Maedi-Visna virus was detected in association with virally exposed IVF-produced early ewes embryos.

The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection. Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8-16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks. This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos. This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 10(3) TCID(50)/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier.

First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning.

Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats. Oocytes were enucleated and coupled to bucardo's fibroblasts by electrofusion. Reconstructed embryos were cultured for 36h or 7d and transferred to either Spanish ibex or hybrid (Spanish ibex malex domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardo's donor cells. To our knowledge, this is the first animal born from an extinct subspecies.

Heterologous in vitro fertility evaluation of cryopreserved Iberian red deer epididymal spermatozoa with zona-intact sheep oocytes and its relationship with the characteristics of thawed spermatozoa.

A heterologous in vitro system, using zona-intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37 degrees C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (approximately 80, 80 and 70% respectively). However, these values significantly decreased after incubation (approximately 59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona-intact sheep oocytes, although the percentage of cleaved oocytes was low (approximately 22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona-intact sheep oocytes.

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.

Measurement of ovine pregnancy-associated glycoprotein (PAG) during early pregnancy in Lacaune sheep.

This study describes ovine pregnancy-associated glycoprotein (ovPAG) concentrations in 20 Lacaune sheep during early pregnancy. Measurements were performed by using semi-purified ovPAG as standard, tracer and immunogens for antibody production in rabbits. Antisera R780 (against ovPAG(57+59kDa)) and R805 (against ovPAG5(58+61kDa)) were used respectively in RIA-780 and RIA-805. Blood samples were collected at days 0, 18, 20, 22 and 25 after artificial insemination. From day 18 after breeding onward, the mean ovPAG concentration was significantly higher (p < 0.001) in plasma samples from pregnant ewes (n = 17) than in non-pregnant ones (n = 3). The specific activity of the tracer was 11 760 Ci/mmol in RIA-780 and 14 900 Ci/mmol in RIA-805. The minimal detection limits for RIA-780 and RIA-805 were 0.2 ng/ml and 0.3 ng/ml, respectively. The intra-assay CV of samples with low (1.0 ng/ml), medium (2.5 ng/ml) and high (4.0 ng/ml) PAG concentrations were 3%, 6% and 9% for RIA-780 and 8%, 9% and 5% for RIA-805. The inter-assay CV in the same samples were 13%, 12% and 7% for RIA-780 and 13%, 11% and 5% for RIA-805. The recovery was higher than 95% in both assays. No cross-reaction was observed with members of aspartic proteinase family as well as with other tested proteins. In both RIA-780 and RIA-805, inhibition of the binding of the tracer by antisera was parallel between standard curve and serial dilutions of pregnant ewe samples. In conclusion, the two homologous RIA systems are suitable for early quantification of ovPAG concentrations in ewe plasma samples from day 18 after breeding.

Laparoscopic ovum pick-up and in vitro production of sika deer embryos: effect of season and culture conditions.

Amongst the 200 deer subspecies worldwide, more than 40 are considered as endangered. In vitro embryo production may represent an efficient way to produce and disseminate offspring from sparse remaining individuals in these species. With a view to establishing a method of in vitro embryo production, we assessed the ovarian response after hormonal stimulation (oFSH), oocyte yield following laporoscopic ovum pick-up (LOPU) and oocyte developmental competence according to seasonal reproductive status in sika deer (Cervus nippon nippon). Twelve adult sika deer hinds were allocated between two groups and submitted weekly to oFSH follicular growth stimulation followed by LOPU. Hinds in Group A (n=6) were treated first during the breeding season (5 weeks), and then during the non-breeding season (3 weeks). Hinds in Group B (n=6) were submitted to similar procedures but in the reverse order (treated first during the non-breeding season). Cumulus-oocytes complexes (COC) recovered from Group B were allowed to mature in vitro for 24 h in TCM-199 medium supplemented with oFSH, goat follicular fluid and 100 microM cysteamine. In vitro fertilization was performed with frozen/thawed semen in SOFaa medium supplemented with 20% estrous sheep serum and presumptive zygotes were cultured in the presence or absence of ovine oviductal epithelial cell monolayer (oOEC) in SOFaa-BSA medium. Mean number of follicles aspirated per hind per session decreased significantly between breeding and non-breeding season in Group A (9.8+/-0.7 versus 3.2+/-0.7, mean+/-S.E.M., respectively, P<0.001) but did not change between the non-breeding and the subsequent breeding season in Group B (5.3+/-0.7 and 5.7+/-0.7, respectively, P>0.05). Irrespective of the season, good quality COC with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Whereas development to the blastocyst stage did not occur in SOF medium alone, high development rates to the blastocyst stage were observed in oOEC co-culture regardless of season (22% and 34% of total oocytes in co-culture during non-breeding and breeding season, respectively).

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.

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.

Experimental infection of Black Belly and INRA 401 straight and crossbred sheep with trichostrongyle nematode parasites.

Compared to INRA 401 lambs reared in France, Black Belly (BB) lambs reared in Guadeloupe (F.W.I.) were highly resistant to both primary and secondary experimental infection with Haemonchus contortus. To investigate this huge inter-breed difference, a nucleus flock of BB was constituted, and experiments were conducted to: (i) confirm this difference in lambs born in France, (ii) check whether it was similar for Trichostrongylus colubriformis and Teladorsagia circumcincta, and (iii) find out whether this difference was age-related. Forty BB lambs, 84 F1 lambs (BB siresxINRA 401 ewes) and 88 INRA 401 lambs born in two cohorts were used in an experimental design involving three host breeds, both genders and two age-groups (3.5- and 7-month-old when first infected). The limited availability of BB lambs made the study incomplete. Infection consisted of the administration of two doses of 10,000 infective larvae of one of the nematode species, separated by an anthelmintic treatment and an interval of 1 week before the second dose was administered. Fecal egg counts (FECs) were done on Days 28 and 35 after each infection; ewe lambs of the INRA 401 and F1 breeds were necropsied, the worm burden was established, the length of the female worms measured and the eggs in utero counted. For H. contortus and T. colubriformis, the FEC was lower in the BB than in the INRA 401 lambs, and the FEC found for the F1 lambs was intermediate. For T. circumcincta, only data for the F1 and INRA 401 lambs were available, and the FECs for these breeds were the same. In all three breeds, the FEC determined after the second dose was significantly lower than that found after the first dose in the ewe lambs, but not in the ram lambs. Infecting the lambs when they were 7- instead of 3.5-month-old significantly lowered egg excretion of both parasites in the F1 (P<0.0001), but not the INRA 401 lambs. Worm numbers in the F1 and in the INRA 401 ewe lambs confirmed the FEC data, many F1 lambs being free of T. colubriformis. The H. contortus female worms were shorter and had fewer eggs in utero in the F1 than in the INRA 401 lambs. A higher proportion of T. circumcincta was at the fourth larval stage in the F1 lambs. In conclusion, the BB breed is much more resistant than INRA 401 to H. contortus, with the F1 lambs being more like the BBs after the second dose. This difference was also found for T. colubriformis and, to a lesser extent, for T. circumcincta.

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.

Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes.

Ewes from the Booroola strain of Australian Mérino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecB(B) allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22-23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-beta (TGF-beta) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecB(B)/FecB(B) ewes were less responsive than granulosa cells from FecB(+)/FecB(+) ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecB(B)/FecB(B) ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles.

Successful in vitro production of embryos in the red deer (Cervus elaphus) and the sika deer (Cervus nippon).

The aim of our study was to define the conditions for IVM and IVF of oocytes in 2 common deer species as models for endangered related subspecies. Immature oocytes were recovered during the breeding season from postmortem ovaries (red deer) or by repeated laparoscopic follicular aspiration (sika deer). Oocytes were cultured for 24 h in IVM medium supplemented with EGF or FSH and follicular fluid. Stag semen was collected by electroejaculation (both species) or by epididymal flushing (red deer) and cryopreserved. For IVF, oocytes were exposed to different concentrations of thawed spermatozoa in a modified Tyrode albumin lactate pyruvate medium supplemented with 20% (v/v) estrus sheep serum for 18 h. After IVF, presumptive zygotes were allowed to develop in vitro for 7 days in synthetic oviduct fluid (SOF) supplemented with fetal calf serum (10%, v/v). In both species, the presence of ovine FSH and follicular fluid improved the in vitro maturation rate. In the sika deer, the optimal sperm concentration for IVF was 10(6)/mL and some fertilized oocytes reached the early morula stage (20 to 25 cells). In the red deer, after IVF with ejaculated or epididymal spermatozoa (2.0 x 10(6)/mL), 20% of zygotes developed to the blastocyst stage (50 to 80 cells).

Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes.

The objective of these experiments was to determine the effect of exogenous addition of insulin-like growth factor-I (IGF-I, 100 ng/mL), epidermal growth factor (EGF, 10 ng/mL) and estradiol (E2, 100 ng/mL) to the maturation medium of sheep oocytes on their subsequent development in vitro. Addition of IGF-I to the maturation medium did not improve nuclear or cytoplasmic maturation of sheep oocytes at the concentration tested. However, EGF improved significantly the resumption of meiosis (84% oocytes in metaphase II stage after IVM vs. 59% in medium alone). Cleavage rate and blastocyst development rates were improved (P<0.01) after addition of EGF (60% and 29%, respectively), as compared with maturation in TCM 199 alone (39% and 19%, respectively), but remained lower than rates observed after maturation in complete medium containing follicular fluid (FF, 10%) and FSH (81% and 35%, respectively). No additive effect of EGF over FSH was observed during these experiments. Addition of FF to FSH containing maturation medium improved significantly both cleavage (P<0.001) and blastocyst rates (P<0.05). Addition of E2 to the IVM medium is not required when medium already contains FF. However, in defined conditions supplementation of maturation medium with E2 had a positive effect. These results suggest that EGF, FSH and E2 may play an important role in the nuclear and cytoplasmic maturation of sheep oocytes in vitro.

Effects of the Booroola Fec gene on ovarian follicular populations in superovulated Romanov ewes pretreated with a GnRH antagonist.

Endocrine control of follicular growth was studied in mature Romanov ewes carrying (RF+) or not carrying (R+2) the Booroola Fec gene during an oestrous cycle after gonadotrophin-dependent follicles were suppressed by treatment with an antagonist of GnRH (Antarelix, 0.5 mg per day) and superovulatory treatment was administered. The left ovary was removed after 10 days of treatment (saline or Antarelix) and the right ovary was removed at the end of the superovulatory treatment. Ewes of both genotypes treated with Antarelix had lower plasma LH concentrations than did controls from day 0 to day 10. The inhibitory effect of Antarelix on LH concentration increased with day of treatment. The variability in FSH concentrations during the initial 10 days was reduced by Antarelix treatment in both genotypes. Plasma FSH concentrations were higher in RF+ ewes than in R+2 ewes. In both genotypes, FSH concentrations varied significantly with day of treatment, with the lowest concentrations at day 8 and the highest concentrations at day 5. RF+ ewes had a greater total and atretic number of antral follicles 0.62-1.12, 1.12-2.00 and 2.00-3.00 mm in diameter (classes 2, 3 and 4) than did R+2 ewes before and after superovulatory treatment. After superovulatory treatment, the total number of atretic and non-atretic follicles > 3.00 mm in diameter (class 5) increased in both genotypes. Superovulatory treatment also increased the number of total and atretic class 4 follicles in RF+ only. Conversely, superovulatory treatment decreased the mean number of class 3 follicles in both genotypes, while the number of atretic follicles was decreased only in R+2 ewes. Antarelix treatment significantly reduced the percentage of follicles > 2.00 mm in diameter in RF+ but not in R+2 ewes. Antarelix treatment before superovulatory treatment increased the total number of class 4 follicles in both genotypes but the increase was more significant in RF+ than in R+2 ewes. These results indicate that Antarelix pretreatment favours a greater superovulatory response in Romanov ewes carrying the Fec gene because ovulatory follicles are recruited from a wider range of follicular size classes.

Effect of GnRH antagonist-induced prolonged follicular phase on follicular atresia and oocyte developmental competence in vitro in superovulated heifers.

A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.

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.

Aspects of follicular and oocyte maturation that affect the developmental potential of embryos.

The ability to mature, be fertilized and finally to develop into a viable embryo is acquired gradually by the oocyte during progressive differentiation throughout folliculogenesis. This process starts with oocyte growth during the first steps of follicular development. As the oocyte is close to its final size, other modifications occur, less spectacular but at least as important in determining the resulting ability of the oocyte to accomplish its reproductive purpose (developmental competence). These modifications, referred to as 'oocyte capacitation', are probably influenced by the follicle. The proportion of developmentally competent oocytes increases with follicular size. However, the relationship between follicular growth and oocyte competence is not very strict, since a given oocyte may acquire its competence at any stage of follicular growth and since some examples of functional disjunction between follicular size and oocyte competence are described. Follicular atresia may impair the acquisition of oocyte competence, as evidenced by the parallel study of follicular characteristics and of the developmental potential of their oocytes treated individually through in vitro maturation, fertilization and development. However, when atresia is experimentally induced in large preovulatory follicles, oocytes remain competent, indicating that once competence is acquired, it is no longer sensitive to atresia. Oocyte maturation represents only the end of this long and progressive process and validates the preparation of the oocyte by conferring its final developmental ability. As evidenced by recent cloning experiments, the cytoplasmic aspects of oocyte maturation are crucial for the acquisition of developmental competence. This cytoplasmic maturation may be activated in vitro by the use of complex media supplement (serum, follicular fluid) but the use of defined media for maturation allowed the identification of some active factors (such as epidermal growth factor, growth hormone, inhibin and activin). The study of some differential models of oocyte competence (follicular size and atresia, Booroola gene, prepubertal oocytes) will provide a better understanding of oocyte capacitation and maturation, and allow the improvement of in vitro methods for oocyte maturation, which represent the most limiting step of in vitro production of embryos in large mammals.

Reduction of the developmental competence of sheep oocytes by inhibition of LH pulses during the follicular phase with a GnRH antagonist.

A GnRH antagonist (Antarelix) treatment was used during the breeding season of Romanov ewes, to investigate whether LH pulses are required the day before the preovulatory surge for normal early embryo development in vivo (Expt 1) and in vitro (Expt 2). In Expt 1, at the onset of oestrus after removal of a fluorogestone acetate sponge, group A0.5 (n = 22) received a subcutaneous injection of 0.5 mg Antarelix, and ovulation was induced with an intravenous injection of 3 mg pig LH 24 h later. The control group (group C, n = 20) were untreated. All ewes were mated naturally at 36 and 48 h after oestrus and embryos were recovered 8 days after sponge removal. There were significant differences in the decrease in LH and in the increase in FSH concentration after Antarelix treatment between treated and control groups. The ovulation rate and embryo recovery rate were not significantly different between the two groups but the blastocyst rate was lower (P < 0.0001) in group A0.5 than in group C, with more unfertilized or degenerated oocytes in group A0.5 (69.2%). In Expt 2, 24 h after sponge removal, group A (n = 10) and group B (n = 10) received one subcutaneous injection of 0.5 mg Antarelix. The control group (group C, n = 10) was left untreated. LH pulsatility was re-established in group B with hourly intravenous injections of 5 micrograms ovine LH for 24 h. Oocytes were collected by flushing the oviducts 28 h after the LH surge, and were fertilized and cultured in vitro for 7 days. Ovulation and cleavage rates were not significantly different among the three groups but a higher rate of blastocysts (P < 0.01) was obtained after Antarelix treatment when LH pulsatility was re-established (group B). Oestradiol concentration was strongly depressed (P < 0.0003) after Antarelix treatment in group A, but was maintained after injection of LH pulses in group B, although at a lower value than before the preovulatory surge in the control group. In conclusion, inhibition of endogenous LH pulses 1 day before the preovulatory surge was not essential for ovulation and in vitro fertilization but was associated with a decrease in plasma oestradiol concentrations and inferior embryo development both in vivo and in vitro. When LH pulsatility was re-established, oestradiol concentrations increased and embryo development was restored.

State of the art in sheep-goat embryo transfer.

Considerable advances have been made in the last 25 yr in sheep and goat embryo production and transfer technology. This presentation covers the procedures used to overcome the variability of ovarian response after treatment with exogeneous gonadotropins, the asynchrony of ovulations, failure of fertilization in females showing a high ovulatory response, and the side-effects of repeated treatments (surgical trauma, gonadotropins and their antibodies). In the ewe, prior antigonadotrophic pretreatment results in a significant gain in ovulation rate due to the elimination of nonresponses and in a two-fold increase in embryo yield. A better comprehension of the relationships between oocyte quality and follicular characteristics after superovulation can be gained using in vitro techniques. This knowledge will subsequently be used for the optimization of embryo production needed for the genetic improvement of livestock and the development of new biotechnologies.