Effects of hyaluronic acid on sperm parameters, mitochondrial function and apoptosis of spermatozoa in Simmental bulls with good and poor freezing ability.

Bulls with varying freezability exhibit substantial variation in semen characteristics after cryopreservation. Sperm freezability is positively correlated with membrane cholesterol content, membrane integrity, mitochondrial activity and antioxidant content. The purpose of this study was to determine the optimal concentration of hyaluronic acid (HA) in bull sperm with different cryotolerances. Simmental bulls (n = 10) semen samples were taken and categorized based on their progressive motility (PM) after freeze-thawing: Group I, consisting of bulls (n = 5) with progressive sperm motility ≥45%, was considered good freezability ejaculates (GF), and Group II, including bulls (n = 5) with progressive sperm motility ≤30%, was considered poor freezability ejaculates (PF) bulls. Semen samples were diluted with a Tris-egg-yolk-glycerol (TEYG) extender containing various concentrations of HA: without HA (control), 1 mM HA, 2 mM HA and 4 mM HA. After the freeze-thaw process, sperm kinematics, plasma membrane and acrosome integrity, mitochondrial activity and apoptotic status were evaluated. The addition of 1 mM HA to the diluent of bulls with GF increased PM and linearity (LIN) compared to the control group (p < 0.05). Normal morphology was improved after thawing in the samples treated with 1 and 2 mM HA in the GF and PF bulls respectively. The membrane and acrosome integrity of GF bulls treated with 1 mM HA was significantly (p < 0.05) greater than that of the control groups. Adding 1 mM HA to the extender of bulls with GF and PF improved the proportion of viable cells compared with the highest concentration (4 mM) of HA. The mitochondrial activity of PF bulls treated with 1 and 2 mM HA was significantly (p < 0.05) greater than that of the controls and 4 mM HA. Finally, it can be concluded that adding low doses of HA (1 mM) to the TEYG extender of GF and PF bulls ameliorated the post-thaw semen quality.

Effect of Bovine Serum Albumin Supplementation in Tris-Soybean Lecithin-Based Extender on Quality of Chilled Ram Epididymal Spermatozoa.

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.

Inclusion of ovine enriched serum with vitamin E and polyunsaturated fatty acids in the freezing medium: a new strategy to improve human frozen-thawed sperm parameters.

The objective was to evaluate the effect of inclusion of 2.5% and 5% ovine serum, enriched with vitamin E (Vit E) and fish oil (FO), in human sperm freezing medium. Serum samples were prepared from sixteen rams (n = 4) feeding on a without supplemented diet, and diets supplemented with Vit E, FO and Vit E + FO. Semen samples, from 60 normozoospermic men, were frozen in: (I) a commercial freezing medium (SpermFreeze™; control medium), (II) the commercial freezing medium containing foetal bovine serum, (III) the commercial freezing medium + nonenriched serum (serum group), (IV) the commercial freezing medium + Vit E enriched serum (Vit E group), (V) the commercial freezing medium + FO enriched serum (FO group) and (VI) the commercial freezing medium + Vit E + FO enriched serum (Vit E + FO group). Sperm total and progressive motility, morphology, viability and plasma membrane integrity were significantly higher (p ≤ .05) in Vit E and Vit E + FO groups compared with the control group. Mitochondrial membrane potential did not differ between treatments (p > .05). It was concluded that ovine serum enriched with vitamin E and vitamin E + FO improved the quality of human spermatozoa but enriched serum containing FO could not improve the sperm cryo-injuries.

A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids.

Extracellular matrix-derived scaffolds provide an efficient platform for the generation of organ-like structures. Successful development of testicular organoids (TOs) with the capability of supporting complete spermatogenesis has not been reported yet. Here, we have developed an optimized method for the decellularization of ram testicular tissue fragments. Our findings showed that testicular fragments treated with a serial combination of Triton X-100 and SDS in PBS for 48 h resulted in the efficient removal of cellular materials and retention of the extracellular matrix (ECM) components. In order to fabricate testis-derived scaffolds (TDSs), the testicular ECM (T-ECM) was digested in acid/pepsin, followed by neutralization of pre-gel solution to form a hydrogel. Then, the hydrogels were freeze-dried and cross-linked using a chemical method. To reach the optimal concentration for the T-ECM in the fabrication of TDSs, the scaffold properties including porosity, pore size, swelling behavior, and degradation were evaluated. Our study suggested that 25 mg ml-1 of the T-ECM is the best concentration for the fabrication of macroporous TDSs for demonstrating lower pore size, homogeneously distributed pores, and a higher swelling ratio. Furthermore, inoculation of neonatal mouse testicular cells onto TDSs resulted in the generation of multicellular TOs in which the differentiation of spermatogonial cells into post-meiotic cells was confirmed. Hormonal analysis of TDSs revealed the functionality of TOs in the secretion of testosterone and inhibin B. The current study also demonstrated that macroporous TDSs could provide a novel platform for testicular tissue engineering and in vitro spermatogenesis.

Effect of extender and equilibration time on post thaw motility and chromatin structure of buffalo bull (bubalus bubalis) spermatozoa.

OBJECTIVE: The aim of the present study was to investigate the effects of four equilibration times (2, 4, 8 and 16 hours) and two extenders (tris or Bioxcell®) on cryopreservation of buffalo semen. MATERIALS AND METHODS: In this experimental study, split pooled ejaculates (n=4), possessing more than 70% visual sperm motility were divided in two aliquots and diluted in Bioxcell® and tris-citric egg yolk (TCE) extenders. Semen was cooled to 4°C within 2 hours, equilibrated at 4°C for 2, 4, 8 and 16 hours, then transferred into 0.5 ml French straws, and frozen in a programmable cell freezer before being plunged into liquid nitrogen. Postthaw motility characteristics, plasma membrane integrity, acrosome morphology and DNA integrity of the buffalo sperm were studied after thawing. RESULTS: There were significant interactions between equilibration times and extenders for sperm motility and membrane integrity. Post thaw sperm motility (PMOT), progressive motile spermatozoa (PROG), plasma membrane integrity (PMI) and normal apical ridge (NAR) measures were lower for sperm equilibrated for 2 hours in both TCE and Bioxcell® extender compared to others equilibration times. PMOT, PMI and NAR for sperm equilibrated for 4, 8 and 16 hours showed no significant differences in either extender, although PROG measures were superior in Bioxcell®compared to TCE at all equilibration times (p<0.05). Kinematic parameters such as average path velocity, curvilinear velocity and linearity in the Bioxcell®extender were superior to those in the TCE extender studied. In contrast to motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected by different equilibration times. CONCLUSION: Equilibration time is necessary for preservation of the motility and integrity of buffalo sperm membranes. Equilibration times of over than 2 hours resulted in the greatest preservation of total semen parameters during cryopreservation. There were no significant interactions between equilibration times over 4 hours and type of extender which lead to greater post thaw sperm survival.

Effect of Different Thawing Rates on Post-Thaw Viability, Kinematic Parameters and Chromatin Structure of Buffalo (Bubalus bubalis) Spermatozoa.

OBJECTIVE: The aim of the present study was to evaluate three thawing rates on the post thaw motility, viability and chromatin structure of buffalo semen frozen in 0.5-ml straws. MATERIALS AND METHODS: In this experimental study semen was collected with artificial vagina (42℃) from four buffalo bulls.Split pooled ejaculates (n=4) were extended at 37℃ with a Bioxcell® extender. Semen was cooled to 4℃ within 2 hours, equilibrated at 4℃ for 4 hours, then filled in 0.5 ml French straws, and frozen in programmable cell freezer before plunging into liquid nitrogen. Straws were thawed at water bath temperatures of 37, 50 or 70℃ for 30, 15 and 6 seconds, respectively. Semen was incubated at 37℃ for 2 hours and evaluated for post thaw motility, viability, acrosomal and DNA integrity of spermatozoa. Analysis of variance (ANOVA) was used for comparisons of means. When the ANOVA test showed statistical differences, the mean of the treatments were compared using Duncan's multiple range tests. RESULTS: The initial postthaw motility (0 hour) averaged 62.7 ± 7.2%, 73.1 ± 9.77%, and 74.9 ± 8.58% for the three thaw rates, respectively. Kinematic parameters such as average path velocity, linearity and beat/cross frequency in the thaw rate of 70℃ for 6 seconds were superior to other rates studied (p<0.05). After 2 hours of incubation, proportions of progressive motility and Kinematic parameters decreased in all groups (p>0.05). A positive correlation was detected between sperm motility and thawing rate after two hours incubation times. The percentage of viable spermatozoa and spermatozoa with an intact acrosome and plasma membrane integrity were not different between the groups of samples thawed at different temperatures (p>0.05). The percentage of spermatozoa with chromatin dispersion forthe thaw rate of 70℃ for 6 seconds was significantly higher than for the to other rates studied (p< 0.05). In contrast with motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected during 2 hours incubation. CONCLUSION: The post thaw motility and kinematic parameters of buffalo spermatozoa were significantly improved immediately after thawing by increasing the thawing rate from 37℃ in 30 seconds to70℃ in 6 seconds. However, this relative advantage had disappeared after incubation in a water bath at 37℃ for two hours.A thaw rate of 70℃ for 6 seconds was associated with higher chromatin dispersion than the other thaw rates studied. Sperm thawing over at 50 degrees could be safely used to improve motility recovery after sperm cryopreservation in buffalo bulls.