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In some horse breeds, such as Argentinian Polo Horses, limiting pregnancies to the desired foal sex has both economic and welfare implications. Biopsy of equine embryos to assess embryonic sex by polymerase chain reaction (PCR) on DNA from sampled cells has been reported under research settings and in a limited number of commercial programs. We previously reported the results of embryo biopsy and PCR of biopsied trophoblast cells for sexing in our commercial equine embryo transfer program. In that study, including one season and results on biopsy of over 450 embryos, there was no significant difference in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the center (73%) compared with those shipped overnight (72%). Initially, sampled cells were probed with both a genomic (confirmatory) DNA probe and a Y-specific probe, however a sizable proportion (14%) of biopsies did not provide a sex diagnosis (no signal for either probe). We later found that determination only of presence of Y DNA was equally or more effective than using the genomic DNA control; it appeared that the division of the DNA isolated from the biopsy into two aliquots (for analysis of genomic and Y-specific DNA) reduced the accuracy.Overall, the accuracy (correct foal sex) was 95% for embryos diagnosed as male and 78% for embryos diagnosed as female. In the present breeding season (2021-22), we used only Y-DNA assessment. At the time of writing, we have biopsied and assessed over 300 embryos. Embryos were recovered at the Center or shipped overnight to the Center. Biopsy was performed via micromanipulation, and the biopsied cells were evaluated for Y-chromosome specific DNA via PCR. Only embryos without a Y-DNA signal were transferred. Pregnancy rates after transfer of biopsied embryos are currently 124/163 (76%). Sexing of fetuses from transferred embryos via ultrasonographic evaluation at 60-70 days gestation is ongoing and at the time of writing, 48 fetuses have been evaluated. The proportion of female fetuses resulting from embryos with a "female" biopsy/PCR diagnosis (no Y-DNA signal) appears to have varied through the season, being 80% for the first 20 fetuses evaluated and 68% for the next 28 fetuses evaluated. We are currently examining the system to determine possible reasons for the reduction in the accuracy of the PCR diagnosis, and updated results will be presented at the time of the symposium. [ABSTRACT FROM AUTHOR]

Intracytoplasmic sperm injection (ICSI) is an important research and clinical tool. However, work with standard in vitro fertilization (IVF) is needed to explore sperm capacitation and the early events of fertilization. Repeatable methods for equine IVF have not yet been reported. Definitive evidence of fertilization, such as presence of the sperm tail within the cytoplasm, is critical when assessing potential methods for IVF. We have previously stained live sperm with the mitochondrial-specific stain, MitoTracker, and successfully visualized the sperm tail in the oocyte cytoplasm after ICSI. In the present study, we evaluated whether staining sperm with MitoTracker would A) be compatible with maintenance of sperm motility in culture and B) allow visualization of sperm tails within the COC or oocyte after co-culture with sperm. Semen was collected from one fertile stallion, and 200 µl of extended semen was layered under 1 mL of MOPS-buffered medium containing 10 or 100 nM Mitotracker Red CMXRos, in a 5-mL tube. The tube was incubated at a 45o angle at 38 °C for 20 min. The uppermost 750 µL of the medium was collected, washed, centrifuged, and the pellet resuspended in modified TALP. Sperm were added to 50-µL droplets of modified TALP to a concentration of 1 million sperm/mL and these were incubated in 5% CO 2 in air. Sperm motility was assessed visually after 1, 6 and 22 h in culture. At 22 h, sperm were removed, counterstained with DAPI, and evaluated under fluorescence microscopy. In some replicates, in vitro-maturedequine cumulus-oocyte complexes (COCs) were added to the sperm droplets and co-incubated for 1 to 9 h. To assess sperm progress toward oocyte penetration, oocytes were denuded, fixed, and counterstained with DAPI. Some COCs were stained intact for confocal assessment of chromatin, sperm mitochondria, and acrosome status. These were fixed, permeabilized with 0.1% Triton-X 100, labeled with fluorescein-conjugated peanut agglutinin, then stained with Hoechst 33342. A minimum of three replicates were performed with each MitoTracker concentration. MitoTracker-stained sperm showed progressive motility after 1, 6 and 22 h incubation (∼80%, 50%, and 15% respectively) but at 22 h appeared to have a higher proportion of immotile sperm, and less vigorous motility, than did unstained sperm (∼25% progressive at 22 h). Staining with 10 nM MitoTracker supported more vigorous motility than did 100 nM. Both concentrations enabled clear visualization of the sperm tail on fluorescence microscopy. In penetrated oocytes, the sperm tail was clearly visualized in the oocyte cytoplasm, associated with sperm chromatin. On confocal microscopy, acrosomal ghosts were visualized at the periphery of the zona pellucida, indicating that sperm underwent the acrosome reaction in response to the zona. We conclude that staining of live sperm with 10 nM MitoTracker during swim-up provides a simple method to allow confirmation of sperm penetration of oocytes after sperm-oocyte coincubation. [ABSTRACT FROM AUTHOR]

Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer, in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell culture, have been developed with the aim of refining breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for human disease and therapy. The following review focuses on presenting important aspects of preimplantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed. [ABSTRACT FROM AUTHOR]

Abstract: Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vitro were assessed after differential staining. A discrete inner cell mass could not be appreciated in blastocysts of either source after staining; this was attributed to the presence of a network of cells within the trophoblastic vesicle. Because increased medium calcium concentrations have been reported to decrease the incidence of trophoblast-only pregnancy after transfer of equine nuclear transfer embryos, we investigated the effect of increased calcium concentrations during oocyte maturation or during embryo culture. Increasing calcium concentration of culture medium from 2 to 5.6mM during in vitro oocyte maturation did not affect maturation rate (75 and 68%, respectively) or blastocyst development after fertilization (23 and 27%). However, increasing calcium concentration (from 1.3 to 4.9mM) of medium used for embryo culture significantly decreased blastocyst development (27% versus 13%, respectively) and adversely affected embryo morphology. More work is needed to optimize culture systems for in vitro production of equine embryos. [Copyright &y& Elsevier]

Abstract: Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and absolute difference) between TMOT and SMI was high (–0.7 and 5.6%, respectively), but decreased after 24 (10.8 and 15.1%, respectively) and 48 h (23.0 and 23.8%, respectively) of cooled storage as motility declined more rapidly than SMI. We concluded that TMOT and SMI measured separate aspects of sperm quality. [Copyright &y& Elsevier]

Abstract: The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30min under average ambient conditions of >30°C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F2α was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38°C to a mean of 39.9°C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8±0.5 compared with 41.5±0.5mm diameter; P <0.05), and had an increased time from PGF2α administration to subsequent ovulation (8.47±0.337 compared with 9.27±0.294 d; P <0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P <0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P =0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery. [Copyright &y& Elsevier]

Abstract: Hexamethyldisiloxane polymer layers obtained at low frequency glow discharge were post-treated by ammonia vapours under the same plasma conditions. Infrared spectroscopic ellipsometry and atomic force microscopy studies were carried out with the aim to elucidate the structural and morphological changes accompanying this modification. The infrared ellipsometry combined with best-fit calculations was used to evaluate the optical constants of the polymer film before and after the treatment, and thus to provide data for the vibrational band analysis. No additional modified layer due to the plasma treatment was detected, but a newly emerged weak band at 1562 cm−1 was observed in the ellipsometric spectra of the ammonia-treated sample, which was ascribed to N–H deformation in amines. The surface morphology was imaged by atomic force microscopy. The data revealed that the NH3 treatment had caused distinct micro-structural modification on the top layer surface. The size of the nodule aggregates was largely reduced and this contributed to a much better developed top surface. [Copyright &y& Elsevier]