Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow-freezing or vitrification.

REASONS FOR PERFORMING STUDY: Equine embryos are cryopreserved by slow-freezing or vitrification. While small embryos (<300 µm) survive cryopreservation reasonably well, larger embryos do not. It is not clear if slow-freezing or vitrification is less damaging to horse embryos. OBJECTIVES: To compare the type and extent of cellular damage suffered by small and large embryos during cryopreservation by slow-freezing vs. vitrification. STUDY DESIGN: Sixty-three Day 6.5-7 embryos were subdivided by size and assigned to one of 5 treatments: control, exposure to slow-freezing or vitrification cryoprotectants (CPs), and cryopreservation by either technique. METHODS: After thawing/CP removal, embryos were stained with fluorescent stains for various parameters of cellular integrity, and assessed by multiphoton microscopy. RESULTS: Exposing large embryos to vitrification CPs resulted in more dead cells (6.8 ± 1.3%: 95% confidence interval [CI], 3.1-10.4%) than exposure to slow-freezing media (0.3 ± 0.1%; 95% CI 0.0-0.6%: P = 0.001). Cryopreservation by either technique induced cell death and cytoskeleton disruption. Vitrification of small embryos resulted in a higher proportion of cells with fragmented or condensed (apoptotic) nuclei (P = 0.002) than slow-freezing (6.7 ± 1.5%, 95% CI 3.0-10.4% vs. 5.0 ± 2.1%, 95% CI 4.0-14.0%). Slow-freezing resulted in a higher incidence of disintegrated embryos (P = 0.01) than vitrification. Mitochondrial activity was low in control embryos, and was not differentially affected by cryopreservation technique, whereas vitrification changed mitochondrial distribution from a homogenous crystalline pattern in control embryos to a heterogeneous granulated distribution in vitrified embryos (P = 0.05). CONCLUSIONS: Cryopreservation caused more cellular damage to large embryos than smaller ones. While vitrification is more practical, it is not advisable for large embryos due to a higher incidence of dead cells. The choice is less obvious for small embryos, as vitrification led to occasionally very high percentages of dead or damaged cells, but a lower incidence of embryo disintegration. Modifications that reduce the level of cellular damage induced by vitrification are required before it can be considered the method of choice for cryopreserving equine embryos.

Effect of alternate day collection on semen quality of Asian elephants (Elephas maximus) with poor initial fresh semen quality.

In captivity, male Asian elephants often yield poor quality semen after transrectal manually assisted semen collection; however, the reasons for the disappointing semen quality are not clear. Here we test the hypothesis that accumulation of senescent spermatozoa is a contributory factor, and that semen quality can therefore be improved by more frequent ejaculation. To this end we investigated the effect of collecting semen five times on alternate days, after a long period of sexual rest, on semen quality in Asian elephants known to deliver poor semen during infrequent single collections. All eight bulls initially displayed a high incidence of detached sperm heads and low percentages of motile (close to 0%) spermatozoa. After semen collection on alternate days, the percentages of detached sperm heads, and head and mid-piece abnormalities, were reduced significantly (p<0.05). In particular, one bull showed markedly improved sperm motility (increased from 0% to 60%) and membrane integrity (increased from 5% to 75%). In addition, advancing age significantly (p<0.01) correlated with lower percentages of sperm with intact membranes and a higher frequency of detached sperm heads. In contrast to sperm accumulation problems in other species, a small ampullary diameter correlated significantly (p<0.05) with reduced semen quality.

Advancing maternal age predisposes to mitochondrial damage and loss during maturation of equine oocytes in vitro.

In many mammalian species, reproductive success decreases with maternal age. One proposed contributor to this age-related decrease in fertility is a reduction in the quantity or functionality of mitochondria in oocytes. This study examined whether maternal age or (in vitro maturation). IVM affect the quantity of mitochondria in equine oocytes. Oocytes were collected from the ovaries of slaughtered mares categorized as young (<12 years) or aged (≥12 years) and either denuded and prepared for analysis immediately (not-IVM) or matured in vitro for 30 hours before preparation (IVM). The mean oocyte mitochondrial DNA copy number was estimated by quantitative polymerase chain reaction and found to be significantly lower in oocytes from aged mares and that had been subjected to IVM than in any other group. Transmission electron microscopy demonstrated that mitochondria in aged mare oocytes subjected to IVM experienced significantly more swelling and loss of cristae than in other groups. We conclude that maternal aging is associated with a heightened susceptibility to mitochondrial damage and loss in equine oocytes, which manifests during IVM. This predisposition to mitochondrial degeneration probably contributes to reduced fertility in aged mares.

Effect of pre-freeze semen quality, extender and cryoprotectant on the post-thaw quality of Asian elephant (Elephas maximus indicus) semen.

Semen cryopreservation and artificial insemination (AI) are potentially valuable methods for supporting the breeding management of endangered species like the Asian elephant. Cryopreservation of Asian elephant semen has however proven problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. In this study, nine ejaculates from three adult bulls were used to compare the influence of extender (TEST versus INRA96®) and penetrating cryoprotectants (3% glycerol, 5% glycerol and 4% methylformamide) on post-thaw semen quality. We demonstrate that not only the freezing process, but also the quality of the semen before freezing, significantly influences the freezability of Asian elephant semen. Pre-freeze motility, viability, semen volume, semen pH, sperm concentration and the incidence of sperm mid-piece and tail abnormalities all significantly (p<0.05) affected post-thaw semen quality. While extender and cryoprotectant did not significantly affect any of the above semen quality parameters post-thaw, the skim-milk based extender (INRA96®) preserved DNA integrity better (p<0.05) than the egg yolk extender (TEST). Considerable between-ejaculate variation in all post-thaw semen quality parameters was also noted. It is concluded that strict criteria for semen quality is essential for the selection of Asian elephant bull ejaculates suitable for cryopreservation; stricter initial selection should improve the mean post-thaw quality.

Effect of cooled storage on quality and DNA integrity of Asian elephant (Elephas maximus) spermatozoa.

Artificial insemination (AI) is a potentially useful tool for breeding captive elephants because it facilitates efforts to minimise inbreeding. However, cooled storage of elephant semen markedly reduces fertility. This study compared the effects on semen-quality parameters, including sperm DNA fragmentation, of storing elephant semen at 4°C or 15°C in a commonly-used diluent (TEST) or a diluent developed to protect against sperm DNA damage (BullMax). Storing elephant semen for >24 h in either extender at either temperature resulted in decreases in sperm motility, viability, acrosome integrity and DNA integrity (P < 0.05); the decrease in motility was especially rapid. A subjective impression of circular sperm movement in TEST was confirmed by a higher curvilinear velocity and amplitude of lateral head displacement, but lower straight-line velocity and linearity than in BullMax. Initial percentages of spermatozoa with fragmented DNA (%SDF) did not differ between extenders or temperatures, but the rate of increase in %SDF during a 48-h incubation at 37°C was higher in TEST than in BullMax (P < 0.05). In conclusion, BullMax allows more linear movement and better preserves DNA stability of stored elephant spermatozoa than TEST. Sperm DNA stability during incubation at 37°C is a promising, discriminative parameter for selecting semen storage conditions of bulls for elephant AI.

The origin of Indonesian cattle and conservation genetics of the Bali cattle breed.

Both Bos indicus (zebu) and Bos javanicus (banteng) contribute to the Indonesian indigenous livestock, which is supposedly of a mixed species origin, not by direct breeding but by secondary cross-breeding. Here, the analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed banteng introgression of 10-16% in Indonesian zebu breeds with East-Javanese Madura and Galekan cattle having higher levels of autosomal banteng introgression (20-30%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Y-chromosomal haplotypes, presumably of French Limousin origin. There was no evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng.

Maturation competence of swamp buffalo oocytes obtained by ovum pick-up and from slaughterhouse ovaries.

This study was designed with the final goal of improving in vitro embryo production in the Thai swamp buffalo (Bubalus bubalis carabensis). Oocytes were collected by ovum pick-up (OPU) from six non-lactating multiparous swamp buffalo twice per week for 10 consecutive sessions followed by once-weekly collection for 10 consecutive sessions without hormone stimulation. In addition, oocytes were collected from slaughterhouse ovaries that were classified as follows: ovaries from non-pregnant cows with a visible corpus luteum (NPCL); pregnant cows with a corpus luteum (P); and non-pregnant cows without a corpus luteum (NP). Follicles in each group of ovaries were categorized as small (2-4 mm), medium-sized (5-8 mm) or large follicles (≥ 9 mm). The quality of the oocytes was assessed by their capacity to undergo in vitro maturation. The total number of observed follicles per session (all sizes combined) was larger in the once-weekly OPU group compared with the twice-weekly OPU group. In particular, the numbers of small and large follicles were higher in the once-weekly OPU group (5.2 ± 0.7 and 0.9 ± 0.2, respectively) than in the twice-weekly OPU group (3.9 ± 0.5 and 0.5 ± 0.1). The number of medium-sized follicles did not differ between the groups. The percentages of oocytes with an abnormal spindle morphology were not different between oocytes from the twice-weekly (30.0%) and the once-weekly (28.6%) OPU groups. A higher percentage of oocytes obtained in vitro (49.5%) exhibited nuclear abnormalities compared with those obtained in vivo (≤34.8%) after in vitro maturation. In conclusion, oocytes can be successfully collected by OPU in the swamp buffalo, without hormonal pretreatment, and per week more good-quality oocytes can be collected by twice-weekly OPU. In addition, oocytes collected from slaughterhouse ovaries can be used with the reproductive status of the cow having no influence on the maturation competence of oocytes.

Follicular dynamics and oestrous detection in Thai postpartum swamp buffaloes (Bubalus bubalis).

This study characterized follicular activity and oestrous behaviour from 5 to 9 days post-calving up to the 4th ovulation postpartum (pp) in 16 multiparous (range 2-7 parities) Thai swamp buffalo cows (Bubalus bubalis), aged 4-12 years and weighing from 432 to 676 kg. Ovarian follicular activity was examined by transrectal ultrasonography (TUS) every morning. Oestrous detection was performed twice daily by direct personal observation of behaviour and for presence of clear cervical mucus discharge and indirectly by video camera recording during 21 h/day. A follicular wave-like pattern was present before the 1st ovulation leading to short oestrous cycles. Growth rates and maximum diameters of the ovulatory follicles did not differ between the 1st and 4th ovulations. However, growth rate for non-ovulatory dominant follicles (DF) before the 1st ovulation was lower than for the ovulatory follicle (p<0.05). In addition, the diameter of all ovulatory follicles (14.3 ± 0.46 mm, n=39) was significantly larger (p < 0.01) than those of the preceding last but one non-ovulatory DF (10.8 ± 0.20 mm, n = 5), but similar to the last preceding non-ovulatory DF diameter (12.92 ± 0.96 mm, n = 14). Short oestrous cycles were most common between the 1st and 2nd ovulations (93.75%, 15/16 cows, 10.2 ± 0.38 days) decreasing in prevalence thereafter (50%, 3/6 buffaloes, 12.0 ± 1.53 days). Oestrous signs were relatively vague around the 1st ovulation pp to become more easily detectable thereafter. This study suggests that properly fed swamp buffaloes could be mated successfully within 2 months pp, at their 2nd spontaneous ovulation, provided oestrous detection is at least performed daily at 06:00-08:00 hour.

A method for immediate comparative assessment of microinjected mammalian oocytes.

Manipulation of mammalian oocytes at the molecular level is hampered by low transcriptional activity and the presence of large stores of mRNA and protein. Microinjection of interfering macromolecules has become an important tool in studying oocyte maturation, although injection success, final concentrations of injected substances and viability after injection remain difficult to assess with current techniques. To address these problems, we developed an epifluorescence microscopy based technique to evaluate oocytes directly after (co-)injection of green fluorescent protein (GFP).

Aspects of the epidemiology, research, and control of lentiviral infections of small ruminants and their relevance to Dutch sheep and goat farming.

In 1862, the veterinarian Loman reported the first sheep in The Netherlands with symptoms associated with lentiviral infection, although at the time the symptoms were ascribed to ovine progressive pneumonia. In the following century, similar cases were reported by South African, French, American, and Icelandic researchers. Extensive research into the pathology, aetiology, and epidemiology of this slowly progressive and ultimately fatal disease was initiated in several countries, including the Netherlands. Studies of the causative agents--maedi visna virus (MVV) in sheep and caprine arthritis encephalitis virus (CAEV) in goats, comprising the heterogeneous group of the small ruminant lentiviruses (SRLV)--prompted the development of diagnostic methods and the initiation of disease control programmes in many European countries including the Netherlands, as a pioneer in 1982, and in the U.S.A. and Canada.

Vaccination against GnRH may suppress aggressive behaviour and musth in African elephant (Loxodonta africana) bulls--a pilot study.

Aggressive behaviour and musth are constant problems in captive and sometimes in free-ranging African elephant bulls. Aggressive bulls are difficult and musth bulls almost impossible to manage without severely restricting their movement either by leg-chaining or using tranquillisers. This study investigated the relationship between faecal androgen metabolites (FAM) and faecal cortisol metabolites (FCM) concentrations and aggressive behaviour and tested a GnRH vaccine as a means of down-regulating aggressive behaviour and musth in 1 free-ranging and 5 captive elephant bulls. The bulls were non-aggressive (n=3), aggressive (n=2) or in musth (n=1) at the onset of the study. The bulls were injected with a GnRH vaccine-adjuvant combination 3 or 4 times at 3- to 7-week intervals. Behaviour, FAM and FCM concentrations were measured during every week prior to vaccination until 4 months after the last vaccination. FAM concentrations were positively correlated with aggressive behaviour before the 1st vaccination. Androgen production, as reflected by FAM concentrations, was down-regulated in 3 of the 6 immunised bulls. At least 2 bulls and possibly a 3rd showed behavioural improvement following GnRH vaccination and in all 3 temporal gland secretion ceased. No further aggressive behaviour was observed until the end of the study in any of the bulls. The results of this 1st GnRH immunisation study suggest that it could be a useful method to control aggressive behaviour and musth in African elephant bulls.

Porcine oocytes are most vulnerable to the mycotoxin deoxynivalenol during formation of the meiotic spindle.

Deoxynivalenol (DON, vomitoxin) is a secondary metabolite and mycotoxin produced by Fusarium species that occurs with a high prevalence in cereals and grains intended for human and animal consumption. Pigs are considered to be the most sensitive animal species and exposure to DON results in reduced feed intake, reduced performance and cause alterations in the expression of markers of inflammation and cell cycle regulation. The objective of this study was to determine how DON possibly affects the oocyte developmental potential in vitro at concentrations which correspond to those observed in practice. To evaluate DON toxicity during specific stages of oocyte meiosis, cumulus-oocyte complexes were exposed to 0.02, 0.2, or 2 microM DON. Exposure to the highest DON concentration inhibited cumulus expansion and induced cumulus cell death. After exposure for 42 h, DON at all concentrations reduced Metaphase II formation and led to malformations of the meiotic spindle. Despite spindle malformations, exposure to different concentrations of DON did not lead to increased percentages of blastomeres with abnormal ploidy in embryos. Spindle malformation occurred by DON exposure during formation of meiotic spindles at Metaphase I and II, but embryo development was also reduced when oocytes were exposed to DON during Prophase I. Together, these results indicate that exposure to DON via contaminated food or feed can affect oocyte developmental competence by interfering directly with microtubule dynamics during meiosis, and by disturbing oocyte cytoplasmic maturation through other as yet undetermined mechanisms.

Y-chromosomal variation confirms independent domestications of swamp and river buffalo.

Y-chromosomal variation in the water buffalo was analysed by sequencing of DBY, ZFY and SRY gene segments. A clear separation of the paternal lineages of the river and swamp types parallels the differences between their maternal lineages and nuclear DNA. Sequence divergence was found to be comparable to the divergence of taurine cattle and zebu, and this divergence predated domestication, confirming that river and swamp buffalo originated from different wild populations. Within a sample of 23 Thai swamp buffaloes, we identified four haplotypes with different geographical distributions, two of which were shared by Thai wild buffaloes.

Genetic parameters for oocyte number and embryo production within a bovine ovum pick-up-in vitro production embryo-production program.

Genetic factors influencing the outcome of bovine ovum pick-up-in vitro production (OPU-IVP) and its relation to female fertility were investigated. For the first time, genetic parameters were estimated for the number of cumulus-oocyte complexes (Ncoc), quality of cumulus-oocyte complexes (Qcoc), number and proportion of cleaved embryos at Day 4 (Ncleav(D4), Pcleav(D4)), and number and proportion of total and transferable embryos at Day 7 of culture (Nemb(D7), Pemb(D7) and NTemb(D7), PTemb(D7), respectively). Data were recorded by CRV (formally Holland Genetics) from the OPU-IVP program from January 1995 to March 2006. Data were collected from 1508 Holstein female donors, both cows and pregnant virgin heifers, with a total of 18,702 OPU sessions. Data were analyzed with repeated-measure sire models with permanent environment effect using ASREML (Holstein Friesian). Estimates of heritability were 0.25 for Ncoc, 0.09 for Qcoc, 0.19 for Ncleav(D4), 0.21 for Nemb(D7), 0.16 for NTemb(D7), 0.07 for Pcleav(D4), 0.12 for Pemb(D7), and 0.10 for PTemb(D7). Genetic correlation between Ncoc and Qcoc was close to zero, whereas genetic correlations between Ncoc and the number of embryos were positive and moderate to high for Nemb(D7) (0.47), NTemb(D7) (0.52), and Ncleav(D4) (0.85). Genetic correlations between Ncoc and percentages of embryos (Pcleav(D4), Pemb(D7), and PTemb(D7)) were all close to zero. Phenotypic correlations were in line with genetic correlations. Genetic and phenotypic correlations between Qcoc and all other traits were not significant except for the phenotypic correlations between Qcoc and number of embryos, which were negative and low to moderate for Nemb(D7) (-0.20), NTemb(D7) (-0.24), and Ncleav(D4) (-0.43). Results suggest that cumulus-oocyte complex (COC) quality, based on cumulus investment, is independent from the total number of COCs collected via OPU and that in general, a higher number of COCs will lead to a higher number of embryos produced. The correlation between the estimated breeding values for Ncoc and PTemb(D7) of sires in this study and the sires breeding index for female-fertility based on the Dutch cattle population was close to zero. This study revealed OPU-IVP traits (Nemb(D7), NTemb(D7), and Ncoc) that could be of potential value for selection. Introduction of such traits in breeding programs would enhance the number of offspring from superior donors as well as improve the cost efficiency of OPU-IVP programs.

Maternal age and parity influence ultrasonographic measurements of fetal growth in Dutch Warmblood mares.

Ultrasonographic examination of the equine fetus in mid-late gestation is usually performed only if there are concerns about fetal or maternal health. Even then it is difficult to determine whether development is 'normal' for gestational age because the reference values include considerable error margins. This study examined maternal factors that influence fetal growth with the aim of producing more precise late gestation fetal growth curves for Dutch Warmblood horses. Fetal development was monitored at 2-week intervals from day 100 of gestation until term in 32 mares ranging from 4 to 18 years in age; seven of the mares were primiparous. Transrectal and/or transabdominal ultrasonographic measurement of the fetal eye orbit, cranium, aorta, heart rate and of the combined thickness of uterus and placenta (CTUP) were performed using a portable ultrasound machine equipped with 6 MHz linear and 3.5 MHz curved array probes. During days 100-250 of gestation, the CTUP was thicker in primiparous than multiparous mares (p<0.05). After day 220 the maximum cross-sectional area, but not diameter, of both the eye orbit and cranium were also greater in primiparous than multiparous mares (p<0.05). Fetal aorta diameter was not influenced by parity but was affected by maternal age, being smaller in mares > or =15 years of age than younger animals (p<0.05). Only biparietal cross-sectional surface area and aorta diameter increased linearly throughout late gestation. However, even allowing for the effects of parity and maternal age, the late gestational variation in fetal size is such that serial measurements may be required to definitively identify abnormal development.

Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes.

Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were labelled for connexin-43 or injected with Lucifer yellow dye to determine whether the integrity of the gap junctions between an oocyte and its cumulus was compromised by vitrification. Finally, the effect of denuding immature and mature oocytes on their ability to withstand vitrification was examined. Cryopreserving immature COCs increased the number of dead cumulus cells (13 vs 2.6% for controls; P<0.05). However, an increased proportion of dead cumulus cells did not affect post-warming maturation rates (approximately 30% MII) presumably because dead cells were located at the periphery of the cumulus mass and cumulus-oocyte gap junction communication was not disrupted. Moreover, cumulus removal prior to IVM or vitrification indicated that while the cumulus does protect immature oocytes during vitrification it does so by mechanisms other than support during maturation. Cumulus presence was also found to protect mature equine oocytes against vitrification-induced damage since cumulus-enclosed MII oocytes preserved their meiotic spindle quality better during vitrification than denuded oocytes (38.1 vs 3.1% normal spindles; P<0.05).

Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig.

Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to several morphological and cell biological properties, and evaluated the survival of in vitro produced, morphologically classified, blastocysts following non-surgical transfer. In vitro and in vivo produced blastocysts were allocated to two groups (classes A and B) on the basis of morphological characteristics. The quality of their actin cytoskeleton, their total cell number, their ability to re-expand after cytochalasin-B treatment and the occurrence of numerical chromosome aberrations were studied and compared. In vivo produced blastocysts were used as a control. Our results indicate that the ability of blastocysts to re-expand after cytochalasin-B-induced actin depolymerization was positively correlated with the morphology of the blastocyst, and associated with the quality of the actin cytoskeleton. Chromosome analysis revealed that mosaicism is inherent to the in vitro production of porcine embryos, but also that in vivo produced blastocysts contained some non-diploid cells. In non-surgical embryo transfer experiments more recipients receiving class A blastocysts were pregnant on Day 20 than those receiving class B blastocysts. One recipient gave birth to six piglets from class A in vitro produced blastocysts, providing a verification of the enhanced viability of blastocysts that were scored as 'good' on the basis of their morphology.

Expression of progesterone and oestrogen receptors by early intrauterine equine conceptuses.

Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently characterised membrane-bound progesterone receptors (PGRMC1 and mPR). Horse conceptuses recovered on days 7, 10 and 14 after ovulation (n=8 at each stage) were examined for steroid receptor mRNA expression using quantitative rtPCR. Where commercial antibodies were available (PR, ERbeta), receptor localisation was examined immunohistochemically in day 10, 12, 14, 15 and 16 conceptuses (n=2 at each stage). mRNA for PR, PGRMC1 and mPR was detected at all stages examined, but while PGRMC1 and mPR expression increased during the day 7-14 period, PR expression decreased. ERalpha mRNA was not detected at any stage examined, whereas ERbeta mRNA was detected in all day 14, some day 10 and no day 7 conceptuses. Immunoreactive ERbeta receptors were localised to the trophectoderm of day 14-16 conceptuses; PR were not detected immunohistochemically in conceptus tissue. In summary, this study demonstrates that equine conceptuses express mRNA and, in the case of ERbeta, protein for steroid hormone receptors during the period encompassing rapid conceptus growth, differentiation and maternal pregnancy recognition.

Presence of pro-lentiviral DNA in male sexual organs and ejaculates of small ruminants.

To be able to predict sexual transmissibility of small ruminant lenti viruses (SRLV), it is necessary to know whether or not the virus is excreted in the semen, and under what circumstances. Thus, this research focussed on establishing the presence of proviral DNA of SRLV in semen and in the male genital tract of small ruminants. After initial results established the presence of SRLV in serum, the emergence of proviral DNA of SRLV in semen and presence in blood in a group of naturally SRLV-infected individuals (13 rams and 4 bucks), was followed temporally using real-time polymerase chain reaction (PCR). The same animals were also systematically serologically monitored by enzyme-linked immuno sorbent assay (ELISA) during the breeding season (August-February). A triple monocyte-macrophage count was performed on both blood and semen using a specific monoclonal antibody in conjunction with flow cytometry. The finding that epididymal semen and tissue samples of the testes, epididymides, ampullary, vesicular, prostate and bulbo-urethral glands all tested positive for the presence of proviral DNA indicates that various male sexual organs may contribute directly to shedding of proviral SRLV DNA in ejaculated semen. Our results suggest that small ruminants show intermittent shedding of proviral SRLV DNA into epididymal as well as ejaculated semen. They also demonstrate that a single PCR-negative semen sample cannot be used as a diagnostic tool to predict that subsequent ejaculates will be SRLV-free. No significant relationship was found between numbers of monocytes and/or macrophages in blood or semen and the detection of proviral SRLV in ejaculates.