Besnoitia besnoiti impact on fertility of cattle exploited in Mediterranean pastures
(Alentejo).
H. Cortes1, Chagas e Silva, J.2, Baptista, M.C. 3, Pereira, R.M. 3, Horta, A.E.M.3,
Vasques, M.I. 3, Barbas, J.P. 3, Marques, C.C3, Leitão, A.4,
1
Laboratório de Parasitologia. Núcleo da Mitra. Universidade de Évora (ICAM),
Portugal.
2
Divisão de Selecção e Reprodução Animal, DGV, Venda Nova, Amadora, Portugal
3
Departamento de Reprodução Animal, Estação Zootécnica Nacional, Santarém,
Portugal
4
Instituto de Investigação Científica Tropical (CIISA), Lisboa, Portugal.
Abstract
Besnoitia besnoiti is a bovine parasite endemic in many tropical and subtropical areas
whose prevalence in the Mediterranean countries such as Portugal seems to be
increasing. Most infections are mild or subclinical, characterized by the formation of
large numbers of cutaneous and sub-cutaneous cysts, lowering the quality of skins for
leather industry. Male sterility or impaired fertility is a common sequela in breeding
bulls, and is one of the most negative aspects of the disease in animals that survive the
acute and chronic stages of infection. Our objective was to investigate if asymptomatic
besnoitiosis leads to bovine infertility, by comparing seminal parameters pre and postthawing and in vitro fertilization and embryo development rates between asymptomatic
infected (n=3) and uninfected (n=5) bulls, under an extensive production system in
Alentejo-Portugal. Skin biopsies were collected and submitted to histopathological
analyses to identify the presence of B. besnoiti cysts in sires. Semen was collected by
electroejaculation and data of sperm quality parameters before and after
cryopreservation were analyzed using ANOVA. The quality of semen collected from
asymptomatic infected and uninfected bulls presented no differences before
cryopreservation. From all the sperm post-thawed quality parameters evaluated
(motility and hypoosmotic swelling tests; motility, activity, concentration and
agglutination post-swim-up; fertilization and embryo development rates) only postthawed (51.0±36.3 vs 42.3±10.6%, P=0.05) and post-swim-up (36.3±18.8 vs 25.1±12.0,
P=0,009) motility were significantly different between asymptomatic infected and
uninfected bulls, respectively.
Semen from asymptomatic Besnoitia infected bull may maintain fertilization ability
although the presence of these animals in herds implies a potential transmission of the
disease leading to further economic losses.
Keywords: Besnoitia besnoiti, Bovine besnoitiosis, infertility, Portugal.
Introduction
Besnoitia besnoiti (Marotel 1912) is a coccidian parasite of cattle that is closely related
to Toxoplasma gondii. B. besnoiti is endemic in many tropical and subtropical areas
where it causes both acute and chronic bovine besnoitiosis, leading to significant
economic constraint to commercial cattle production (Bigalke & Schoeman, 1967,
Shkap et al., 1994). In Europe, it has been reported only in the Mediterranean countries,
France (Besnoit & Robin, 1912, Bourdeau et al., 2004) Spain (Juste et al., 1990,
Irigoien et al., 2000), and Portugal. In the latter country, prior to 1991 (Malta & Silva,
1991), the disease was seldom recognized or reported (Franco & Borges, 1915, Leitão,
1949). At present its prevalence may be increasing (Cortes et al., 2003, Cortes et al.,
2005).
All breeds, both sexes, and animals of all ages are affected by this parasitosis, although
clinical disease occurs rarely in calves less than 6 months of age (Bigalke, 1968). The
occurrence is usually sporadic, and only a small proportion of infected animals develop
clinical disease (Pols, 1960, Bigalke, 1968). In the acute stage of disease, parasitic
tachyzoites invade blood vessels of the skin, subcutaneous tissues, fascia and testes
causing widespread vasculitis and thrombosis. The result is a severe generalized
reaction which is accompanied by oedema of the skin and acute orchitis. During the
chronic stage, large numbers of tissue microcysts containing bradyzoites are formed in
the skin (fig. 1). The most striking features of this stage are thickening, wrinkling and
hair loss of the skin (fig. 2), accompanied by anorexia and severe weight loss. The case
fatality rate is approximately 10% (McCully et al., 1966, Basson et al., 1970).
Irreversible male infertility or impaired fertility is a common sequela in breeding bulls,
and is one of the most negative aspects of the disease in animals that survive the acute
and chronic stages of infection (Basson et al., 1970, Kumi-Diaka et al., 1981, Ferreira et
al., 1982, Cortes, et al., 2005).
The main objective of this experimental work was to compare fertility parameters
between uninfected and infected bulls where at histopathology, cysts where identified in
the skin, without showing any other clinical signs of disease and, within predict some
implications of their presence on farm reproduction.
Material and Methods
Animals.
Farms with historical cases of bovine besnoitiosis were chosen for this study. All the
farms were located in Évora region (Portugal) and exploit beef cattle extensively. Eight
animals, seven Limousine and one Charolais bull, aged 2-9 years, were selected in 3
different farms from this region.
Skin samples were collected from the neck of several bulls and fixed in 10% formalin
solution, to identify the presence of Besnoitia cyst in it. Skin samples were stained with
Hematoxilin/Eosin and the presence of cysts was searched with 100X amplification in a
light microscope. The target was finding males without any sign of disease, but
histopathology revealing the presence of Besnoitia cysts. This was achieved in three
bulls.
Semen collection and cryopreservation.
Semen was collected by electroejaculation to asymptomatic infected (n=3) and
uninfected bulls (n=5) and immediately stored in a 32ºC water bath. Before
cryopreservation, semen morphology was evaluated by determination of gross (1 to 5)
and individual progressive (0 – 100%) motility, percentage of abnormal and of live
(supravital stain eosin–nigrosin) spermatozoa (spz). After sperm concentration
determination using a single Neubauer hemacytometer, dilutions were performed using
Triladyl (ref. 13500/0250, Minitub GmbH) with egg yolk and distilled water,
previously warmed at 32ºC, to obtain progressively motile 20x106 spermatozoa per
seminal dose. Extended semen was immediately loaded into 0.25 ml straws. All the
straws were vertically stored in appropriate device and maintained at 5ºC for 4 hours
and then frozen by vertical freezing method (Jondet, 1972 modified by Chagas e Silva,
1992). The rackets with straws were kept inside the RCB4 Container (Air Liquide,
France) in the nitrogen vapours (on the position that, over a 4 cm of liquid nitrogen, the
devise touches the liquid phase) for 20 minutes with the lid closed, before being placed
into appropriate cryogenic containers and immersed into liquid nitrogen (-196ºC) for
storage.
Post-thawed semen evaluation
Post-thawed semen samples from all ejaculates were subjected to evaluation of motility,
hypoosmotic swelling, post-swim-up tests, in vitro fertilization, and embryo
development. The effect of B. Besnoiti on these parameters was investigated according
to the following experimental design: In experiment 1 (6 replicates), semen from
uninfected bulls (n=5) was tested. In experiment 2 (5 replicates) semen was used from
two uninfected bulls, with the worst and the best fertilizing ability selected in
experiment 1, and from three asymptomatic infected bulls. For the hypoosmotic
swelling test only two straws from each infected and uninfected animals were evaluated.
Post-thawed motility and Host
Individually progressive semen motility after thawing from infected or uninfected sires
was evaluated. To perform the host test two straws from each bull were thawed. The
hypoosmotic solution consisted of tri-sodium citrate (10.4g L-1) with100 mOsm L-1 of
final osmolarity. Aliquots (100 µl) of semen were incubated with 1 ml of the
hypoosmotic solution for 5, 25 and 40 minutes. After each incubation period, a droplet
of this mixture was pippeted on to a slide and fixed with 2% glutaraldeyde in BL-1
medium (Pursel et al., 1972) and 100 spermatozoa were examined in each preparation.
Post-swim-up tests
To perform the post-swim-up tests semen was thawed and incubated for 1 hour in
TALP medium without calcium or antibiotics supplemented with 48,55 l mL-1 caffeine
(Sigma, ref. C-0750) in an humidified atmosphere of 5% CO2 at 39ºC. Spermatozoa
from the upper layer were taken to evaluate their individual motility (0 – 100%), vitality
(1 to 5), concentration and agglutination (number of agglutinated heads in 100 observed
spermatozoa) post-swim-up.
In vitro fertilization and embryo development
In vitro maturation, fertilization and co-culture procedures have been described
previously (Pereira et al., 2005). Briefly, follicles with 2-6 mm diameter were aspirated
from slaughterhouse bovine ovaries. Selected cumulus oocyte complexes were matured
in TCM199 (GibCo, ref.22340-020) with 10% superovulated oestrus cow serum
(SOCS), 10 µg mL-1 FSH (Follotropin, vetrepham Inc.) and antibiotics (Sigma, ref.
P0781) during 22-24 hours. For fertilization, frozen-thawed semen was submitted to
swim-up as above. Spermatozoa (106 spz mL-1) and 10 oocytes were placed in 40 µL
droplets of TALP medium supplemented with 5.4 USP mL-1 heparin (Sigma, ref. H3393), 10 mM penicillamine (Sigma, ref. P-4875), 20 mM hypotaurine (Sigma, ref. H-
1384) and 0.25 mM epinephrine (Sigma, ref. E-1635). Following co-incubation for 22
hours the presumptive zygotes were randomly placed in 100 µL droplets of a granulosa
cell monolayer cultured with TCM199, 10% SOCS and antibiotics under paraffin oil,
where embryo culture proceeded. Atmospherical conditions for IVM-IVF and embryo
co-culture were 39º C and 5% CO2 with humidified atmosphere. Cleavage was assessed
48 hours after insemination and embryos were morphologically evaluated on day 7 and
8 post insemination (pi) and embryos were morphologically evaluated on day 7 and 8
pi.
4. Statistical analysis.
All data are presented as means ± standard deviations. The mean values were compared
by using the ANOVA and LSD, and P<0.05 was considered significant.
Results
Besnoitia besnoiti diagnosis
Three bulls, 2 Limousine and 1 Charolais, presented B. besnoiti cysts with live
bradoyzoites in the skin (figure 1).
Semen evaluation
Before cryopreservation, there were no differences (P>0.05) in morphological
evaluation (table 1) of semen from asymptomatic infected with Benoitia besnoiti and
uninfected bulls.
Table 1. Evaluation (mean±SD)
asymptomatic infected with Benoitia
n=5) before cryopreservation.
Volume
Gross
Bulls
(mL)
motility
control
5.2 ± 2.4
3.0±0.7
INF
6.7 ± 2.8
3.3±0.6
of semen collected by electroejaculation to
besnoiti (INF, n=3) and uninfected bulls (control,
Individual Concentration
motility (%) (105spz mL-1)
82.0±11.0 10.6 ± 3.7
76.7±11.7
8.4 ± 3.6
Dead spz Morphological
(%)
abnormalities (%)
23.6±13.7
23.4±3.2
16.0±1.7
25.0±1.0
After cryopreservation, there were no significant differences between results from the 2
uninfected bulls running simultaneously in both experiments. Consequently, postthawing results from experiment 1 and 2 were treated together (table 2). From all the
sperm quality parameters evaluated, only post-thawed (P=0.05) and post-swim-up
(P=0,009) motility were significantly different between asymptomatic infected and
uninfected bulls, respectively. Cleavage and embryos rates obtained with semen from
uninfected and asymptomatic infected bulls presented no differences (P>0.05).
Table 2. Evaluation (mean±SD) of post-thawed semen collected to asymptomatic
infected with Benoitia Besnoiti (INF) and uninfected (control) bulls
(Host25=hypoosmotic swelling test, 25 minutes).
Bulls
control
INF
Post-thawed
Motility n Host25
Post-swim-up tests
n
n Motility
Activity Agglutinat. Concentrat
(%)
(%)
(%)
(106spzmL-1)
a
a
45 43.3±10.6 10 54.1±9.6 45 25.1±12.0
2.4±0.7 28.4±12.0 83.6±82.9
b
b
15 51.0±18.6 6 45.4±19.1 15 36.3±18.9
2.6±1.1 27.2±10.3 100.5±63.8
Discussion
Besnoitia Besnoiti infections are economically important to cattle owners in endemic
areas due to mortality, loss of general health condition, low market value and sterility
which may be temporary or permanent (Bigalke, 1968, Kumi-Diaka et al., 1981).
Prevalence of bovine besnoitiosis in Portugal, especially the asymptomatic form, is not
yet clearly determined but may be increasing. In the present study live bradyzoites
within Besnoitia cyst were identified although all the bulls tested were asymptomatic.
Previous studies (Cortes et al., 2003, 2005) in farms located in Évora region diagnosed
42% of B. Besnoiti infected Limousine bulls without clinical signs of the disease.
Asymptomatic B. besnoiti infected bulls presence in herds implies a potential
transmission of besnoitiosis although as demonstrated sire fertilitlity is not affected.
Infertility or sterility seems to be a late consequence of B. besnoiti infection (Sekoni et
al., 1992). Semen quality pre- and post-cryopreservation was not diminished in infected
asymptomatic bulls. Neither single parameters of semen quality (motility, sperm
number and morphology) having moderate value as predictors of absolute fertility levels
(Parkinson, 2004), nor supravital stain and Host tests which can be considered
important variables for normal sperm function (Tartaglione & Ritta, 2004) were
affected. Also, IVF and embryo rates presented no differences between asymptomatic
infected bulls and control. IVF and embryo rates are a useful indicator of bull fertility,
as the majority of reports correlated its results with both non-return rates with
cryopreserved semen (Marquant-LeGuienne et al., 1990, Ward et al., 2001) and
conception rates in natural service (Brahmkshtri et al., 1999).
These results suggest that in herds carrying the parasite for a long period, there are no
implications on the fertility of subclinically infected bulls. Notwithstanding, we strongly
disagree with the transport of infected animals, even if subclinically, to other areas or
herds without any contact with this parasite. In fact, due to the mechanical transmission
of B. besnoiti by blood-sucking insects or iatrogenicaly from infected to susceptible
cattle (Bigalke, 1968), due to the high number of tabanids and Stomoxis calcitrans
during summer season, moving infected animals to Besnoitia free herds will contribute
to the disease dissemination.
Figure 1. Hematoxilin Eosin Stain of a skin, where large number of cysts containing
bradizoites are present.
Figure 2. Clinical besnoitiosis with thickening, wrinkling and hair loss of the skin.
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