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.

Staining, as a valuable method for sperm morphological assessment, has been used to determine sperm abnormalities, fertilization capability and sperm suitability during freezing‐thawing process. Synthetic dyes have been used for sperm viability and morphological evaluation. However, most of them have been made from chemical substances and have a perilous effect on the environment. In the current study, we evaluated three different natural dyes as the natural sources of dye for sperm staining. Bull frozen semen was used and prepared on slides for staining. Aqueous extract dye of black mulberry (BM), henna (HA), safflower (SA) and eosin‐nigrosine (control group) were used for sperm staining. Additionally, the effect of staining dyes on viability and some morphological parameters (head area: HR, head abnormality: HB and tail abnormality: TA) were evaluated. Although none of the natural dyes could detect viability of the sperm cells, safflower stain (HR: 26.55 µm, HB: 0% TA: 28%) and black mulberry stain (HR: 25.07 µm, HB: 2% TA: 3%) compared to control group (HR: 34.29 µm, HB: 4%, TA: 4%) provoked a strong reaction in the sperm cells, so that the sperms were observed yellow and red respectively. The reaction of sperm cells to the henna dye was very poor and it did not stain the sperm cells. Thus, the present study demonstrated that SA and BM dyes are able to stain the spermatozoa and with further modification could be used as alternative dyes for sperm staining in the study of sperm morphology, but not viability. Staining with these dyes can be an alternative to current costly chemical staining methods.

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

Here, we present a protocol for in vitro embryo production (IVEP) in sheep using slaughterhouse-derived ovaries. We describe steps for ovary collection and transportation under controlled conditions. We then detail procedures for oocyte retrieval, selection, and in vitro maturation. Mature oocytes undergo fertilization with swim-up-processed sperm, and the resulting zygotes are cultured in a commercial medium. This approach ensures high reproducibility and developmental success, yielding cleavage and blastocyst rates over 90% and 60%.