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Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they differ from their ICSI counterparts. To address this, we performed IVF using frozen-thawed sperm from five different stallions following sperm selection and a prolonged capacitation period of 10 h, on a total of 109 oocytes. We then analyzed the cleavage rate (cleaved/initial oocytes), blastocyst rate (blastocyst/initial zygotes), and blastocyst development (blastocyst/cleaved zygotes) of the IVF cycles, and compared them with those of the clinical ICSI cycles during the same period. We also evaluated time-lapse images of the developed embryos to assess developmental time points such as time to morula compaction and blastocyst expansion, as well as morula and blastocyst sizes. Overall, developmental rates were not different between IVF and ICSI cycles (blastocyst rate 41.1 % IVF and 41.8 % ICSI, p > 0.05). However, development proceeded faster in IVF cycles (blastocyst expansion IVF 155.5 ± 18.5 h; ICSI 167.2 ± 19.6 h; p < 0.05) and IVF embryos were also larger (blastocyst area IVF 22608 ± 2857 μm 2 ; ICSI 20806 ± 1505 μm 2 ; p < 0.05). The faster development and larger size might suggest a more advanced developmental stage. The implications of these findings need to be further evaluated to assess their association with pregnancy potential. The successful developmental rates achieved in IVF cycles demonstrate the potential of this technique for clinical application, although the amount of frozen-thawed semen required is significantly higher in IVF than in ICSI, which is an important consideration for mare and stallion owners. Nonetheless, the use of frozen-thawed semen in equine IVF, coupled with comparable blastocyst rate, presents promising potential for broader clinical adoption of the IVF technique., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Once diagnosed pregnant with ultrasound at an early stage of gestation, mares are usually not examined before foaling. The objective of this case report was to highlight the importance of transrectal ultrasound screening examination and to report a unique case of fetal congenital cataracts associated with other feto-placental abnormalities in a mule pregnancy, its in utero ultrasound diagnosis and outcome. A 17-year-old Thoroughbred research mare carrying a mule fetus was examined by transrectal ultrasonography at 186 days of gestation for a routine pregnancy examination. Ultrasonography allowed in utero diagnosis of fetal congenital cataracts, hyperechogenic bowels, intrauterine growth restriction (IUGR), hydramnios and placental abnormalities. The mare was monitored bi-monthly to observe the progress of the pregnancy. At 258 days of gestation, the abnormal chorioallantois detached at the cervical star and at 272 days, fetal asystole was diagnosed. Abortion was induced and the fetus was delivered uneventfully. Post-mortem gross and histologic findings confirmed the prenatal ultrasonographic diagnosis. This case highlights the diagnostic value of a complete fetal ultrasound examination to detect equine fetal abnormalities., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene ( FSHR ) and luteinizing hormone/choriogonadotropin receptor gene ( LHCGR ) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 μg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 μg/ml (H60, n = 196), 164 μg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32-76%; median, min-max) in comparison to H60 and H164 (24%,11-33% and 13%, 8-44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained., Competing Interests: Declaration of competing interest The authors do not have any conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)