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In guinea pigs (Cavia porcellus), dystocia is a common occurrence. Several factors have been identified in the literature, including the ossification of the pubic symphysis following failure to breed before 9-12 months of age. The objective of this study was to investigate the ossification of pubic symphysis and its evolution during growth in two groups of females. The first group consisted of non-breeding females, while the second group comprised females introduced to breeding at 4-6 months of age. Twelve pairs of sows were selected for comparison, with one non-breeding and one breeding sow in each pair. Symphysis width and tissue density were assessed using micro-computed tomography. Measurements included the distance between the acetabula, width and bone density of the pubic symphysis. Serial computed tomography scans were performed on each sow over several months, both before and after parturition. The results revealed a significantly higher symphysis width in females that had bred. In addition, symphysis ossification was absent in both breeding and non-breeding sows, contrary to previous descriptions of this species. Therefore, dystocia in guinea pigs may not be attributable to ossification of the pubic symphysis., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 °C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations ( p ≤ 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations ( p ≤ 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant ( p ≤ 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity.

ProAKAP4 is identified within the flagellum of spermatozoa in various mammalian species, serving as a structural protein associated with motility parameters. This investigation focuses on the presence of proAKAP4 in donkey sperm, elucidating its localization, molecular characteristics, and its correlation with motility descriptors and mitochondrial membrane potential. Twelve ejaculates from Catalan donkeys were analyzed in this study. The initial steps involved proAKAP4 sequencing and detection through Western blotting and immunofluorescence. Post-thaw assessments were conducted at 0, 1, and 3 h, encompassing proAKAP4 levels, sperm motility analyzed via Computer-Assisted Sperm Analysis (CASA), and mitochondrial membrane potential determined by flow cytometry using the JC-1 stain. The findings reveal that proAKAP4 in donkeys exhibits a characteristic localization at the principal piece of the flagellum, consistent with observations in other mammals. The molecular weight of proAKAP4 is determined to be 100 kDa. Significantly, a positive correlation (p ≤ 0.05) is established between proAKAP4 concentration and both total and progressive motility. The presence of cryoprotectant is associated with a lower proAKAP4 concentration. Notably, proAKAP4 experiences a substantial decrease (p ≤ 0.05) during the initial hour post-thawing. In conclusion, proAKAP4 is identified in donkey sperm, akin to its presence in other mammals. It exhibits a positive correlation with total and progressive motility, its concentration is notably affected by the presence of cryoprotectant with significant consumption observed during the initial hour following thawing. These findings contribute to our understanding of proAKAP4 dynamics in donkey sperm, providing insights that may have implications for semen preservation and reproductive technologies in equids., Competing Interests: Declaration of Competing Interest Maryse Delehedde and Nicolas Sergeant, co-authors of this manuscript, are cofounders of SPQI., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abortions in horses represent an important health and economic challenge for equine industry. Primary causes of abortion are divided in non-infectious and infectious. Non-infectious causes include abnormalities of foetal appendices (umbilical cord and placenta essentially), abnormalities of gestation, maternal and foetal origins. Infectious abortions are caused in almost cases by bacterial infections, followed by viruses, fungi and parasites. New abortive pathogens (as Leptospira, Neospora caninum, Coxiella burnetii, Chlamydophila abortus, and) have been confirmed in equines by comparison already known for their abortive properties in human or in other species. Despite an increasing number of autopsies and continuous improvements in diagnostic tools, in management and surveillance, 20%-40% of the causes of equine abortion remain unknown depending on the country. To increase the likelihood of a definitive diagnosis in cases of abortion and stillbirth in horses, new diagnostic approaches are needed., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06−35.61 ng/10M spz; n = 23) or high (39.92−82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen.

A table was generated, based on foetal ultrasonographic measurements in light breed mares, for each day of gestation beginning with day 100, to provide the predicted value of four biometric parameters: biparietal diameter (BPD), eye approximated volume (EyV), foetal aortic diameter (AortD) and femur length (FL). Using this table, day of gestation was successfully predicted in 23 Quarter Horses (QH) with known mating or ovulation dates. BPD, EyV and FL were the best foetal age predictors between 100- and 200-days gestation predicting within 2 weeks of the actual day of gestation, while BPD and EyV were best between 200 and 300 days (within 3 weeks), and EyV was best after 300 days (within 3 weeks)., (© 2022 Wiley-VCH GmbH.)

Functional sperm quality markers to predict bull fertility have been actively investigated. Among them, proAKAP4, which is the precursor of AKAP4, the main structural protein in the fibrous sheath of spermatozoa; appears to be promising, especially since spermatozoa lacking AKAP4 expression were shown to be immotile, abnormal, and infertile. In this study, the objective was to evaluate proAKAP4 concentration values with the classic sperm motility descriptors and fertility outcomes (NRR at 90 days) in post-thawed conditions of 10 bulls' semen. ProAKAP4 expression was confirmed by Western blotting and proAKAP4 concentrations were determined by ELISA. Variations in proAKAP4 concentrations were observed independently of the motility sperm descriptors measured using computer-assisted semen analysis (CASA). A ProAKAP4 concentration of 38.67 ± 8.55 ng/10 million spermatozoa was obtained as a statistical mean of all samples. Threshold values of proAKAP4 were then determined between 19.96 to 96.95 ng/10 million spermatozoa. ProAKAP4 concentrations were positively correlated with progressive motility and the linearity coefficient. The sperm showing the lowest progressive motility were the samples exhibiting proAKAP4 concentrations below 20 ng/10 million spermatozoa. Furthermore, proAKAP4 concentrations were significantly higher in bulls with a higher NRR in the field. Our results demonstrate a correlation between the semen concentration of proAKAP4 and NRR-90d ( p = 0.05) in post-thawed bull semen, highlighting the potential of proAKAP4 as a predictive marker of bull fertility.

The aim of this study was to assess the efficacy of different doses of buserelin acetate and another GnRH agonist, triptorelin acetate, in saline solution in a single subcutaneous injection, to induce ovulation of growing pre-ovulatory follicle in mare and compare it with the classical treatment of a single injection of hCG. The study is split into 3 experiments over different breeding seasons in the same stud with a random distribution of treatment. The first one was to compare the injection of 6 mg of buserelin with 1,500 IU of hCG; the second one consisted of comparing different doses of buserelin (6 mg and 3 mg); and the third one compared three different doses of buserelin (3, 2 and 1 mg), 0.1 mg of triptorelin with 1,500 IU of hCG as a control group. The results of all experiments showed the same efficacy between all treatments with mares ovulating between 24 and 48 hr after injection: experiment 1: hCG (78% n = 41) and buserelin 6 mg (90% n = 50); experiment 2: buserelin 6 mg (78,1% n = 192) and buserelin 3 mg (78% n = 341); and experiment 3: hCG (87% n = 106), buserelin 3 mg (84,7% n = 137), buserelin 2 mg (82,7% n = 104), buserelin 1 mg (87% n = 54) and triptorelin 0.1 mg (84,7% n = 72). In conclusion, this study contributes to erasing the dogma that has been established since 1975 that a single injection in solution without any long-acting excipient of a GnRH agonist cannot induce ovulation in the mare. This study also shows that a injection of 0.1 mg of triptorelin in solution is a good alternative for ovulation induction and is comparable to small doses of buserelin acetate in solution (1 mg) and 1,500 IU of the gold standard trigger hCG, mainly in countries where human formulation of buserelin is not available., (© 2020 Blackwell Verlag GmbH.)

Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n = 31-39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pregnancies (5/7 for one, 11/12 for the other) were obtained at day 14 post-ovulation (D 14 ), and 12 (3/7 for one; 9/12 for the other) foals were born. One cloned mare was a less efficient donor mare than the other (p < .05), In control donor mares, 623 embryo collections were performed, with a recovery rate (80%-496/623) significantly higher than for cloned mares. The recovery rate in the subpopulation of 2-5-year-old control donor mares (same age of cloned mares) (89%-127/143) and The recovery rate in the subpopulation of 12 control mares bred with the seven same stallions as clones (55%-17/31), were both higher than for cloned mare (p < .05). The success rate of transfer was not different between embryos produced by cloned mares (84%-16/19) and those produced by control donor mares (79%-392/496). However, the foaling rate per embryo collection was significantly lower for cloned mares (28%-12/43) than for control donor mares (52% - 325/623) (p < .05)., (© 2019 Blackwell Verlag GmbH.)

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. The experimentation was made twice. For each replicate 100 (8-16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 10 7 Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO 2 , the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000×g. Each batch of washed embryos and each wash pellets were tested using PCR. C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ± 9 × 10 3 bacteria/mL) than for batches of ZP-intact embryos (0.47 ± 0.19 × 10 3 bacteria/mL). These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 10 9 Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria. C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches. After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration. This study clearly demonstrates that C. burnetii, after in vitro infection at 10 9 Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Since the availability on the European market of the vaccine Improvac® dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone-releasing hormone or gonadotropin-releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvac® vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti-GnRH antibodies was detected up to 200 days after the first injection. Anti-GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme-linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10(7)Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main source of infection for humans. The objectives of this study were to determine (1) whether C. burnetii would adhere to the intact zona pellucida (ZP-intact) of early in vitro-produced bovine embryos; (2) whether the bacteria would adhere to or infect the embryos (ZP-free) after in vitro infection; and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. One hundred and sixty, eight- to 16-cell bovine embryos produced in vitro, were randomly divided into 16 batches of 10 embryos. Twelve batches (eight ZP-intact and four ZP-free) were incubated in a medium containing C. burnetii CbB1 (Infectiologie Animale et Santé Publique, Institut National de Recherche Agronomique Tours, France). After 18 hours of incubation at 37 °C and 5% CO2 in air, the embryos were washed in 10 successive baths of a PBS and 5% fetal calf serum solution in accordance with the IETS guidelines. In parallel, four batches (two ZP-intact and two ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. Ten washing fluids from each batch were collected and centrifuged for 1 hour at 13,000× g. The embryos and wash pellets were tested using conventional polymerase chain reaction. C. burnetii DNA was found in all ZP-intact and ZP-Free embryos after 10 successive washes. It was also detected in the first four washing fluids for ZP-intact embryos and in the 10th wash fluid for two of the four batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adheres to and/or penetrates the early embryonic cells and the ZP of in vitro bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP and to verify if similarly results are obtained with in vivo-derived embryos., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The detection of significant bacterial loads of Coxiella burnetii in flushing media and tissue samples from the genital tracts of nonpregnant goats represents a risk factor for in utero infection and transmission during embryo transfer. The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo-fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and polymerase chain reaction, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9-9, 11-11, and 4-4 in replicates 1, 2, and 3, respectively) were placed in 1 mL minimum essential medium containing 10(9)C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37 °C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3-3, 5-5, and 2-2 in replicates 1, 2, and 3, respectively) were subjected to the same procedures, but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 hour at 13,000 × g. The washed embryos and pellets were tested by polymerase chain reaction. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first five washing fluids for ZP-intact embryos and in the first eight washing fluids for ZP-free embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly indicate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to adhere to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The aim of the present study was the detection and quantification of Coxiella burnetii DNA in the flushing media (oviducts and uterine horns) and genital tract tissues of non pregnant goats from 20 goats chosen at random from 86 goats originating from 56 different breeding herds in south-west France. The serological prevalence rate of C. burnetii in the study population was 70.3%. The DNA of C. burnetii was identified using conventional PCR in the flushing media from the oviducts and uterus in 8/20 goats (40%) and in genital tract tissues (oviduct, uterus and ovary) in 5/20 goats (25%). This study clearly shows for the first time that the media used to flush the oviducts or uterine horns, collected using the standard embryo harvesting technique in goats, are susceptible to infection with C. burnetii. The 16 conventional PCR-positive samples were also analyzed using real-time PCR. The bacterial load of the oviduct and uterine flushing media varied from 2.9×10(4) to 7.5×10(6) bacteria per flushing medium, while the bacterial load of the tissue samples varied from 1.0×10(2) to 1.5×10(5) bacteria per mg of tissue. The infection of genital tract flushing media and tissues is a risk factor for the transmission of C. burnetii from donor to recipient during embryo transfer or to the embryo and fetus when gestation is pursued to term., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pyometra in an inguinal hernia was diagnosed in a 10-year-old intact cross-bred bitch which had had dysorexia, depression and inguinal distension. The hernia contained caudal portions of the two uterine horns, uterine cervix and cranial part of the vagina. As the organs were enlarged and full of pus, manual attempt to push back the uterine horns and the vagina in the abdominal cavity through the inguinal canal was unsuccessful. Herniated uterine horns were ligated and cut in their median portion, so it became possible to remove the cervix and the caudal portion of the horns through the hernial orifice, and the ovaries and the cranial part of the horns through a peritoneal midline incision. This bitch was not intended for breeding purposes and, given the presence of a huge pyometra associated with an inguinal hernia, an ovario-hysterectomy was recommended. Uterine herniation should be considered as a differential diagnosis of a caudal lateral inguinal mass. When pushing the uterus back in the abdominal cavity is impossible, a surgical procedure should be performed to detect ischemia–reperfusion injury and/or a septic risk.

In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility. Samples of stallion semen (n=390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test. EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p<0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs. There was a significant difference (p<0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing. In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.

The objective of this study was to determine whether the 10 wash cycles proposed by the International Embryo Transfer Society (IETS) for bovine embryos efficiently decontaminated equine embryos exposed to equine herpes virus 1 (EHV-1) in vitro. Donor mares and stallions were individually screened and shown to be negative for the virus by PCR detection of EHV-1 DNA in blood leukocytes, semen, and uterine lavages in which embryos were recovered. Twenty embryos were recovered and randomly assigned to one of two groups: 10 embryos were exposed for 24h to infectious EHV-1 at 10(6)TCID(50)/ml, and 10 embryos were used as negative controls. Exposed embryos were washed in accordance with IETS recommendations for ruminant and porcine embryos, before being incubated for 24 h with semiconfluent rabbit kidney (RK13) cells to detect any cytopathic effects (CPE), and finally tested for the presence of EHV-1 viral DNA by PCR. The embryo washing media were also assayed for the virus on RK 13 cells and by PCR. Control embryos were neither exposed to the virus nor washed. EHV-1 was not found in the control embryos, or in the last five washes of the exposed embryos. However, the virus was detected in 7/10 of the embryos exposed to EHV-1 for 24h, as well as in the first five washes of the embryos. The gradual disappearance of EHV-1 from the 10 successive wash solutions from the exposed embryos and the detection of viral DNA in 7/10 washed embryos by PCR, demonstrated that the washing procedure was unable to remove EHV-1 and suggested that EHV-1 could be attached to the acellular layer surrounding embryos (zona pellucida or capsule) or had penetrated the embryo.

Rules of horse racing stipulate that pregnant mares may compete under definite conditions of date, because early pregnant status may be misused for the sake of enhancing physical performance by putative anabolic steroid action. Screening for pregnancy is generally performed by plasma equine gonadotrophin (eCG) immunoassay, which covers the period between Days 40 and 120. In common screening for urinary anabolic steroids performed by gas chromatography-mass spectrometry, inclusion of two complementary criteria, i.e. the evaluation of total conjugates of 5(10)-estrene-3beta,17alpha-diol (EED) and estrone (E1), can easily be performed. Although EED and E1 have no anabolic property per se in the horse, assessing these two markers may be helpful in the period comprised between Days 70 and 250, thereby prolonging the detection period behind that of eCG. Peak values of EED and E1 are then attained, so that visual inspection of chromatographic tracings remains in general sufficient as a diagnostic tool. Comparison of EED and E1 during pregnancy and in an estrus cycle indicates a drastic difference in the attained excretion values, attributable to either the placenta or the ovarian follicle. The identity of EED has been proven by GC-MS(n) in urine and in placental tissue.

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.

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.

The cryoprotective effect of l-glutamine and an approach of its mechanism of action, in preserving motility of stallion spermatozoa during the freezing-thawing process, were studied. In Experiment 1, thirty-six ejaculates were collected from six stallions (two good, two middle, and two of poor sperm freezability) and were diluted with 10 different freezing media derived from INRA 82 medium supplemented with 20 mM HEPES and 2% (v/v) centrifuged egg yolk (BM). After thawing, sperm motility was evaluated by a computer-assisted semen motility analyser. The effects of glutamine and glycerol at different concentrations on post-thaw sperm motility were studied. A possible interaction between medium and semen freezability was investigated. Only the 50 mM glutamine + 2.5% glycerol medium significantly improved sperm motility compared to classical freezing medium (2.5% glycerol). The presence of glutamine at 50 mM was not sufficient to offset the need to use glycerol in the freezing extender. The use of glutamine at a higher concentration >100 mM in the presence of 2.5% of glycerol was toxic. Reducing the glycerol proportion from 2.5% to 2 or 1.5% in the presence of glutamine at 50, 75, and 100 mM had no influence on post-thaw motility of semen of middle and good freezability. Moreover, the substitution of 2.5% glycerol by 50 mM glutamine in BM, did not significantly change the post-thaw motility of semen of good freezability. In Experiment 2, 3H-glutamine and 3H-glycerol were used to study the kinetics of penetration of glutamine and glycerol in sperm cells. The radioactivity of each radio-labelled semen pellet was measured at different times (0, 15, 30, 60, 90, 120 min), by using a Packard tri-carb 4530 apparatus. The percentages of incorporated radioactivity (%IRA) in semen pellets were calculated at different times. The %IRA of 3H-glycerol in semen pellets were significantly higher than the %IRA of 3H-glutamine. The %IRA of 3H-glycerol in semen pellets increased greatly from time 0 to 60 min, and then it is stabilized from 60 to 120 min. In contrast, the %IRA of 3H-glutamine in semen pellets increased slightly from 0 to 60 min, then it stabilized until 120 min. These experiments demonstrate that glutamine has a synergistic cryoprotective effect with glycerol on cryopreservation of stallion spermatozoa, and suggest that glutamine acts at the extra-cellular level, independently of glycerol.

The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fragmentation (TUNEL). Finally, all the fixed embryos were re-stained with DAPI to determine the total number of cells. The percentage of cells stained with both TUNEL and DAPI or TUNEL-only or DAPI-only were determined. The percent of dead cells (DAPI-labelled) per embryo increased with duration of storage, but no differences were detected between the storage media. The percentage of early apoptotic cells (TUNEL+/DAPI-) in fresh and stored embryo for 6 h or 24 h did not differ significantly (P > 0.05). There was a significant correlation between the percentage of cells labelled by TUNEL and DAPI (R = 0.87) (P < 0.001). These results suggest that cooled storage increases cell death but this does not appear to occur by induction of apoptosis and that DAPI staining proves to be a quick and reliable method for assessing embryo viability.

Equine embryos have been successfully transferred after 24h cooled storage in Ham's F-10. The aim of this study was to compare the viability of equine embryos in vitro and in vivo after 6 and 24h cooled storage using three media and to examine the relationship between embryo size and viability after 24h cooled storage. In Experiment 1, the viability of embryos was evaluated using DAPI-staining after 0, 6 or 24h in Ham's F-10, 24h in Emcare embryo holding solution (EHS) or 24h in ViGro holding plus (VHP) (n=10/group). The mean number of dead cells was similar for embryos stored in Ham's F-10, EHS and VHP for 24h. Larger Day 7 embryos appear to withstand 24h cold storage better than small Day 7 embryos. The embryo quality for 24h cold storage was negatively correlated with size. In Experiment 2, 40 embryos were stored (n=20/group) either in Ham's F-10 or in EHS then transferred as pairs in recipient mares. Fifteen of the 20 recipient mares (75%) were pregnant. Out of 17 surviving embryos, 9 embryos (53%) were stored in Ham's F-10 and 8 (47%) in EHS. These results suggest that EHS and VHP offer a good alternative to Ham's F-10 for 24h cooled storage of equine embryos and that larger embryos may have a better viability after 24h of cooled storage than smaller embryos.

The aim of this study was to investigate whether cells of early goat embryos isolated from in vivo-fertilized goats interact with the caprine arthritis-encephalitis virus (CAEV) in vitro and whether the embryonic zona pellucida (ZP) protects early embryo cells from CAEV infection. ZP-free and ZP-intact 8-16 cell embryos were inoculated for 2 h with CAEVat the 10(4) tissue culture infectious dose 50 (TCID50)/ml. Infected embryos were incubated for 72 h over feeder monolayer containing caprine oviduct epithelial cells (COECs) and CAEV indicator goat synovial membrane (GSM) cells. Noninoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. Six days postinoculation, infectious virus assay of the wash fluids of inoculated early goat embryos showed typical CAEV-induced cytopathic effects (CPE) on indicator GSM monolayers, with fluids of the first two washes only. The mixed cell monolayer (COEC + GSM) used as feeder cells for CAEV inoculated ZP-free embryos showed CPE. In contrast, none of the feeder monolayers, used for culture of CAEV inoculated ZP-intact embryos or the noninoculated controls, developed any CPE. CAEV exposure apparently did not interfere with development of ZP-free embryos in vitro during the 72 h study period when compared with untreated controls (34.6 and 36% blastocysts, respectively, P > 0.05). From these results one can conclude that the transmission of infectious molecularly cloned CAEV-pBSCA (plasmid binding site CAEV) by embryonic cells from in vivo-produced embryos at the 8-16 cell stages is possible with ZP-free embryos. The absence of interactions between ZP-intact embryos and CAEV in vitro suggests that the ZP is an efficient protective embryo barrier.

Inducing ovulation in a cyclic mare is often necessary. For this purpose, hCG has been used commonly, but the response can be reduced after successive administrations. The aims of this study were to test the effectiveness of buserelin in hastening ovulation in estrus mares, and its influence on fertility; and to investigate the effect of treatment on LH secretion. Five crossover trials were designed to compare the effect of two treatments: buserelin (40 microg in 4 doses i.v. at 12 h intervals) vs placebo (Experiments 1 and 2); buserelin 40 microg (in 4 doses i.v.) vs 20 microg (Experiment 3); buserelin (4 doses of 20 microg i.v.) vs hCG (1 dose of 2,500 IU i.v.) (Experiment 4); or buserelin (3 doses of 13.3 microg at 6 h interval) vs hCG (Experiment 5). In Experiment 2, blood samples were taken hourly until ovulation, for LH measurements. In Experiment 1, buserelin treatment significantly hastened ovulation. Reduction of the dose by half (Experiment 3) did not alter the effectiveness. In Experiments 4 and 5, buserelin was as effective as hCG in inducing ovulation between 24 and 48 h after initiation of treatment. Buserelin treatment induced a rise in LH concentration during the 48 h period of the experiment, and LH concentrations before ovulation were significantly higher in buserelin treated cycles than in placebo cycles. These experiments demonstrated the usefulness of two new protocols of administration of buserelin, as an alternative to hCG for induction of ovulation. One hypothesis explaining the mechanism of action is that the persistant rise in LH concentration could modify the ratio of biological/immunological LH, as it occurs physiologically, thereby hastening ovulation.

Parturition was induced in ten Beagle bitches by injecting them subcutaneously with 15 mg aglepristone kg-1 (Alizine) at day 58 of gestation and 24 h later and subsequently at 2 h intervals with either 0.08 mg alfaprostol kg-1 (Alfabedyl) (group 1; five bitches) or 0.15 iu oxytocin kg-1 (Ocytocine S) (group 2; five bitches). Blood samples were collected every 4 h until the end of parturition to assay plasma concentrations of progesterone, dihydro-keto prostaglandin F2 alpha (PGFM), oxytocin, prolactin and cortisol. Parturition occurred in all bitches. The mean time of onset of parturition for both groups was not significantly different (32.6 +/- 3.7 h for group 1 versus 31.6 +/- 3.6 h for group 2), although the mean expulsion time for bitches from group 2 (4.5 +/- 1.8 h) was significantly shorter than that of bitches in group 1 (9.1 +/- 2.0 h). At birth, 93% of the pups were alive in group 2 compared with 86% in group 1. Peripheral plasma concentrations of progesterone increased significantly after the administration of aglepristone, but direct or indirect luteolysis was not induced, and plasma concentrations of oxytocin or cortisol did not change during the first 24 h after administration of aglepristone. PGFM concentrations increased significantly after 4 h of aglepristone administration. During the first 20 h after aglepristone administration, prolactin concentrations increased significantly. At parturition, bitches in group 2, which had the shorter expulsion time of pups, were characterized by significantly higher concentrations of oxytocin and PGFM than bitches in group 1.

Seven bitches in early pregnancy (12.8 +/- 3.8 days after ovulation; group 1) and seven bitches in mid-pregnancy (32.0 +/- 1.53 days after ovulation; group 2) were used in this study. For each group, five bitches were treated with 0.10 mg aglepristone (Alizine) kg-1 and this dose was repeated 24 h later. Two control bitches received a placebo. Blood samples were collected at 6 h intervals to determine plasma concentrations of progesterone, dihydro-keto prostaglandin F2 alpha (PGFM), oxytocin, prolactin and cortisol. Parturition occurred in the four control bitches. All bitches treated with aglepristone aborted. In group 1, embryonic death occurred; in group 2, fetal expulsion occurred 60-132 h after administration of aglepristone. After pregnancy termination, the interoestrous interval of aglepristone-treated bitches was significantly shorter than that before treatment. Treatment with aglepristone did not modify plasma concentrations of progesterone, prostaglandin, oxytocin or cortisol within 24 h after its administration, but it induced, in mid-pregnancy (group 2) a discharge of prolactin within 12 h after its administration. As an abortifacient, aglepristone acted on the uterus and, therefore, did not have direct or immediate luteolytic properties. Termination of pregnancy occurred with high plasma progesterone concentrations. Fetal expulsion was characterized by an increase in the concentration of PGFM, but oxytocin and cortisol remained at basal concentrations.

Equine embryos were collected at exactly 156 +/- 0.5 (n=8) and 168 +/- 0.5 h (n=11) after ovulation. The embryos were fixed in glutaraldehyde, sectioned serially and observed using light microscopy. In the 156 h group, all embryos were early blastocysts except for one, which was a morula. The morula and one early blastocyst had no capsule. The capsules of the other embryos were thin. The mean +/- SD total number of cells was 275 +/- 105 (range 117-417). The mean +/- SD proportions of mitotic and pycnotic cells were 2.5 +/- 1.2 and 1.1 +/- 1.8%, respectively, and there were no differences between each inner cell mass (ICM) and trophoblast. The mean +/- SD proportion of ICM cells was 36.5 +/- 5.2%. In the 168 h group, there were early, mid- and expanded blastocysts, all of which had a capsule. The mean +/- SD total number of cells was 1093 +/- 666 (range 272-2217). The mean +/- SD proportions of mitotic and pycnotic cells were 3.5 +/- 1.4% and 0.1 +/- 0.03%, respectively, and there were no differences between each ICM and trophoblast. The mean +/- SD proportion of ICM cells was 21.1 +/- 9.7%. In the 168 h group, there was a significant correlation (P < 0.01) between the total number of cells and the diameters and proportions of ICM cells. The 168 h embryos were composed of significantly (P < 0.01) more cells (approximately four times) than were the 156 h embryos but had lower proportions of ICM cells. These results indicate that in equine embryos there is: (i) an individual (perhaps seasonal) variability in the rate of development; (ii) a doubling in the number of cells every 6 h between 156 h and 168 h after ovulation; and (iii) a decrease in the proportion of ICM cells between the early and expanded blastocyst stages of development.

Early (day 6) equine embryos (n=23) were assigned to four treatment groups to assess the cryoprotectant properties of glycerol and ethylene glycol and the effect of adding sucrose during removal of the cryoprotectant: (i) group GG (n=5) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as the cryoprotectant, which was added at 22 degrees C in four steps (0.375, 0.75, 1.125 and 1.5 mol glycerol l(-1)), and removed after thawing in five steps (1.5, 1.125, 0.75, 0.375 and 0.0 mol glycerol l(-1)); (ii) group GS (n=6) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as for group GG, except that 0.25 mol sucrose l(-1) was added during removal of the glycerol; (iii) group EE (n = 6) embryos were frozen and thawed using 1.5 mol ethylene glycol l(-1) as the cryoprotectant, which was added in three steps (0.5, 1.0 and 1.5 mol ethylene glycol l(-1)) and removed after thawing in four steps (1.5, 1.0, 0.5, 0.0 mol ethylene glycol l(-1)); and (iv) group ES (n = 6) embryos were frozen and thawed using 1.5 mol ethylene glycol l(-1) as for group EE, except that 0.25 mol sucrose l(-1) was added during removal of the ethylene glycol. After thawing, the embryos were incubated at 37 degrees C in Ham's F10 medium supplemented with 10% (v/v) fetal calf serum and antibiotics, in 5% CO2 in air for 6 h. The embryos were fixed in glutaraldehyde, serially sectioned and observed using light microscopy. None of the frozen-thawed embryos treated with ethylene glycol (groups EE and ES) had any viable cells. There were no lysed cells in the frozen-thawed embryos treated with glycerol (groups GG and GS) and the proportion of cells with pyknotic nuclei was low (group GG = 1.1 +/- 0.8% and group GS = 2.5 +/- 1.5%). There were no differences between embryos treated with cyroprotectant diluted with or without sucrose. The embryos were morulae or early blastocysts and either did not have a capsule or had a very thin capsule. The results of the present study confirm that ethylene glycol is a poor cryoprotectant for early equine embryos and that the use of sucrose during dilution of the cryoprotectant after thawing does not improve the morphology of the embryos. The results of this study also indicate that glycerol is an effective cryoprotectant for freezing equine embryos with intact zonae pellucidae and with either very thin or no capsules.

The purpose of this study was to evaluate the exact age when the equine embryo reaches the uterus. The time of ovulation was determined by hourly ultrasound examinations starting 32 h after an injection of crude equine pituitary gonadotrophin or human chorionic gonadotrophin, or after the first of 4 injections of buserelin. Nonsurgical uterine flushings were carried out 144 h (Day 6), 156 h (Day 6.5) or 168 h (Day 7) after ovulation. Induction of ovulation was attempted in 101 oestrous cycles and 61 of 101 mares (60.4%) ovulated 32-44 h post injection. Sixty embryo collections were performed which yielded: 0/20 embryos at 144 h, 9/17 embryos (53%) at 156 h and 12/23 embryos (52%) at 168 h. The mean (+/- s.e.m.) diameter of the embryo was significantly greater (P<0.01) at Day 7 (244 +/- 15 microm) than at Day 6.5 (186 +/- 9.1 microm), and variability in size was observed among embryos collected from the same mare after synchronous natural multiple ovulations. These results suggest that; i) horse embryos enter the uterus between 144 and 156 h after ovulation, and ii) the time interval between ovulation and fertilisation in mares is inconsistent and/or embryonic development rate may differ between individual embryos.

Seventeen horse embryos recovered on the sixth day after spontaneous ovulation were; 1) washed in PBS (n = 6), 2) treated with 1.5 M 1-2 propanediol (n = 6) or, 3) frozen and thawed using 1.5 M propanediol as the cryoprotectant (n = 5). After treatment, the embryos were incubated for 6 h in medium before they were fixed, serially sectioned and examined microscopically to count the total numbers of interphase, mitotic and pycnotic nuclei. Significant differences were measured only in the mean proportions of pycnotic cells (+/- s.d.), both between the control (9.2 +/- 7.3%) and frozen-thawed embryos (52.8 +/- 37.1%; P<0.05) and between the propanediol-treated (10.8 +/- 4.6%) and the frozen-thawed embryos (P<0.01). Propanediol appears to be minimally toxic to equine embryos but it is a poor cryoprotectant.

Sixty-seven pregnant bitches were given atropine sulphate (0.025 mg kg-1), prifinium bromide (0.1 ml kg-1) and metopimazine (0.5 mg kg-1) and 15 min later 2.5 micrograms cloprostenol kg-1 s.c., three times at 48 h intervals (day 1, day 3, day 5). After one treatment, 53 of the 67 bitches had aborted, and after a second treatment, 62 of the 67 bitches had aborted. In 18 bitches, progesteronemia kinetics were followed-up: the first injection of cloprostenol resulted in a significant (P < 0.01) fall in progesteronemia. In 12 of the 18 bitches that had aborted following the first protocol, this rapid fall in progesterone was noteworthy as it decreased progesterone concentration on average from 17.07 +/- 8.20 ng ml-1 on day 1 to 1.31 +/- 0.34 ng ml-1 on day 3. The premedication administered 15 min before the injection of prostaglandins, prevented the appearance of side effects in 39 of the 67 bitches (58.2%).

The SDS-PAGE method was used to determine the composition of isolated bovine zona pellucida (ZP). This egg extracellular coat appears to be characterized by 3 major glycoproteins (ZP1, ZP2, ZP3), with apparent molecular weight (MW) of 80-70 kD, 66-63 kD and 60 kD, respectively, as revealed by 1-dimensional SDS-PAGE. After 2-dimensional SDS-PAGE, the zona pellucida electrophoretic pattern indicates a fourth glycoprotein, thus called ZP4. SDS-PAGE analysis of ZP isolated from oocytes and embryos after transit through the genital tract of A23187 pretreated oocytes allowed the description of modifications in glycoproteinic composition.

The decrease in embryo viability caused by cryopreservation may be due, in part, to metabolic disturbances. To determine the effect of cryopreservation on metabolism, Day -6.5 horse embryos were either frozen and thawed using glycerol as the cryoprotectant, given only the glycerol treatment or washed an equal number of times in phosphate buffered saline (PBS). Before and after treatment, individual embryos were incubated with L-[14C(U)]-glutamine, to measure Krebs cycle activity, and D-[5-3H]-glucose, to measure Embden-Meyerhof pathway activity. Before treatment, glucose metabolism ranged from 110-625 pmol/2 h and glutamine metabolism from 4.1-15.9 pmol/2 h, both being highly correlated with embryo volume. Mean glucose metabolism in the control group increased 76% between the pre-treatment and post treatment measurements compared with 1% in the pooled treated groups, whereas mean glutamine metabolism increased only 10% in the control group but 50% in the treated embryos. Before treatment, there was no difference in mean ratio of glucose to glutamine metabolism between groups, but after treatment this ratio was almost 2-fold greater in the control group than in the treated group. These results indicate that cryopreservation inhibits anaerobic glucose metabolism and stimulates aerobic glutamine metabolism. However, this is an effect of the cryoprotectant, rather than of freezing and thawing.