Assessment LOPU-IVF in Japanese sika deer (Cervus nippon nippon) and application to Vietnamese sika deer (Cervus nippon pseudaxis) a related subspecies threatened with extinction.

In mammals, recovery of oocytes by laparoscopic ovum pick-up (LOPU) coupled with in vitro production (IVP) of embryos represents a promising strategy for both amplification and genetic management of sparse animals from captive endangered wild species. As integrated technique developed mainly for domestic livestock, LOPU-IVP requires several studies to set up protocols for follicular stimulation or optimization of IVP before envisaging successful transposition to wild species. In deer, many endangered subspecies would be potentially concerned by applying such an approach using common subspecies for protocols optimization. The aim of the present study was to assess efficiency of follicle stimulation using ovine FSH (oFSH) for recovery of oocytes by LOPU in common sika deer (Cervus nippon nippon) before transposition of an optimized methodology for IVP of embryos from endangered Vietnamese sika deer hinds (Cervus nippon pseudaxis). In common sika deer, two doses of oFSH (0.25 and 0.5 U) and two frequencies of administration (12 and 24 h) were compared by monitoring of subsequent ovarian response, quality of oocytes recovered by LOPU, and in vitro developmental competence. In a first experiment, the dose of oFSH administered did not significantly affect the total number of follicles aspirated per hind per session (8.6 ± 1.0 vs. 8.2 ± 1.6 with 0.5 vs. 0.25 U oFSH, respectively; not significant). In a second experiment, frequency of 0.25 U oFSH administration did not affect ovarian response. Efficiency of IVP determined on blastocysts rates after in vitro maturation, fertilization, and development in oviduct epithelial cells coculture was increased when FSH was administered at 12-h intervals. Immune response after several follicular stimulations was detected against exogenous oFSH in plasma from the majority of sika deer hinds but was not associated with decreased ovarian response. When 0.25 U oFSH was administered at 12-h intervals to Vietnamese sika deer (N = 4), good quality cumulus oocyte complexes with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Development to the blastocyst stage occurred in a high proportion (30% of oocytes) after coculture with ovine epithelial cells allowing cryobanking of transferable embryos from Vietnamese sika deer. These results confirm that LOPU-IVF after ovarian stimulation with oFSH may be a successful tool for cryobanking transferable embryos from endangered sika deer subspecies.

Can caprine arthritis encephalitis virus (CAEV) be transmitted by in vitro fertilization with experimentally infected sperm?

For each of the five fertilization trials of the experiment, frozen semen was prepared for in vitro capacitation at a concentration of 1 × 10(7) spz/ml and divided into three groups. One group was used as a control, while the two others were inoculated with 100 µl/ml of either culture medium from non-infected cells (placebo group) or cell culture medium containing virus at a concentration of 10(5) TCID(50)/ml (infected group). A total of 789 oocytes were used for IVF. For each of the five trials a group of oocytes were used as a non-infected control and were found to be caprine arthritis-encephalitis virus (CAEV) free. The other oocytes were divided in two equal batches. Oocytes in the first batch were in vitro fertilized with CAEV infected sperm (infected group) and the second batch were fertilized with CAEV non-infected sperm (placebo and control groups). After IVF, the zygotes of each group were washed 12 times. The CAEV genome was not detected (using RT-PCR) in the washing media of either the control or placebo groups from each trial. In contrast, the first three washing media from the infected group were consistently found to be positive for the CAEV genome (5/5), whereas subsequent washing media were CAEV-free (P < 0.05). Zygotes obtained using all semen groups tested negative for both the provirus and genome of CAEV. These results clearly show that the first four washes were sufficient to remove viral particles from CAEV infected fertilization media and that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV.

[In vivo imaging of ram spermatozoa in the ewe genital tract using fibered confocal microscopy]. / Visualisation in vivo des spermatozoïdes de bélier dans le tractus génital de brebis par microscopie confocale fibrée.

Sperm transit in the female tract is one of the key factors in the success of fertilization after artificial insemination in sheep species. However, its study is limited by the absence of in vivo imaging methods. The imaging of ram sperm in the female genital tract was made possible using the confocal fibered microscopy and fluorescent stains adapted to spermatozoa. Our results show the active role of the uterotubal junction in the selection of sperm during their transit.

Sex and PRNP genotype determination in preimplantation caprine embryos.

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.

Time of ovulation in nulliparous and multiparous goats.

Fifteen nulliparous and nine multiparous Serrana goats were used, through two successive oestrous cycles, in order to characterize their ovulation time with regard to the number of ovulations after induced and natural oestrus during the breeding season. The onset of oestrus was detected by the amount of vasectomized bucks after oestrus synchronization with prostaglandin, given 10 days apart, and in the following two expected natural oestrus. The preovulatory LH peak was determined from blood samples collected 0, 4, 8, 12, 16, 20 and 24 h after onset of oestrus. A transrectal ovarian ultrasound scanning was performed 20, 24, 28, 32, 36, 40, 44 and 60 h after onset of oestrus, for the detection of ovulations by means of the disappearance of large follicles (>4 to 5 mm). Single ovulations were observed in 76% of oestrous periods in nulliparous goats and in 18% of nulliparous goats. The onset of oestrus to LH peak interval was lower in nulliparous (12.1 ± 0.9 h, n = 38) than in multiparous (15.6 ± 1.0 h, n = 22, P < 0.05) goats with no oestrus interaction effects (P > 0.05). The LH peak to first ovulation interval was higher after natural (18.9 ± 0.7 h, n = 36) than after induced (15.8 ± 1.2 h, n = 24, P < 0.05) oestrus. The onset of oestrus to total ovulation interval was influenced by parity (P < 0.01) and oestrus type (P < 0.05) with a length of 30.1 ± 1.1 h (n = 15) and 33.4 ± 1.5 h (n = 9) for induced oestrus of nulliparous and multiparous goats, respectively, and 32.5 ± 1.0 h (n = 23) and 36.5 ± 1.1 h (n = 13) for natural oestrus of nulliparous and multiparous goats, respectively. The onset of oestrus to first ovulation interval was not influenced by parity, but an interval of 8.0 ± 1.6 h was observed between the first and second ovulations in polyovulatory oestrus. Consequently, nulliparous goats that are predominantly monovular ovulate earlier than multiparous goats that are predominantly polyovulatory. In conclusion, significant differences occurred in the number and time of ovulations between nulliparous and multiparous goats. More research is necessary for a deeper understanding of the mechanisms regulating monovularory and polyovulatory oestrous cycles regarding the parity of goats.

Lack of risk of transmission of caprine arthritis-encephalitis virus (CAEV) after an appropriate embryo transfer procedure.

The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.

Effect of coculture with oviduct epithelial cells on viability after transfer of vitrified in vitro produced goat embryos.

This study evaluates the effect of coculture with goat oviduct epithelial cells (GOEC) on the pregnancy rate, embryo survival rate and offspring development after direct transfer of vitrified/thawed caprine in vitro produced (IVP) embryos. Oocytes were recovered from slaughterhouse goat ovaries, matured and inseminated with frozen/thawed capacitated semen, and presumptive zygotes were randomly cultured in synthetic oviduct fluid (SOF) (n=352) or GOEC (n=314). The percentage of cleaved embryos reaching the blastocyst stage was 28% and 20% in SOF and GOEC, respectively (P<0.05). Overall, 26 blastocysts of SOF were transferred freshly in pairs to recipient goats, whereas 58 of SOF and 36 of GOEC were vitrified and transferred directly in pairs to recipient goats after thawing without removal of cryoprotectants or morphological evaluation. The kidding rate was 92% for SOF fresh, 14% for SOF vitrified (P<0.001) and 56% for GOEC vitrified (P<0.05); the difference was also significant between vitrified groups (P<0.01). The embryo survival rate was 62% for SOF fresh, 9% for SOF vitrified (P<0.001) and 33% for GOEC vitrified (P<0.05) with a significant difference between vitrified groups (P<0.01). The results showed that the coculture of IVP goat embryos with GOEC significantly improves the pregnancy and embryo survival rates and leads to the birth of healthy offspring. However, further research using more defined GOEC coculture is required to confirm its capacity to increase the success rate of IVP embryo technology in goat.

Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes.

We determined whether kisspeptin could be used to manipulate the gonadotropin axis and ovulation in sheep. First, a series of experiments was performed to determine the gonadotropic responses to different modes and doses of kisspeptin administration during the anestrous season using estradiol-treated ovariectomized ewes. We found that: 1) injections (iv) of doses as low as 6 nmol human C-terminal Kiss1 decapeptide elevate plasma LH and FSH levels, 2) murine C-terminal Kiss1 decapeptide was equipotent to human C-terminal Kiss1 decapeptide in terms of the release of LH or FSH, and 3) constant iv infusion of kisspeptin induced a sustained release of LH and FSH over a number of hours. During the breeding season and in progesterone-synchronized cyclical ewes, constant iv infusion of murine C-terminal Kiss1 decapeptide-10 (0.48 mumol/h over 8 h) was administered 30 h after withdrawal of a progesterone priming period, and surge responses in LH occurred within 2 h. Thus, the treatment synchronized preovulatory LH surges, whereas the surges in vehicle-infused controls were later and more widely dispersed. During the anestrous season, we conducted experiments to determine whether kisspeptin treatment could cause ovulation. Infusion (iv) of 12.4 nmol/h kisspeptin for either 30 or 48 h caused ovulation in more than 80% of kisspeptin-treated animals, whereas less than 20% of control animals ovulated. Our results indicate that systemic delivery of kisspeptin provides new strategies for the manipulation of the gonadotropin secretion and can cause ovulation in noncyclical females.

Assessment of luteal function by ultrasonographic appearance and measurement of corpora lutea in goats.

In order to characterize the evolution pattern of the corpora lutea (CL) and to compare luteal function with their ultrasonographic appearance, 37 estrous cycles of Serrana goats (n=22) were studied during breeding season. A daily transrectal ultrasound scanning was performed through two successive estrous cycles. Both solid and fluid-filled CL were observed and measured in both ovaries of each goat. Additionally, each CL was classified as CL(ICHE) (CL with irregular contours and heterogeneous echotexture) or CL(RCGE) (CL with regular contours and granular echotexture). Ovarian cyclic activity and luteal function were evaluated by biweekly plasma progesterone (P4) determination. The CL (n=60) were first visualized on day 2.9+/-1.0 after the day of ovulation (day 0), showing 7.1+/-1.8mm of diameter and reach their maximum size (12.5+/-1.6mm) on day 10.7+/-3.2 (P<0.001). Two days before the following ovulation (day -2), the CL regressed to 8.4+/-1.3mm (P<0.001). The central cavity was found in 78.3% of CL, and had a persistence of over 50% until the last days of estrous cycle. The ratio CL length/cavity length was low during the first-third and high during the remaining two-thirds of estrous cycle. On day 2, the percentage of CL(ICHE) was 33.3%, and began to decrease to 16.7% on day 6, reaching the minimum of 3.3% on day 10 (P<0.001). This proportion increased on day -3 to 48.3% and reached 90% on day -1 (P<0.001). The correlation between CL size and plasma P4 levels was r=0.63 (n=87; P<0.001). A negative correlation between the daily proportion of CL(ICHE) and plasma P4 levels was found (r=-0.95; n=18; P<0.001). These results suggest that the ultrasonographic appearance of CL is a reliable parameter for the assessment of luteal function in goats. Both the characterization of echotexture and size of central cavity could be valuable tools to differentiate between phases of normal estrous cycles.

Improved vitrification method allowing direct transfer of goat embryos.

The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goat embryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35% versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (direct transfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfer method (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%). Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%, respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4M sucrose was added with cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56% versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment: vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after direct transfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus 21%). In conclusion, our results indicate that addition of 0.4M sucrose in association with direct transfer improves significantly the viability of goat vitrified embryos.

Follicular dynamics in Serrana goats.

Twenty-two Serrana goats were studied through two successive estrous cycles in order to characterize their follicular dynamics during the breeding season. The ovaries of the goats were scanned daily by real-time ultrasonography and all follicles >or=3mm were measured and classified. The data were classified by the number of follicular waves per goat to test the hypothesis that temporal and morphological differences between the last follicular wave of an ovary, irrespective of ovulation, will affect the selection of the next ovulatory wave. The mean interovulatory interval was 20.7+/-1.0 days (mean+/-S.D.). Three to five waves per estrous cycle were observed and 61.3% (19/31) of cycles had four waves. In estrous cycles with four waves, the day of onset of the first, second, third and fourth wave was 1.4+/-1.0, 6.9+/-1.4, 11.6+/-1.8 and 16.8+/-1.6, respectively. No differences (P>0.05) were found between the day of onset of the first and second waves for estrous cycles with three, four or five waves. However, the day of onset of the third and fourth waves occurred later when the number of waves per estrous cycle increased (P<0.001). The duration of the interwave interval (time between the day of onset of two consecutive waves) was longer when the second wave was ovulatory. The length of the growth phase (2.4+/-0.9 days) and size (5.9+/-0.7 mm) of the dominant follicle in the second wave were lower (P<0.01) than for the first wave (3.3+/-1.2 days and 6.6+/-0.9 mm, respectively) and the fifth wave (4.1+/-1.2 days and 7.5+/-1.0mm, respectively). Within pairs of ovaries, the onset of the last wave occurred later (P<0.05) and was less variable in ovulatory ovaries (day 16.8+/-1.4, n=20) than in anovulatory ovaries (day 15.1+/-3.7, n=20). The length of the growing phase was longer (P<0.001) in the last waves of ovulatory ovaries (3.1+/-0.9 days) than in the last waves of anovulatory ovaries (1.7+/-0.8 days). These results support the hypothesis that the day of onset of the ovulatory wave is related to or, at least, conditioned by the luteolysis and the decrease in plasma progesterone. In summary, the estrous cycle of Serrana goats is characterized by sequential follicular wave growth with a great variability in their onset and duration, with the exception of the ovulatory wave. The temporal and morphological differences observed in the last wave of estrous cycle provide strong evidence for the role of progesterone in their regulation.

Successful use of oviduct epithelial cell coculture for in vitro production of viable red deer (Cervus elaphus) embryos.

Techniques for in vitro production (IVP) of viable embryos have been thoroughly developed in several domestic species in view to improve breeding efficiency. When applied to wild life, these techniques may also help the maintenance of biodiversity through amplification of sparse animals offspring and facilitation of genetic material exchange. During the successive steps of IVP, i.e. oocyte in vitro maturation (IVM), fertilization (IVF) and early embryo development (IVD) to the blastocyst stage, gametes and embryos are faced with unusual environment, including oxidative stress, known to be detrimental to their survival. In the present study, starting from methods developed in domestic species, we have adapted IVP to produce viable red deer embryos. In a first experiment, cumulus cells were removed from in vitro matured oocytes either before or after IVF. The presence of cumulus cells during IVF did not affect final cleavage or development rates. In a second experiment, in vitro matured oocytes were fertilized in the presence of cumulus cells and cultured in SOFaaBSA medium alone or in the presence of ovine oviduct epithelial cell (oOEC) monolayer. Whereas, oviduct cells did not improve the cleavage rate, they significantly increased the rate of embryos reaching the blastocyst stage (from 3 to 25% of total oocytes). Ten blastocysts from oOEC coculture were transferred after freezing and thawing to five recipient hinds and gave rise to three pregnancies. The three pregnant hinds gave birth to three live and normal calves.

Proviral DNA of caprine arthritis encephalitis virus (CAEV) is detected in cumulus oophorus cells but not in oocytes from naturally infected goats.

The aim of this study was to determine whether oocytes taken from ovarian follicles in 123 naturally infected goats were carrying the proviral CAEV genome. Examination of DNA isolated from 190 batches of oocytes with intact cumulus cells and 190 batches of oocytes whose cumulus cells had been removed, taken from follicles of the same ovaries, demonstrated that 42/190 batches of oocytes with intact cumulus cells had the proviral CAEV genome, whereas none of the 190 batches of oocytes without cumulus cells were positive for the provirus. To confirm that the proviral genome was present in the cumulus cells and not in the oocyte cells, 586 oocytes from 56 different ovaries, were separated from their cumulus cells. The DNA was then extracted from each fraction and examined. The purity of the oocyte fraction was verified by searching for granulosa cell-specific mRNA, using RT-PCR; this was negative in all the batches of oocytes in which the cumulus cells had been removed. PCR analysis demonstrated that none of the oocytes without cumulus cells were positive, whereas 22/56 of the batches with cumulus cells were found to be positive. This study clearly demonstrates that despite being surrounded by infected cumulus cells, the oocytes are not infected, and that the enzymatic and mechanical technique for removing the cells surrounding the oocyte, as used in this study, is effective, thus enabling CAEV-free oocytes to be obtained from infected goats.

Comparison of cell proliferation index in equine and caprine embryos using a modified BrdU incorporation assay.

The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min at 39 degrees C to permeabilise the capsule, and then embryos were rinsed in PBS containing 10% of foetal calf serum. After washing, embryos were immediately fixed in 2.5% paraformaldehyde with 0.3M NaOH during 15 min at ambient temperature. The S-phase was detected by immunocytochemistry technique. In caprine embryos, BrdU was visualised by the same technique but without the trypsin treatment. The percentage of cells (+/-S.E.M.) with BrdU incorporated into newly synthesised DNA strands was significantly higher in equine embryos (74+/-1) than in caprine (38+/-2). Our results demonstrated that BrdU incorporation assay can be used in equine embryos. This assay allows the determination of the proliferation index of live cells and could be used as an additional tool for evaluating the viability of embryos. The high percentage of cells incorporating BrdU during 1h of incubation with BrdU suggests that in comparison with the caprine embryos the cellular activity of proliferation is more intense in equine embryos and suggests that the cellular cycle is shorter in equine embryos.

Morphometry of ovarian structures by transrectal ultrasonography in Serrana goats.

The accuracy of transrectal real-time ultrasonography (RTU) scanning technique to detect ovarian structures (follicles and corpus luteum) of Serrana goats was compared to the data obtained by observation of ovarian sequential slices. This slicing technique (SLI) was considered as reference method. The laparoscopy and laparotomy techniques were also used for corpora lutea identification. For this purpose the ovaries of 14 females were observed, 7-8 days after ovulation, by transrectal ultrasonography followed by laparoscopic examination. Then ovaries were removed and studied by slicing. In the sliced sections of each ovary (n=28), follicles and corpus luteum (CL) were identified and counted. CL and follicular diameters were measured using a millimetre scale. The total number of follicles, counted by RTU, was significantly lower than that observed by SLI (P <0.01). This difference was mainly due to the under estimation of <2 mm follicles category. The correlation coefficient between category data obtained by RTU and SLI methods for the number of follicles > or =3 mm was high (r2=0.95, P <0.001), which highlights the use of UTR as a potential methodology to study the follicular dynamic of goats. There were no significant differences (P >0.05) between the average number (mean +/- S.D.) of corpus luteum identified per ovary by RTU (0.71 +/- 0.75), laparoscopy (0.58 +/- 0.71), laparotomy (0.67 +/- 0.76) or SLI (0.83 +/- 0.76) methods. The accuracy for the identification of ovulation, validated by CL detection on D7-D8 by SLI (100%), was 91.7%, 87.5% and 83.3% by RTU, laparotomy and laparoscopy, respectively. The negative predictive value of RTU, laparotomy and laparoscopy to verify the absence of a CL in the ovary was 81.8%, 75.0% and 69.2%, respectively. The specificity of all three methods for the CL identification was 100%. No significant differences (P >0.05) were found in the probability to detect the exact number of CL (0, 1 or 2) counted in each ovary between the RTU (87.5%), laparotomy (83.3%) and laparoscopy (75.0%) methods when compared with the reference method. The diameter of spherical CL could be estimated with reliability (r2=0.86; P <0.001). The real-time ultrasonographic scanning proved to be a highly accurate method for detection and measurement of several categories of follicles and CL size in Serrana goats. The results of the present study show that laparoscopy and RTU are similarly reliable techniques for CL detection. However, the RTU represents a non-traumatic technique with advantages to animal welfare both in experimental and reproductive evaluation of the size of ovarian structures.

Current status of embryo technologies in sheep and goat.

This review presents an overview of the technical bases of in vivo and in vitro embryo production in sheep and goat. The current limitations of in vivo production, such as variability of response to the hormonal treatment, fertilization failure in females showing a high ovulatory response, and the importance of premature regressed CL in the goat, are described along with possibilities for improvement. The new prospects offered by in vitro embryo production, by repeated ovum pick-up from live females and by juvenile breeding, are presented along with their limiting steps and research priorities. The recent improvements of embryo production and freezing technologies could be used for constitution of flocks without risks of disease transmission and will allow wider propagation of valuable genes in small ruminants populations in the future.

Successful direct transfer of vitrified sheep embryos.

The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 min in 10% glycerol, for 5 min in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20 degrees C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos.

Four years of induction/synchronization of estrus in dairy goats: effect on the evolution of eCG binding rate in relation with the parameters of reproduction.

Ninety-eight Alpine goats of two herds were followed over 4 years in a program of annual artificial insemination after estrus induction/synchronization, including progestagen administration (vaginal sponge) followed by prostaglandin analog and equine chorionic gonadotrophin (eCG) 48 h before sponge removal. Goats were sampled every 4 hours from the 16th to the 56th following sponge removal, for determination of LH surge and tested for estrus by the presence of a buck. Seven days after AI, endoscopic examination of the ovaries was performed to determine the number of corpus lutea. Pregnancy diagnosis was performed at day 21-22 post AI by determination of plasma progesterone and at day 40-45 by ultrasonography. Parturition, number and sex of kids were recorded. All the goats were sampled before and after each treatment, for anti-eCG antibodies screening. Statistical analysis of the results clearly established a significant effect of the treatments on anti-eCG antibodies. Time of estrus and LH surge were significantly different between herd. The antibodies significantly delayed the time of coming out of estrus as well as the time of LH surge. Two antagonistic effects were evidenced: first, the delayed of time of estrus and time of LH surge in relation with the immune reaction to eCG; secondly, the ahead of time of estrus and time of LH surge during the years of treatment, identical to both herd. The antibodies negatively influenced the percentage of ovulating females as well as kidding rate. Finally, no effect of antibodies on prolificacy was found.

Implications of recent advances in reproductive physiology for reproductive management of goats.

The control of reproduction in goats is interesting for technical reasons (synchronization of kiddings, adjustment to forage availability or to economy), and for genetic reasons (identification and dissemination of improved genotypes). The use of short-light rhythms leads to markedly increased production of semen per buck and prevents occurrence of a 'resting' season. Recent identification of a bulbourethral lipase in goat spermatozoa opens new perspectives in sperm preservation. Light plus 'short day' treatments also allow induction of out-of-season oestrous cycles and ovulations leading to enhanced fertility. Repeated use of eCG provokes the production of antibodies, delays the timing of ovulation and causes a reduction in fertility after fixed-time artificial insemination. All steps of embryo production, freezing and transfer are now controlled and allow the attainment of satisfactory numbers of kids born per donor female, which are compatible with the development of the technique for exchanging genotypes between countries. In vitro production of embryos allows high development rates to be achieved after in vitro maturation and fertilization of oocytes, and will ensure the production of synchronous populations of one-cell zygotes at the stage required by new biotechnologies.