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.

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.

Cryptorchidism is the most common non-lethal congenital defect of the male reproductive system in sheep, with potential economic consequences for flock management. This study investigated the incidence, testicular morphology, ultrasonographic characteristics, semen quality, and sexual behavior of bilateral cryptorchid Sarda rams. Slaughterhouse inspections of 2360 lambs showed an incidence of 0.87% cryptorchidism. Cryptorchid testes were significantly rounder and lighter than intact testes, indicating impaired development in affected animals. Ultrasonography of 15 adult bilateral cryptorchid rams showed that retained testes were markedly undersized and that the left testis was less frequently visualized. No significant association with age was detected within the studied age range. All ejaculates recovered from bilateral cryptorchid rams were azoospermic. Nevertheless, behavioral trials suggested that bilateral cryptorchid males retained sexual interest and the ability to identify estrous ewes. These findings confirm the infertility of bilateral cryptorchid Sarda rams while highlighting their preserved sexual behavior, suggesting a potential zootechnical use as teaser rams for heat detection. Repurposing cryptorchid males in this way could represent a potential alternative to surgically modified teaser rams or the use of aprons on intact rams.

This study investigated the combined effects of seminal plasma (SP) and Sonchus oleraceus L. aqueous extract on post-thaw sperm quality and fertilizing ability in rams. Semen collected from seven adult rams was pooled and divided into six experimental groups according to the presence (SP+) or absence (SP−) of SP and supplementation with 0, 0.5, or 2 g/L of Sonchus oleraceus extract. Samples were cryopreserved in a Tris–egg yolk–citric acid extender. Post-thaw sperm quality using computer-assisted sperm analysis (CASA), hypo-osmotic swelling (HOS) test, and oxidative stress biomarkers, including reactive oxygen species (ROS), malondialdehyde (MDA) and total thiols as antioxidative parameter were evaluated. Removal of SP significantly enhanced several kinematic parameters (VCL, ALH, DCL) and reduced abnormal morphology (p < 0.05), whereas its presence improved motion quality indicators such as beat cross frequency (BCF), linearity (LIN), and straightness (STR) (p < 0.05). However, SP removal induced a marked oxidative imbalance, characterized by increased ROS and MDA levels and decreased total thiols (p < 0.001). Supplementation with S. oleraceus extract improved membrane integrity and restored redox balance by increasing thiols levels (p = 0.0001) and reducing lipid peroxidation (p < 0.001), with significant SP × dose interactions. Based on these results, in vitro fertilization (IVF) was performed only using SP+ groups, as the presence of SP ensured a more physiologically relevant environment and maintained oxidative balance necessary for fertilization. No significant differences were detected in cleavage or blastocyst rates among the three S. oleraceus extract doses. These findings highlight an interaction between SP and Sonchus oleraceus aqueous extract, suggesting that combining endogenous protein-rich components with exogenous plant-derived antioxidants may enhance post-thaw sperm functionality and its oxidative status in rams.