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We hypothesized that nutrient restriction from day 50-90 of gestation decreases umbilical blood flow and that umbilical blood flow would recover to control values upon realimentation during late gestation (d 90 to 130) or remain reduced in ewes that continued to be nutrient restricted. On d 50 of gestation, young nulliparous whiteface ewes (6-8 mo; n = 41) carrying singletons were randomly assigned to two dietary treatments: 100% of NRC recommendations (CON) or 60% of CON (RES). On d 90 of gestation, ewes either remained on CON or RES until d 130, or CON ewes were RES from d 90 to 130, or RES ewes were realimented to CON from d 90 to 130. This resulted in 4 treatment groups on day 130: CON-CON, CON-RES, RES-RES, RES-CON. Umbilical blood flow and fetal and placental measurements were obtained via ultrasonography every 10 days from day 50-110. Non-survival surgeries were performed on days 50, 90, and 130 (n = 6-7 ewes/group) where uterine artery and umbilical blood flows were measured during surgery via ultrasonography. Conceptus weights were recorded and placentomes collected to determine binucleate cell numbers. The study was conducted as a completely randomized design arrangement with repeated measures. Data were analyzed using the MIXED procedure of SAS. There was a nutritional treatment by day interaction (P < 0.01) with CON ewes having greater umbilical blood flow compared with RES by d 90. Fetal biparietal distance, abdominal width, and kidney area increased (P < 0.05) in CON-RES with all these measurements increasing during late gestation. We partially accept our hypothesis as nutrient restriction during mid gestation decreased umbilical blood flow. However, blood flow did not return to control levels upon realimentation. By d 130, fetal and placental weights were similar between RES-RES and CON-CON. Binucleate cell numbers in the fetal trophoblast were not influenced by nutritional treatments. Our findings suggest that refeeding previously nutrient restricted pregnant adolescent ewes to control levels does not reestablish umbilical blood flow. Adequate placental development during mid gestation could protect the fetus from a decreased umbilical blood flow later in gestation when nutrients were limited by 40%., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ovarian steroids play an important role in increasing plasma volume in pregnant females and preparing the uterus for implantation. We hypothesized that a short duration of increased estradiol-17β (E2) would increase plasma volume and uterine cell proliferation in ovariectomized ewes. Adult non-pregnant Romanov ewes (n = 15) were ovariectomized. After ovariectomy, ewes were individually housed and were offered water at ad libitum intake and were fed a pelleted diet at maintenance once daily according to body weight. After at least 30 days post-ovariectomy ewes were fasted and received an implant placed in the axillary region that contained 100 mg of E2 (E2; n = 8) or a sham implant with no E2 (CON, n = 7). After 24 h, ewes were weighed prior to plasma volume measurement procedures. Plasma volume was determined using the Evans blue dye method. Blood samples were taken at 0 (pre dye injection), 5, 10, 15, 20, 25, 30, 40, 50 and 60 min after dye injection. After the final blood collection, ewes were euthanized with an overdose of sodium pentabarbital and uterine weights were recorded. Uterine cross-sections were fixed in formalin for immunohistochemical localization of Ki67 (a marker of proliferating cells) followed by image generation of luminal epithelium and endometrial stroma (5 areas each/tissue section) and analysis to determine the proportion of proliferating cells. Plasma volume tended to be greater in E2 vs CON (2.75 ± 0.11 vs. 2.54 ± 0.12 L, P = 0.07) and uterine weights were greater in E2 vs CON (27.25 ± 2.35 vs. 17.35 ± 2.51 g, P < 0.01). Water intake after implant placement was similar in E2 and CON (3.85 vs. 4.87 ± 0.67 L; P = 0.28). Cell proliferation in the luminal epithelium was greater in E2 vs CON (6.55 vs. 1.2 ± 1.75%, P = 0.02) and stromal cells tended to be greater in E2 vs CON (0.59 vs 0.37 ± 0.06%, P = 0.07). Our results demonstrate that E2-treatment tends to increase plasma volume acutely and increases uterine cell proliferation in ewes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris' Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin, respectively (Diff-Quik>Hemacolor>Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.