In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts (n = 3) and blastocysts (n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro (n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

The horse as a natural model to study reproductive aging-induced aneuploidy and weakened centromeric cohesion in oocytes.

Aneuploidy of meiotic origin is a major contributor to age-related subfertility and an increased risk of miscarriage in women. Although age-related aneuploidy has been studied in rodents, the mare may be a more appropriate animal model to study reproductive aging. Similar to women, aged mares show reduced fertility and an increased incidence of early pregnancy loss; however, it is not known whether aging predisposes to aneuploidy in equine oocytes. We evaluated the effect of advanced mare age on (1) gene expression for cohesin components, (2) incidence of aneuploidy and (3) chromosome centromere cohesion (measured as the distance between sister kinetochores) in oocytes matured in vitro. Oocytes from aged mares showed reduced gene expression for the centromere cohesion stabilizing protein, Shugoshin 1. Moreover, in vitro matured oocytes from aged mares showed a higher incidence of aneuploidy and premature sister chromatid separation, and weakened centromeric cohesion. We therefore propose the mare as a valid model for studying effects of aging on centromeric cohesion; cohesion loss predisposes to disintegration of bivalents and premature separation of sister chromatids during the first meiotic division, leading to embryonic aneuploidy; this probably contributes to the reduced fertility and increased incidence of pregnancy loss observed in aged mares.

The Effect of Different Flushing Media Used to Aspirate Follicles on the Outcome of a Commercial Ovum Pickup-ICSI Program in Mares.

The in vitro production of embryos by ovum pickup (OPU) and intracytoplasmic sperm injection (ICSI) is gaining popularity among horse breeders and veterinarians. Various collection media are available for flushing follicles during OPU. The objective of this study was to determine whether the type of flushing media used to aspirate follicles and collect oocytes influences the outcome of a commercial equine OPU-ICSI program. Two commercial embryo flushing media (EFM1 and EFM2) supplemented with heparin were compared with a flushing media designed specifically for the collection of oocytes (oocyte flushing media [OFM]) on the outcome of OPU-ICSI parameters in 234 Warmblood mares. The OPU-ICSI performed in mares using one of the EFM1 resulted in a lower (P < .05) blastocyst rate and blastocysts per OPU-ICSI session (11.9 ± 13.2%, 0.88 ± 1.3) than the OFM (19.2 ± 15.2%, 1.24 ± 1.2). Unlike the EFM2 solution, the heparin used to prepare the EFM1 contained preservatives including benzyl alcohol, a component known to alter the oocyte membrane, which might have been responsible for the lower developmental competence of oocytes collected with EFM1. In conclusion, exposure of oocytes (<1.5 hours) to one of the flushing medium tested in this study affected negatively the outcome of the OPU-ICSI commercial program when compared with flushing media designed for collection of equine oocytes. Care should be taken when choosing the components of the flushing media used to collect oocytes. Further research should be carried out to confirm the potential negative effect of the preservatives used in multidose heparin vials.