivo under field conditions.
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Buffalo Bulletin (December 2013) Vol.32 No.4
Original Article
PREVALENCE AND ANTIBACTERIAL SUSCEPTIBILITY IN MASTITIS IN BUFFALO AND
COW IN DISTRICT LAHORE-PAKISTAN
Yasser Saleem Mustafa*, Farhat Nazir Awan and Tooba Zaman
ABSTRACT
INTRODUCTION
A total of 450 milk samples including
both 272 buffalo and 178 cow were randomly
collected in and around District Lahore to study
the incidence of mastitis and antibiotic sensitivity
by performing culture and sensitivity test. The
prevalence of mastitis in buffalo was found to
be 20.98% while that in cowwas 24.71%. The
prevalences of both clinical and subclincal mastitis
in buffalo were 40.35% and 59.64% , respectively,
and those in cow were 61.26% and 30.63%
respectively. The milk samples mixed with both
mucus and blood in buffalo and cow were 5.51%
and 4.49%, respectively. Quarter-wise prevalence
was 47.72%, 11.36%, 36.36% and 4.54% in the left
fore, left hind, right fore and right hind quarters in
cow while in buffaloes, the prevalence was 0%,
68.96%, 11.49% and 19.54% in the left fore, left
hind, right fore and right hind quarters, respectively.
Ciprofloxacin was found highly sensitive in buffalo
while gentamicin was found highly sensitive in cow.
Buffalo and cattle are mostly reared for
milk production, and the disease mastitis renders
them useless for this purpose. Milk production
usually decreases and blood alone or mixed with
mucus come with the milk. It is one of the most
important reasons for termination of lactation and
unwanted culling of dairy buffalo (McDowell et
al., 1995). Mastitis is considered to be the most
costly disease of dairy animals worldwide. This
disease complex is the outcome of interaction of
various factors associated with the host, pathogens
and the environment. The productive efficiency of
dairy animals is adversely affected by suboptimal
management, poor nutrition and various diseases,
in particular mastitis, which is one of the most
important impediments confronting economic
milk production in Pakistan. It is the most costly
disease of the dairy industry throughout the world
(DeGraves and Fetrow, 1991) that affects both
quality (Barbano, 1989) and quantity of milk
(Arshad et al., 1995). Field surveys of major
livestock diseases in Pakistan have indicated that
mastitis is one of the most important diseases of
dairy animals in the country (Hussain et al., 2005).
Mastitis is the outcome of the interaction of various
Keywords: antibiotic, buffaloes, Bubalus bubalis,
cow, antibiotic, incidence, mastitis
Provincial Diagnostic Laboratory, Livestock and Dairy Development, 16-Cooper Road, Lahore-Pakistan,
*E-mail: yasserbutt1@yahoo.com
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Buffalo Bulletin (December 2013) Vol.32 No.4
factors associated with the host, the pathogen(s)
and the environment.
In Pakistan and other developing countries
owing to small herd sizes, dairy animals are
predominantly hand-milked. Infectious agents of
mastitis may be transmitted from infected to uninfected animals through the milker’s hand (Oliver,
1975) especially because milk is often used as a
lubricant for milking. Mastitis in hand-milked cows
was nearly twice as frequent as in machine-milked
ones (25.1 VS 14.6%) Motie et al. (1985).
The infection originates either from the
infected udder or the contaminated environments.
The major sources of pathogens and means of
transmission include infected quarters and soiled
udder, contaminated milking machines, teat cups,
milker’s hands, washing clothes, flies and surgical
instruments. Moreover, the stage of lactation,
lactation number, trauma to udder, teat and teat
canal, loose teat sphincters, lesions on teat skin,
immunological status of each mammary gland, bulk
of infection in the environment and managemental
conditions are amongst the determinants which
dictate the level of mastitis incidence (Radostits et
al., 2000).
Clinical mastitis is an individual problem
and it is characterized by changes in the udder and
milk drawn from it. Whereas, subclinical mastitis
The present study was, therefore designed
to determine the frequency distribution of mastitis
in dairy buffaloes and cows and to determine the
association of some host and pathogen(s) related
determinants with the disease.
MATERIALS AND METHODS
A total of 450 animals (n=272 buffaloes
n=178 cattle) of 50 randomly selected livestock
farmers were screened to find the epidemiology
of clinical and sub-clinical mastitis in the study
area. Milk samples were also brought to the
laboratory from diseased animals not treated with
antibiotics, immediately cooled, and transported
to the Provincial Diagnostic Laboratory, L&DD,
16-Cooper Road, Lahore in an ice box for
microbiological examination. Clinical mastitis
was diagnosed when there were visible or palpable
signs of udder inflammation along with the
changes in milk secretions whereas subclinical
mastitis was diagnosed by using the Surf Field
Mastitis Test (SFMT) (Muhammad et al., 1995).
A comprehensive questionnaire focused on data
related to cattle and buffaloes, host and managerial
determinants/risk factors associated with mastitis
was completed in the presence of each livestock
farmer whose animal was selected for the present
study.
is herd problem because it constituents a reservoir
of infection which could be transmitted to other
animals of the herd. The frequency, severity, and
economic impact of mastitis are known to depend
upon the preventive and management approaches.
It has also been observed that the incidence and
the patterns of causative agents markedly differ
from place to place, herd to herd, and time to time.
Studies conducted in different states within India
reflect high incidence of the disease for past seven
decades.
Microbiological examination
Microbiological examination of milk
samples began within 8 h of collection. The
procedure described by National Mastitis Council
Inc., USA (1990) was followed for the collection
of milk samples. After discarding the first few
streams, about 10 ml of milk was collected
aseptically. The procedures described by National
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Buffalo Bulletin (December 2013) Vol.32 No.4
hindquarters as compared to the forequarters and
slightly higher in right quarters than left ones. In
case of forequarters, both species were equally
affected as also reported by Rehman (1995).
The prevalence of clinical mastitis in cow
was reported to be 61.36% while in buffaloes, the
prevalence of clinical mastitis was 40.35% (Table
2). These findings are in close alignment with the
findings of Nooruddin et al. (1997) and Bilal et al.
(2004). The prevalence of sub-clinical mastitis was
also found higher in buffaloes (59.64%) than in
cows (30.63%). Dangore et al. (2000) and Allore
(1993) reported low prevalence of subclinical
mastitis in dairy cows, which is in accordance with
the findings of present study.
In mastitis, there is drastic change in the
milk, taste and consistency. In sub-clinical mastitis,
there was bad taste and odor; in the second stage,
there was a watery discharge; in the third stage,
mucus mixed with milk, and in the fourth stage,
blood mixed with milk from the affected teat,
which resulted in culling of animal if not properly
treated. The changes in the milk due to mastitis are
shown in Table 3. Milk with bad taste and odor was
found 8.08% in buffalo and 6.74% in cow. Milk
mixed with mucus and blood was recorded 6.61%
and 7.35% in buffalo and in cow 7.35% and 5.61%
while milk with mixed mucus and blood was
5.51% in buffalo and 4.49% in cow, respectively.
These findings are in agreement to those reported
by Khan and Muhammad (2005).
Quarter-based prevalence of clinical
mastitis in cow and buffaloes were also determined.
The prevalence of clinical mastitis in relation
to quarters was determined, it was found that
prevalence was higher in fore quarters than in rear
quarters in cow and it was higher in rear quarters
than in fore quarters in buffaloes. Prevalence was
47.72%, 11.36%, 36.36% and 4.54% in the left-
Mastitis Council Inc., USA (1987) were followed
for culturing the milk samples and identification of
mastitis pathogens. The samples were shaken eight
times to get a uniform dispersion of the pathogens.
Using a platinum-rhodium loop, 0.01 ml of milk
sample was streaked each onto MacConkey’s agar
plate. Milk samples were cultured on a 100 mm
plate by plating and incubated at 37°C for 48 h.
The Guidelines of National Mastitis Council Inc
(1987) on the significance of colony numbers in
pure or mixed cultures were used to categorize a
sample as infected or contaminated. The colonies
of the microorganisms were isolated and with
platinum loop mixed in distilled water and then
spread on Petri dishes with antibiotic disks. Eight
different antibiotics, i.e. gentamycin, ciprofloxacin,
norfloxacine,
ampicillin,
streptomycine,
chloramphenicol, pencillin and amoxicillin were
used for the treatment of mastitis and their efficacy
was studied. These antibiotics were injected intramuscularly at the dose rate of 1 ml/10 kg live body
weight of the animal. The data was statistically
analyzed by applying percentage.
RESULTS AND DISCUSSION
In the present study, the overall prevalence
of mastitis was found 22.44% including 24.71% in
cow and 31.75% in buffaloes (Table1). The overall
prevalence of mastitis was lower in the buffaloes
as compared to the crossbred cows. This lower
prevalence might be attributed to the tighter teat
sphincter of buffaloes as compared to that of cows
(Uppal et al., 1994). There was higher incidence
in hindquarters in buffaloes than crossbred cows
and among hindquarters, right hindquarters were
found to be more susceptible. Iqbal (1992) reported
that the prevalence of hind quarters was higher in
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Buffalo Bulletin (December 2013) Vol.32 No.4
Table 1. Prevalence of mastitis in buffalo and cow in district Lahore.
Species
No. of animals
examined
No. of affected
animals
Mastitis
Prevalence (%)
Buffalo
Cow
Total
272
178
450
57
44
101
20.95
24.71
22.44
Table 2. Types of mastitis in buffalo and cow in district Lahore.
Species
Buffalo (n=57)
Cow (n=44)
Total (N=101)
Clinical
23 (40.35%)
27 (61.36%)
50 (49.50%)
Sub clinical
34 (59.64%)
17 (30.63%)
51 (50.49%)
Table 3. Physical characters of the milk.
Species
Normal
Bad Taste
and Odor
Watery
Mucus
Blood
Mucus mix
with Blood
Buffalo
(n=272)
185 (68.01%)
22(8.08%)
12 (4.41%)
18(6.61%)
20(7.35%)
15(5.51%)
Cow
(n=178)
122(68.53%)
12 (6.74%)
8(4.49%)
18(4.49%)
10(5.61%)
8(4.49%)
Total
(N=450)
307 (68.22%)
34 (7.55%)
20(4.44%)
36(8%)
30(6.66%)
23(5.11%)
Table 4. Quarter-wise incidence of mastitis in buffalo and cow.
Species
Left Fore
Quarter
Right Fore
Quarter
Left Hind
Quarter
Right Hind
Quarter
Buffalo (n=87)
Cow (n=44)
- (0%)
21 (47.72%)
10 (11.49%)
16 (36.36%)
60 (68.96%)
5 (11.36%)
17 (19.54%)
2 (4.54%)
310
S
H.S
Buffalo
(n=87)
Cow
(n=44)
S
H.S
Ciprofloaxcin
CST = Culture and Sensitivity test
HS = Highly Sensitive
S = Sensitive
R = Resistant
Gentamicin
Species
S
S
Norfloxacine
R
R
Enorfloxacin
R
R
Ampicillin
R
R
Streptomycine
Table 5. Antibiotic Response using CST for the treatment of Mastitis in buffalo and cow.
R
R
Chloramphenicol
R
R
Pencillin
R
R
Amoxicillin
Buffalo Bulletin (December 2013) Vol.32 No.4
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Buffalo Bulletin (December 2013) Vol.32 No.4
fore, left-rear, right-fore and right-rear quarters,
respectively, in cow. In buffaloes, the prevalence
was 0%, 68.96%, 11.49% and 19.54% in the left
fore, left rear, right fore and right rear quarters,
respectively (Table 4).
Prevalence of hind quarters was higher in
buffaloes than in cow. It was 1.11% and 1.41% in cow
and buffaloes, respectively. When the prevalence
of hind quarters in relation to anatomical location
of quarters was determined, it was found that
prevalence was higher in fore quarters than in rear
quarters in cow and it was higher in rear quarters
than in fore quarters in buffaloes. Prevalence was
0.46%, 0.19%, 0.27% and 0.19% in left fore, left
rear, right fore and right rear quarters, respectively,
in cow. In buffaloes, the prevalence was 0.20%,
0.47%, 0.27% and 0.47% in left fore, left rear,
right fore and right rear quarters, respectively.
The slightly higher prevalence of hind quarters in
buffaloes might be due to the high incidence of
clinical mastitis in buffaloes as advanced untreated
cases of mastitis could lead to teat blindness
Shukla et al. (1997) reported that forequarters were
more affected than hind quarters in the case of
cows where in buffaloes hind quarters had higher
prevalence of mastitis than forequarters, which
supported the findings of present study. Similar
findings were observed by (Bilal et al., 2004;
Allore,1993; Premchand et al.,1995) who reported
a higher prevalence of mastitis in hind quarters of
buffaloes than in fore quarters. The findings of the
present study do not correlate with the findings of
Ahmad et al. (1991).
Ciprofloxacin was found to have high
sensitivity in buffalo, and gentamicin was found
to have high sensitivity in cow while norfloxacin
was found to have sensitivity in both buffalo and
cow by performing the culture and sensitivity
test. It was found that all other antibiotics shown
resistant to the bacteria (Table 5). These findings
are in agreement with findings of Mustafa et al.,
2007. Sumathi et al., 2008 also found genatmicin
effective while Guerin et al., 2002; Gianneechini
et al., 2002; Ebrahimi et al., 2002; Erskine et al.,
1986 found gentamicin resistant.
CONCLUSION
It was concluded from present the study
that prevalence of clinical and subclinical mastitis
was higher in hindquarters than forequarters and
among hindquarters, left hindquarters were more
susceptible than the right.
With the advent of improved diagnostic
tests, more understanding of the disease and
availability of third generation antibiotics, and
improved ways and means to upkeep the hygiene
and management, the opportunities for clean milk
production in periurban areas are increasing.
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Original Article
Buffalo Bulletin (December 2013) Vol.32 No.4
LAPAROSCOPIC BIOPSY TECHNIQUE OF LIVER AND SPLEEN IN BUFFALO CALVES
K. Srinivasa Rao, Makkena Sreenu*, K.B.P. Raghavender and P.V.S. Kishore
ABSTRACT
on fasting for 24 to 48 h prior to laparoscopy
and they were bathed and dried before being
allowed into operating room. The animals were
administered with xylazine hydrochloride 0.05 mg/
Kg body weight intra muscularly. Local infiltration
of portal sites with 8 to 10 ml of 2% lignocaine
hydrochloride (Xylocaine® Astra IDL Bangalore)
Exploratory surgery is one of the diagnostic
procedures followed to detect various abdominal
disorders in bovine practice. Laparoscopy is a
minimally invasive surgical technique using an
endoscope inserted transabdominally to observe
organs within the abdominal and pelvic cavities.
The advantages of the use of laparoscopy-guided
biopsy techniques are the direct visualization of the
target organ and the selection of the exact biopsy
site. In this way, obtaining biopsy specimens of the
wrong organ is avoided, and possible hemorrhages
are identified and controlled. The direct view of
the target organ can provide additional information
concerning the condition and eventually its
prognosis.
was done in all the layers of the abdominal muscles
for flank approach and subcutaneously for mid
ventral approach prior to introduction of cannuals.
The two portal sites selected to perform laparoscopy
on the left side were at the middle and lower
paralumbar fossa. For the mid-ventral approach,
the portal sites selected were 2 inches lateral to
mid-ventral line anterior to umbilicus. The biopsy
specimens of the organs like the liver and spleen
were collected under laparoscope guidance and
after introducing a second cannula equidistant
parallel or opposite to the first cannula introduced
for a particular approach. To avoid injury to the
abdominal structures, instrument cannulas are
introduced under laparoscopic guidance and the
instrument was pushed slowly into the cannula
and entered the abdominal cavity. The jaws
were kept closed until the instrument reached
the required site if forceps or scissors were used.
The collected tissue specimens were subjected to
histopathological examination as per the method
of Singh and Sulochana (1997) to study the tissue
artifacts if any and to ascertain the suitability of the
Keyword: buffalo calves, Bubalus bubalis, liver,
spleen, laparoscopic biopsy technique
MATERIALS AND METHODS
A total of twelve male buffalo calves
aged about one and a half to two years presented
to clinics were utilized to perform laparoscopy.
Laparoscopy equipment along with accessories
manufactured and supplied by Karlstorz (Germany)
were used for this study. All the calves were kept
Department of Veterinary Surgery and Radiology, NTR College of Veterinary Science, Gannavaram Sri
Venkateswara Veterinary University, Tirupati, Andhra Pradesh, India,*E-mail: drmakkena@yahoo.co.in
315
Buffalo Bulletin (December 2013) Vol.32 No.4
effectively with swabbing of adrenalin using biopsy
forceps after collection of tissue for about two
minutes. Meticulous care is needed while catching
the edge of the spleen to crush the surface of the
spleen to avoid multiple attempts (Figure 2).
In this technique, the laparoscope and
instrument portals are created equidistance from
the linea alba at xiphoid level on left and right sides
to visualize the liver through midventral approach.
Biopsy specimens of the liver were collected using
biopsy forceps through instrument portal under the
illumination of the midventral laparoscopy. The
laparoscope was directed parallel to the right side
cranially to identify the lobe of liver. The edge of
the liver was caught with the biopsy forceps and the
piece of about 1mm size could be collected. There
was haemorrhage from cut surface of liver which
was controlled by a swab of adrenalin as described
earlier during the spleen biopsy Biopsy specimens
of the liver were collected using biopsy forceps
through the instrument portal under the illumination
of the mid ventral laparoscopy satisfactorily yielded
in sample size. The search and visibility of the liver
was good, and the edge of the liver was caught with
the biopsy forceps on first instance in almost all the
cases (Figures 3 and 4).
All the histological sections obtained from
the laparoscopic guided biopsy specimens revealed
a normal microscopic picture except for a few
artifacts. The yielded sections of spleen revealed
a dense connective tissue capsule from which
connective tissue trabeculae extend deep in to the
spleen interior and characterized by the presence
of numerous aggregations of lymph nodules
(white pulp) and surrounded by a diffuse cellular
meshwork intermeshed with trabeculae (red pulp)
along with arterial and venous structures (Figure
5). The sections of liver have shown the connective
tissue from the liver hilus extends between the liver
yielded laparoscopic guided biopsy specimen for
determining cellular architecture.
RESULTS AND DISCUSSION
Biopsy is a method aiding in the
determination of a precise diagnosis and disease
prognosis. Diagnostic evaluation of many different
medical conditions can be assisted by obtaining
biopsy samples from multiple abdominal organs.
The sample collection has traditionally been
performed several ways like fine-needle aspiration
biopsy, percutaneous biopsy, biopsy under the
guidance of ultrasonography, biopsy under
endoscopic / otoscopic guidance, biopsy at the
time of exploratory laparotomy (Mayhew, 2009).
In cattle, the first reports on an organ biopsy by
laparoscopy guidance involved the kidney (Naoi
et al., 1985) and the liver (Whitehair, 1998) while
Klein et al. (2002) described an intestinal biopsy
technique in calves and sheep.
Biopsy specimens of the spleen were
collected using biopsy forceps through the
instrument portal under the illumination of the left
flank laparoscopy. The laparoscope was directed
parallel to the spine cranially to identify the body
of the spleen. The edge of the spleen was caught
with the biopsy forceps, and a piece of about 1
mm size could be collected (Figure 1). There was
haemorrhage from the cut surface of the spleen.
A small swab of ear bud size was imbibed with
adrenalin and placed on the bleeding area using
forceps provided with the instrument for about 2
minutes to arrest the bleeding. Biopsy specimens
of the spleen were collected in all the calves under
the illumination of the left para lumbar laparoscopy.
The technique is easy to perform and yielded an
accurate sample size. Haemostasis was achieved
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Buffalo Bulletin (December 2013) Vol.32 No.4
lobes as indistinct interlobular septa dividing in
to hepatic lobules. The interlobular septa had the
branches of the portal vein, bile duct and hepatic
artery (portal triad). In the centre of each lobule a
central vein, cords of hepatic cells (the at periphery)
and between hepatic cords, hepatic sinusoids are
seen (Figure 6). Artifacts like separation of tissue,
detachment of surface, vacuolization, cracking and
mild congestion were observed at the edges of the
tissue. (Figures 7-10).
Laparoscopic guided spleen and liver
biopsy is a minimally invasive alternative to the
biopsy methods by use of sharp cutting or grasping/
shearing instruments. The technique selected
depends on the surgeon’s preference, stability of
the animal and available equipment. Hidiroglou
and Ivan (1993) conducted liver biopsies in
sheep in sternal recumbency. Steve (2000) used
laparoscopic techniques for biopsy collection
from the spleen, liver, and kidney in horses and
the hemostasis was achieved by using endoscopic
bipolar cautery forceps.
As in open surgery, uncontrolled bleeding
during laparoscopy is a major surgical pitfalls. A
variety of techniques and instruments have been
transferred from open surgery and adapted to the
specific needs of laparoscopy to gain adequate
haemostasis. Boure (2005) stated that control
of intraoperative bleeding is most important in
laparoscopic surgery and even a small amount of
blood can obscure the laparoscopic surgical site
because it absorbs the light and even cover the lens.
The procedure adopted in the present i.e. swabbing
of the cut surface with adrenalin following biopsy
collection satisfactorily controlled the hemorrhage
which might due to the vasoconstrictive property
of the adrenalin.
Specimens obtained by laparoscopic guided
biopsy techniques in the present study had minimal
distortion of tissue as evaluated microscopically
and were considered an accurate representation of
the organs and histological structures are similar
to the observations of William and Linda (2000).
All biopsy methods evaluated produced minimal
immediate hemorrhage and resulted in adequate
tissue samples for histological evaluation,
(Vasanjee et al., 2006). Damage was defined as
any disruption of the normal cellular architecture
at the incised margins and extent was determined
by measuring the furthest edge of the damage
perpendicular to the incised margin. Harmoinen et
al. (2002) observed some inflammatory changes
in sample collected through laparoscopic assisted
biopsy around the biopsy sites
The biopsy forceps caused collateral
damage, and two distinct forms of damage were
apparent. Sharp cutting and grasping methods/
instruments (biopsy punch, biopsy needle, ligature
method, laparoscopic biopsy forceps) resulted
in crushing of the tissue. The degree of crushing
that occurs is a function of the instrument and
handling of the tissues. For instance, an instrument
like the laparoscopic biopsy forceps, where tissue
is crushed and torn, would be expected to cause
more collateral damage when performing a biopsy,
the resultant tissue sample size is a function of
the instrument used to obtain the biopsy. The
biopsy needle, biopsy punch, and laparoscopic
biopsy forceps are restricted in the amount of
tissue obtained by the instrument size and design.
Laparoscopy-guided biopsy of the liver and spleen
yielded normal cellular architecture with minimum
artifacts at the edges of the tissue collected due to
the crushing effect of the forceps edges.
317
Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 1.
Note the biopsy forceps holding
the splenic edge.
Figure 2. Hemorrhage from cut surface of
the spleen following biopsy.
Figure 3. Collection of biopsy specimen
from liver through ventral
approach.
Figure 4. The hemorrhagic surface of the liver
following the biopsy procedure.
Figure 5. Cellular architecture of the spleen
H&E 10 X.
Figure 6.
318
Cellular architecture of the liver
H&E 10 X.
Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 7. Cellular detachment and
hyperemia– spleen. H&E 10 X.
Figure 8. Folding and stain precipitate– -spleen. H&E 10 X.
Figure 9. Detachment of the capsule - spleen.
H&E 10 X.
Figure
319
10.
Cracking of the
liver. H&E 10 X.
specimen-
Buffalo Bulletin (December 2013) Vol.32 No.4
REFERENCES
144-147.
Boure, L. 2005. General principles of laparoscopy.
Veterinary Clinics Food Animal Practice.,
21: 227-249.
Harmoinen, J., S. Saari and M. Rinkinen. 2002.
Evaluation of pancreatic forceps biopsy by
laparoscopy in healthy beagles. Vet. Ther.,
3: 31- 36.
Hidiroglou, M. and M. Ivan. 1993. Liver biopsy in
sheep. Vet. Res., 24: 260-265.
Klein, C., S. Franz, A. Leber, Z. Bago and W.
Baumgartner. 2002. A new technique of
laparoscopicbiopsy sampling of the small
intestine in calves and sheep [German].
Wien Tierarztl Monatsschr., 89: 291-301.
Mayhew, P. 2009. Techniques for laparoscopic and
laparoscopic assisted biopsy of abdominal
organs. Compendium:Continuing Education
for Veterinarians, 170-179.
Naoi, M., E. Kokue and Takshakshi. 1985.
Laparoscopic assisted serial biopsy of the
bovine kidney. Am. J. Vet. Res., 46: 699702
Singh, U.B. and S. Sulochana. 1997. A Practical
Manual
of
Histopathological
and
Histochemical
Techniques,
Kothari
Publications, Bombay. pp. 154.
Vasanjee, S. C., L.J. Bubenik, G. Hosgood and R.
Bauer. 2006. Evaluation of
hemorrhage, sample size and collateral damage for
five hepatic biopsy methods in dogs. Vet.
Surg., 35: 86-93.
William, J.B. and M.B. Linda. 2000. General
principles of histology, p. 1-8. In Colour
Atlas of Veterinary Histology, 2nd ed.
Lippincott Williams and Wilkins. USA.
Whitehair, C.K., R.B. Dasilva and N.K. Anes. 1988.
Live biopsy in cattle. Bovine Practice., 23:
320
Original Article
Buffalo Bulletin (December 2013) Vol.32 No.4
IMPACT ON HEMATOLOGICAL PARAMETERS IN YOUNG AND ADULT MURRAH
BUFFALOES EXPOSED TO ACUTE HEAT STRESS
N. Haque1,*, A. Ludri2, S.A. Hossain2 and M. Ashutosh1
ABSTRACT
to adult, indicating that young animals were more
susceptible to heat stress compared to their adult
counterparts. This study concludes that acute heat
stress evokes a series of drastic changes in the
animal’s hematological functions.
The present study was designed to
investigate the effect of acute heat stress on some
hematological parameters in which young and adult
Murrah buffaloes (n=6) were exposed to 40°C,
42°C, 45°C for 4 h duration in climatic chamber
and thermoneutral temperature (22°C). Blood
samples were collected by jugular vein puncture
in sterile vacutainer tubes containing ethylene
diamine tetra acetic acid (EDTA) from animals after
4 h exposure to different temperatures. In packed
cell volume (PCV) and hemoglobin, there was an
increasing trend with increase of temperature, but
there was no effect of age on these parameters.
In case of total erythrocytic count (TEC), there
was no effect of temperature but adult animals
had higher TEC. Total leukocytic count (TLC)
was significantly increased at 40°C and 42°C in
young, but there was no effect of temperature on
TLC in adult animals. Mean corpuscular volume
(MCV) and mean corpuscular hemoglobin (MCH)
values were similar in all the four temperatures
groups in both young and adult animals. There
was no effect of temperature and age on mean
corpuscular hemoglobin concentration (MCHC)
values. The intensity of changes in all parameters
were more pronounced in young animals compared
Keywords: young and adult Murrah buffalo, heat
stress, hemoglobin, packed cell volume, total
erythrocytic and leukocytic count
INTRTODUCTION
Stress is the state manifested by a specific
syndrome, which consists of all the non-specifically
induced changes within a biological system. Both
external and internal stressors cause pronounced
behavioral, physiological and hematological
alterations in tropical livestock. Swamp buffaloes
(Bubalus bubalis) are found in hot-humid climates
and are important livestock in east and southeast
Asia. Hafez et al. (1955) reported that in Egyptian
buffaloes, glandular surface of sweat gland per cm2
of skin surface was 1.07 in buffaloes and 3.08 in
cattle and that the skin thickness of buffaloes was
about twice that of cattle. The sweat glands in
buffaloes are underdeveloped (Koga, 1999). This
indicates less efficiency in sweating in buffaloes
than cattle, placeing buffaloes at a disadvantage
Dairy Cattle Physiology Division, National Dairy Research Institute, Karnal, Haryana, India,
*E-mail: haquenilufar@gmail.com
2
Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal, Haryana, India
1
321
Buffalo Bulletin (December 2013) Vol.32 No.4
were brought to the laboratory for evaluation of
hematological parameters.
PCV estimation was done by the Wintrobe,
or macrohematocrit, method . Hemoglobin
concentration was estimated with Drabkins solution,
and the optical density was measured at 540 nm on
a spectrophotometer. TEC and TLC were done in a
Neubauer chamber under a compound microscope
at 40X. Differential leukocyte count (DLC) was
done using Leishman stain of 0.15% in methyl
alcohol. The cells were counted using a high power
oil emersion lens in a strip running the whole length
of the film. The erythrocyte indices such as MCV,
MCH and MCHC were calculated based on TEC,
hemoglobin and PCV.
All the data generated were statistically
analyzed by two-way ANOVA using SigmaStat
(3.1 software package) Statistical Analysis
System (Jandel Scientifiic, San Rafael, CA, USA),
according to Snedecor and Cochran (1994).
under solar radiation as enhanced reabsorption of
solar radiation interferes with heat loss resulting
in higher heat storage. So, buffaloes have poor
capacity to withstand high temperatures and so
need greater attention to protection against adverse
climatic conditions. The physiological responses of
these animals to environmental stress during winter
and summer and their energy balance showed that
seasonal heat and cold stress have a profound effect
on some biochemical, hematological parameters
(Nazifi et al., 1999). Therefore, the present study was
undertaken to measure the comparative influence of
heat stress on hematological parameters at different
temperatures in young and adult buffaloes. This
information is likely to further help to minimize
stress in Murrah buffaloes.
MATERIALS AND METHODS
Two groups of healthy Murrah buffaloes,
young (1-2 yrs) and adult (3-4 yrs), were selected for
the experiment. Each group contained six animals.
An insulated climatic chamber (22’26”x 10’10”
x 8’) fitted with thermostatically controlled heat
convector was used for exposing animals to heat.
The temperature of of the chamber was maintained
at 40.0 ± 1.0°C, 42.0 ± 1.0°C and 45.0 ± 1.0°C
prior to the experiments. After exposure of animals
for 4 h at the above mentioned temperatures, blood
was collected. Blood samples were also collected
from the animals in the month of March, when
average environmental temperature was 22°C,
which is considered to be the temperature of
thermoneutral environment for tropical animals
and used as control. Blood samples were drawn
in sterile vacutainer tubes containing EDTA (1mg/
ml) anticoagulant by jugular vein puncture posing
minimum disturbances. Immediately the tubes
RESULTS AND DISCUSSION
The data on hematological parameters
of young and adult Murrah buffaloes exposed to
different temperatures are presented in Table 1. The
PCV increased with the increase of temperature in
both young and adult buffaloes. In young animals,
PCV (%) was increased from 33.83 at 22°C to
38.17 at 45°C whereas the values were 33.17 and
37.33, respectively, in adult animals. The results
also showed a significant increase (P<0.05) of PCV
at 40°, 42° and 45°C as compared to 22°C in young
animals whereas in adults, significant differences
were only observed at 22°C and 42°C; and 22°C
and 45°C interactions only. At other temperature
interactions, the values were statistically the same.
There was no effect of age on PCV since the values
322
323
22°C
33.83ax
12.52ax
7.65ax
7.93ax
72.33
24.00
2.83
0.83
0.00
44.36y
16.41y
37.06x
40°C
36.67 bx
13.54bx
7.94ax
10.75bx
75.00
22.17
2.33
0.50
0.00
46.73y
17.26y
36.94x
42°C
36.83 bx
13.84bcx
8.19 ax
11.54by
77.17
20.17
1.83x
0.83
0.00
45.26y
17.00y
37.60x
Young
Temperature (°C)
45°C
22°C
bx
38.17
33.17ax
14.50cx
11.94ax
8.89 ax
9.59 ay
8.92ax
8.08ax
74.00
72.67
24.00
22.50
x
1.33
3.67
0.67
1.17
0.00
0.00
x
43.01
35.05x
16.36x
12.57x
38.06x
36.08x
40°C
35.08abx
13.41bx
9.89 ay
9.73ax
75.00
20.67
3.33
0.83
0.17
35.69x
13.64x
38.46x
a, b, c
42°C
36.00 bx
13.70bx
10.01 ay
9.17ax
74.67
21.67
2.83y
0.83
0.00
34.76x
14.16x
38.12x
Adult
45°C
37.33 bx
14.23bx
9.68 ay
8.40ax
72.83
22.33
3.67y
1.00
0.17
39.14x
14.89x
38.15x
0.062
0.118
<0.001
0.005
0.377
0.246
0.002
0.260
0.165
<0.001
<0.001
0.692
Age
<0.001
<0.001
0.034
<0.001
0.261
0.593
0.385
0.696
0.577
0.857
0.409
0.445
Temp.
0.922
0.776
0.546
0.045
0.162
0.202
0.429
0.986
0.577
0.358
0.414
0.668
Age x
Temp.
Significance of effects (p)
indicate significant difference between temperatures; x,y indicate significant difference between age groups.
Means with different superscripts in rows for a parameter differ significantly (P<0.05).
PCV (%)
Hb conc. (g%)
TEC (106/μl)
TLC (103/μl)
Lymphocyte
Neutrophil
Monocyte
Eosinophil
Basophil
MCV (fl/cell)
MCH (pg/cell)
MCHC (g/dl)
Parameters
Table 1. Comparision of hematological parameters in young and adult Murrah buffaloes exposed to different temperatures.
0.722
0.246
0.408
0.442
1.319
1.351
0.537
0.310
0.0833
2.056
0.764
1.011
SEM
Buffalo Bulletin (December 2013) Vol.32 No.4
Buffalo Bulletin (December 2013) Vol.32 No.4
age groups, it was found that the adult animals had
significantly higher levels (P<0.05) as compared to
young at all the temperatures. These observations
are similar to the findings of El-Nouty et al. (1990),
Mayengbam (2008) and Broucek et al. (2009).
TLC was found to be increased 35.56%
when temperature was increased from 22°C to 40°C
and 45.22% when increased from 22°C to 42°C in
young buffaloes whereas the values were 20.42
and 13.49 in adults. The TLC had significantly
(P<0.05) increased at 40° and 42°C temperature
as compared to 22°C, which is considered as the
thermo-neutral temperature. In case of effect of
age on TLC, it was significantly higher (P<0.05)
in young as compared to adult animals only at
42°C but no difference (P<0.05) was found at other
temperatures. The increasing trend in TLC with
increase of temperature may be attributed to the
fact that leukocytes are generally engaged in the
immune system. So, when an animal is exposed
to thermal stress, the immune system becomes
activated, and as a result, TLC may increase.
Abdel-Samee (1987) also reported that the white
blood cell (leucocytes) count values increased
by 21–26% in Friesian cattle under heat stress
conditions. Lallawmkimi (2009) reported the same
trend in buffalo heifers and lactating buffaloes. This
may be due to thyromolymphatic involution under
heat stress. After 42°C, there was again a decline
in TLC. This may be due to destruction of blood
cells at higher temperature. In adult animals, there
was no effect of temperature on TLC. These results
are in consistent with the findings of El-Nouty
et al. (1990), Mayengbam (2008) and Broucek
et al. (2009) who did not observe any effect of
temperature on total leukocytic count. The higher
value in younger animals might be due to the fact
that they are easily affected by thermal stress as
compared to adults.
were statistically the same in both the age groups.
The result is consistent with the observation of
Fagiolo et al. (2004) who reported that the PCV
was higher at the higher environmental temperature
(summer - 40.75% vs winter - 32.63%.). However,
in comparision of age groups, a higher value for
young was also reported by Ciaramella et al. (2005)
who found that PCV was higher in heifers than in
adult buffaloes. The increase in PCV may be due to
the hemoconcentration and dehydration of plasma
because when any animal faces severe heat stress,
it tries to maintain its body temperature through
evaporative water losses, which ultimately lead to
the hemoconcentration.
The average hemoglobin concentrations (g
%) in young Murrah buffaloes were 12.52, 13.54,
13.84 and 14.50 and those of adults were 11.94,
13.41, 13.70 and 14.23 at 22°, 40°, 42° and 45°C,
respectively, and showed an increasing trend with
increase of temperature. There was a significant
increase (P<0.05) in hemoglobin concentration at
40°, 42° and 45° C as compared to 22°C in both
young and adult animals. The age had no effect on
the concentration of hemoglobin. The observations
are in agreement with Fagiolo et al. (2004) who
reported higher Hb in lactating buffaloes during the
summer season (13.62 g/dl) than in the winter season
(11.37 g/dl). Like PCV, the increase in hemoglobin
also may be due to hemoconcentration. The increase
may also be attributed to the fact thatan animal
requires more oxygen in any stressful condition
and as a consequent, hemoglobin concentration
may rise.
Our findings demonstrated that the average
TEC (106/μl) showed an increasing trend with
elevation of temperature (7.65 vs. 8.89 in young
and 9.59 vs. 9.68 in adult at 22°C and 45°C,
respectively) but statistically, temperature had no
effect on TEC in either age group. In comparision of
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Buffalo Bulletin (December 2013) Vol.32 No.4
All DLCs were statistically similar (P<0.05)
in all temperature interactions and in both age
groups except for monocytes, which were found to
be increased in adult buffaloes compared to young
at 42° and 45° C. There was no significant effect
of temperature or age group on the count, which
was found consistent with the result of Broucek
et al. (2009), who also did not find any significant
differences in the percentage of basophils,
monocytes, and neutrophils when Holstein calves
were allotted to different temperature groups.
Fagiolo et al. (2004) found seasonal changes in
early lactating buffaloes in terms of neutrophil
percentage (from 64% in summer to 7% in winter).
Early lactating buffaloes showed a decrease in
lymphocytes during summer (41%) with respect to
winter values (77%). Ciaramella et al. (2005) also
reported significant reduction in the absolute values
of lymphocytes in buffaloes above eight years of
age. Canfield et al. (1984) found eosinophils were
significantly higher compared to those in immature
females, and Ciaramella et al. (2005) reported that
buffaloes over ten years of age show higher absolute
values of eosinophil levels. Lallawmkimi (2009)
observed that lymphocytes, monocytes, eosinophils
were decreased after buffalo heifers were exposed
to 42°C for 3 h whereas neutrophil values showed
the opposite trend. In contrast, Mayengbam (2008)
showed that changes in eosinophil and basophil
counts due to thermal exposure were not significant
in Sahiwal and Karan-Fries after exposure to 40°C
and 45°C. The lymphocyte counts decreased
whereas neutrophil counts increased after thermal
exposure. May et al. (1977) also reported a
significant increase of neutrophils and decrease of
eosinophils when environmental temperature rose
from 32°C to 52°C by direct solar radiation for
6 h.
The MCH and MCV values were statistically
similar (P<0.05) at all the four temperatures in
both young and adult. But, in case of age group,
the values were higher in young animals compared
to adult at all temperatures except 45°C, where the
MCH and MCV were statistically similar. But, in
case of MCHC, there was no effect of temperature
and age. These results are in agreement with the
observations of Fagiolo et al. (2004) who reported
nonsignificantly different mean values of MCV
(53 vs. 56 femtolitres), MCH (17.8 and 19.5
picograms), MCHC as (33.5 vs. 34.8 g/dl) during
different seasons in lactating buffaloes. In adults,
the low MCH could be due to smaller than normal
cells with normal Hb concentration or normal sized
cells with lower than normal Hb concentrations.
But in contrast, Ciaramella et al. (2005) found that
it was normally lower in heifers. El-Nouty et al.
(1990) reported that hot summer weather resulted
in significant reductions in mean cell volume and
mean cell hemoglobin but had no significant effect
on mean cell hemoglobin concentration of calves.
Hence, it is very clear that when buffaloes
are exposed to heat stress even for a very short
duration (acute heat stress), the total impact on
hematological functions may be severe. Young
animals, in this study, showing higher intensity of
changes of hematological parameters indicates that
they are more susceptible to heat stress. Therefore,
these animals need greater attention towards
protection against adverse climatic conditions.
REFERENCES
Abdel-Samee, A.M. 1987. The role of cortisol in
improving productivity of heat-stressed farm
animals with different techniques. Ph.D.
Thesis, Faculty of Agriculture, Zagazig
University, Zagazig, Egypt.
325
Buffalo Bulletin (December 2013) Vol.32 No.4
Haryana, India.
Snedecor, G.W. and W.G. Cochram. 1980.
Statistical Methods, 7th ed. The Iowa State
University Press, Ames, Iowa, USA. 593p.
Broucek, J., P. Kisac and M. Uhrincat. 2009. Effect
of hot temperatures on the hematological
parameters, health and performance of
calves. Int. J. Biometeorol., 53: 201-208.
Canfield, P.J., F.G. Best, A.J. Fairburn, J. Purdie and
M. Gilham. 1984. Normal haematological
and biochemical values for the swamp
buffalo (Bubalus bubalis). Aust. Vet. J.,
61(3): 89-93.
Ciaramella, P., M. Corona, R. Ambrosio, F. Consalvo
and A. Persechino. 2005. Haematological
profile on non-lactating Mediterranean
buffaloes (Bubalus bubalis) ranging in age
from 24 months to 14 years. Res. Vet. Sci.,
79(1): 77-80.
El-Nouty, F.D., A.A. Al-haidary and M.S. Salah.
1990. Seasonal variations in haematological
values of high and average yielding Holstein
cattle in semi-arid environment. J. King
Saud. Univ. Agric. Sci., 2(2): 173-182.
Fagiolo, A., O. Lai, L. Alfieri, A. Nardon and R.
Cavallina 2004. Environmental factors
and different managements that influence
metabolic, endocrine and immuno responses
in water buffalo during lactation, p. 24-26.
In Proceedings of the 7th World Buffalo
Congress, Manila, Philippines.
Lallawmkimi, C.M. 2009. Impact of thermal stress
and vitamin-E supplementation on Heat
shock protein 72 and antioxidant enzymes
in Murrah buffaloes. Ph.D. Thesis, NDRI
University, Haryana, India.
May, J., I. Mannoiu and C. Donta. 1977.
Untersuchungen über die Wärmebelastung
beim Kalb. Zbl. Vet. Med. A. 24: 153-159.
Mayengbam, P. 2008. Heat shock protein 72
expression in relation to thermo- tolerance
of Sahiwal and Holstein-Friesian croosbred
cattle. Ph.D. Thesis, NDRI University,
326
Original Article
Buffalo Bulletin (December 2013) Vol.32 No.4
COMPARATIVE EVALUATION OF DIFFERENT SURGICAL APPROACHES OF
CAESAREAN SECTIONS IN BUFFALOES UNDER FIELD CONDITIONS
G.G. Chandore1, S.P. Meshare and M.V. Ingawale
ABSTRACT
INTRODUCTION
The present research was conducted
to evaluate the most suitable of three surgical
approaches to caesarean section, namely caudal
paramedian incision, right ventro-lateral oblique
incision and left ventro-lateral oblique incision, for
caesarean section in cases of dystocia in buffaloes
under field conditions. In the study, the average
age of dystocia affected buffaloes was between
5 and 6 years while 83.33 percent of the affected
cases were pleuriparous and 16.66 percent were
primiparous. The commonest cause of dystocia was
irreducible uterine torsion (10 cases - 55.55%) as
well as incomplete dilatation of cervix (three cases
- 16.66%). Caesarean section in dystocia affected
buffaloes could be successfully carried out in lateral
recumbent surgical restraint. Left ventro-lateral
oblique incision approach was observed to be the
most preferred caesarean section approach in lateral
recumbent restraint and right ventro-lateral oblique
incision was better if intestinal evisceration could
be avoided. Caudal paramedian incision approach
was successfully used for relieving dystocia due to
dead emphysematous and abnormally developed
foetuses.
India is an extremely rich gold mine of
buffalo germplasm resources and harbours all the
recognized, high-producing breeds of this species.
The buffalo forms the backbone of India’s dairy
industry and is rightly considered as the ‘bearer
cheque’ of the rural flock considered as India’s
milking machine (Balain, 1999). India, with
106 million tons, is word’s topmost buffalo milk
producer accounting for 64 percent of world’s
total of 49 million tons. According to FAO (2005)
statistics on livestock, there are 98 million buffaloes
in India, which is about 50 percent of the world
buffalo population.
Parturition is a stressful process for
buffaloes. In dairy farming, there are various factors,
including under-nutrition, periparturient disorders,
and improper housing management,which put
together make this process difficult. Amongst the
several parturition maladies faced by the dairy
farmers, dystocia constitutes a major reproductive
disorder of vital economic importance because
there may be loss of calf and dam together or either
of them (Pearson, 1971) as well as subsequent
effects on the production potential and fertility of
the animals.
Dystocia is a serious and occasionally
fatal gynaecological malady. Whenever dystocia
is not relieved manually, caesarean section, or
Keywords: buffaloes, Bubalus bubalis, caesarean
sections, surgical approaches, dystocia, India
Indraprashta Building, Honest Housing Co. Society, Near Housing Corporation, Near Adarwadi Chowk,
Kalyan (West)-421302. Maharashtra, India, E-mail: goraksh.chandore@gmail.com
1
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Buffalo Bulletin (December 2013) Vol.32 No.4
MATERIALS AND METHODS
foetotomy, is the only alternative to save the life of
dam as well as the calf. Fetotomy is impracticable
in cases of irreducible uterine torsion, rupture of
uterus, constriction of birth canal etc. Fetotomy is
also associated with some disadvantages such as
possibility of uterine injuries, uterine infections,
and inability to obtain the calf in live condition.
These disadvantages can be avoided by undertaking
a prompt caesarean operation. Caesarean section
is potentially indicated in cases of dystocia when
a calf cannot be delivered by foetal mutation and
extraction
Caesarean operation is considered as
surgery of highest magnitude due to extent of stress
involved both due to dystocia and surgical trauma
(Cox, 1987). The fact that the level of plasma
cortisol increases three to four fold in dystocia
suggests lot of stress to the animal (Prabhakar et
al., 2002). Bovine practitioners are often presented
with dystocia cases that require a caesarean section.
Many bovine practitioners perform this surgery using
the same approach each time due to their comfort
with one specific approach or lack of familiarity
of other available options (Schultz et al., 2008).
A paramount goal of caesarean section should be
to limit the contamination of the peritoneal cavity
with uterine contents. The challenge of performing
successful caesarean section in buffaloes is often
directly related to proper choice of incision
approach. So, various incisional approaches have
been suggested by several workers (Verma et al.,
1974; Noordsy, 1979; Saxena et al., 1989). Hence,
present research was conducted to evaluate the
most suitable among three surgical approaches
to caesarean section, namely caudal paramedian
incision, right ventro-lateral oblique incision and
left ventro-lateral oblique incision for caesarean
section in cases of dystocia in buffaloes under field
conditions.
The present study of caesarean section
operations was carried out on eighteen clinical
cases of dystocia in buffaloes at the Veterinary
Polyclinic, Miraj, Dist. Sangli, in the area of
operation of Kolahapur Zilha Sahakari Dudh
Utpadak Sangh Ltd., Kolhapur, Maharashtra State,
and the Teaching Veterinary Clinical Complex,
Post Graduate Institute of Veterinary and Animal
Sciences, Akola, MAFSU, Nagpur (M.S.).
These buffaloes were divided into three
groups comprising six buffaloes each and were
operated by following incision sites of caesarean
section.
Group A: Caudal paramedian incision (Figure
1)
Group B: Right ventro-lateral oblique incision
(Figure 2)
Group C: Left ventro- lateral oblique incision
(Figure 3)
Pre-operative treatment and restraining of
buffaloes
As these were protracted and emergency
cases of dystocia, most of the buffaloes suffered
from dehydration, septicemia, toxemia etc.
After assessment of dehydration of case fluid
therapy using 5% dextrose normal saline along
with broad spectrum bactericidal antibiotic was
given intravenously. The buffaloes were given
antihistaminic 30 to 50 mg /animal i/m, meloxicam
0.2 to 0.3 mg/kg b.wt. i/v and a broad spectrum
antibiotic 10 to 20 mg/kg b.wt. i/v before caesarean
section. For performing caesarean section in the
lateral recumbent position, the buffalo was cast on
platform prepared from grass covered with sterile
plastic drape. Precautions were taken to prevent a
dusty environment. The legs were secured by tying
328
Buffalo Bulletin (December 2013) Vol.32 No.4
the two front legs together with a single rope and
stretching the legs forward. The rear legs were tied
together and were stretched backward. The head of
the buffalo was controlled by one person.
lateral recumbancy to slight sternal position taking
due care that no foetal fluids entered the peritoneal
cavity of the mother. The incised uterine wall was
pulled out and was grasped firmly on either side
by the assistant until the foetus and fluid were
removed. The foetus was exteriorized by grasping
both legs (fore or hind) and was removed gently
avoiding uterine tear. The placenta was removed
and uterine cavity was cleaned thoroughly with
normal saline. Blood clots and any debris of
placenta were removed during caesarean in all the
cases and 3-4 antiseptic boluses were inserted into
the uterine cavity.
The uterine wall was sutured with No. 1/0
chromic catgut by Cushing and Lambert sutures.
The suturing was started from the cervical end and
was continued towards ovarian end in all the cases.
The uterus was cleaned thoroughly with normal
saline and blood clots and any debris of placenta
were removed from the uterus. About 30 I.U. of
oxytocin was injected in all the buffaloes. The
uterus was repositioned in the normal location.
The peritoneum, abdominus transversus and intra
abdominal oblique muscles were sutured together
with simple interrupted suture using chromic catgut
No 2. The external abdominal muscle was sutured
as a second layer with interrupted sutures and this
was followed with sub cuticular suture. The skin
was sutured using modified vertical mattress.
The skin suture was dressed with tincture iodine
and was sealed with sterile cotton plug soaked in
Compound Tincture Benzoin .
Anaesthesia and preparation of the site of
operation
All buffaloes were given triflupromazine
as pre-anaesthetic 0.2 mg/kg body weight before
anaesthesia by the intramuscular route. Local
infiltration anaesthesia was produced with 120 to
130 ml of 2% xylocaine (Lignocaine) hydrochloride
injection on the respective site of caesarean section
depending upon the health status of the buffaloes
and requirement in the field condition. The site of
operation was prepared for aseptic surgery. First,
clipping of hair was performed in a wider area
around the site of incision followed by shaving
with a razor. The surgical area was scrubbed with
antiseptic solution and was painted with tincture
iodine. The site of incision was then draped.
Operative procedure
A skin incision of 25 - 30 cm long was taken
at the respective site of incision after checking the
bleeding points, subcutaneous tissues and muscles
were incised in the direction of their lay. The
peritoneum was incised and omentum was pushed
anteriorly and was packed off with a towel soaked
in physiological sterile saline. Every attempt was
made to exteriorize the uterus outside the surgical
wound in each case of caesarean. The exteriorized
part of the uterus was draped with sterile drape
and was sealed off from rest of the organs. The
incision was given from ovarian end and extended
towards the cervix avoiding the cotyledons and
every attempt was made to prevent uneven tear
of uterus and hemorrhage. While removing the
foetus, the position of the dam was changed from
Post-operative Care
The position of the buffalo was changed
from lateral to sternal position. A sufficient quantity
of fresh drinking water was provided to the buffalo.
Fluid therapy was continued post operatively for
two to three days using Inj. 5% dextrose saline
329
Buffalo Bulletin (December 2013) Vol.32 No.4
pleuriparous buffaloes as compared to primiparous
was reported by Singh et al. (1978).
The commonest cause of dystocia was
uterine torsion (10 cases - 55.55%). Holy et al.
(1960), Verma et al. (1974), Saxena et al. (1989)
and Shiv Prasad et al. (2000) also reported uterine
torsion as commonest cause of dystocia. The second
important casuse of dystocia was incomplete
dilation of cervix (3 cases-16.66%). Similar
observations were also recorded by Parkinson
(1974) and Iyer et al. (1989) (16% cases). Other
causes of dystocia in the present study include one
case each of breech presentation, arthrogryposis
foetus, deviated head and neck, foetus with leg
defects and dead emphysematous foetus.
intravenous. From the 2nd day onwards injection
of broad spectrum antibiotics in proper doses was
given for 7days, if required. The analgesic and antiinflammatory injections was given for 5 days. The
dressing was continued with fly repellent antibiotic
ointment till wound healing was achieved. The
skin suture was removed only after the 13th day or
after conforming complete healing. The buffaloes
were examined per rectum periodically to assess
the involution of the uterus and any adhesions.
The buffaloes were post-operatively observed for
a period of 15 days for any complications such
as fever, anorexia, vaginal discharges, wound
dehiscence, infection, herniation etc. The buffaloes
were given broadspectrum antibiotic 10 to 20 mg/
kg b.wt. i/v for 7 days and meloxicam 0.2 to 0.3
mg/kg b.wt. i/v for 5 days, if required.
RESULTS AND DISCUSSION
Restraint in Lateral Recumbancy
In this study, all eighteen caesarean section
operations were performed in recumbent restraint
position. Animals were restrained properly and
were easily controlled in lateral recumbency. In this
method of restraint the exteriorization of uterus was
easier. With restraint in lateral recumbency uterine
spillage was minimum in caudal paramedian
incision.
In the present study the mean age of
dystocia affected buffaloes was between five and
six years. Iyer et al. (1989) reported dystocia in
cows and buffaloes with average age of 2-5 years.
The health status of eleven dystocia-affected
buffaloes operated for caesarean section was good
while in seven buffaloes, the health status was poor
and were recumbent at the time of examination.
Parkinson (1974) also reported in his study that
75 percent of the buffaloes were recumbent at first
sight. Out of eighteen affected buffaloes, fifteen
were pleuriparous, while three were primiparous.
A similar finding, of high incidence of dystocia in
Group A: Caudal paramedian incision
In this Group A, six dystocia affected
animals were operated with caudal paramedian
approach of caesarean section. The technique
of performing caesarean section through this
approach has been successfully used by Deore
(1973). In this approach operative haemorrhage
was of small degree. The exteriorization of uterus
was intractable. At this approach the operative
haemorrhage was seen to a very small degree.
Exteriorization of uterus was facile. The average
time for removal of sutures in all other cases was
12 days.
Statistical analysis
The data collected in the present study in
were statistically analysed by using analysis of
variance as per Snedecor and Cochran (1994).
330
Buffalo Bulletin (December 2013) Vol.32 No.4
Right ventro-lateral oblique incision
In this Group B six dystocia affected
animals were operated with right ventro-lateral
oblique incision approach of caesarean section. The
technique of performing caesarean section with
approach has been successfully used by Noordsy
(1979) in cow.
In this incision approach the operative
haemorrhage were of a moderate degree. The
exteriorization of uterus was easier. The abdominal
closure was easy in all cases. Post-operative
herniation was not seen in any case from this
group. The average time for suture removal in this
group was 12 days. In this approach the operative
haemorrhage was to a moderate degree but Verma et
al. (1974) observed minimum haemorrhage at this
site. The exteriorization of uterus was facile. The
prolapse of intestines was seen more as compared
to left ventro-lateral oblique incision because rumen
was preventing the prolapsed of intestine. Similar
observations were recorded by Milne (1952) and
Noordsy (1979). Muscle relaxation was adequate
hence abdominal closure was easy. Milne (1952)
has reported that adequate muscle relaxation is
must for efficient abdominal closure.
Left ventro-lateral oblique incision
In this Group C, total six dystocia affected
animals were operated with left ventro-lateral
oblique incision approach of caesarean section.
Caesarean section at this approach has been
successfully carried out by Verma et al. (1974) and
Saxena et al. (1989) and Shiv Prasad et al. (2000).
With this approach operative haemorrhage
was greater as compared to other approach. The
exteriorization of uterus was facile in all cases.
Abdominal closure was easy in all cases. With this
approach, operative haemorrhage was greater as
compared to other approaches. This is in agreement
with Saxena et al. (1989) but Verma et al. (1974)
have described minimum haemorrhage at this site.
The exteriorization of uterus was facile. Similar
observations are recorded by Milne (1952) and
Saxena et al. (1989). However, Verma et al. (1974)
reported that exteriorization of uterus was difficult.
The prolapsed of intestine was not seen in any of
the cases and spillage into peritoneal cavity was
also rarely seen. The post operative infection was
Table 1. Incidence and causes of dystocia.
Sr.
No.
1
2
1
2
3
4
5
Codition causing dystocia No. of buffaloes Percentage
Maternal causes
Uterine torsion
Incomplete dilation of
cervix
Foetal causes
Arthrogryposis foetus
Breech Presentation
Deviated neck & head
Legs Defect
Dead emphysematous foetus
Total
10
72.22%
55.55%
3
16-66%
27.77%
1
1
1
1
1
18
Condition of calf
Sex
Live
Dead Male Female
6
12
9
9
6
12
9
9
5.55% each
331
Buffalo Bulletin (December 2013) Vol.32 No.4
Figure 1. Showing approach of caudal paramediam incision.
Figure 2. Showing approach of right ventro-lateral oblique incision.
Figure 3. Showing approach of left ventro-lateral oblique incision.
332
Buffalo Bulletin (December 2013) Vol.32 No.4
CONCLUSION
not seen in any of the cases Present observations
correlated with the observations recorded by Verma
et al. (1974), Saxena et al. (1989).
The average age of dystocia-affected
buffaloes was between 5 and 6 years. In this
study 83.33 percent of the affected cases were
pleuriparous, and 16.66 percent were primiparous.
The commonest cause of dystocia was irreducible
uterine torsion (10 cases - 55.55%) as well as
incomplete dilatation of cervix (3 cases - 16.66%).
Caesarean section in dystocia affected buffaloes
could be successfully carried out in lateral
recumbent surgical restraint. The comparative
evaluation of different approaches of caesarean
section it was observed that the left ventro-lateral
oblique incision is the most preferable approach
while the right ventro-lateral incision is better
provided effective care of prolapse of intestine is
taken and caudal paramedian approach can be used
for removal of dead and emphysematous fetus.
Comparative evaluation of different approaches
of caesarean section
Eighteen clinical cases of dystocia in
buffaloes were operated with three different
incision approaches of caesarean section. The
operative haemorrhage was minimum in caudal
paramedian incision. It was maximum at left
ventro-lateral oblique incision and moderate at right
ventro-lateral oblique incision. The exteroriztion of
uterus was easier in caudal paramedian incision but
was difficult in some cases of right ventro-lateral
oblique incision. Spillage was not observed in
left ventro-lateral oblique and caudal paramdian
incisionwhile it was seen to a small extent in
right ventro-lateral oblique incision. Evisceration
of intestines was seen to a moderate degree in
caudal paramedian and right ventro-lateral oblique
incision, but it was not seen in left ventro-lateral
oblique incision. Abdominal closure was difficult
in caudal paramedian incision while it was facile
in right and left ventro-lateral oblique incision
approaches. Post operative infection was seen
in two cases operated by the caudal paramedian
incision approach and in one each operated by
the right ventro-lateral oblique incision approach
and the left ventro-lateral oblique incision; these
were due to unhygienic conditions in stables under
field conditions. Post operative herniation was not
seen in any of the cases operated by either of the
three approaches of caesarean section. The average
healing of wound in all three approaches was 12
days.
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