Worldwide over 5 million children have been conceived using assisted reproductive technology, and research has concentrated on increasing the likelihood of ongoing pregnancy. However, studies using animal models have indicated undesirable effects of in vitro embryo culture on offspring development and health. In vivo, the oviduct hosts a period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of (epi)genetic marks. We designed an oviduct-on-a-chip platform to better investigate the mechanisms related to (epi)genetic reprogramming and the degree to which they differ between in vitro and in vivo embryos. The device supports more physiological (in vivo-like) zygote genetic reprogramming than conventional IVF. This approach will be instrumental in identifying and investigating factors critical to fertilization and pre-implantation development, which could improve the quality and (epi)genetic integrity of IVF zygotes with likely relevance for early embryonic and later fetal development.

Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X–Y gametologs recorded in eutherians, in addition to novel Y-born and transposed genes. Horse, donkey and mule testis RNAseq reveals several candidate genes for stallion fertility. A novel testis-expressed XY ampliconic sequence class, ETSTY7, is shared with the parasite Parascaris genome, providing evidence for eukaryotic horizontal transfer and inter-chromosomal mobility. Our study highlights the dynamic nature of the Y and provides a reference sequence for improved understanding of equine male development and fertility.

MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date, most studies that have examined miRNA expression levels have used the global mean expression of all expressed genes or the expression of reference mRNAs or nuclear RNAs for normalization. We analyzed the suitability of a number of miRNAs as potential expression normalizers in bovine oocytes and early embryos, and porcine oocytes. The stages examined were bovine oocytes at the germinal vesicle (GV) and metaphase II stages, bovine zygotes, 2, 4 and 8 cell embryos, morulae and blastocysts, as well as porcine cumulus oocyte complexes, GV, metaphase I and II oocytes. qRT-PCR was performed to quantify expression of miR-93, miR-103, miR-26a, miR-191, miR-23b, Let-7a and U6 for bovine samples and miR-21, miR-26a, miR-93, miR-103, miR-148a, miR-182 and miR-191 for porcine oocytes. The average starting material for each sample was determined using specific standard curves for each primer set. Subsequently, geNorm and BestKeeper software were used to identify a set of stably expressed miRNAs. Stepwise removal to determine the optimum number of reference miRNAs identified miR-93 and miR-103 as the most stably expressed in bovine samples and miR-26a, miR-191 and miR-93 in porcine samples. The combination of miR-93 and miR-103 is optimal for normalizing miRNA expression for qPCR experiments on bovine oocytes and preimplantation embryos; the preferred combination for porcine oocytes is miR-26a, miR-191 and miR-93.

Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were labelled for connexin-43 or injected with Lucifer yellow dye to determine whether the integrity of the gap junctions between an oocyte and its cumulus was compromised by vitrification. Finally, the effect of denuding immature and mature oocytes on their ability to withstand vitrification was examined. Cryopreserving immature COCs increased the number of dead cumulus cells (13 vs 2.6% for controls; P<0.05). However, an increased proportion of dead cumulus cells did not affect post-warming maturation rates (∼30% MII) presumably because dead cells were located at the periphery of the cumulus mass and cumulus-oocyte gap junction communication was not disrupted. Moreover, cumulus removal prior to IVM or vitrification indicated that while the cumulus does protect immature oocytes during vitrification it does so by mechanisms other than support during maturation. Cumulus presence was also found to protect mature equine oocytes against vitrification-induced damage since cumulus-enclosed MII oocytes preserved their meiotic spindle quality better during vitrification than denuded oocytes (38.1 vs 3.1% normal spindles; P<0.05). Abstract Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were labelled for connexin-43 or injected with Lucifer yellow dye to determine whether the integrity of the gap junctions between an oocyte and its cumulus was compromised by vitrification. Finally, the effect of denuding immature and mature oocytes on their ability to withstand vitrification was examined. Cryopreserving immature COCs increased the number of dead cumulus cells (13 vs 2.6% for controls; P<0.05). However, an increased proportion of dead cumulus cells did not affect post-warming maturation rates (∼30% MII) presumably because dead cells were located at the periphery of the cumulus mass and cumulus-oocyte gap junction communication was not disrupted. Moreover, cumulus removal prior to IVM or vitrification indicated that while the cumulus does protect immature oocytes during vitrification it does so by mechanisms other than support during maturation. Cumulus presence was also found to protect mature equine oocytes against vitrification-induced damage since cumulus-enclosed MII oocytes preserved their meiotic spindle quality better during vitrification than denuded oocytes (38.1 vs 3.1% normal spindles; P<0.05).

This study retrospectively examined the degree to which success within a commercial ovum pick-up (OPU)–intracytoplasmic sperm injection (ICSI) program varied between individual mares and stallions. Over 2 years, 552 OPU sessions were performed on 323 privately owned warmblood mares. For mares that yielded at least one blastocyst during the first OPU-ICSI cycle, there was a 77% likelihood of success during subsequent attempts; conversely, when the first cycle yielded no blastocyst, the likelihood of failure (no embryo) in subsequent cycles was 62%. In mares subjected to four or more OPU sessions, the mean percentage of blastocysts per injected oocyte was 20.5% (range 1.4–46.7%), whereas the mean number of blastocysts per OPU-ICSI session was 1.67 (0.2–4.2). Age did not differ significantly between mares that yielded good or poor results. The number of recovered oocytes per OPU was positively associated with the likelihood of success (P < 0.001). Although there were considerable between-stallion differences, most stallions (14/16) clustered between 15.6% and 26.8% blastocysts per injected oocyte, and the number of blastocysts per OPU (mean 1.4; range 0.2–2.2) was less variable than among mares. In conclusion, although both mare and stallion affect the success of OPU-ICSI, mare identity and the number of oocytes recovered appear to be the most reliable predictors of success.