Sperm storage is a crucial reproductive adaptation that ensures fertilization success by maintaining viable sperm until ovulation. Birds and mammals have evolved anatomically distinct yet functionally analogous structures, sperm storage tubules (SSTs) in the avian female reproductive tract and the epididymis in the mammalian male reproductive tract, that represent a striking example of convergent evolution. These systems prolong sperm lifespan and regulate fertilization timing through shared physiological strategies. While each system has been studied independently, a direct comparison between SSTs and the epididymis has not been thoroughly explored. This review proposes that, although structurally distinct, SSTs and the epididymis exhibit shared physiological strategies such as metabolic suppression, pH and ion regulation, oxidative stress control, and hormonally mediated sperm release. By highlighting these parallels, we present a novel perspective on sperm storage as a case of evolutionary convergence in reproductive physiology. Understanding these shared mechanisms provides new insights into sperm viability regulation and offers practical implications for assisted reproductive technologies (ARTs), such as improved cryopreservation strategies and biomimetic sperm storage platforms designed to mimic SST or epididymal conditions.

Background: The poultry industry experiences genetic losses due to recurring infectious diseases, necessitating effective preservation strategies. Nitric oxide plays a crucial role in male reproduction, and optimal NO (nitric oxide) levels may enhance sperm viability. This study investigated the effects of SNAP (S-nitroso-Nacetylpenicillamine) on the longevity of rooster sperm. Methods: Semen was diluted with Beltsville Poultry Semen Extender-I containing 0 or 25 μM SNAP and stored at 10°C. Sperm motility and acrosome integrity were assessed at 1, 3, and 7 days. NO levels were quantified by DAF-FM diacetate and AI trials were evaluated by fertility and hatchability. Results: On day 1, sperm motility in the SNAP 25 μM-treated group was significantly higher than in the control. NO quantification confirmed that SNAP-treated semen exhibited higher NO levels. For fertilization and hatchability assessment, hens were divided into two groups based on the presumed duration sperm resided in sperm storage tubules. Before artificial insemination, the sperm was preserved at low temperature (10°C) to maintain viability. Fertilization rates were significantly higher in the SNAP-treated group in both short-term and long-term SST storage conditions. However, hatchability was only significantly improved in the SNAP-treated group when fertilization occurred after extended storage. Conclusions: These findings suggest that NO enhances sperm viability and fertility in poultry semen stored at low temperatures. SNAP 25 μM enhances AI efficiency by maintaining sperm viability and extending fertilization potential. Further research is needed to refine NO-based fertility enhancement strategies for avian species.

Background: Hanwoo cattle farmers aim to improve calf production and reproductive efficiency. Recovery of the reproductive tract postpartum is a critical factor influencing the postpartum period and conception of breeding cows. This study aimed to precisely analyze the recovery process of the reproductive tract in primiparous Hanwoo postpartum and to establish recovery criteria. Methods: Ten primiparous Hanwoo cows were used in this study. After parturition, estrus was examined daily using visual observations and estrus detection patches. Ovarian recovery, cervical diameter, and uterine horn diameter were examined using ultrasonography four times per week. Results: The analysis revealed that the first estrus occurred at 19.1 ± 6.5 days postpartum, the first ovulation at 27.1 ± 4.5 days, and the first normal estrus cycle at 39.2 ± 6.4 days. The ovulation rate during the first estrus was 40%. A normal estrus cycle occurred in 11.1% of patients at the first ovulation. The cervix diameter recovered to 42.0 ± 3.5 mm and the uterine horn diameter to 34.4 ± 7.1 mm by 24 days postpartum, with the difference in uterine horn diameter recovering to 2.6 ± 1.2 mm by 31 days postpartum. Conclusions: This study can aid in determining the optimal breeding time for postpartum primiparous Hanwoo cow and provide foundational data for Hanwoo breeding studies.

The acrosome cap allows sperm to penetrate the egg membrane and produce male pronuclei within female chicken eggs, facilitating successful fertilization. Given this, it is important to establish practical methods for evaluating the integrity of the acrosome cap and thus the quality of the rooster’s sperm. There are several established methods for evaluating the acrosomes of mammalian sperm, but none of these methods are suitable for evaluating the acrosome status of rooster spermatozoa. Therefore, a simplified method for evaluating the rooster acrosome is needed. Here we evaluated the usefulness of CBB (coomassie brilliant blue) staining of the acrosome at concentrations of 0.04%, 0.08%, and 0.3% CBB solutions. Our data revealed a clear staining pattern for intact acrosome caps at 0.04% and 0.08% CBB but not at 0.3% CBB. This protocol revealed differences in acrosome integrity between fresh and frozen rooster sperm smears suggesting that CBB staining may facilitate easier semen evaluation in roosters. This protocol allows for the accurate differential staining of acrosome cap in rooster spermatozoa.

Tight junctions are constituents of the blood–epididymis barrier that play roles in regulating the unidirectional transcellular transport of ions, water, and solutes to maintain optimal conditions for sperm maturation and storage. Claudin 1 (Cldn1) and 4 (Cldn4) are known as tight junction proteins and are expressed in the basolateral membranes as well as tight junctions in the epididymis of rodents. Here, we examined the expression and localization of Cldn1 and 4 to determine the function of these proteins in the pig epididymis. Cldn1 was highly expressed in the basolateral membrane of epithelial cells in the caput and corpus regions of the epididymis. In the cauda region, however, Cldn1 labeling was significantly decreased in the basolateral membrane of epithelial cells. In contrast, labeling indicated that Cldn4 was expressed in the basolateral membrane in the cauda region of the epididymis and was present at punctate reactive sites in the caput and corpus regions. However, in no region of the epididymis did we detect colocalization of Cldn1 and 4 with labeled ZO-1, the distribution of which is restricted to the tight junctions. Our results indicate that Cldn1 and 4 were region-specifically expressed in the pig epididymis but not present in the tight junctions of epididymal epithelium. In addition, reciprocal regulation in specific regions of the epididymis between Cldn1 and 4 may play an important role in generating an optimal luminal environment for sperm maturation and storage in the pig epididymis.

The initial segment (IS) in rodents is functionally and structurally distinct from other epididymal segments and plays an important role in sperm maturation. We previously showed that, in the mouse epididymis, basal cells (BCs) extend a narrow luminal-reaching projection only in the IS, while in all other regions, they mainly nestle at the base of the epithelium. We also found that BC projections are regulated by testicular luminal factors, and the present study was aimed at characterizing the signaling pathway involved in their formation and elongation. Previous studies reported that testicular luminal factors maintain the extracellular signal-regulated kinase (ERK) in a highly phosphorylated state in the IS. We report here that the BC projections, which we call axiopodia, periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement is controlled by MAPK/ERK signaling pathway. Our results suggest that ERK phosphorylation plays a key role in the formation and elongation of BC projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment.