This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

The aim of this study was to evaluate the motility, morphology, and antioxidant status of European red deer sperm stored in a liquid state (variant I) and in the epididymides (variant II). Spermatozoa were harvested post-mortem from the cauda epididymis. Sperm samples in both variants were stored for up to six days (D6) at 5 °C. Spermatozoa were assessed for motility, viability, morphology, activity of antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx; catalase, CAT), and lipid peroxidation (malondialdehyde, MDA, content). Sperm samples were analyzed on storage days 0, 2, 4, and 6 (D0-D6). Storage time and storage method significantly (p ≤ 0.05) influenced the examined variables. On D2, a decrease in motility and acrosomal integrity was observed in both storage variants, whereas a decrease in viability and an increase in MDA content were noted in spermatozoa stored in the epididymides. On D4, higher values of SOD and GPx activity and MDA content were noted in variant I than in variant II. Catalase activity was very low. GPx is the key enzyme that participates in the reduction of hydrogen peroxide in sperm cells. Spermatozoa stored in a liquid state were characterized by higher motility and viability, improved morphology and antioxidant status than those stored in the epididymides; therefore, liquid storage is more recommended for short-term preservation of epididymal spermatozoa.

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

UNASSIGNED: Epididymal sperm preservation is a simple conservation approach that can help prevent the loss of high genetic quality of farm animals. The chance of loss increases, especially during disease outbreaks or other interruptions to normal reproduction function.
UNASSIGNED: This study looked into the ability of preserved ram epididymal sperm to fertilize oocytes. Due to motility becoming an issue following sperm storage for fertilization, the sperm microinjection known as intracytoplasmic sperm injection approach was employed.
UNASSIGNED: The study was divided into two parts. First, involved the preservation of epididymal sperm at 5°C for 12 days. During preservation, sperm quality parameters namely motility, viability, intact membrane, acrosome, and Deoxyribonucleic acid (DNA) are evaluated every three days. For the fertility test in the second experiment, matured oocytes were injected with immotile sperm discovered in the last days of preservation. The presence of pronucleus development following in vitro culture is used as an indicator of sperm\'s ability to activate and fertilize oocytes.
UNASSIGNED: All sperm quality parameters significantly (p < 0.05) declined during preservation time. On day 12, motility was discovered to be 0%, but viable sperm, sperm with intact membrane, acrosome, and DNA remained at 41.86% ± 9.30%, 31.18% ± 5.15%, 21.88% ± 1.93%, and 33.35% ± 8.74%, respectively. On the fertility test, we inject immotile sperm from day 12 of preservation, which has the lowest motility found, into matured oocytes. Those sperms are able to activate (52.05% ± 7.15%) and fertilize (31.37% ± 1.75%) the injected oocytes, but their fertilizing ability is significantly lower (p < 0.05) when compared to the sperm derived from the ejaculate.
UNASSIGNED: In this study, simple preservation of epididymal sperm reduces all sperm quality criteria, particularly motility. Using the microinjection approach preserved sperm which had no motility, still demonstrated its ability to activate and fertilize the oocytes. According to that, this study provides potential approaches and tools for using genetically superior animals that have lost their ability to execute regular fertilization, and also prolong reproduction function.

In laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits.
In mice, sperm quality is usually assessed in sperm collected from the epididymis (organ where ripe sperm is stored) of euthanized males. However, there is one non-terminal and minimal invasive alternative to collect sperm, called percutaneous epididymal sperm aspiration (PESA), which allows repeated sample collections from the same individual. Given that individual sperm quality is variable and can change according to various factors, PESA could allow to track sperm quality over time and would be highly appreciated in different research fields. Here, we tested the suitability of PESA to determine sperm quality by comparing sperm samples collected by PESA vs the commonly applied terminal epididymis dissection. We used computer-assisted sperm analysis to determine various sperm quality traits. Surprisingly, we found that sperm collected by PESA showed significantly reduced motility, swimming velocity and more morphological abnormalities compared to sperm samples collected by epididymis dissection. Thus, we cannot recommend PESA as a suitable method to determine sperm quality traits as the procedure itself seems to affect collected sperm cells.

Techniques for the cryopreservation of epididymal sperm was are widely used in clinical practice. However, given the unique characteristics of sperm from patients with obstructive azoospermia, epididymal sperm cryopreservation is more difficult because of low count and weak motility; therefore, conventional methods of sperm cryopreservation may not result in the best outcomes. We used the micro-straw method to store small quantities of sperm obtained from patients with severe oligozoospermia or azoospermia and achieved successful deliveries in the previous study. This retrospective study of ICSI cycles included the first ICSI cycles of fresh or frozen/thawed epididymal sperm that were performed in patients suffering from obstructive azoospermia who were admitted to the CITIC-Xiangya Hospital of Reproduction and Genetics of China from June 1, 2015 to June 31, 2019. A total of 2441 patients with obstructive azoospermia were divided according to the use of fresh (n = 2342) or frozen/thawed (n = 99) epididymal sperm. The results showed that the fertilisation rate was higher with fresh epididymal sperm than that with frozen/thawed epididymal sperm (85.14% vs. 79.26%, respectively; p = 0.000). However, the rates of embryo cleavage, high-quality embryos, clinical pregnancy, miscarriage, singletons and birth defect were similar between fresh and frozen/thawed epididymal sperm (98.28% vs. 99.13%, 60.34% vs. 57.29%, 67.90% vs. 70.51%, 8.12% vs. 10.91%, 57.76% vs. 49.09%, 1.59% vs. 1.45%respectively; p = 0.088, 0.109, 0.628, 0.462,0.203 and 0.686). In addition, the short-term cryostorage of small quantities of epididymal sperm did not affect clinical outcomes. The results indicated that in cases of obstructive azoospermia, cryostorage of small quantities epididymal sperm is a reliable option.

BACKGROUND: The industrial revolution has resulted in increased synthesis and the introduction of a variety of compounds into the environment and their potentially hazardous effects have been observed in the biota. The present study was aimed to evaluate the potential endocrine-disrupting effects of chronic exposure to the low concentrations of bisphenol S (BPS) in male rats.
METHODS: Weaning male Sprague-Dawley rats (22 days old) were either exposed to water containing 0.1% ethanol for control or different concentrations of BPS (0.5, 5, and 50 μg/L) in drinking water for 48 weeks in the chronic exposure study. After completion of the experimental period, animals were dissected and different parameters (hormone concentrations, histology of testis and epididymis, oxidative stress and level of antioxidant enzymes in the testis, daily sperm production (DSP), and sperm parameters) were determined.
RESULTS: Results of the present study showed a significant alteration in the gonadosomatic index (GSI) and relative reproductive organ weights. Oxidative stress in the testis was significantly elevated while sperm motility, daily sperm production, and the number of sperm in epididymis were reduced. Plasma testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations were reduced and estradiol levels were high in the 50 μg/L-exposed group. Histological observations involved a significant reduction in the epithelial height of the testis along with disrupted spermatogenesis, an empty lumen of the seminiferous tubules, and the caput region of the epididymis.
CONCLUSIONS: These results suggest that exposure to 5 and 50 μg/L of BPS for the chronic duration started from an early age can induce structural changes in testicular tissue architecture and endocrine alterations in the male reproductive system which may lead to infertility in males.

Objective: This study was designed to assess the effects of using tris-soybean lecithin (TSL)-based extender supplemented with bovine serum albumin (BSA) on the quality of ram epididymal spermatozoa during refrigerated storage. Method: Epididymal sperm were collected from 22 Zandi rams, diluted in TSL-based extender at different concentrations (0%, 2.5%, 5%, 7.5%, and 10%) of BSA, and stored for 5 days at 4°C. Sperm parameters including motility, viability, plasma membrane integrity, chromatin protamination, and malondialdehyde (MDA) content were evaluated at 0, 24, 72, and 120 hours of refrigeration. Results: The addition of 10% BSA to the extender significantly improved sperm viability at 24 and 120 hours of refrigerated liquid storage (p < 0.05). An enhancement in plasma membrane integrity was observed along with a decrease in MDA level by increasing the concentration of BSA from 0% to 10% (p > 0.05). Sperm motion characteristics were higher in the BSA-free group at 120 hours of preservation (p < 0.05). No statistical difference was found for nuclear protamination between experimental groups (p > 0.05). Conclusion: BSA supplementation in TSL-based extender can preserve the viability of epididymal ram spermatozoa during liquid storage at 4°C.

Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls\' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time-PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p < .05), while in the parameters of fast progressive motility, VSL, LIN, WOB and STR there were not any significant variations in the frozen sperm compared to fresh (non-frozen) counterpart. The assessment of abundance of transcripts encoding motility (TSSK6) and fertility (PRM1 and PRM2)-related genes in epididymal sperm, showed that these transcripts were affected by freezing especially in slow progressive motility status (p < .01). Furthermore, cleavage and blastocyst rate did not present any significant differences between bovine embryos produced in vitro by fresh or frozen-thawed epididymal sperm. It can be concluded that epididymal sperm has enough freezability after overnight testes storage, and cryopreservation could not affect the percentage of in vitro produced embryos in spite of the changes of relative abundance of some transcripts and direction progressive motility pattern of sperm.

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca2+ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.