Reproduction in Male Animals
Anatomy of male reproduction:
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The male reproductive tract consists of primary sex organs (testes), secondary
sex organs (efferent ducts of the testes, epididymises, vasadeferentia, urethra
and penis) and accessory sex glands (ampulla, vesicular glands, prostate
gland, and bulbourthral glands or Cowper gland).
The reproductive organs of the male animal have three major functions:
1. Production of spermatozoa in the testis.
2. Maturation, storage and transport of spermatozoa within the duct
system.
3. Deposition of semen within the female genital tract via the penis.
The testis is oval in shape with two borders (anterior and posterior), two
surfaces (medial and lateral) and two extremities (proximal and distal).
The size divers according to deferent species. The largest testis weight is for
the bull (300-400 g), followed by the ram (250-300 g), buffalo (200-300 g),
stallion (150-250 g) buck (100-150 g), and camel (60-100 g).
The testicles of all domestic animals are normally located in the scrotum
outside the body. They lies in the inguinal region, with their longitudinal axises
in a vertical position for the bull, buffalo, ram and buck, but divers in the
stallion that their axises are in a horizontal position. For camel, the testes are
located in the perineal region just under the anus, with their longitudinal axises
in an oblique position. Male cats have two testicles, which in an uncastrated
cat sit just below the anus.
Each testis is suspended by a spermatic cord which contains the blood supply
(internal spermatic artery) and venous drainage in the anterior portion and the
vas deferens in the posterior portion.
Two agents produced by the fetal testis are responsible for this differentiation
and development. Fetal androgen causes development of the male
reproductive tract. Anti Mullerian hormone or Mullerian inhibiting substance is
responsible for suppression of the Mullerian ducts.
The testes develop in the abdomen, medial to the embryonic kidney and
descend from their site of origin. There are differences among farm animals
concerning the time of testicular descent.
The testicular descent occurs in mid gestation for the bull, buffalo, ram and
buck and just before or after birth for the stallion, camel, dog and tom cat (2-5
days after birth). The descent is probably under hormonal control
(Testosterone and insulin3 are promote testicular descent and oestrogens
inhibit it). The late descent of the stallion and camel gonads as compared to
cattle can be ascribed to the high oestrogen content of the blood of the
pregnant mare and female camel before the time of descent.
The scrotum is a two-lobed sac, which houses both testes. It keeps the testes
out side animal’s body as production of spermatozoa requires temperatures
below that of the body.
The wall of the scrotum is composed of four layers; skin, tunica dartos muscle,
scrotal fascia and parietal layer of vaginal tunic. The tunica dartos forms the
median septum which divides the scrotum into two distinct pouches.
The plexus of ducts within the testis becomes connected to the mesonephric
duct (Wolffiain duct) to form the epididymis, ducts deference, and vesicular
gland.
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The epididymis is convoluted tube connecting the testis to the vas deferens
and covered by an extension of the tunica albuginea testis.
Three anatomic parts of the epididymis are recognized. The head (caput), in
which a variable number of efferent ductules. It continues as the narrow
corpus epididymis (body), which terminates at the opposite pole in the
expanded cauda epididymis (tail). The epididymis (head, body and tail) is less
clearly differentiated in the stallion than in other farm species.
There are two vasa deferentia, each one is continuous with corresponding
epididymis.
Each one is a thick duct extending from the cauda epididymis to the pelvic
portion of the urethra. It runs dorsomedial to the urinary bladder to enter the
pelvic urethra.
In the most species, the terminal part of the vas deferens is furnished,
branched tubular glands to form the ampulla of the ductus deferens. The
ampulla is absent in the boar and tom cat, but well developed in the stallion.
Vesicular glands (seminal vesicle) occupy a dorsal position in the urogenital
fold lateral to the terminal part of the ductus deferens. The duct of the seminal
vesicles and the ductus deferens may share a common ejaculatory duct that
opens into the urethra. They are pair of compact lobular glands that are easily
identified because of their knobby appearance in the bull, ram and buck. In the
stallion, they are large pyriform glandular sac that evades identification
because of their thin wall. The vesicular glands are absent in the dog, cat and
camel.
The prostate and bulbourethral glands form from the embryonic urogenital
sinus.
Prostate gland is a single gland with 2 parts, a body that stretches across the
dorsal surface of the neck of the urinary bladder and disseminate or internal
part that surrounds the pelvic urethra. The body of the prostate is small in the
bull and large in the boar and camel, but absent in the ram and buck. The
prostate gland in the stallion is bilobed and wholly external that can be
identified by palpation per rectum.
Bulbourethral glands (Cowper) are paired round or oval compact bodies
located above the urethra near its exit from the pelvic cavity. The bulbourethral
glands are absent in the dog.
The penis form by tabulation and elongation of a tubercle that develops at the
orifice of the urogenital sinus.
The penis either fibro-elastic (with sigmoid flexure) such as the bull, buffalo,
rams, buck and camel or vascular, musclocavernosus and haemodynamic in
the stallion.
The sigmoid flexure is post-scrotal for the bull, buffalo, ram and buck, but prescrotal for the camel and boar.
The fibro-elastic and vascular penises have retractor penis muscles, a pair of
smooth muscles which relax to permit extension of the penis and contract to
draw the penis back into the prepuce.
Male cat have a barbed penis and its points backwards, which will be painful
for the female during mating, while the dog have an os penis.
The penis is cylindrical in shape and composed of root, body and glans penis.
The root is attached to the bony pelvis by two lateral branches, the right and
left crus penis, which are covered by ischiocavernosus muscles. The body
contains three erectile rods or cavernous bodies the two dorsal rods (corpora
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cavernosa) unite to form expansive cavernous body called corpus cavernosum
penis 'CCP'. The ventral rod called corpus spongiosum penis 'CSP') which lie
parallel to each other. In the stallion the cavernous bodies contain large
cavernous space (increase in size during erection). In bull, boar and ram the
cavernous spaces of are small.
The glans penis, the free end of the penis, is supplied with sensory nerves and
is homologous to the clitoris in the female.
The urethra opens into a twisted groove in the glans penis of the bull and
buffalo. The ram and buck have urethral process known as a filiform
appendage extending beyond the glans penis. The glans penis of the camel is
transverse, cartilaginous and hooked-shaped with a definite neck between the
glans and the body of the penis. It has a small urethral process present on the
lateral aspect of the external urethral orifice on the left side of the glans penis.
The glans penis of the stallion is flattened, mushroom shaped, with a small
urethral process extending beyond the flattened end. The dog has a fibrocartilagenous end of os penis ventrally grooved, the glans penis includes the
bulb, the long part of the head, and the crown,while the tom cat has spines on
glans penis supposedly enhances vaginal stimulation to induce ovulation.
The penis of the camel is directed caudally, so that urination is towards the
rear. However, when erection of the penis occurs, the cranial preputial
muscles pull the prepuce and penis forward.
The prepuce is an invagination of the skin which completely encloses the free
end of the penis. It sheds the non-erected penis. Has outer (prepenile) and
inner (penile) folds which straighten out during erection.
In all species the head of the penis and the free part of the penis are within the
preputial cavity, but in the dog, the head of the penis only is in the preputial
cavity.
The orifice of the prepuce for the males of ruminants is surrounded by long
and tough preputial hair.
The prepuce of the stallion is formed by a double fold (external and internal
sheath).
The prepuce of the camel is pendulous, flattened from side to side and
triangular shaped sheath, which open to the rear.
Anatomy of reproductive system of male chicken:
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The avian male reproductive system is all inside the bird - unlike the males of
mammalian species which have their reproductive systems outside of the
body. This is one of the really remarkable things about birds; the sperm remain
viable at body temperature.
 The male chicken possesses two testes, located along the chicken’s back,
near the top of the kidneys (cranio-ventral). The testes are elliptical shaped
and light yellow in color and increase dramatically in size during the breeding
season.
 The vas deferens emerges medially and passes caudally to the cloaca where
it has a common opening with the ureter (opens into a small bump, or papilla).
The terminal vas deferens is swollen as a storage organ: the seminal glomus.
 Poultry have no external penis, but rather a protuberance termed a
"rudimentary phallus" is located on the middle and front portion of the cloaca.
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Physiology of male reproduction:
Puberty:
 Is the age at which a growing male is able to mount, copulate and successfully
impregnate a female. Practically, puberty is defined as the age at which an
ejaculate first contains 50 million spermatozoa among them ≥10 % is
progressively motile.
 The age of puberty is influenced by genetic, nutritional, and environmental
factors.
 In the normal breeding conditions puberty occurs at 10-12 months for bulls, 46 months for rams and bucks, 13-18 months for stallions, 4-8 months for boars
24-40 months for camels, 6-8 months for dog and 4-5 months for cock
(roosters).
 When puberty is attained, the male exhibit external and behavioural changes,
including changes in body conformation, increased aggressiveness, sexual
desire, rapid growth of penis and testes, and separation of the penis from the
prepuce for extension of the penis due to rupture of the penile frenulum.
 Sexual maturity is a gradual process and it is may take years after puberty.
Thus, puberty should not be confused with sexual maturity, as spermatogenic
potential and size of the testes continue to increase when puberty is attained.
For example, efficiency of spermatogenesis in the stallion reaches adult levels
by 2.5-3 years of age, whereas daily spermatozoal production per testis and
testicular weight continue to increase until 4-5 years of age.
 As rule, mating is delayed few months after puberty for the bull, buffalo, ram
and buck, or even years for the stallion and camel.
Libido and mating behavior:
 Libido or sexual desire is the male interest in sexual congress or mating.
 Libido is primarily dependent upon androgenic steroid hormones, which allow
mating, and aggressive behaviour to occur, as well as maintaining the function
of all parts of the male reproductive system.
 Despite the dependency of male behaviour upon androgen, there has been
much debate over the relationship between absolute concentrations of
androgen and libido.
 Some have argued a permissive role of androgen, while others have
demonstrated positive correlations between testosterone concentrations and
measures of libido.
 Oestrous females of many species secrete pheromones to attract males, while
others, notably the cow and some breeds of pig, exhibit homosexual behaviour
as a signal to the male of the presence of oestrus. All males smell the perineal
region of the female, and the odour of the oestrous female induces the so
called ‘flehmen’ reaction in the bull, ram and stallion: a characteristic raised
posture of the head an elevation of the upper lip.
 Females that are not in oestrus signal their objection to the advances of the
male and, at the least, will respond by moving away or, perhaps, by attacking
the male in the offensive manner peculiar to the species.
 Oestrous females signal receptivity by squatting, urinating, moving the tail to
one side and remaining stationary. During this foreplay the male becomes
progressively aroused; there are frequent erections of the penis, with emission
of accessory fluid and many unsuccessful attempts to mount the female.
Finally, mounting and copulation occur.
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Ruminants:
 Copulation in all of the domestic species of ruminants is brief. After detecting
an oestrous female, mounting is followed quickly by the single ejaculatory
thrust.
 The male then immediately dismounts, but frequent subsequent matings
occur.
Stallion:
 Following intromission, the stallion performs a succession of copulatory
movements of the hindquarters which, within a minute, culminate in
ejaculation.
 During ejaculation, successive waves of urethral peristalsis can be palpated
on the lower surface of the penis, while the stallion exhibits a characteristic
‘flagging’ movement of the tail. The stallion then dismounts.
Dog:
 The dog achieves intromission by vigorous thrusting of the hindquarters. Once
intromission has been achieved, the bulbus glandis swells considerably, while
the constrictor vulvae muscles of the bitch contract behind it, thus forming the
‘copulatory tie’.
 The sperm-rich fraction of canine semen is ejaculated within as little as 80
seconds of intromission, so that conception may occur even if copulation does
not proceed through to its second stage. In this second stage, the dog
dismounts but remains connected and faces away from the bitch.
 This change of position causes the penis to become bent through an angle of
180°; the efferent veins of the penis are thereby occluded and the penis
remains turgid.
Cat:
 During mating, the tom mounts the queen and grasps her neck with his teeth.
As the tom adjusts his position the queen paddles her hindlegs, continuing to
do so at an increasing frequency during the 10 seconds or so for which coitus
lasts.
 The queen cries out during copulation and, as the tom dismounts, she may
strike out at him, displaying the typical ‘rage’ reaction. This is followed by a
period of frantic rolling and licking at the vulva.
 As soon as the postcoital reaction has ceased, the tom will attempt to mount
again. Several matings may therefore occur within the first 30–60 minutes.
 The cat is an induced ovulator , so the number and frequency of matings are
important in ensuring that the LH surge is of sufficient magnitude to cause
ovulation.
chickens:
 The completed mating in chickens is the culmination of a sequence of
behaviours.
 The rooster will initiate mating by exhibiting courtship behaviour: dropping one
wing and dancing in a circle (the lowered wing will be on the inside of the circle
dance).
 The hen will crouch (dip her head and body) to indicate receptiveness to the
male.
 The rooster will then mount the hen and grab her comb, neck feathers, or the
skin on the back of her head or neck to help hold onto the hen's back.
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 The next behaviour is the tread (the rooster walks quickly in place on the hen's
back) and finally the completed mating of the behavioural sequence. The
completed mating occurs when the rooster dips his tail to the side of the hen's
tail and spreads his tail feathers so that their cloacae come into contact
('cloacal kiss'). At this point the rooster's ejaculate is released directly into the
hen's vagina via her cloaca.
 A rooster may mate from 10 to 30 or more times per day, depending on the
availability of hens and competition from other roosters.
 The rooster has a small phallus that becomes engorged with lymph to form a
copulatory organ. The copulatory organ is rudimentary and at the time of
mating there is practically no penetration. The hen everts her vagina during
copulation, which helps to transfer the semen into the oviduct.
Hormonal control of male reproduction:
 All aspects of male reproductive physiology are under the endocrine control of
two major gonadotrophins LH and FSH.
 The actions of LH are primarily upon the Leydig cell (adenylate cyclase). It
promotes steriodogensis.
 Testosterone is required for the production of sperm in testis and their
subsequent maturation in the epididymis, for function of the accessory sex
glands and for development of masculine secondary sexual characteristics.
 The main target of FSH is the Sertoli cell (through adenylate cyclase – linked
enzyme) to secret androgen binding protein (ABP) (maintain high androgen
concentrations and prolong life span of epididymal sperm) and aromatize
testosterone into oestrogens. Adequate FSH stimulation is also required to
permit Sertoli cells to support spermatogenesis.
 The substance of testis is composed of two main tissues: seminiferous
tubules and interstitial tissue.
I.
Seminiferous epithelium composed from two cell lines: somatic Sertoli cells
and sperm-producting germinal cell line.
II. Interstitial tissue which consists of steroid producing Leydig cells, blood
vessels and lymphatics.
 The endocrine role of the testis is production of testosterone and estradiol, and
other hormones such as inhibin and actvin. However, the exocrine role of the
testis is directed to the production of spermatozoa.
 The process of Spermatogenesis occurs in the convoluted portion of the
seminiferous tubules, which constitute more than 70% of the testicular
parenchyma. It is divided into three steps:
1. Spermatocytogenesis:
 Cell division of spermatogonia, located at the periphery of seminiferous
tubules. Cell move progressively towards the lumen of the tubules as they
develop.
 Spermatogonia undergo several mitotic cell divisions (where the number of
chromosomes remain the same) eventually forming the large primary
spermatocytes.
 A meiotic (reduction of cell division) then occurs in which secondary
spermatocytes are formed. The number of chromosome is reduced from
diploid to haploid.
 The secondary spermatocytes divide, further to form smaller cells, the
spermatids.
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Spermiogenesis:
 Spermatids undergo differentiation to form spermatozoa.
 The differentiation includes formation of acrosome, head and tail
 The proximal cytoplasmic droplet is retained between the head and
midpiece until maturation of spermatozoa.
Spermiation: Is the release of spermatozoa into lumen of the
seminiferous tubues.
Maturation of spermatozoa occurs during transmit through the epididymis;
motility increase as spermatozoa enter the corpus epididymis (they traverse in
8-14 days).
Spermatozoa stored in the epididymis (Cauda) retain fertilizing capacity for
about 60 days.
Transportation of spermatozoa from the epididymis to the ampulla ductus
deferens by peristaltic movements.
The main function of the ampulla is to act as reservoirs of sperm. Also it is
contribute slightly to the seminal plasma
The prostate, vesicular and bulbourethral pour their secretions in the urethra,
where, at the time of ejaculation (seminal plasma). Fructose and citric acid are
important components of vesicular gland secretions of ruminants.
The prostate contributes the greatest part of seminal plasma in the camel. Its
secretion is milky, viscid and rich in Na, Cl, Ca, P and Zn.
The physiological functions of the various constituents of seminal plasma
remain a matter of debate. Provision of energy, maintenance of osmotic
pressure, chelation of free calcium ions and buffering are some of the
suggested functions. while other possibilities include immuno-suppression in
the female genital tract and regulation of spermatozoon motility.
Seminal plasma is also responsible for the coagulation of semen.
Fertility and infertility in Male Animals:
Examination for breeding soundness:Examination of male animals is made for two mains purposes: either to
ascertain normal fertility can be expected from the animal, or for the diagnosis of
infertility. The requirements for examination are a history of the animal, a general
examination, a detailed examination of genital tract, observation of copulation, and
collection and evaluation of semen.
Observation of the normal environment of the sire is important. Seeing how
the animal is handled, how it is housed, fed and cleaned. How it moved and how it is
handled during service.
The general examination of sire must take into consideration its age and likely
sexual experience, body condition and animal's temperament.
Any systemic illness can affect reproductive performance. Disease of the
locomotor system causing pain and pyrexia may leads to incompatible with normal
mating behavior. Furthermore, stress of prolonged pain may cause corticosteroid
mediated impairment of spermatogenesis. Systemic illness causing prolonged
pyrexia can result in increased temperatures within testis (impairment of
spermatogenesis).
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Reproductive Examination:A complete examination of the reproductive system require physical
examination of genital system, observation of response of the animal to an oestrus
female, observation of mating and collection of semen.
During examination of the genital tract, all parts of the genitalia that are
accessible externally should be palpated. When examining the contents of the
scrotum, the temperature, size, texture, resilience and evenness of the testes and
epididymis should be determined. It is generally possible to palpate the head and tail
of the epididymis. The vasa deferntia should be palpated throughout the scrotal neck
(ram).
The spermatic cord should be palpated up to level of the inguinal ring.
Measurement of scrotal circumference is useful in animals with a pendulous scrotum
(in the stallion and dog by calipers or ultrasonography). Scrotal circumference of
yearling bulls should exceed 30 cm, while mature bulls should be over 36 cm (British
beef breeds).
In bull and stallion all accessory glands can be palpated (particularly vesicular
glands).
Libido testing:Assessment of libido and serving ability is widely used in the examination of bulls for
breeding soundness. Libido is considered to be highly heritable in cattle, so early
selection of animals for high libido is likely to result in an overall increase in this
aspect of reproductive performance. There are different methods to evaluate and
measure the intensity of male sexual desire:
 Reaction time: it is the time elapsing between bringing the male to the teaser
until ejaculation is completed.
 Number of copulations per unit time.
 Number of copulations before sexual exhaustion (loss of libido). The average
number of copulations to exhaustion is 20 in cattle, 10 in sheep 7 in goats, 3 in
horse and swine, and 5 in camels.
 Recovery time (refractory period) after exhaustion.
 Libido index. which has 6 grades as follow:
1) No interest of the male in the female.
2) Little interest in sniffing and mounting.
3) Mounting the female after obvious and repeated hesitation.
4) Comparatively quick mounting without showing obvious eagerness.
5) Quick mounting with the male attention concentrated on the female.
6) Uncontrolled extremely eager mounting with hyper sexuality and
viciousness.
Many of the factors such as age, sexual experience and social dominance, can affect
libido.
Collection and assessment of semen: Semen collection is the process which involves obtaining the maximum
number of sperm at highest possible quality in each ejaculate.
 Aims to investigate the fertility status of a certain male and/ or make maximum
use of superior sires.
 Preparation of the male animals; including clipping the long tuft of hair around
the prepuce and cleaning, as well as teasing the male by making approach to
the female with false mount. The general health condition of the male should
be taken in consideration.
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
The methods of semen collection from different male animals include artificial
vagina, electro ejaculation, manual massage, vaginal recovery and condom.
Collection from the bull: Samples of semen can be obtained from most bulls by means of the artificial
vagina (AV), which consists of a strong outer rubber cylinder containing a latex
liner. At one end of the AV, a latex extension cone carrying a gradual
collecting tube is attached. The space between the rubber cone and latex liner
is filled with warm water, so that the temperature in the lumen of the AV is
between 45 and 48˚C. The main stimulus to ejaculation is the temperature of
the AV. A little inert lubricant (liquid paraffin or gynaecological jelly) is placed
in the lumen of the AV just prior to use.
 Control of the bull and the safety of all personnel are paramount importance
during semen collection. A halter- trained, oestrous cow is the ideal object for
a bull to mount.
 The bull is led up to the cow (teaser) with the collector standing to the right of
the bull.
 As the bull mounts, full erection is achieved and the bull usually makes a
single ejaculatory thrust, during these the collector grasps the prepuce with left
hand and deflects the penis to entrance to the AV. The bull will then normally
make the ejaculatory thrust into the AV.
 The main alternative to collection by an AV is collection of semen by electroejaculator. Electrodes, which are either in the form of a rectal probe or are
worn on two fingers over a gloved hand, are placed over the accessory glands
(ampula) via the rectum after evacuation of faeces from it.
 Erection of the penis usually occurs at lower voltage while ejaculation often
occurs at higher voltage levels.
 If performed by unskilled operators or with poor equipment, distress can be
caused. Over stimulation can cause harm to valuable male due to over
excitement and exhaustion, and shock may be followed.
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Semen collected by this method is usually of larger volume, but with lower
sperm concentration than the artificial vaginal. Advantages of this method
include:
a) Semen can be collected without sexual response.
b) Males that are unable to mount due to injury fracture or aging can be
handled.
c) Males that can not be trained for collection by AV.
 Semen can also be collected by penile or rectal massage of the internal
genitalia (vesicular glands and ampullae). This method is used if the male
animal is trained for the massage.
 For rectal massage, a lubricated, gloved hand is passed through the rectum.
The side of the ampulla, vesicular glands, and prostate is located and a
downward pressure is exerted with milking caudally. This stimulates and
mechanically causes the release of semen through the urethra with some
degree of penis erection. However, the semen is usually of poor quality.
 Manual massage of the penis can also be used to collect semen from the boar
and camel.
 In some animals, semen can only be obtained by aspiration of the ejaculates
from the vagina of a freshly served cow (vaginal recovery). The quantity and
quality of semen obtained by this method is poor.
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Collection from the stallion: In the stallion, semen can be obtained by the use of an AV (larger than that
used for a bull). The stallion is prepared for semen collection with an AV in
same manner as it would be natural mating. The penis should be washed with
warm water and if a jump mare is to be used, up to three handlers will be
needed, one to hold the stallion, one to collect the semen and one to hold the
mare. All handlers should stand on the same side of the stallion.
 The stallion is allowed to mount and collector diverts the penis, when erect,
towards the AV.
 Ejaculation is noted by flagging of the tail or by feeling contractions of the
urethra.
 The condom is more applicable for the stallion. It is a thin rubber sleeve used
to cover the glans penis after erection and removed after dismount. (But
leakage of semen is possible).
Collection from the camel: The camel AV is about 30 cm in length, supplied with a cervix-like cork
(inserted at the distal end of the inner liner).
 A teaser female is tied up in sternal recumbency and the male camel is
allowed to enter the collection area, sniff, perform flehmen and mount. When
the male attempts to introduce his penis in the vagina, the handler at the right
side of the camel directs AV towards the penis.
 The time required for ejaculation varies, but usually lasts for 5-20 minutes.
Collection from the dog:
Semen can be taken fairly readily from most dogs by digital manipulation
(masturbation) or, less commonly, by the use of an AV.

It is generally considered that semen collected by digital manipulation is of
better quality and quantity, probably due to deleterious effects of the latex of
the AV upon canine sperm.

Erection can be induced by applying encircling pressure with the thumb and
forefinger behind the bulbus glandis. When erection is obtained, the penis can
be deflected into an AV, or digital collection continued.

Where an AV is used, rhythmic changes in pressure are applied until the dog
attempts to tie. The AV is then repositioned to allow the penis to be directed
backwards, as in the tie, while ejaculation continues.
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In order to induce ejaculation by digital manipulation, the bulbus glandis may
be rhythmically compressed, although many dogs will induce ejaculation by
their own thrusting.
Frequency of semen collection:
o
The number of collections per unit time varies according to species and
season of the year.
o
Semen from bulls and buffaloes is collected twice a day 2-3 days per week.
o
Semen from ram is collected 3-4 times a day for several weeks.
o
Semen from buck is collected 2-3 times a day for several weeks.
o
For a normal stallion, one ejaculate every 2-3 days can maintain normal
semen quality.
o
Semen is collected from the camel every other day, but if daily collection is
needed for several days, rest for 2-3 days is recommended.
Semen examination for ascertain the number of functionally normal spermatozoa
present in an ejaculate are sufficient to cause pregnancy and the sire has an
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adequate capacity to produce enough spermatozoa to achieve pregnancies
amongst all the females he is required service.
Reproductive Abnormalities of Male Animal:
The fertility of male is related to several phenomena: Sperm production,
viability and fertilizing capacity of the ejaculated sperm, sexual desire (libido) and
ability to mate.
Reproductive abnormalities causing absolute or relative infertility in male
animals may classically divide into two main classes:
 Conditions causing failure of normal service (impotentia coeundi).
 Conditions causing failure of conceptions after normal service (impotentia
generandi).
The impotentia coeundi can be divided to: conditions causing an unwillingness to
mount and conditions that prevent normal copulation from occurring, despite normal
libido.
Conditions causing a lack of libido: Lack of libido may be hereditary or may originate from psychogenic
disturbances, endocrine imbalance or environmental factors.
 The lack of libido may presented in many animals are either young or of
advanced age. Where immaturity is suspected as the cause of low libido, little
can be achieved by exercise and provision of a plentiful supply of oestrous
females. Hormone therapy may be required giving large doses of hCG (500010000 iu) or GnRH, in attempt to stimulate libido. High doses of these
hormones may cause aggression as on libido and testicular oedema.
 Unwillingness to copulate can also result from poor service management
(slippery floors, too low roofs and intensive handling by stock persons).
 Most lesions affecting locomotion impair ability and willingness to copulate.
 In dogs and in aged animals of all species, lesions of the joints of the hind
limbs are important causes of impaired libido. Animals with overgrown
hooves, are frequently unwilling to mount or, if they do mount are unwilling to
remain mounted for long enough for successful copulation to occur. Any
lesion of the trunk affects ability to mate. In young bulls (15-21 months old)
that are over-zealous in their attempt to mate, the lumbo-dorsal fascia may
rupture, and leads to pains prevent the forelimb from raised in preparation for
mounting 'honeymoon back'
Failure to copulate:Inability to copulate is a relatively frequent cause of infertility in animals.
Conditions cause failure to copulation includes: failure of erection, abnormalities of
erection that prevent intromission and lesions of the penis and prepuce that prevent
protrusion of the penis.
(1) Failure of erection:Erection is achieved by the action of the ischiocavernosus muscles pumping
blood into the corpus cavernosum penis (CCP). If any aspect of the vascular system
of the CCP is perturbed, failure of erection ensures. Two main classes of
abnormalities occur:i.
Abnormal venous drainage of the CCP:
It is commonly in young bulls which are presented with normal libido, ability to
mount but never achieving erection or intromission due to invariably failure of
occlusion of the veins that drain the CCP during foetal life. Thus, because of
the presence of veins draining the CCP, it is not a closed vessel, the high
11
blood pressures required to produce erection cannot be achieved and the
penis therefore remains flaccid.
ii.
Occlusion of the longitudinal canals of the penis:
 In ruminants, the blood pressure during erection, which are initiated by
ischiocavernosus activity, are transmitted throughout the CCP by the
longitudinal canals.
 Congenital absence or acquired blockages of these canals therefore
prevent erection.
 In young bulls, the condition is diagnosed by observation of mating
behaviour.
 It can be differentiated from abnormal drainage of the CCP by palpation of
the base of the penis. Although the great majority of the penis is flaccid, a
short length of turgid tissue is present, in the part of the penis proximal to
the occlusion.
Rupture of the CCP:
The condition has many names, including ruptured penis, fractured penis and
broken penis. Rupture of the tunica albuginea occurs spontaneously if pressures
within the CCP rise substantially above the pressures achieved during normal
copulation. Such abnormal increases in pressure can occur if the penis is suddenly
subjected to shearing forces – for example, by the cow moving suddenly at the
moment of ejaculation.
Rupture occur (in bulls) most commonly either in the region of the insertion of
the retractor penis muscle, or on the dorsal aspect of the distal sigmoid flexure.
In the rams, the rupture of CCP occurs near to the roof of the penis, above the
proximal sigmoid flexure.
Clinical signs:1) In most cases, will immediately refuse to make further attempt to mate.
2) Shortness of gait and general indications of mild discomfort.
3) Haemorrhage occurs from the site of rupture, with haematoma collecting in the
surrounding tissues (cranial to the scrotum in ruptures of the distal sigmoid
flexure, behind the scrotum with proximal ruptures).
4) Distal ruptures are also characterized by preputial oedema may leads to
eversion of the preputial mucosa or prolapse of penis.
5) If the haemotoma untreated, may develop abscesses (infection) or fibrous
adhesions between the penis and prepuce.
Treatment:1) Conservative treatment, consisting of sexual rest for 90 days, with initial
antibiotic therapy to prevent abscess formation and daily massage of affected
area to limit formation of peri-penile adhesions.
2) In the larger haematoma with abscess formation and resulting adhesions,
surgical evacuation should be indicated.
(2) Abnormalities of erection:i.
Persistence of the penile frenulum:It is frequently in the young bulls, in which it either limits the amount of penis
can be protruded or causes the protruded penis to be deviated ventrally.
ii. Congenital abnormalities of the penis preventing protrusion:Such as shortness of the penis and failure of development of retractor penis
muscles (inability to protrude).
12
iii.
Deviation of the penis:Ventral deviation of penis, often referred to as 'rainbow' deviation, can be due
to persistence of the penile frenulum, defects in the tunica albuginea
(congenital or injury).
True ventral deviation of the penis must be
differentiated from partial failure of erection. Lateral deviation of the penis is
also occurs due to injuries to the tunica.
The most common deviation of bovine penis is the spiral deviation. Spiraling
of the tip of the penis is a normal part of the process of ejaculation in the bull,
occurring after intromission. If the spiraling occurs before intromission, it may
consider pathogenic. Premature spiraling occurs in most bulls as the tip of the
penis touches the hindquarters of the cow. Continual premature spiraling can
result in the trauma and pain leads to ulcer on the glans penis, may in time,
impair the libido.
The spiral deviation can be alleviated by suturing the dorsal apical ligament to
the tunica albuginea (catgut and stainless steel).
(3) Lesions of the prepuce:Adhesions between the peri-penile tissues can arise from localized trauma,
haemorrhage and/or abscessation in and around the prepuce. Infection of the penis
(balanitis) or prepuce (posthitis) may result in unwillingness to copulate, development
of adhesions between two organs and preventing protrusion of penis.
Preputial injuries:In bull, intermittent protrusion of varying lengths of preputial mucosa is a
normal occurrence: Pathological eversion of the prepuce is associated with aplasia or
hypoplasia of the retractor muscles of the prepuce, which normally stabilize the
preputial mucosa during penile movement. The damage to the prepuce occurs
commonly in the segment of preputial mucosa that is closest to the preputial orifice.
In this cases, eversion of the preputial mucosa followed by acute inflammation
(hyperemia and oedema) and if not soon replaced, the mucosa may become
permanently prolapsed, with severe and defuse fibrosis and thickening with chronic
tissue formation and granulation tissue.
Treatment:1)
Acute cases may be treated with application of emollient dressings and
replacement of the everted organ.
2)
Chronic cases may be requires surgical removal (resection or amputation).
Balanoposthitis: Infection of the penis and prepuce are common in the dog, bull and ram. In
the dog, mild balanopasthitis with a mild sero-purulent exudates. Sometimes,
a canine herpes virus causes a more severe balanoposthitis, which is
characterized by ulceration of the penis and unwillingness to copulate.
 In the bull, many organisms are able to colonise the preputial cavity. Most of
these do not cause any clinical problems, although there are also a number
that produce venereally transmitted diseases. Most of these diseases do not
cause any gross lesions of the penis, with exception the condition caused by
the infectious bovine rhinotracheitis – infectious pustular vulvovaginitis (IBR –
IPV) virus, leads to an acute ulcerative inflammation of the penis and prepuce.
Secondary bacterial infection of ulcers results in a severe, purulent
balanposthitis.
 Treatment is symptomatic and consists of sexual rest and infusion of oily
suspensions of antibiotics into the prepuce. In neglected cases, fibrinous
13


adhesions may develop, resulting in impaired the penis from protrude.
Pustular vulvovaginitis usually occurs in cows served by bulls in the early
stage of the disease.
In the ram (pizzle rot), bacterial balanoposthitis is caused by a number of
organisms, of which corynebacterium renale, the disease is more common in
wethers than in rams, due to incomplete development of the penis and
prepuce in castrates. Ulcers and scabs develop over the preputial area,
followed by necrosis. Eventually the orifice of prepuce may become blocked
(urine retention).
The most serious cause of balanoposthitis in the stallion is the disease of
dourine is caused by Trypansoma equiperdum. Infection is predominantly
venereal, but can also be transmitted through infected AI equipment and can
be passed to foals through vaginal discharges from the mare. The initial sign
is oedema of prepuce, penis scrotum and surrounding skin (paraphimosis).
Inguinal lymph nodes are enlarged and mucopurulent urethral discharge may
also be present. Death occurs as the consequence of vascular degeneration
causing peripheral nerve degeneration (paralysis). Treatment in early stages
by trypanocidal drugs.
Phimosis:Phimosis indicates a stricture of the preputial orifice that prevents the penis
from being protruded. It may arise from the injuries and congenital detect particularly
in the dogs (German shepherd and golden retriever breeds). Affected puppies may
be unable to urinate adequately with balanoposthitis leading to septicaemia and
death.
Treatment may by removing a wedge of preputial skin, fascia and mucosa
from behind the ventral aspect of orifice of prepuce. Mucosa and skin are then
sutured together.
Paraphimosis: Inability to withdraw the penis into the prepuce results from congenital or
acquired strictures of the prepuce, paralysis of penis and from balanoposthitis.
The condition is most common in the dog and the stallion (may occur in the
other domestic species).
 In the dog, paraphimosis following copulation or spontaneous erection. It may
also occur when the preputial opening becomes constricted by a band of hair,
preventing return of penis to the prepuce. In the early cases, the penis can be
returned to the prepuce with careful manipulation and plentiful lubrication. In
the neglected cases, when the penis becomes oedematous, swollen inflamed,
the preputial orifice may be enlarged surgically. If the condition is left
untreated the penis can become strangulated with short time.
 In the stallion, prolapse of the penis is the sequel to many conditions. For
examples: after the administration of phenothiazide tranquilizers, follow
exhaustion and severe systemic illness, terminal stages of disorders of the
CNS, secondary to the preputial oedema follows castration and commonly
occurs after injury to the penis. In the early stages, oedema may be relieved
by use of cold packs and exercise, where as in the later stages the use of antiinflammatory drugs and diuretics may also be helpful. The surface of the
penis must be protected by the use of ointments to prevent drying. The penis
must be supported by made of nylon stocking material or U – section plastic
14
guttering, appropriately slung around the horses hindquarters. If condition fails
to resolve, amputation of the penis may become necessary. (*priapism is a
prolonged and painful erection that can last from several hours up to a few days and
occurs without sexual stimulation. The condition develops when blood in the penis
becomes trapped and unable to drain. If the condition is not treated immediately, it
can lead to scarring and permanent erectile dysfunction).
Strangulation and necrosis of the penis:Strangulation may occur as a consequence of paraphimosis or result of
constriction of the penis by hair. It is most common in long-haired breeds of dog and
long-wolled breeds of sheep.
Necrosis of the penis which occurs in the ram following obstruction of urethra
by urethral calculi. In the neglected cases, urethral rupture may occur, leading to
infiltration of urine into perineal tissues, the prepuce and the scrotum. Treatment by
amputation of necrotic tissue.
Penile neoplasia: Virally induced fibro-papillomata of the skin, genitalia and alimentary tract are
common in young cattle (penile integument). The tumour may be single or
multiple, sessile or penduculated.
 Clinical effects vary according to the size and morphology of the lesions.
Haemorrhage and ulceration are the most common sequelae, the pain may
leads to impair libido.
 Large lesions can prevent retraction of the protruded penis (traumatized and
infected). Rapid growth of penile tumours within the preputial cavity can result
in compression of the urethra, which may even rupture with infiltration of urine
into per penile tissues. The tumour may be removed surgically.
 Squamous cell carcinoma of glans penis and sarcoid are also common in the
horse.
Conditions causing failure of fertilization:Fertilization failure, despite normal copulation generally characterizes
diseases of the testis, epididymis and accessory glands. Many of the conditions
causing fertilization failure can be diagnosed by an examination of the external
genitalia of sires, but most of cases can only be diagnosed by semen evaluation.
Conditions affecting the testis and epididymis
Cryptorchidism: It is one or both testes fail to complete their descent into the scrotum.Unilateral
cryptorchidism occurs more commonly than the bilateral condition.
 Spermatogenesis is generally impaired or absent in testes that are not scrotal,
due to high intra-testicular temperature.
 Animals that have a single cryptorchid testis are usually fertile, but when both
testes are cryptorchid, the ejaculate is either aspermic or very severely
oligospermic. Testosterone secretion is unaffected, so that the libido of
affected animals is normal.
 Cryptorchidism occurs most commonly in stallion, boar and in some breeds of
dog. In a newborn puppy or kitten, the testes are small, soft and can move
between scrotum and inguinal canal, especially when the pup is stressed or
frightened. It is recommended to wait up to about six months of age before
declaring a dog or a cat cryptorchid. The reason for the six months wait is that
the inguinal rings of most dogs are closed by 6 months of age.
15
 Cryptorchidism is heritable and is a sex-limited autosomal recessive trait in
dogs. The incidence of cryptorchidism seems to be higher in purebred and
inbred dogs than in mixed-breed dogs.
 It is uncommon in others species, except as an iatrogenic condition of bulls
and rams. In these species, unskilled use of rubber rings for castration can
result in one testis being forced back into the inguinal canal or into a S/C
position cranial to the scrotum. Testes may be retained in the abdomen or
inguinal canal.
 For diagnosis, visual examination and careful digital palpation of the scrotum
and inguinal area is helpful. However, scrotal fat and inguinal lymph nodes
may be confused with the retained testis. Abdominal testes are difficult to
palpate or visualized by ultrasonography.
 Castration or removal of both testes is the treatment of choice for
cryptorchidism. The most common medical treatment is the use of drugs
providing luteotrophic hormone (LH) activity, such as HCG hormone, or use of
GnRH to induce an increase in endogenous LH.
Testicular degeneration: The seminiferous epithelium of the testis is highly susceptible to damage, with
a wide variety of agents causing reversible or irreversible degeneration.
 Testicular degeneration occurs in response to raised intra-testicular
temperature, toxins, endocrine disturbances and infection. Degeneration may
result from inguinal or scrotal hernias. The most severe infectious cause of
degeneration is orchitis.
 Clinical signs of infertility and oligospermia occur 4 – 8 weeks after onset of
the cause of the degeneration. Ejaculate volume is usually unaffected, but
number and motility of spermatozoa fall with abnormal morphology. In severe
cases, the ejaculate may become virtually aspermic. Resolution of the acute
degeneration occurs over a period of weeks or months (some of abnormal
sperm may be present).
In more severe cases, permanent loss of
seminiferous tubules occurs, with fibrosis and calcification of the testis
following. Such animals never regain normality.
 Testicular biopsy can be a valuable aid to prognosis.
Orchitis and epididymitis: Orchitis ranges from a mild infection of the testis, through to gross supurative
or necrotic destruction of the organ. Orchitis can arise from a primary infection
or by haematogenous spread of bacteria into the testis. Primary testicular
damage can arise from ECBO (enteric cytopathic bovine orphan viruses) or
BEV (Bovine enterovirus) infection in bulls. Brucella species cause orchitis in
many domestic animals. Orchitis is more commonly unilateral than bilateral
and may involve the epididymis.
 Acute orchitis is characterizes by hyperaemia, heat, pain and swelling (3 times
up normal size). The pain may be severe to produce abnormal gait. The
chronic orchitis is associated by fibrosis, shrinkage and adhesion to the
scrotum (abscess may develop). Treatment in early stage by removal of
affected testis, to limit degeneration of the unaffected testis. If bilateral orchitis
occurs, the prognosis is hopeless, and castration should be performed.
 Epididymitis can also occur as a primary infection or by spread from infected
testis. The general signs of epididymitis are similar to those of orchitis.
Unilateral epididymitis result in reduced fertility, where as bilateral obstruction
16
result in sterility. Ovine epididymitis also occur as consequence of infection
with Actinobacillus seminis and Haemophilus sommis.
Testicular hypoplasia:Testicular hypoplasia implies an incomplete development of the germinal
epithelium of seminiferous tubules, due to inadequate numbers of germinal cells
within the testis. Lack of germinal cells may arise through partial or complete failure
of the germinal cells to develop in the yolk sac, failure to migrate to the genital ridge,
failure to multiply in the developing gonad or wide spread degeneration of embryonic
germinal cells within the primitive gonad. Mild cases may exhibit moderate
oligospermia or poor sperm morphology, but severe cases may be aspermic.
Klinefelter's syndrome (XXY) is a sporadic cause of testicular hypoplasia in
bulls, rams, boars and dogs. The semen of such animals is aspermic, although the
Leydig cells being unaffected, libido is normal.
Diagnosis of the condition is by measurement of scrotal circumference.
Palpation of testis reveals one or both to be small and flabby, but regular in outline
and freely movable in the scrotum. Semen analysis may reveal aspermic or
oligspermic ejaculates, with abnormal morphology or motility.Treatment by castration
and slaughter (meat animals).
Lesions of the accessory glands:Vesicular glands: Infection of vesicular glands is common in bulls. Primary causative organisms
may include B. abortus Chlamydia spp and the Epivag, ECBO and IBR-IPV
viruses. Seminal vesiculitis occurs most commonly in young bulls of less than
2 years old and in aged bulls. Secondary infection of vesicular glands may
occur by C. renale, Actinobacillus actinoides, E. coli, Pseudomonas
aeruginosa, Streptococci and Staphylococci.
 Infection of vesicular glands also occurs in the stallion from which a similary
organisms.
 In bulls, during the acute phase, localised peritonitis may occur in the caudal
abdomen. Later animals generally present as being infertile despite normal
service. Occasionally abscesses form in infected gland with fistulae generally
into the rectum.
 The main consequence of infection is decline in semen quality a decrease in
motility accompanied by elevated pH, low fructose concentration. In severe
cases, the semen may appear purulent, brownish, due to the presence of
degenerating blood from the damaged gland. Diagnosis by rectal palpation of
glands may reveal enlargement, tense and pain in acute phase/or fibrosis and
shrinkage in the chronic case.
 Treatment is possible in early stage by the administration of very large I/V
dose of bactericidal antibiotics. If ineffective, amputation of unilaterally
infected gland is required. In bilaterally vesiculitis, the prognosis is hopeless.
Prostate:Prostatic disease is rare in species other than the dog. Infection and
hypoplasia are common. The infection is generally ascending with B. canis, E. coli,
proteus spp and streptococcus spp.
Prostatic hyperplasia is a common age – related change, with a numerous
small, or a few large cysts. Constipation, but not pain or signs of systemic illness,
characterises this condition.
17
Prostatitis may be treated with broad – spectrum antibiotics, whereas
hypoplasia is best treated by the administration of oestrogens or by castration.
Abnormalities of semen:Semen examination:Assessments of semen are a valuable part of evaluation of potentially infertile
sires. Semen evaluation aims to determine the biological value of semen for
fertilization.
Much care is needed in the handling of semen for an accurate result of it is
examination. Semen must be maintained at temperature close to that of the body (at
30 ˚ C for the ram and buck, 35 ˚ C for the bull, and 37 ˚ C for the stallion and camel)
prior to and during assessment.
Gross examination:
1- Volume: there are differences in semen volume among farm animals. The
normal volume of semen is 5-8 ml for the bull, 0.8-1.2 ml for the ram and buck,
50-100 ml for the stallion, 5-18 ml for the camel ,2-19 ml for the dog and 0.51ml for the rooster . The normal sperm concentration (million/ ml) is 800-1000
for the bull, 2000-3000 for the buck and ram, 100-300 for the stallion, 200-350
for the camel 125 for the dog and 100 million to 5 billion for the rooster .
Generally, semen volume changes according to age, size, general or specific
health and frequency of collection.
2- Colour and consistency: depend on the sperm concentration and extraneous
bodies. The colour can be grayish white as in the stallion, to milky whitish as in
the bull, buffalo and camel, to white creamy as in the ram and buck.
The consistency should be fairly uniform. Gel or gelatinous-like fluid is
normally present in the semen of camel and stallion.
Semen is initially inspected for the presence of urine( urospermia), fresh or
changed blood (hemospermia), pus and extraneous material. The watery
samples are usually oligospermic (chronic epididymitis) and samples that are
not homogeneous often contain pus (seminal vesiculitis).
3pH: fresh semen has an alkaline shift for the stallion, camel (7.2-7.8) or an
acidic shift for the bull, buffalo, ram and buck (6.4-6.8).
Microscopic examination:
(1) Motility:Sperm motility is influenced by temperature. Ideally, this is achieved by using prewarmed slides on a heated microscope stage. The use of a small burner to warm the
slides or glass bottle full of warm water on the microscope stage beneath the slide,
have effective methods. For the bull and ram, a drop of semen is placed on the slide
and examined under low power for observed mass sperm movement (swirling
waves). Mass sperm movement is generally poor in the stallion, camel and boar
semen unless the ejaculate is constitute exclusively by the sperm rich fraction.
The mass motility can be grade from 0 to 5 in raw semen as follow:
Grade
Description
0
Immotile.
1
Stationary or weak rotatory movements
2
Very slow wave movements
3
Slow wave movements
4
Rapid wave movements
5
Extremely vigorous wave movements
18
A subjective evaluation of motility using raw (for thin semen) or extended by a good
quality extender (for thick semen), so that the individual sperm can be seen. Only
sperm that have forward or progressive movements are counted, but sperm with
backward, vibrating or circling movements are not included.
A drop of fresh diluted semen is placed on a warm slide and covered with a warm
cover-slip. The individual motility is examined with high-power heated microscopic
stage (38 ˚ C). Parameters of individual motility include:
 Percentage of motile sperm (normally 70-90). Very Good (VG) :80-100%
motile, Good (G): 60 - 79% motile, Fair (F): 40 - 59% motile and Poor (P):
<40% motile
 Longevity of sperm motility in raw semen at 38 ˚ C.
 Longevity of sperm motility in extended semen at 38 ˚ C.
 Longevity of sperm motility in extended semen at 4 ˚ C.
(2) Sperm count: Sperm density can be estimated most readily using a haematocytometer
(Neubaur counting chamber)). Bull and ram semen should be diluted 1: 100 in
0.9 saline / 0. 02% formalin solution the total sperm count is derived as the
product of volume and density (dilution rates of semen are 20 for the stallion
and camel and 200 for the buck and ram).
 The red blood cells diluting pipette is used for higher concentrations, suitable
for ram, buck, bull and buffalo. The white blood cells diluting pipette is used for
lower concentrations, suitable for the stallion and camel.
 The number of sperm/ ml ejaculate can be calculated by the following formula:
No. sperm/ ml = no. sperm in 0.1mm3 (25 squares) X10 X dilution rate X
1000.
 Although the haematocytometer is considered accurate, it is time consuming
and tedious.
 In the large number of semen samples require evaluation (AI stud); estimation
of sperm density can be facilitated by the use of spectrophotometery. It is a
fast method so that determination of sperm concentration can be performed in
about one minute.
 The procedure is based on light absorbtion by the sperm in the semen sample.
The equipment consists of a light sources which passes through a series of
lenses and filters and then through a diluted sample of semen. The light
passes through the semen is measured with a galvanometer (optical density).
 The disadvantage of this method that abnormal semen containing foreign
bodies can give false results.
(3) Sperm morphology:
 Every semen sample contains some abnormal sperm cells. These
morphological abnormalities of sperm have the greatest relationship to fertility
of livestock.
 A large number of staining materials and techniques have been adopted for
sperm morphology examination.
 Eosin- nigrosin stain is prepared by dissolving eosin (1%) and nigrosin (5%) in
sodium citrate dehydrate buffer.
 A drop of eosin- nigrosin stain is placed and mixed with a small drop of semen
on a slide. Another slide is used to make a thin film from the mixture. The slide
is then dried on heated plate (55-60 C) and examined under high- power oil
emersion lens. At least 200 spermatozoa are examined for the presence, type
and incidence of each morphological defect.
19

Defects of the acrosome are often difficult to see in stained preparations,
although specialised stains such as that of Wells and Awa are used to
visualise acrosomal vacuoles.
 More commonly, differential interference contrast microscopy of wet
preparations is used to examine acrosomal defects.
(4) Live-dead sperm ratio:

Estimate of the proportion of dead sperm in an ejaculate can be obtained by
the use of a vital stain, such as eosin B (eosin-nigrosin).

A small drop of semen mixed with a drop of the stain on a warm slide is mixed.
A thin film is made, quickly dried and examined under a high power lens.
Partially stained sperm should be included with totally stained sperm
representing the number of dead sperm in sample.
(5) Sperm function tests:The simplest of such tests incubate semen at various temperatures and by
relating the duration of sperm survival under these conditions to survival in the female
genital tract, produce reasonable correlations with fertility.
Others tests may be used such as pH, adenosine triphosphate (ATP) content
or aspartate transaminase concentration. Failure of mucous penetration is frequently
a sign of failure of sperm function and occurs in sperm that have been damaged by
cryopreservation or in the presence of anti-sperm antibodies. Of more widespread
use in veterinary practice is computer – assisted analysis of sperm swimming
characteristics.
Assessment of the proportion of sperm with intact acrosomes has been highly
correlated with fertility.
The most recent innovation in assessment of sperm function has derived from
development of in vitro fertilization (IVF) procedure.
Abnormalities of spermatozoa:There are three main classifications of spermatozoal abnormalities:1) According to their site on the sperm (head, midpiece and tail defects and
sperm bearing protoplasmic droplets).
2) According to the site within the genital tract where the sperm defect has
arisen. By this classification, defects are divided into primary defects, which
arise during spermatogenesis, secondary defects, which arise within the
epididymis and tertiary defects, which arise after ejaculation: Thus defects of
the head and midpiece are mostly primary, protoplasmic droplets secondary,
looped tails tertiary.
3) According to their effects on fertility, into major and minor defects. Major
include most defects of the head, proximal protoplasmic droplets and
congenital acrosomal defects (minor defects such as detached normal heads).
More recent concept of compensable (tail defects, detached/loose heads )
and uncompensable (pyriform heads, proximal cytoplasmic droplets).
Abnormalities of the sperm head: Two aspects of the morphology of the sperm head appear to be essential for
normal fertility; the shape of the sperm head and the morphology and stability
of the acrosome.
 Most abnormalities of head are major defects upon the fertility. The majority of
such defects arise within the testis as abnormalities of spermatogenesis
(primary defects). Such defect include: narrow at base, pear-shaped, small
abnormal head, decapitated sperm and misshapen. Less serious defects of
20
the head include giant heads, double heads, narrow heads and small. The
diadem defect represents pouches in the nuclear material.
 Many acrosomal defects arise as primary abnormalities of spermatogenesis
may be present at a high percentage in ejaculate (inherited) or at low
percentages in most ejaculates (spontaneously). The significance of these
defects depends upon the species. For example the fertility of bulls is
impaired by single-figure percentages of the knobbed acrosome defect, but in
the stallions and boars may require higher percentage for impaired fertility.
Abnormalities of midpiece and the tail and attachment of the head:Abnormalities of attachment of head are generally primary and may be
acquired defects (detached heads). Detached head may occur during testicular
degeneration or in the semen of aged bulls. Semen with fractures of attachment
between head and tail (fractured neck) may arise from senescent changes, or due to
congenital weakness of the attachment.
The most defects of the midpiece and tail are: Coiled tail defect, dag defect
and tail stump defect. The minor defects of tail may include looped tails.
Protoplasmic droplets:
 The residual cytoplasm that remains at the end of the spermiation is removed
during maturation (epididymis). The presence of sperm with protoplasmic
droplets indicates that epididymal maturation is incomplete by the time of
ejaculation.
 The droplets may be proximal (close to head) or distal (at the distal end of the
midpiece).
 Protoplasmic droplets are often observed in sires that are overused.
21
Reproductive biotechnology
Biotechnology: A broad term generally used to describe the use of biology in
industrial processes such as agriculture, brewing and drug development. The term
also refers to the production of genetically modified organisms or the manufacture of
products from genetically modified organisms.
Assisted reproductive technology (ART): The use of medical techniques, such as
drug therapy, artificial insemination, embryo transfer, or in vitro fertilization, to
enhance fertility.
Artificial Insemination
Artificial insemination is a method of breeding in which semen is obtained from
the male, processed and introduced into the female reproductive tract by means of
instruments with no direct contact between the male and female.
The successfully of artificial insemination (AI) as a means of animal breeding
depending upon three major premises:1) The spermatozoa can survive outside the body.
2) They can be reintroduced into the female genital tract in a way that results in
an acceptable conception rate.
3) The fertile period of the female can be identified.
AI is routinely practiced in cattle, sheep, pigs, goats, fowl, turkeys, dogs,
horses and even bees. AI in salmon farming is also very widespread.
Advantages and Disadvantages of AI:Advantages:1) Genetic improvement:
 Enables superior genes from outstanding sires to be spread more widely.
 The longstanding insemination of inferior females with semen of superior
sires over years will make the female gain superior genetic traits.
 Of great value in cross breeding to change production trait.
 Facilitates progeny test under a range of environmental and managerial
conditions, thereby further improving accuracy of selection
2) Reduce large number of sires and may be used to breed a very large number
of females.
3) Control of venereal disease (trichomoniasis and complyobacteriosis).
4) Economic service by accelerate the introduction of new genetic material
through the export or import of semen and reduces international transport
costs.
5) Provides a necessary tool for investigating many aspect of male and female
reproductive physiology.
6) Improvement of performance and potential of the national herd.
7) Persist crossbreeding to change the production line, such as changing from
milk to beef.
8) Any male animal can be potentially dangerous; the safety aspect is one of the
major stimuli to set up AI programme.
9) Enables use of deep-frozen semen after a donor is dead, aiding in the
preservation of selected lines.
10) Allows evaluation of seminal quality before insemination and assists in early
detection of infertility problems in the male.
22
Disadvantages:1) Detection of fertile period in female oestrous cycle is less easy in some
species such as ewes (AI requires either the presence of vasectomised rams
to detect oestrus or pharmacological manipulation of oestrus).
2) The process of insemination requires trained personal and separation of the
female from the herd.
3) The genetic faults can be widely disseminated if they are present in an AI sire.
4) Uncontrolled use of sires in AI can disseminate disease.
5) AI has ruined the bull market.
6) There is certain wastage of semen in an AI project due to the necessity of
each inseminator having on hand each day sufficient supply of semen to breed
the maximum numbers of females in heats.
Handling of semen:
 Ejaculate semen should be promptly transported to the laboratory for
evaluation and processing.
 Upon arrival, semen should be placed in water bath at 30˚C for rams and
bucks, and 35 ˚C for the bulls and buffaloes or 37-38˚C for stallions and
camels.
 Once semen is evaluated, it can be used in four ways:
1. Used immediately undiluted to inseminate one or two females.
2. Diluted and used immediately for insemination of several females.
3. Diluted and refrigerated for use over the next 3 days (cooled and chilled
semen).
4. Diluted and frozen for use at a later date (frozen semen).
Preparation of semen for use in AI:Dilution:The ejaculates of most domestic animals contain more sperm than are needed
for achieving a pregnancy. Hence, by diluting the semen, it can potentially be used
for several inseminations in the food animals species, the maximum degree of
dilution is determined from the minimum number of spermatozoa and the volume of
inseminate that is required to achieve acceptable pregnancy rates. These factors
are determined by the site of insemination and the survival of sperm in diluent.
The major properties of semen diluents (extenders) are:1) Addition of volume. Insemination doses must be prepared in a volume, which
is compromise between ease of handling and an appropriate volume for the
site of insemination.
2) Buffers. Spermatozoa have a narrow range of tolerance to change in pH, so
provision of buffering capacity is necessary (has a pH similar to that of the
seminal plasma). Buffering is especially important where the semen is only to
be chilled and not cryopreserved, as the metabolic activity of cooled
spermatozoa remains appreciable. Simple buffers are effective, with citrate
being widely used. Phosphate buffered saline is rather less suitable. More
recently, organic buffers have been used is TRIS {organic compound tris
(hydroxymethyl) aminomethane}.
3) Maintenance of osmotic pressure. Sperm can tolerate a moderate range of
tonicity. Sugar and protein/ which are added to provide nutrition for the sperm
or to contribute to cryprotective properties of the diluent.
23
4)
Energy substrate. Most diluents make some provision of energy substrate for
sperm (glucose, fructose and mannose). The provision of energy is relatively
less important where sperm are to be frozen. However if semen is to be
sustained for several days, provision of energy is important.
5) Antimicrobial activity. Antibiotic are added to most semen diluents as a
prophylactic measure against the transmission of pathogenic bacteria and to
reduce the load of non – pathogenic that contaminate the semen. In cattle AI,
benzyl penicillin and streptomycin are the most widely used.
6) Protect the sperm against injury during freezing and thawing.
7) Free of deleterious substances (such as toxic material) harmful to the sperm
or fertilization process.
8) With a reasonable price or easy to prepare.
The dilution process involves:
a- Both semen and extenders are placed in a water bath at 30-35 ˚C.
b- Semen is diluted by gradually adding the extender to the semen. Never add
the semen to the extender.
c- Semen to extender dilution ratios are 1:1 to 1:10 depending on the species,
semen volume and sperm-cell concentration.
d- The recommended antibiotics (500µg gentamicin, 100µg tylosin per ml) are
add and allowed to stand (holding time) for 5 minutes with raw semen or 2
hours diluted semen to increase the antibiotic action on microorganism.
The life span of spermatozoa at ambient temperature is generally short, but
can be extended by inhibiting their metabolism and motility with carbon dioxide (by
cooling). Cooling sperm, result in considerable damage to the cells, with leakage of
intracellular potassium, enzymes, lipoprotein and ATP occurring (cold shock).
Lowering temperature causes:
• Membrane phospholipids to change from fluid to a gel phase.
• Membrane proteins become irreversibly clustered, leading to loss of function.
• Membrane becomes more permeable to Ca and more fusogenic.
The most effective way of protecting sperm against the detrimental effects of
cooling is by the inclusion of proteins, lecithins, and lipoproteins (egg yolk and milk).
Further more some of the constituents of egg are toxic to the sperm of some species
(goat).Whole milk is also toxic to sperm, for it contains a protein (lactenin), which is
spermicidal. Thus, milk for use as a semen diluent most be treated by heat
(skimming process).
The fertility of bovine semen stored at 5˚C in such a diluent remains
acceptable for 2-4 days (in ram only persists for 12-24 hrs).
Cryopreservation:Long term storage of semen is achieved through cryopreservation. For sperm
to survive freezing they need to be extended in a diluent that contains not only
substances that protect them against cold shock, but also cryopretectants, such as
glycerol. Cryoprotective agents may either penetrate or remain outside the cell, but
both act by binding water either for dehydrative loss or for ice crystal formation.
Glycerol is the main primary cryoprotectant used in preparing mammalian
semen for freezing, despite the fact that it has some directly toxic effects upon sperm.
Concentrations of glycerol depend upon the species and the other components of the
diluent.
24
For example, diluents for bovine semen that contain disaccharides can utilize
lower percentages (3-4%) of glycerol than diluents that lack such disaccharides,
which have a final glycerol concentration of at least 7%.
Freezing and storage:


Liquid nitrogen (LN, -196˚C) is the refrigerant of choice.
LN container is a double-wall stainless steel or aluminum with a vacuum
between the walls. A container of 20 litres nitrogen capacity holds nitrogen for
90 days before charging, taking into consideration the ambient temperature
and the frequency of use.

The LN container must be checked periodically for topping up with LN.

Diluted semen is packed into thin plastic tubes of 0.25 or 0.5 ml capacity,
(straws).

The standard procedure for freezing is to place a single layer of straws on a
tray exposed to LN vapour (5.5 cm above the LN level at -120˚C) in this area
freezes semen in about 10 minutes.

The straws are then placed in aluminum canes and plunged into the liquid
nitrogen (by canthter).
New methods of cryopreservation:
1. Vitrification: a process in which a liquid turns into a solid without the formation
of ice crystals by rapid cooling of water to a glassy state through extreme
elevation of viscosity.
2. Multi thermal gradient: is a mean of controlling and optimizing the formation of
ice during the freezing process.
Thawing of frozen semen:
o
Thawing of the semen needs to be rapid; slow thawing allows recrystallisation
of ice within the cells, causing membrane damage.
o
The process of thawing is simply immersing the straws, ampule or pellets in
water at 35 ˚C for only 7 seconds.
o
Care must be that semen in improperly sealed ampule or straw will be
damaged at thawing.
Microencapsulation:
 Sperm encapsulation is a mean of preserving sperm without the need for
recourse to cryopreservation.
 Sperm encapsulation is the process of enclosing spermatozoa within a semipermeable membrane.
 After extension in an ambient diluent, sperm are suspended in a sodium
alginate. Spraying into a high calcium buffer solidifies droplets of diluent
sperm, after which the semi-permeable membrane is constructed on the
surface of the droplet.
 Calcium is then chelated, which return the gel core of the microcapsule back
to a liquid phase.
AI of cattle:Semen is usually collected by an AV. After assessment of semen, it is diluted
into insemination doses. For cryopreservation or for use at 4˚C, the semen is first
extended with a diluent based upon either egg yolk or skimmed milk, which
contains antibiotics for the control of contaminating bacteria.
25
If the semen is destined for cryopresevation, glycerol is also added and the
semen packed into 0.25 or 0.5 ml straws. The semen is then frozen in the vapour
of liquid nitrogen or in a microprocessor – controlled freezer.
The ability to perform an intrauterine insemination in cattle is requiring low
dose of sperm to achieve acceptable pregnancy rates (10-25 million sperm).
Insemination:Cows ovulate at about 12 hours after the end of the oestrus period. The ideal
time for insemination is therefore 6 – 24 hours prior to ovulation. Practically, if the
cow first seen in heat in the morning, she should be inseminated in the afternoon of
the same day, or the cow first seen in heat in the afternoon, she should be
inseminated on the next day morning.
Cows are insemination just into the short uterine body. Insemination into the
cervix produce a lower fertilization rate, while insemination deeper into the uterus
runs the risk of either inseminating into the uterine horn contra-lateral to the
ovulation site, or scoring the endometrium with the tip of the insemination catheter.
The standard technique of insemination is to grasp the cervix through the
rectum with the left hand. A catheter into the tip of which a straw of semen has been
inserted, is then passed in to the vagina and manipulated into and through the cervix
by the right hand. The vulval lips are opened by downwards pressure from the arm in
the rectum, while the circular folds of vaginal mucosa are obliterated by pushing the
cervix forward.The catheter is initially inserted pointing upwards at angle of about 30˚
or 45˚ to avoid entering the urethral meatus or fossa, and is then moved horizontally
until it engages in the cervix. Entry into the external os is accompanied by a
characteristic ‘gritty’ sensation. The catheter is then introduced through convoluted
cervical canal by manipulation of cervix through the rectal wall. One finger is placed
over the internal os of the cervix; so that the tip of the catheter can be palpated at it
emerges from the cervical canal. As soon as the catheter has emerged, deposition
of semen into the uterus begins. No forward pressure should be exerted on the
catheter with right hand, for the uterine wall is friable and easily penetrated if catheter
moves suddenly. The most common fault of insemination is twisting the cervix in the
left hand, so that one uterine horn is partly occluded.
Management of insemination:
Insemination can be performed at an observed or induced oestrus. The former
is more common in dairy cattle, for considerable opportunity exists for the
observation of oestrus, but in beef cattle and dairy or beef heifers the time required
for observation makes the use of induced oestrus relatively more attractive. Oestrus
can be induced and synchronised by the use of prostaglandin (PGF 2α) or its
analogues, progesterone-like hormones, or combinations of progesterone and a
luteolytic agent. Most such regimens require the use of fixed-time insemination,
although, particularly in the case of PGF2α, the accuracy with which the timing of
oestrus can be predicted is sufficiently imprecise for some observation to be
advisable, and reinsemination performed if animals exhibit signs of oestrus after
fixed-time insemination has occurred.
Fertility to AI is generally very similar to that achieved at natural service, with a
calving rate to a single insemination of around 50%.The true fertilization rate is much
higher than this, at around 90%, but subsequent embryonic losses bring the apparent
figure to the lower value. The proportion of the cows that are not re-presented for
insemination after initial service is closely related to the proportion which actually
have become and remained pregnant (non-return rate NRR). AI centres therefore
26
use the ‘non-return rate’ to monitor both the fertility of their bulls and the results
obtained by their technicians.
Control of infectious diseases:Most of the serious viral diseases of cattle (foot and mouth, rinderpest, etc.)
can potentially be transmitted through AI. A number of bacterial diseases are
transmissible in semen, including tuberculosis, brucellosis leptospirosis and possibly
Johne's disease. In cattle control of these diseases rests upon three major
strategies:1) Diseases that can be detected by serology, such as brucellosis, IBR, Q fever
are controlled by exclusion of seropositive bulls from AI studs; tuberculosis is
controlled by exclusion of bulls that react to tuberculin testing.
2) The antibiotics are added to semen diluents to kill both pathogenic bacteria
and the contaminant bacteria that originate from the penis and prepuce during
semen collection.
3) Quarantine of semen after its collection. After semen has been frozen, it is
placed in a container where it remains untouched for 28 days. If during that
period, the donor bull develops any disease, the semen is destroyed. If not it
is release for use.
AI of Mare:






The technical demands of AI of horses differ somewhat from those of cattle.
Firstly, it might be considered that the collection of semen from stallions is
more difficult than from bulls, for coitus is both more prolonged and more
violent in horses than in cattle. Secondly, the stallion produces a fractionated
ejaculate, from which the viscous, post-sperm-rich fraction has to be
separated and discarded. Thirdly, stallion semen has proved more difficult to
store than has that of bulls, with cryopreservation proving difficult to achieve
consistently. Finally, the peculiarities of the oestrus period of the mare make
pinpointing the moment of peak fertility more difficult than in the cow.
Mares do not reliably display visible signs of oestrus in the absence of a
stallion and, furthermore, the oestrous period is prolonged, so that unless the
ovaries of the mare are palpated to determine the time of ovulation, repeated
insemination is likely to be required to ensure that the fertile period is covered.
The mare ovulates about 24 hours before end of oestrus. It is recommended
to inseminate every other day during oestrus.
However, insemination of the mare is much easier than insemination of the
cow, for the equine cervix is soft and does not have a convoluted luminal
canal.
Mares are generally inseminated with 250×106 to 500×106 progressively
motile sperm in 10-25ml extended chilled semen (100-200×106 for frozen
semen).
By the vaginal methods, a 50-ml syringe connected to a plastic insemination
catheter is used to deposit the semen inside the uterus through the easily
dilated cervix.
AI of Ewe and doe:

The sheep is less amenable to artificial insemination than is the cow, since
oestrus cannot readily be detected without the presence of rams, insemination
is less straightforward and ovine semen is less easy to freeze than bovine
semen.
27

1-
2-
3-
4



There are different methods of AI:
Intra-vaginal method, a small stainless steel speculum and by the aid of light,
the inseminating catheter is used to deposit semen in the anterior vagina and
around the external cervical os. Intravaginal insemination is best suited to use
after oestrus detection during the natural breeding season.
Intra-cervical method, an inseminating catheter fitted with a 10 cm long, balltipped, 17 gauge needle is used to deposit semen through the cervix in the
anterior part, if possible. Intra-cervical insemination is best achieved with the
hindquarters of the ewe elevated (it is common used in the doe goat)
Laparoscopic intrauterine method, a laparoscopic is used to deposit semen
inside the uterus. In this method, ewes are restrained in a cradle and
laparoscopy is performed close to the udder. After inflation of the abdomen
with CO2, the uterus is located and semen injected into the uterine lumen via
small stab.The semen can be introduced to the uterus via a simple pipette or
by the use of specialized insemination equipment.
Trans-cervical intrauterine method, it is difficult to achieve in the ewe than in
the doe goat, because the cervical canal of the ewe is tortuous.
Most inseminations of ewes are performed using semen that was collected on
the day of insemination. Such semen is normally extended by the addition of
simple diluents, although direct insemination of raw semen is still practiced in
some regions.
In goats, the major problem with semen preservation is that components in
seminal plasma that impair the viability of sperm stored in media containing
milk and egg yolk. The toxic interaction with egg yolk is due to egg yolk
coagulating enzyme (EYCE) that secreted by bulbo-urethral gland. Toxic
interaction with milk is BUSgp60.
Ovulate towards the end of the oestrus phase. Therefore, insemination is
carried out 12-18 hours from the start of oestrus phase.
Heat is detected through a vasectomized ram or buck, or applying
synchronization of oestrus.
AI of Camel:
o
o
o
o
o
o
o
Progress in the application of artificial insemination (AI) to camel breeding had
been slow because of two main factors:
Firstly, the nomadic, pastoral nature of typical camel husbandry in arid regions
militates against frequent herding, enclosure and restraint of animals.
Secondly, she-camels in natural circumstances ovulate only in response to
mating.
However, despite these two constraints, it has been shown that AI is possible
in camels kept intensively and by using: (1) service by a vasectomised male,
(2) an injection of seminal plasma, or (3) an injection of human chorionic
gonadotrophin (hCG) or a GnRH analogue.
The inseminating catheter is used to collect semen from the ampules and
deposited inside the uterus. The cervix easily allows the catheter through.
The insemination gun is used to inseminating with semen packed in the plastic
straws to be deposited inside the uterus.
A minimum of 50-100 million motile sperm per insemination are recommended
(good conception rates are obtained by 300 motile sperm).
28
o
o
Oestrous female camel is detected by teaser male. However, for accurate
records, rectal examination or ultrasound scanning is made to examine to
ovarian activity.
The female camel is inseminated when she has an ovulatory follicle between
1.0 and 1.9 cm in diameter. Insemination of the female camel with smaller or
larger follicles would have poor conception rate.
AI of dog:
Semen is collected from the dog by digital manipulation or by artificial vagina.
The pre-ejaculatory fluid, sperm-rich fraction and a little of the post-ejaculatory
(prostatic) fluid are collected.
The whole ejaculate may be immediately inseminated into the bitch’s vagina,
but it is more common to dilute the semen so that multiple inseminations can
be performed.
The bitch has a prolonged period of receptivity to the male, but a relatively
short fertile period. For successful AI, much closer attention to the time of the
fertile period is needed, with the timing of ovulation predicted from vaginal
cytology or the preovulatory rise in circulating progesterone concentrations
(ideal time of insemination 3-4 day after ovulation).
Where the reason for AI is failure of copulation, semen is collected from the
dog by digital manipulation, and the whole ejaculate may be inseminated into
the vagina of the bitch immediately after collection. It may, however, be
preferable to dilute the semen, inseminating one portion immediately and the
remainder 48 hours later.
When fresh or chilled semen is used, intra-vaginal insemination is undertaken,
with the semen deposited as close as possible to the external os of the cervix.
The semen may be deposited through a shortened bovine insemination
catheter, which may require the use of a speculum to be guided into the
correct site. Once inseminated, the hindquarters of the bitch should be raised
for a few minutes, to prevent retrograde loss of semen.
Where frozen semen is used, timing of insemination is critical and, as with
most species, there is very considerable sire-to-sire variation in the ability of
sperm to survive cryopreservation.
The site of insemination is also important; intra-vaginal insemination with
cryopreserved sperm gives poor conception rates, so an intra-cervical or
intrauterine route is required.
AI in Poultry:


The main Objectives of artificial insemination in poultry are:
1. To place the required dose of semen into the oviduct of the female so that it is
deposited near the sperm storage glands and
2. To carry out the AI process with due regard to the best health and welfare of
the breeder females thereby achieving the highest fertility levels possible.
Why we do AI in poultry???
A. Increased mating ratio: In a flock usually one cockerel mated six to ten hens.
With artificial insemination it is claimed this ratio could be increased four fold.
B. Use of older males with outstanding performance: Older male birds that have
been flock improvers can be used for several generations. Whereas under
natural mating their useful life is limited.
29
C. Able to use an injured bird: Valuable male birds that have been injured in the
leg can still be used for artificial insemination.
D. Successful cross breeding: Very successful under natural conditions but some
times there is a kind of color discrimination. Some hens will not mate with a
male of a different colour unless they have been reared together. In AI cross
breeding is quite easy and successful.
 For collection of semen, two people needed; One for handling, vent opening
and other for collection of semen.
 The person collecting the semen sits on a stool and holds the male on his lap.
He or she stimulates the male by stroking his back from the middle towards
the tail, while at the same time stroking the abdomen towards the vent with the
other hand. After doing this several times, with the help of thumb and the index
finger of the right hand massage the pubic bones lightly. This causes the male
to extrude the phallus and, if the bird is producing semen, results in
ejaculation.
 For insemination, pressure is applied to the left side of the abdomen around
the vent. This causes the cloaca to evert and the oviduct to protrude so that a
syringe or plastic straw can be inserted into the oviduct and the appropriate
amount of semen delivered.
 As the semen is expelled by the inseminator, pressure around the vent is
released, which assists the hen in retaining sperm in the vagina or the oviduct.
 Biologically, after deposition of semen in the oviduct the semen will enter the
sperm storage gland, situated at the junction of the vagina and the shell gland
and from here the spermatozoa will make their way up the oviduct to a second
storage site situated at the junction of the magnum and infundibulum. The
passage of an ovum into the infundibulum stimulates spermatozoa activity and
fertilization of the ovum by one sperm takes place.

In order to increase the number of hens that can be inseminated from the
same rooster, the semen maybe diluted with a solution known as modified
Ringer's solution.
30
Embryo transfer (ET)
Embryo transfer (ET) is a technique by which fertilized ova (embryos) are
collected from a genetically outstanding dam (donor) and transferred to
genetically less distinguished females (recipients) to serve as surrogate mothers
for the remainder of pregnancy.
The first successful embryo transfer was carried out over 100 years ago in rabbits
by Walter Heape (1890) in England.
The first embryo transfer in domestic animals was reported in sheep and goats by
Warwick and Berry (1949). In 1951, the first calf was born via the surgical
technique of embryo transfer by Willett. In the early 1970s, embryo transfer has
been applied most extensively in cattle; consequently the technology has
advanced most rapidly in this species.
The first successful equine embryo transfer was reported in England by Allen and
Rowson (1975).
In dromedary camels, the first successful embryo transfer was conducted by
skidmore (1992).
Applications of embryo transfer:
1) Obtaining a large number of offspring from a single donor per year.
Approximately 12-15 calves can be produced annually from each donor cow.
2) A valuable research tool. It has been used exclusively in studies of uterine
capacity, on the uterine environment, the maternal recognition, embryo uterine
relationships and endocrinology of pregnancy.
3) Genetic improvement:
i. The ET can be used to influence genetic variation by introduction of a
breed genetically superior for the desired traits.
ii. The ET can be used to increase the reproductive rate of the best cows;
selection can be restricted to the top 5 – 10% of females.
4) ET has also been used to expedite the screening of both dams and sires for
genetic defects.
5) Disease control. There is increasing evidence to suggest that embryos are
unlikely to spread viral and bacterial diseases when transferred into recipients
(zona pellucida of embryo appear to be an effective barrier to infection of the
embryonic cells from uterine environment).
6) Ease of import and export of embryos. The development of efficient methods
for cryopreservation of embryos provides the following advantages:
 Possibility of transferring an entire herd in the form of frozen embryos in
a small liquid nitrogen container. This will greatly reduces the expenses
of transport.
 Importing frozen embryos prevents the risk of introduction of new
diseases.
7) Induction of twinning. Embryo transfer has been routinely used in sheep and
goats to increase the number of offspring per delivery. In cattle, the genetic
selection for twinning has been largely unsuccessful. However, it has been
estimated that embryo transfer provides a real alternative in the production of
twins by:
a. Transferring of two embryos.
b. Transferring of one embryo to a previously inseminated recipient.
31
8) ET can be used to increase the population of rare or endangered breeds or
species.
9) Related to advanced techniques:
Embryo transfer technology is considered a prerequisite for the following
techniques:
i. Embryo splitting.
ii. Embryo sexing.
iii. In vitro fertilization (IVF).
iv. Intracytoplasmic sperm injection (ICSI).
v. Gamete intrafallopian tube transfer (GIFT).
vi. Nuclear transfer (Cloning).
vii. Genetic engineering.
The sequences of ET can be summarized as follows:
1. Donor selection.
2. Donor treatment (super-stimulation).
3. Donor breeding.
4. Embryo recovery from the donors.
5. Embryo handling and evaluation.
6. Recipient selection.
7. Recipient treatment (synchronization).
8. Transfer of embryos.
9. Embryo preservation.
Donor selection:Usually the owner selects his donor animals on the basis of performance and genetic
merit. The donor must be non-pregnant, either nulliparous or parous female. In
addition to genetic merit, the donor should be characterized by:
 A good body condition and preferably gaining weight.
 Free from diseases.
 Successfully completed the postpartum period.
 Regularly cycling.
 No history of reproductive problems.
 Absence of any genital abnormalities.
 Vaccination against the enzootic infectious diseases.
Donor super-stimulation (super-ovulation):
 Super stimulation is the treatment of a donor female with gonadotrophic
hormones to produce more ova than normal.
 During the last 20 years, hormonal regimens for super-ovulation in cattle have
been refined and combined with subsequent transfer of fresh or cryopreserved
embryos, a technique called multiple ovulation and embryo transfer (MOET).
 Based upon previous information, super-ovulation can be induced by injection of
extra-follicle stimulating hormone near the end of the luteal phase when
progesterone concentration is dropping naturally. For example, super-ovulation is
performed between day 16 to 19 of oestrous cycle for the cow and 14-17 for the
ewe. However, this regimen of super-ovulation faces two difficulties:
a) There is an individual variation in oestrous cycle length, so it is difficult to
optimize the timing of FSH treatment.
b) Scheduling ET procedures is frequently inconvenient with the natural
oestrous cycle.
 To overcome these problems, artificial shortening of oestrous cycle can be
induced by injecting PGF2α.
32

Around day 9-11 after oestrus/ ovulation, the donor receives one injection of
either eCG or FSH administered twice daily for 3-4 days. 24-48 hours after the
initial injection of treatment, oestrus is induced by injection of PGF2α and oestrus
will normally occur within 48 hours afterward. Insemination should be performed
twice, at 12 and 24 hours after onset of oestrus.
 Methods of super-stimulation:
1) FSH (short half-life, 6 hours) is usually administered twice daily for 4 – 5 days
(40 – 50 mg) in cattle.
2) eCG has a longer biological half – life (5 days) in the cow than either FSH or
hMG; consequently, a single dose of 2000 – 3000 IU will induce super
ovulation. The advantages of eCG: it is relatively cheap and administer only
once. The disadvantages:
 It is a variable and unpredictable FSH: LH ratio.
 The residual amount of eCG may a have a continuous superstimulatory effect after ovulation and thus cause development of
postovulatory-producing oestrogen. The prolonged and elevated
oestrogen may disrupt fertilization and early embryonic development.
3) Human menopausal gonadotrophin (hMG) is an FSH-like substance, but less
widely used than FSH and eCG in the veterinary practice.
4) Horse anterior pituitary extract (HAP) is usually administered as three equal
subcutaneous injections on consecutive days.
5) Gn-RH and hCG can improved the ovulation rate and reduce the percentage
of unovulated follicles when given at oestrus following FSH or eCG.
6) Recombinant bovine somatotropin (rbST) is injected as co-treatment with
FSH preparations and it enhances the superovulatory response and embryo
yield in cattle and buffalos.
 The same gonadotrophic preparations used for cow are adopted for the ewe
and doe goat. Gonadotrophin treatment is usually initiated mid- to late cycle,
eCG is given as a single I/M injection on day 12 or 13 of cycle. FSH is
administered in 2 to 6 injections, starting on day 12 of the cycle. PGF2α injected
24-72 hours from the start of the gonadotropin treatment induces oestrus within
24-36 hours. Insemination is commonly achieved by natural or artificially (using
intra-uterine insemination technique to increase the fertility rate).
If the ewes and does are not cycling, oestrus and ovulation can be induced by
intra-vaginal sponge containing progesterone for 12-14 days in sheep and 14-18
days in goats. FSH or eCG given 24-48 hours before or at the time of sponge
withdrawal. Oestrus occurs 24-36 hours after the sponge removal.
 The camel is an induced ovulatory, and hence CL is formed only during
pregnancy. Consequently, PGF2α can not be used during the super stimulatory
regimen. FSH or eCG is injected after a period of 7 days progesterone priming
(PRID or CIDR). A better stimulation of the ovaries using eCG or FSH occurs
when the ovaries have lower follicular activity. In order to achieve a good
ovulation rate, donors should be monitored by ultarasonography to detect
follicles of suitable size (1.3-1.8 cm in diameter) before breeding. This is
expected to occur 8-12 days post gonadotrophin treatment. Mating is allowed
twice 12 hours apart. An intravenous injection of Gn-RH analogue is
administered at the time of first mating.
 Generally, it is difficult to super ovulate mares, probably due to the anatomical
and histological features of equine's ovary, in addition to the marked resistance
of its ovary to exogenous gonadotrophic stimulation. eCG, equine pituitary
33
extract with or without hCG and porcine follicle stimulating hormone have all
been tried with limited success.
 Currently, the most effective protocol to induce multiple ovulations in the mare is
a regimen with twice daily injections of eFSH (3-5 days), initiated 5-8 days after
ovulation. After the 2nd day of treatment, PGF2α is administered to induce
luteolysis. After 36 hours, the mare is treated with hCG to induce ovulation.
 The major uses of ET in equine are:
i.
Production of foals from sub fertile mares.
ii.
Removal of the risks of gestation and parturition from older valuable brood
mares, which may no longer be able to give birth.
iii.
Production of foals from sport mares while they are in competion.
iv.
Production of foals from a very young donor mares (about 2 years old), which
have not yet reached their full growth and development.
v.
Production of foals from chronic disease carrier's valuable mares.
Donor breeding:
 In the presence of hormonally stimulated ovulations, fresh semen gives higher
fertilization rates than frozen semen.
 Following PGF2α injection or removal of progesterone releasing devices,
donors show oestrus within two to three days. Donors should be put in a group
of females not less than ten and carefully observed for oestrous behaviour, or
a vasectomised male is used.
 Ova are randomly shed from super ovulated ovaries over a period of 6 to 12
hours. For this reason, the donor must be inseminated at least twice; the first
breeding should be done 12 hours after the onset of standing heat, followed
by the second breeding in 12 hours interval. If the donor is still in standing heat
after the second insemination, she should be injected with GnRH, the bred 12
hours later.
Embryo recovery:
 Embryo recovery techniques include surgical and non surgical methods. The
non-surgical embryo recovery is now routinely used with great success
(particularly in large animals).
 The first step in non-surgical recovery is to palpate the ovaries per rectum to
estimate the number of corpora lutea. Ultrasonography provides more
accurate information about responses than palpation.
 Embryos descend into the uterus at day 4 to 5 and shed their zona pellucida
(hatch) at day 9 after the onset of oestrus. Consequently, non surgical embryo
recovery is performed between day 6 and 8 in cattle and horses.
Procedures in cows and buffaloes:
a) The donor is restrained and may need to be sedated with I/M injection of 1-2
ml xylazine before embryo recovery (buffaloes).
b) Faeces is carefully removed from the rectum to avoid aspiration of air.
c) Epidural analgesia is performed using 4 to 6 ml 2% lidocaine to prevent
defecation and straining.
d) The vulva is thoroughly washed and disinfected with 70% alcohol, and the tail
is tied out the way.
e) A 2(ebb & flow) - or 3-way (continous flow) round-tip embryo catheter (such as
Foley, Stewart and Neustadt) is used. A sterile stylet is inserted through the
full length of the catheter to render it sufficiently rigid for introduction into the
uterus under guidance per rectum.
34
f) The lips of the vagina are parted and the embryo catheter, with the stylet in
place, is inserted through the vagina and cervix. It is then manipulated into
appropriate uterine horn until the inflatable balloon is situated at the base of
the horn.
g) The balloon is slowly inflated with 15-25 ml of air or phosphate buffered saline
(PBS) in adult cows and 10-20 ml in heifers. Care should be taken to avoid
over distension of the balloon, since the endometrium is easily split, resulting
in heamorrhage and escape of the flushing solution into the mesometrium
from which it can be recovered.
h) After putting the catheter in position, the stylet is removed and the catheter is
connected via a Y junction by sterile tubing to a 1000 to 1500 ml bottle of
flushing solution. The remaining arm of the Y junction is connected to a free
piece of tubing (close-circuit method). The flow medium in both pieces of
tubing is controlled by quick release clamps. While the outlet tubing is
occluded, the flushing solution enters the uterus by gravity flow with bottle
suspended one meter above the level of the uterus.
i) The horn of the uterus is extended by elevating its tip anteriorly. When the
inflow stops, the inlet tubing is clamped off and the clamp on the outlet tubing
is released. The fluid is channeled directly through an embryo filter.
j) The filter should never be allowed to run completely dry leaving the embryos
on the filter dish exposed to the air.
k) The procedure is repeated for the opposite horn, using a separate sterile
catheter. The balloon is then deflated and the catheter is withdrawn from the
vagina. The remaining fluid in the catheter and tubing must be directed to the
filter.
l) Some operator prefer placement of the catheter with balloon in the cervix and
the tip in the body of the uterus, which enables them to flush the both horns
simultaneously.
m) During the final collection of the flushing solution, 50 IU oxytocin might be
given I/V to aid in the recovery of the last residual portion of the solution from
the uterus.
Procedures in camels:
o In the camel and mare, easily dilated cervix and straight uterine horns make
the technique of embryo recovery relatively easier than in the cow and
buffaloes.
o The donor camel can either be placed in a stock or restrained sitting on the
ground.
o Faeces are removed and the tail wrapped before the thorough cleaning of the
perineal region.
o By a sterile gloved hand the catheter is guided through the vagina, the cervix
is then dilated manually and the catheter inserted.
o Once the catheter is through the cervix the cuff is inflated with 30-40 ml of air
and pulled back against the internal os of the cervix to seal it.
o The uterus is then flushed either by the close-circuit method with 60-120 ml of
flushing medium using a total volume of approximately 500ml or open-circuit
method.
o After each flush, the medium is collected into sterile beakers and filtered
through an embryo filter until only 20-30 of medium remains where it will be
subjected for microscopical examination for embryo searching.
35
Procedures in mares:
 An extended 2-way embryo catheter (French size 30) is used with simple in/
out irrigation.
 The donor mare is confined in stocks. Faeces is removed and the tail wrapped
before the thorough cleaning of the perineal region.
 The sterile catheter with a stylet in place is introduced per vaginam and
passed through the cervix until it is approximately 5cm into the uterine body.
Its position is verified per rectum and the balloon is inflated with 75 ml air after
which it is drawn back against the internal cervical os to form a seal preventing
the escape of the flushing medium from the uterus.
 One litre of the flushing solution is infused into the uterus by gravity flow. Both
uterine horns and uterine body are flushed simultaneously.
 It has been reported that an increased recovery rate can be achieved by
allowing the medium to remain in contact with the uterus for 3 minutes during
the flushing attempts. It may also be increased by manipulation of the uterus
per rectum during the flushing procedure.
 The return fluid is collected in a pre-warmed sterile, one litre graduated
cylinder or embryo filter placed well bellow the level of the uterus. The
procedure is repeated twice for a total of 3 litres per collection attempt.
 Mares return to oestrus 3-5 days after the flushing procedure in response to
the endometrial stimulation.
Embryo recovery from small animals:
Surgical method (under general anaesethia) of embryo recovery is still the most
common technique used in sheep, goats and pigs. However, two promising
techniques are now developing; the laparoscopical and the transcervical.
Surgical method:
 There are two methods of surgical recovery; the uterine and the oviductal
flush.
 When flushing the oviduct, the medium is passed from the uterine lumen
through the uterotubal junction and along the oviduct before it is collected into
a dish through a cannula.
 The oviductal approach is appropriate for all stages of embryo development
from day 3 to day 7 after oestrus.
 When flushing the uterus only, a medium is introduced into the uterine lumen
near the uterotubal junction and recovered through a catheter inserted into
the uterus near the external bifurcation.
 The uterine procedure is successful only with embryos recovered on day 4 or
later, by which time the embryos have passed into the uterus. Flushing the
uterus is more common and described as follow:
1) The donor is placed on its back on the operating table and its limbs are tied to
the table. The table is constructed to be inclined at an angle of about 30°, so
that the hind quarter is upwards and the head is downwards.
2) The abdominal area in front of the mammary gland is clipped, shaved,
washed, and disinfected in preparation for surgery.
3) The mammary vein is held to one side and the abdomen is opened by a midline incision some 12 cm long. The incision begins as close to the mammary
gland as possible to facilitate the withdrawal of uterus.
36
4) The forefingers of the right hand are inserted into the abdominal cavity and
directed downwards and backwards to locate the uterus which recognized by
its firm horns and its pink colour. The uterus is then withdrawn through the
incision. The number of corpora lutea and follicles in each ovary are counted.
5) A blunt perforation is made by forcing the tip of artery forceps through the
uterine wall just above the external bifurcation. An 8 FG Foley catheter is
inserted into the uterine lumen through the blunt perforation for a distance of
3-5 cm towards the oviduct before inflating the balloon with air.
6) The flushing medium is introduced into the uterine lumen to rinse the uterine
horns, through a blunt needle inserted through the uterotubal junction and
connected with a 50 ml syringe.
7) Each uterine horn is rinsed with 40 ml of the medium and collected again
through the embryo catheter into a searching grid dish. Efficient recovery of
the embryos is assumed to depend upon a free flow of medium which is able
to rinse the uterine lumen thoroughly and this is achieved by a gentle massage
of the uterine horn.
8) The hole made to allow access for the Foley catheter is closed with a catgut
suture.
 The surgical method has the advantage of direct manipulation of the genital
tract, accurate evaluation of the ovarian response and higher embryo recovery
rate. However, the main problem of using this method is the damage caused
to genetically valuable ewes due to adhesions, which limits the reuse of the
donor ewes. To minimize these adhesions may use dextran and heparinised
saline into the peritoneal cavity following the process of embryo recovery.
Identification of the embryos:
Embryos can be identified in the uterine flush by the following features:
A. Spherical in general appearance.
B. Presence of zona pellucida which is gelatin-like shell surrounding the
embryonic cells. It is spherical and translucent.
C. The diameter of the bovine embryo including zona pellucida is constant (150180µm) until day 7.5-8.0, when expansion of the embryonic mass begin.
D. The 7-day embryo is darker than other uterine debris.
E. Knowing the age of the embryos is an important for their identification in the
searching dish. The 8-day embryo (fully expanded blastocyst) possesses a
thinner zona pellucida and pale. A spontaneously hatched embryo (>8 days)
is difficult to identify, because the embryonic cells are morphologically similar
to other uterine debris.
Development of the early embryos (cleavage):
It is development of the embryo from day one of fertilization up to day 8. Embryos
recovered 6 to 8 days post fertilization are classified into:
1. Morula: embryonic mass contains 32-64 cells. Blastomeres are round in shape
and are not tightly connected to each other.
2. Compacted Morula: the shape of a tight morula is similar to golf ball. Individual
blastomeres are no longer distinguishable.
3. Early blastocyst: general morphological appearance of an early to mid-stage
blastocyst is comparable to a ring (trophoblastic cell layer) with jewel (inner
cell mass) suspended inside.
4. Blastocyst: two groups of cells are present and clearly recognized. The
perivitelline space is still visible and the blastocoele cavity constitutes more
than 70% of the volume of the embryo.
37
5. Expanded to extended blastocyst: there is no perivitelline space between the
layer of trophoblastic cells and the inside of the zona. The zona pellucida
becomes thinner as the blastocyst expands.
6. Hatched blastocyst: when a blastocyst expands sufficiently, the embryo finally
escapes from the zona. This usually occurs after day 8.
Embryo handling:
 Following embryo recovery, the embryo filter or the graduated cylinder
containing embryos is transferred to the laboratory for embryo searching.
 If the flushing solution is collected in a graduated cylinder, it should be left for
about 20 minutes to allow the embryos to settle. Then the supernatant layer is
discarded, keeping the last 80-100 ml of the flushing solution, which is then
transferred to searching dishes.
 When embryo filter is used, it must be thoroughly rinsed using PBS plus 1%
heat inactivated bovine serum in a 30 ml syringe with a 21-guage needle. The
filter should be rinsed several times in searching dishes until no mucus or
tissue debris is left on the filter.
 Dishes are searched under stereomicroscope, when embryos are identified,
they are picked up by a micropipette attached to an insulin syringe and
transferred into small Petri dishes containing sterile holding media.
 Embryos are tentatively classified as good or bad, which is recorded on the
cover of the holding dish. This allows quick estimate of the total number of
embryos found.
 Embryos are then serially rinsed through at least 3 different dishes containing
fresh sterile media, using a new sterile pipette for each step. Embryos for
export must be rinsed through 10 different dishes containing sterile media
under some circumstances.
 Finally, embryos are placed into the last dish awaiting transfer or
cryopreservation.
Embryo evaluation:
The following methods are useful to evaluate the viability of embryos:
1- Morphological appearance:
Embryos are usually classified into five different groups, based on gross
morphological appearance:
 Excellent embryos (quality 1; score 1.0-1.9) have no visible imperfections.
 Good embryos (quality 2; score 2.0-2.9) have few recognizable imperfections,
such as poor compaction, variance of cell size or few extruded cells.
 Fair embryos (quality 3; score 3.0-3.9) show more disarrangement, such as a
small embryonic mass, an irregular shape and large numbers of extruded
cells.
 Poor embryos (quality 4; score 4.0-4.9) show signs of cellular degeneration,
very small embryonic mass and a lot of extruded cells or disintegrated
cytoplasm.
 Very poor embryos (quality 5; score 5.0-5.9) contain either absolutely dead
cells or only few live cells or a very tiny cells mass, which is extremely
disorganized in appearance.
The unfertilized oocytes (UFOs) may assume different shapes and sometimes
appear degenerate, or may contain degenerated, unevenly distributed
ooplasm.
38
2- In vitro culture (IVC):
If embryos are of doubtful quality, observing further development in vitro is helpful.
3- Staining method:
Identification of both live and dead cells under the fluorescent microscope can be
made through incubation of embryos with fluorescent dyes, such as fluorescein
diacetate (FDA) for staining live cells or diamidine-2-phenylindole (DAPI).
4- Measurement of metabolic activity:
The rate of glucose incorporation into the embryo during culture can be examined.
5- Transferring into animals:
Pregnancy rate after transfer can be a tool of evaluation. However, many factors
such as the transfer method and synchrony affect the pregnancy rate.
Recipient selection:
It has been common in embryo transfer programmes to overlook the quality of the
recipients. Good recipient should be:
 Large-framed.
 Healthy and has no reproductive problems.
 Mature heifers or young adults.
 A minimum of two normal cycles should have been recorded.
 Should not be fat and should preferably gain weight.
 Should be vaccinated against enzootic diseases.
Recipients can be supplied (sources) by three ways:
a. From cycling females of the herd, since about 5% of the herds are in heat on
any given day.
b. Large commercial dairy farms sometimes rent recipients from other farms.
c. Synchronization of small number of females.
Synchronization of oestrus in the recipients:
 It has been proved that pregnancy rates higher when the oestrous cycle of the
donor and recipient are synchronized within 24 hours.
 Several treatments are used for synchronisation of the oestrous cycle which
differs according to the species.
 In the cows, prostaglandin F2α or its analogue is most common method for
oestrous synchronisation. They can be used as follow:
a. Animals with a palpable CL are injected with 25 mg dinoprost
tromethamine. Treated cows are expected to come oestrus in 2-4 days.
b. All recipients can be injected with PG regardless of the presence or
absence of a CL. A second injection is given 11 days later and oestrus
peaks on the 3rd day after the second injection.
 It is reasonable to prepare 8 recipients cows for each donor.
 In the ewes and does, injecting prostaglandin F2α or its analogue one day
before the donor.
 If the animals are non-cycling, progesterone treatment can be adopted.
Sponge withdrawal should be 24 hours earlier in recipients than in donors.
 In the camels, recipients should be less than 12 years of age and have had at
least one normal pregnancy with a normal delivery (4 recipients for each
donor).
 Results in the dromedary suggest that the best recipients should have been
ovulated 24-48 hours after donor.
 A group of cycling recipients is examined 24 hours after the donor bred and
all females that have mature follicle are treated with GnRH or hCG.
39
 The recipients are daily treated with progesterone (100 mg/ day) for 10 to 15
days and on the last day of treatment, 1500-2500 IU eCG is injected to induce
follicular development. Progesterone treatment is scheduled to end on the day
of injection of gonadotrophin in the donor.
 In the mares, synchronisation of oestrus in cycling animals can be achieved by
a double prostaglandin treatment. Mares with a 4 to13-day old corpus luteum
will come into oestrus on the 3rd day after treatment and given 2500 IU hCG
I/V on the 6th day.
Transfer of embryos:
 Embryos can be transferred either surgically or non-surgically. Since the
surgical method has no longer been used in the large animals.
 The non-surgical transfer of embryos is performed through the cervix similar to
artificial insemination.
 The embryos can be transferred either directly after collection or be
crypreserved and transfer later.
Transfer of the cow and buffalo embryos:
1) Faeces is evacuated from the rectum and the side of the CL is determined.
2) Epidural analgesia is induced to prevent defecation and to minimize straining.
The perineal region is thoroughly washed and disinfected with 70% alcohol.
3) Prior to transfer, the embryos are washed 10 times and each embryo is then
aspirated into 0.25 ml French straw between two air pockets and two columns
of culture medium (according to the manual of the IETS). The straw inserted
into the AI gun and shortened to fit even with the end of the gun. A sterile
sheath is fitted over the AI gun and fixed in place. A second, sterile large
sheath, which is closed at the distal end is fitted over the first to serve as a
cannula and permits passage of the gun through the vagina without coming in
contact with the vaginal flora.
4) The tip of the AI gun, with the double sheath, is placed into the external os of
the cervix and then pushed through the sheath before being guided as gently
as possible through the remainder of the cervical canal and on into the uterine
horn of the side of the CL.
5) The embryo is deposited approximately one-third of the way up the uterine
horn and the gun is withdrawal slowly.
6) It should be noted that the introduction of the catheter through the cervix is
guided and facilitated by the other hand via the rectum.
Transfer of the camel embryos:
a. The embryo is loaded into a 0.25 or 0.5 ml sterile plastic straw and placed in
the gun for transfer (covered by two sheaths).
b. The recipient is prepared in the same manner described for embryo collection
from the donor. The inseminating gun is introduced into the vagina and guided
towards the cervix using a sterile gloved hand.
c. The sanitary sheath is perforated after passage of the first cervical ring, by
pulling the plastic sheath backwards towards the operator, and the gun further
guided into one of the uterine horns with a hand in the rectum.
d. The plunger of the transfer pipette is pushed home and the embryo deposited
into the uterus.
40
Transfer of the mare embryos:
i. The recipient mare is prepared in the same manner described for embryo
collection from the donor mare.
ii. The embryo is aspirated into a sterile 55-cm insemination pipette in a 5-cm
column of medium. The pipette is protected by double sterilized sleeves.
iii. The lips of the vulva are parted and the hand is inserted into the vagina until
the index finger has entered the cervix.
iv. The pipette is pushed through the outer plastic sleeve and inserted 5 cm into
the body of the uterus. Then the embryo is slowly discharged into the uterus.
v. Upon the withdrawal of the pipette and the hand from the vagina, the lips of
the vulva are held closed to prevent the aspiration of the air into the vagina.
Transfer of the embryos in small animals:
 Surgical transfer under general anaesthesia via a mid-ventral approach is
performed. The uterus is exteriorized from the abdominal wound, and then a
blunted 18 FG needle is passed into the uterine lumen, 10-20 cm from the
uterotubal junction.
 The embryo is loaded in a larger Pasteur pipette fitted to a syringe. The
blunted needle is replaced by the pipette, passed into the lumen for a distance
of 5-10 cm and directed towards the uterotubal junction, where the fluid is
expelled.
Donor and recipient after care:
 The donor is treated immediately with prostaglandin after collection of
embryos. This treatment, not only rapidly reduces the greatly enlarged ovaries,
but also terminates any unwanted pregnancies, should an occasional embryo
not have been flushed out.
 The donor should not bred on the induced heat. The induced heat will have a
useful effect on uterine health, should any contamination have been
introduced at the time of the collection.
 Pregnant recipients should basically be managed like other pregnant females.
 Pregnancy of the recipient should be checked 4-6 weeks post transfer.
Embryo preservation:
1- Short term preservation:
 Embryos can be stored at room temperature for not more than 12 hours
before transfer to the recipients.
 Bovine embryos can survive for 2 to 3 days at 4-5 ° C. Basically; embryos in
culture medium placed in a closed tube are transferred to the refrigerator in a
water bath for gradual cooling (embryos can survive up to 48 hours).
2- Long term preservation:
Embryos can survive for many years when stored frozen in liquid nitrogen at -196 °
C. If embryos are frozen without precautions, the water turns into ice and expands,
resulting in ruptured cell membranes and death of embryo. For successful freezing,
the following steps must be undertaken (slow rate freezing)
 Excellent to good graded embryos must be washed through at least 3 sterile
drops of medium with a new sterile pipette each time to prevent transmission
of diseases.
 The medium used for freezing is a holding medium (PBS+20% fetal calf serum
(FCS) and antibiotic).
41
 Addition of cryoprotectans such as glycerol 10% or ethylene glycol for
protecting embryos from the damage caused by very salty solution produced
by ice formation during the cooling process.
 Slow cooling to remove most of the water content of the embryo before
freezing. Embryos, in straws, are cooled slowly in a rate of 0.5 ° C/ minute
from – 6 ° C for 5 minutes (seeding) to around -35 ° C and then plunged into
liquid nitrogen.
 Vitrification is alternative method for cryopreservation requires addition of high
concentrations of CPA and ultra-rapid freezing.
Thawing procedures:
 The thawing process must be performed very rapidly to avoid the formation of
the large ice crystals inside the embryo.
 Embryos are thawed by subjecting straws in air for 12 seconds followed by 12
seconds in a water bath at 25-30 ° C.
 Glycerol is removed from the thawed embryos by one of the following
dilutions:
A. Six decreasing dilutions of glycerol and medium (8.3, 6.7, 5.0, 3.3, 1.7 and
0 percent glycerol).
B. Three decreasing dilutions of glycerol and medium (6, 3 and 0 percent
glycerol) but about 10% of sucrose is included in each dilution.
C. No glycerol is included but 10 to 30 % solution of sucrose.
 These dilutions are kept in culture dishes or plates. The embryo is removed
from the straw after thawing where it is left for 5 minutes in each dilution to
remove glycerol.
 The final dish is used as a container to hold the embryo until transfer, which
should be done within 5 minutes.
42
Animal cloning
Cloning describes the processes used to create an exact genetic replica of
another cell, tissue or organism. The copied material, which has the same genetic
makeup as the original, is referred to as a clone (A group of genes, cells or
organisms derived from a common ancestor. Each clone is genetically identical).
There are three different types of cloning:
1- Gene cloning, (recombinant DNA technology, DNA cloning and molecular
cloning), which creates copies of genes or segments of DNA.
2- Reproductive cloning, which creates copies of whole animals (Scottish sheep
named Dolly).
3- Therapeutic cloning, which creates embryonic stem cells (undifferentiated cells
in a tissue. These cells can grow into any of the types of specialized cells in that
tissue). Researchers hope to use these cells to grow healthy tissue to replace
injured or diseased tissues in the human body.
1- Recombinant DNA technology or DNA cloning:
The transfer of a DNA fragment of interest from one organism to a self-replicating
genetic element such as a bacterial plasmid. The DNA of interest can then be
propagated in a foreign host cell.
Scientists studying a particular gene often use bacterial plasmids to generate multiple
copies of the same gene. Plasmids are self-replicating extra-chromosomal circular
DNA molecules, distinct from the normal bacterial genome.
To "clone a gene," a DNA fragment containing the gene of interest is isolated from
chromosomal DNA using restriction enzymes and then united with a plasmid that has
been cut with the same restriction enzymes. When the fragment of chromosomal
DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA
molecule." Following introduction into suitable host cells, the recombinant DNA can
then be reproduced along with the host cell DNA.
Besides bacterial plasmids, some other cloning vectors include viruses, bacteria
artificial chromosomes (BACs), and yeast artificial chromosomes (YACs).
2- Reproductive Cloning:
Reproductive cloning is a technology used to generate an animal that has the same
nuclear DNA as another currently or previously existing animal. Dolly was created (at
the Roslin Institute in Edinburgh, Scotland, in July 1996. Dolly was a mother to six
lambs and was died in February 2003) by reproductive cloning technology. In a
process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic
material from the nucleus of a donor adult cell to an egg (IVM oocyte) whose nucleus,
and thus its genetic material, has been removed (enucleation). Enucleated oocyte is
called cytoplast and karyoplast meaning somatic cell derived from either early stage
embryos, cultured somatic cells from a fetus or udder.
The reconstructed egg containing the DNA from a donor cell must be treated with
chemicals (Ca ionophore or dimethyl aminopurine) or electric current in order to
stimulate cell division. Once the cloned embryo reaches a suitable stage, it is
transferred to the uterus of a female host where it continues to develop until birth.
Dolly or any other animal created using nuclear transfer technology is not truly an
identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is
the same as the donor. Dolly's success is truly remarkable because it proved that the
genetic material from a specialized adult cell, such as an udder cell programmed to
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express only those genes needed by udder cells, could be reprogrammed to
generate an entire new organism.
3- Therapeutic Cloning:
Therapeutic cloning, also called "embryo cloning". It involves replacing the nucleus of
an egg cell with the nucleus from a cell from a patient's body and allowing it to
develop to form a blastocyst. The embryonic stem cells from the inner cell mass are
then harvested and used to establish a cell line that has the same genetic makeup of
the patient. These cells can then be directed to develop into the tissue needed for
transplant.
Many researchers hope that one day stem cells can be used to serve as replacement
cells to treat heart disease, Alzheimer's, cancer, and other diseases.
Uses of cloning technologies:
 Recombinant DNA technology is important for learning about other related
technologies, such as gene therapy, genetic engineering of organisms, and
sequencing genomes.
 Gene therapy can be used to treat certain genetic conditions by introducing
virus vectors that carry corrected copies of faulty genes into the cells of a host
organism.
 Reproductive cloning can be used to develop efficient ways to reliably
reproduce animals with special qualities.
 Reproductive cloning also could be used to repopulate endangered animals or
animals that are difficult to breed. In 2001, the first clone of an endangered
wild animal was born, a wild ox called a gaur. Other endangered species that
are potential candidates for cloning include the African bongo antelope, the
Sumatran tiger, and the giant panda.
 Therapeutic cloning technology may some day be used in humans to produce
whole organs (organ transplants) from single cells or to produce healthy cells
that can replace damaged cells in degenerative diseases such as Alzheimer's
or Parkinson's.
 Cloning could provide a means of cultivating plants that are stronger and more
resistant to diseases, while producing more. The same could happen to
livestock as well where diseases such as foot and mouth disease could be
eradicated.
The risks of cloning:
 High failure rate: cloning animals through somatic cell nuclear transfer
is
simply inefficient. The success rate ranges from 0.1 percent to 3 percent, which
means that for every 1000 tries, only one to 30 clones are made. Here are some
reasons:
a) The enucleated egg and the transferred nucleus may not be compatible.
b) An egg with a newly transferred nucleus may not begin to divide or develop
properly.
c) Implantation of the embryo into the surrogate mother might fail.
d) The pregnancy itself might fail.
 Problems during later development: cloned animals that do survive tend to be
much bigger at birth than their natural counterparts. Scientists call this "Large
Offspring Syndrome" (LOS). Clones with LOS have abnormally large organs.
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This can lead to breathing and blood flow problems. Some clones without LOS
have developed kidney or brain malformations and impaired immune systems,
which can cause problems later in life.
 Abnormal gene expression patterns: in a naturally-created embryo, the DNA is
programmed to express a certain set of genes. Later on, as the embryonic
cells begin to differentiate, the program changes. In cloning, the transferred
nucleus doesn't have the same program as a natural embryo. Complete
reprogramming the nucleus is needed for normal or near-normal development.
Incomplete programming will cause the embryo to develop abnormally or fail.
 Telomeric differences: as cells divide, their chromosomes get shorter. This is
because the DNA sequences at both ends of a chromosome, called
telomeres, shrink in length every time the DNA is copied. On the other hand,
Dolly the sheep's chromosomes had shorter telomere lengths than normal.
This means that Dolly's cells were aging faster than the cells from a normal
sheep.
What animals have been cloned?
 Scientists have been cloning animals for many years. In 1952, the first animal,
a tadpole, was cloned.
 Dolly is the first mammal to have been successfully cloned from an adult cell
(udder cells).
 Since Dolly, researchers have cloned a number of large and small animals
including sheep, goats, cows, mares, mice, pigs, cats, rabbits,dogs, and a
gaur. All these clones were created using nuclear transfer technology (donor
cells obtained from oviductal epithelial cell, fetal fibroblasts, granulose cells
and skin fibroblasts).
 Hundreds of cloned animals exist today, but the number of different species is
limited. Attempts at cloning certain species have been unsuccessful.
 Ferrets are animals to be cloned in 2006. The researchers say that the
domestic ferret is an ideal animal model to study human diseases such as
influenza and cystic fibrosis.
 Buffalo calf is the most recent animal to be cloned, through the new and
advanced ‘Hand-guided Cloning Technique’ (hand-made cloning HMC) was
born at National Dairy Research Institute (NDRI), Karnal (India) on August 22,
2010. In this case the used donor cell was embryonic stem cell. However, in
earlier cloning, the donor cell was from somatic cells. The donor embryonic
stem cell was isolated from the 8 day old blastocyst.
 The hand-guided cloning technique developed at NDRI, is an advanced
modification of the “Conventional Cloning Technique CCT”. In this technique,
immature oocytes were isolated from ovaries and were matured in vitro (MII
plate and first polar body to avoid ploidy abnormalities and arrest
development). These were then denuded and treated with an enzyme to
digest the outer layer of oocytes called ‘zona pellucida’. The oocytes were
then treated with chemicals to push their genetic material to one side of the
oocyte. This protruded side was then cut off with the help of “hand held fine
blade” for removing the original genetic material of the oocyte. The enucleated
oocyte was then electrofused with single cell taken from colony of embryonic
stem cells. The resulting embryos were cultured (SOFaaci medium + 5% cattle
serum) and grown in the laboratory for seven days to develop them to the
stage of blastocyst. The blastocysts were transferred to recipient buffaloes.
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Ethics of Animal Cloning:
While most scientists consider the process of animal cloning as a major break
through and see many beneficial possibilities in it, many people are uncomfortable
with the idea, considering it to be 'against nature' and ethically damning, particularly
in the instance of cloning human beings.
In recent times, there have been spurts of new laws banning or regulating
cloning around the world. In some countries, animal cloning is allowed, but not
human cloning. Some advocacy groups are seeking to ban therapeutic cloning, even
if this could potentially save people from many debilitating illnesses.
Artificial Embryo Twinning (micromanipulation and bisection):
 Artificial embryo twinning is the relatively low-tech version of cloning. As the
name suggests, this technology mimics the natural process of creating
identical twins.
 In nature, twins occur just after fertilization of an egg cell by a sperm cell. In
rare cases, when the resulting fertilized egg, called a zygote, tries to divide
into a two-celled embryo, the two cells separate. Each cell continues dividing
on its own, ultimately developing into a separate individual within the mother.
Since the two cells came from the same zygote, the resulting individuals are
genetically identical.
 Artificial embryo twinning uses the same approach, but it occurs in a Petri dish
instead of in the mother's body. This is accomplished by manually separating
a very early embryo into individual cells (twin, triplet or quadruplet), and then
allowing each cell to divide and develop on its own. The resulting embryos are
placed into a surrogate mother, where they are carried to term and delivered.
Again, since all the embryos came from the same zygote, they are genetically
identical.
Somatic Cell Nuclear Transfer (SCNT):
 A method of cloning a living organism. The process involves removing the
nucleus of an egg cell and replacing it with a nucleus from any cell of the
organism being cloned.
 Somatic cell nuclear transfer, (SCNT) uses a different approach than artificial
embryo twinning, but it produces the same result: an exact clone, or genetic
copy, of an individual.
 To make Dolly, researchers isolated a somatic cell from an adult female
sheep. Next, they transferred the nucleus from that cell to an egg cell from
which the nucleus had been removed. After a couple of chemical tweaks, the
egg cell, with its new nucleus, was behaving just like a freshly fertilized zygote.
It developed into an embryo, which was implanted into a surrogate mother and
carried to term.
 The fertilization of an egg by a sperm and the SCNT cloning method both
result in the same thing: a dividing ball of cells, called an embryo.
 An embryo is composed of cells that contain two complete sets of
chromosomes.
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 The difference between fertilization and SCNT lies in where those two sets
originated.
 In fertilization, the sperm and egg both contain one set of chromosomes.
When the sperm and egg join, the resulting zygote ends up with two sets - one
from the father (sperm) and one from the mother (egg).
 In SCNT, the egg cell's single set of chromosomes is removed. It is replaced
by the nucleus from a somatic cell, which already contains two complete sets
of chromosomes. Therefore, in the resulting embryo, both sets of
chromosomes come from the somatic cell.
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