The potential of using long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) from early antral follicles (EAFs) as an assisted reproductive technology in cattle has shown promising results. This study explored the feasibility of applying this technology to sheep as seasonal breeding animals. Ovaries from sheep were collected during both the breeding and non-breeding seasons. COCs were isolated from EAFs (350-450 μm) and cultured in TCM199 medium supplemented with 0.15 μg/mL Zn sulfate, 10^-4IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 μM Cilostamide. After five days of LIVC, the COCs were submitted to an in vitro maturation procedure. The results indicate successful in vitro development of COCs, evidenced by a significant increase in oocyte diameter (p < 0.000) and the preservation of gap junction communication between oocyte and cumulus cells. The gradual uncoupling was accompanied by a progressive chromatin transition from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) (p < 0.000), coupled with a gradual decrease in global transcriptional activity and an increase in oocyte meiotic competence (p < 0.000). Maintenance of oocyte-cumulus investment architecture, viability, and metaphase II capability was significantly higher in COCs collected during the breeding season (p < 0.000), suggesting higher quality than those obtained during the non-breeding season. In conclusion, our study confirms LIVC feasibility in sheep, emphasizing increased effectiveness during the breeding season in isolating higher-quality COCs from EAFs. These findings can influence improving the LIVC system in mammals with seasonal reproduction.

Purpose: This study aimed to evaluate the effectiveness of single versus group culture strategies for cumulus-oocyte complexes (COCs) derived from early antral follicles (EAFs), with the goal of optimizing culture conditions to increase oocyte availability for assisted reproductive technologies. Methods: COCs isolated from EAFs (350–450 µm) from sheep ovaries were cultured in TCM199 medium supplemented with 0.15 µg/mL Zn⁺⁺ as zinc sulfate, 10⁻⁴ IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 µM Cilostamide. After 5 days of long in vitro culture (LIVC), COCs underwent in vitro maturation. This study investigated the effects of single and group culture conditions on COCs, focusing on morphology (integrity of oocyte-granulosa cell complex), viability, oocyte diameter, chromatin configuration, and ultrastructure. Additional factors influencing developmental competence were assessed, including global transcriptional activity, gap junction communication, and meiotic competence. Intracellular reactive oxygen species (ROS) levels and mitochondrial activity were also measured. Results: No significant differences were observed between groups in terms of morphology, viability, oocyte diameter, chromatin configuration, ROS levels, or mitochondrial activity. However, group culture resulted in ultrastructural changes, with a notable reduction in global transcriptional activity, an increase in active gap junctions, and a higher rate of meiosis resumption (p < 0.01). Conclusion: Overall, group culture of COCs derived from sheep EAFs promoted meiosis resumption, suggesting that this approach could improve in vitro culture techniques, increase the availability of mature gametes, and support fertility preservation programs. Graphical

Cryptorchidism is a genital defect in which ram testicles fail to descend, causing azoospermia, while maintaining normal behavior towards females. We investigated whether cryptorchid rams can induce a ram-effect in ewes that would then be subjected to artificial insemination (AI). Therefore, ewes were isolated from any contact with rams for 6 weeks, then exposed to cryptorchid rams for 14 days. From day 15 to day 24, estrus was checked using a cryptorchid teaser four times daily (at 08:00, 12:00, 16:00, 20:00). Ewes detected in estrus were inseminated 24 h later. Experiment 1 included ewes (n = 31) all exposed to the cryptorchid ram-effect (CRE): 70.9% showed estrus, lambing rate after AI was 45.5%, and prolificacy was 1.40. Experiment 2 compared CRE (n = 80) with a control group with no prior exposure to males (n = 39). Estrus occurrence differed significantly (75.0% vs. 23.1%, respectively, p ≤ 0.001). Lambing rate from AI was 44.1% and prolificacy 1.27. These results show that cryptorchid rams effectively induce and synchronize estrus in Sarda ewes. AI fertility results on natural estrus following CRE yields outcomes comparable to those previously reported after hormonal synchronization for this breed.

This study analyzed the effects of dietary supplementation with by-pass linseed oil (LO; rich in α-linolenic acid) on maternal antioxidant systems at Days 14 and 16 of pregnancy in Sarda ewes. This trial used sixteen dry ewes. Eight ewes (CT group) were fed with a control diet without LO, and eight ewes (LO group) were fed with a diet supplemented with LO (10.8 g of α-linolenic acid/ewe/day). Both diets had similar crude protein and energy levels. The experiment included 10 days of an adaptation period and 31 days of a supplementation period. This supplementation period was divided into Period −2 (from Day −15 to −8), Period −1 (from Day −7 to −1; before synchronized mating period/Day 0), Period +1 (from Day +1 to + 7 after mating), and Period +2 (from Day +8 to +15 after mating). Estrous synchronization was induced in all the ewes using an intravaginal sponge (45 mg fluorgestone acetate) for 14 days and equine chorionic gonadotropin (350 UI/ewe) at the end of the treatment. On Days 14 (CT, N = 4; LO, N = 4) and 16 (CT, N = 4; LO, N = 4) after mating, the ewes were slaughtered. Samples of plasma, uterine, and luteal tissues were collected. Thiols, total antioxidant activity (TEAC), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were measured. On Day 16, thiol and TEAC in luteal tissues were higher in the LO group when compared with the control one (p < 0.05). Moreover, TEAC was higher for the LO group in uterine tissues on Days 14 and 16 (p < 0.05). SOD activity was higher in the LO group in luteal and uterine tissues on Day 14 and Day 16, respectively (p < 0.001). On Day 16, uterine MDA content was lower for the LO group (p < 0.001). No differences were found between groups at the plasmatic level. However, the by-pass LO supplementation enhanced the analyzed antioxidant parameters in luteal and uterine tissues. In conclusion, these results demonstrate that by-pass LO supplementation exerted a positive effect on antioxidative defenses on maternal structures during the embryo-maternal recognition period in ewes. Thus, this could contribute to improving the maternal environment during the embryo-maternal recognition period in mammals.

Liquid storage is an important tool used to prolong fresh semen shelf-life while protecting spermatozoa from damage, conserving their overall functionality, and ensuring better fertility than frozen semen from sheep. The increased production of reactive oxygen species (ROS) during sperm storage leads to a decline in sperm quality, particularly with regard to sperm nuclear DNA damage and mitochondrial membrane potential (MMP). This study evaluated the effect of storing Sarda ram semen at 15 °C for 7 h on its redox status, motility, morphology, acrosome integrity, ATP content, mitochondrial potential membrane, and in vivo fertility after artificial insemination. Two different extenders were compared: a lab-made skimmed milk (SM)-based extender and a commercial extender (OviXcell^®, IMV-Technologies, France). Lower ROS levels in the SM (p < 0.001) indicated that its oxidative status was better maintained compared to the commercial extender (CE). Antioxidant defenses (total antioxidant capacity, TEAC; superoxide dismutase, SOD; total thiols) were higher in the SM (p < 0.01) than in the CE. SM also had higher MMP (p < 0.05), acrosome integrity (p < 0.05), ATP content (p < 0.01), and in vivo fertilizing capacity (p < 0.05) compared to the CE, which indicated higher semen quality. In conclusion, the SM extender, while maintaining a better oxidative/antioxidant balance, ensured higher semen quality after 7 h of storage at 15 °C in vitro compared to the CE.