Effects of alpha lipoic acid supplementation on post-thaw quality of drone semen.

This study aimed to determine the effect of alpha-lipoic acid (ALA) on post-thaw quality of bee semen. In the study, semen from sexually mature drone were collected. A series of experiments were carried out in which the retrieved semen was diluted with diluents containing different ALA concentrations or without ALA supplement (control). Cryopreserved sperm were thawed, and evaluated for motility (phase-contrast microscope), plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fregmantation. The results obtained showed that the highest motility after thawing was observed in the groups containing ALA 0.25 mmol (P < 0.05). Likewise, plasma membrane integrity was found to be better preserved in the ALA 0.25 mmol-added group than in other groups. Acrosomal integrity were also higher in the ALA-containing groups than in the control group (P < 0.05). The results of this study show that ALA supplementation especially at 0.25 mmol improved post-thawed sperm motility, plasma membrane functionality, and mitochondrial membrane potantial quality of honeybee semen.

Pregnancy and birth rate outcomes in alpacas (Vicugna pacos) inseminated with frozen semen using two commercial extenders.

The aim of this study was to evaluate alpaca pregnancy outcomes and birth rates of females inseminated with frozen semen using two commercial extenders. A total of 18 ejaculates from 8 adult alpaca males were obtained with artificial vagina, and macroscopic and microscopic semen characteristics were assessed. Afterwards, samples were divided into two aliquots, diluted with Biladyl® B or AndroMed®, and cooled for 2 h at 5°C. At that moment, sperm motility was evaluated, and samples were frozen through a gradual descent of temperature using a liquid nitrogen tank. To analyse frozen sperm quality, samples were thawed at 38°C for 30 s. Even though a significant decrease in sperm motility and viability was detected when thawed (p < .05), no superiority was found between the two commercial extenders (Biladyl® B vs. AndroMed®). A total of 36 alpaca females were artificially inseminated (AI) between 30 and 34 h post-injection of a GnRH analogue, administered when a growing dominant follicle was detected through transrectal palpation and ultrasonography. Obtained pregnancy rates were similar between Biladyl® B (33.3%, 6/18) and AndroMed® (22.2%, 4/18). No significant differences were detected in birth rates between the two tested extenders, obtaining 4 and 3 births for Biladyl® and AndroMed®, respectively. In conclusion, alpaca pregnancies and alive offspring can be obtained through AI with frozen semen at similar efficiency rates using commercial diluents, Biladyl® B or AndroMed®.

Protective effects of different doses of MitoQ separately and combined with trehalose on oxidative stress and sperm function of cryopreserved Markhoz goat semen.

The mitochondria-targeted antioxidant MitoQ has been regarded as an effective antioxidant agent against cryo-induced oxidative cellular damage. This study aimed to evaluate the use of different doses of MitoQ combined with trehalose to minimize mitochondrial impairment and oxidative stress during sperm cryopreservation of Markhoz goat. For this, semen samples (n = 50) were collected by electroejaculation every 5 days from 5 bucks in 10 replicates. On each collection day, 5 ejaculates (one ejaculate for each buck) were pooled and then diluted in eight different Tris-based extenders as follows: no additives (control), 20, 200, 2000 nM of MitoQ (MT20, MT200, MT 2000, respectively), 150 mM of trehalose (Tr), MT20+Tr, MT200+Tr, MT2000+Tr. The semen samples were frozen using a standard protocol, and sperm function and oxidative stress were evaluated after thawing. The semen extender supplemented with MT200+Tr had higher (P < 0.05) total and progressive motility, acrosome and membrane integrity, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and lower (P < 0.05) DNA fragmentation, malondialdehyde and intracellular hydrogen peroxide levels than the all other groups except MT200; meanwhile, MT200 was also improved (P < 0.05) in these parameters than in the control group. Furthermore, MT200 and MT200+Tr showed higher (P < 0.05) percentages of live cryopreserved sperm with high mitochondrial activity than other groups. However, abnormality percentage and catalase activity of frozen-thawed sperm were not affected by treatments (P > 0.05). To conclude, we have found that supplementation of 200 nM MitoQ alone or in combination with 150 mM trehalose to semen extender improved the quality of cryopreserved sperm in goats, which is associated with enhanced antioxidant enzymatic defense and mitochondrial activity and reduced DNA fragmentation.

Soybean lecithin and cholesterol-loaded cyclodextrin in combination to enhances the cryosurvival of dairy goat semen.

The objective of the study was to examine the effect of soy lecithin (SL) and cholesterol loaded cryclodestrin (CLC) on cryo-survival of sperm cryopreserved in the presence or absence of seminal plasma in Saanen dairy goats. Tris-based dilutions containing various concentrations of SL (0, 0.5%, 1.0% or 2.0%) and CLC (0, 2.0 g/L, 4.0 g/L or 6.0 g/L CLC) were used to cryopreserve Saanen dairy goat sperm. The quality of frozen-thawed sperm, including progressive motility, viability, acrosome and plasma membrane integrity, as well as fertility were detected. Results found that the optimal combination of the two cryoprotectants was 1.0% SL+4.0 g/L CLC, which significantly increased progressive motility, viability, acrosome and plasma membrane integrity of frozen thawed sperm. The impact of the two cryoprotectants in combination was not affected by the presence of seminal plasma. The conception rates obtained after artificial insemination using sperm cryopreserved with and without seminal plasma were 88.89% and 91.67% (P > 0.05), respectively. The respective values for average number of litter sizes were 1.55 ± 0.17 and 1.56 ± 0.21 (P > 0.05). Therefore, this study improved the cryopreservation efficiency of goat semen, enhanced the sperm cryosurvival, and layed a foundation for the wide application of frozen goat semen.

Filtration techniques are advantageous over colloidal centrifugation in improving freezability of low-quality buffalo bull (Bubalus bubalis) ejaculates.

The study compared efficacy of three sperm selection techniques in improving freezability of low-quality Murrah buffalo bull ejaculates. Sephadex (SEP), Sephadex ion-exchange filtration (SIE), and 40/80% BoviPure™ (BP) gradient centrifugation protocols were standardized (ejaculates, n = 24). In Experiment-I, Sephadex G-75, G-100, and combined Sephadex G (75-100) column filtrates were compared. In Experiment-II, BP protocols: 200 g-10 min, 250 g-5, and 10 min, 300 g-10, and 15 min were compared. In fresh semen, Sephadex G (75-100) filtration and 250 g-5 min BP protocol improved sperm functions and were used in Experiment-III, where SEP G (75-100), SIE G (75-100), and 250 g-5 min BP processed ejaculates (n = 48) were cryopreserved and compared at post-thaw stage. The mean recovery rate differed in order: SEP > SIE > BP. SIE filtration significantly improved progressive motility, livability, membrane integrity, bovine cervical mucus penetration and live non-apoptotic sperm. Compared with control, all three techniques equally reduced post-dilution and post-thaw lipid peroxidation (LPO) rate. SEP post-thaw filtrates observed lower cryocapacitation-like changes, LPO (C11-BODIPY581/591), and higher active mitochondria than other treatments. SIE and SEP equally improved post-thaw acrosome-intact sperm over BP. Filtration techniques, preferably, Sephadex ion-exchange filtration can most efficiently process low-quality buffalo bull ejaculates for cryopreservation and improve freezability.

Recent approaches in the use of antioxidants and proteomic modifications in ram semen preservation.

Sperm preservation is a well-established technique in reproductive biotechnology that is widely used to maintain the genetic quality of male individuals. However, there are several factors during the preservation process that can affect the vitality, functionality, and quality of sperm, thereby reducing their fertility potential after thawing. One of these factors is the synthesis of high levels of oxidative stress (OS) during semen preservation, which can have detrimental effects on sperm health and functionality. To counter the negative impact of OS on sperm, researchers have explored the supplementation of several exogenous antioxidants in the extenders used to preserve ram sperm. This approach has shown promising results in improving sperm health, functionality, and fertility potential in ram. Additionally, the preservation process can induce modifications in the ram sperm proteome. By employing targeted proteomics techniques, researchers have been able to identify and modify specific proteins in cryopreserved ram sperm, potentially offering further improvements in the quality of the cryopreserved ram sperm. In summary, this review provides a comprehensive overview of the antioxidants and targeted proteomics modifications that have been investigated for enhancing ram sperm preservation. These advancements aim to mitigate the negative effects of OS and optimize the techniques used in preserving ram sperm.

Preservation of male fertility in patients undergoing pelvic irradiation.

As the number of cancer survivors increases, so does the demand for preserving male fertility after radiation. It is important for healthcare providers to understand the pathophysiology of radiation-induced testicular injury, the techniques of fertility preservation both before and during radiation, and their role in counseling patients on the risks to their fertility and the means of mitigating these risks. Impaired spermatogenesis is a known testicular toxicity of radiation in both the acute and the late settings, as rapidly dividing spermatogonial germ cells are exquisitely sensitive to irradiation. The threshold for spermatogonial injury and subsequent impairment in spermatogenesis is ~ 0.1 Gy and the severity of gonadal injury is highly dose-dependent. Total doses < 4 Gy may allow for recovery of spermatogenesis and fertility potential, but with larger doses, recovery may be protracted or impossible. All patients undergoing gonadotoxic radiation therapy should be counseled on the possibility of future infertility, offered the opportunity for semen cryopreservation, and offered referral to a fertility specialist. In addition to this, every effort should be made to shield the testes (if not expected to contain tumor) during therapy.

Gamma-oryzanol supplemented in extender enhances the quality of semen cryopreservation and alters proteomic profile in Thai swamp buffalo.

Reactive oxygen species (ROS) exert an adverse effect on sperm quality during the freezing process. Gamma-oryzanol is an effective antioxidant and has the ability to inhibit lipoperoxidation in various cells. Therefore, this study aims to investigate the effect of gamma-oryzanol supplementation in extender on post-thawed motility and proteomic profiles of swamp buffalo spermatozoa. Each ejaculate of an individual bull was divided into four equal aliquots. Gamma-oryzanol was supplemented at 0 (control), 0.1, 0.25, and 0.5 mM in tris-citrate egg yolk extender. The parameters of sperm motility were evaluated using computer assisted semen analyzer (CASA). The results showed that the progressive motility was significantly higher in 0.5 mM of gamma-oryzanol supplementation group when compared with the control group (p < 0.05), but no significant differences were observed among the treatments. In addition, a proteomic approach was applied to analyze the differentially expressed proteins in post-thawed sperm with or without gamma-oryzanol supplementation in extender. We confirmed that 2-phospho-d-glycerate hydro-lyase (ENO1), glutathione s-transferase mu 1 (GSTM1), phospholipid hydroperoxide glutathione peroxidase (GPX4), outer dense fiber protein 2 (ODF2), tektin-4 (TEKT4), tubulin beta-4B chain (TUBB4B), and ATP synthase subunit beta (ATP5B) were up-regulated in 0.5 mM of gamma-oryzanol supplementation group, which might be associated with the improved post-thawed motility observed in this treatment group. These results demonstrate the beneficial effect of gamma-oryzanol on post-thawed survival of swamp buffalo spermatozoa and help advance the understanding about molecular metabolism of sperm in this species.

Effects of extenders and cryoprotectants on cryopreservation of Thai red junglefowl (Gallus gallus gallus) spermatozoa.

Semen cryopreservation is crucial maintain genetic diversity of avian species. Little is known about suitable extenders for post-thaw survival of spermatozoa from Thai red junglefowl (Gallus gallus gallus). Therefore, the present study aimed to compare the suitability of the modified Thai red junglefowl extender (TRJFE) for cryopreservation of Thai red junglefowl spermatozoa with other extenders, including the Schramm extender, the red junglefowl extender (RFE), and the HS1 extender, in terms of sperm viability, motility, and fertility. First, the effects of adding 6% and 9% (v/v) N,N-dimethylacetamide, N,N-dimethylformamide (DMF), or N-methyl acetamide to these extenders on the post-thaw sperm quality of pooled ejaculates from 25 male fowls. The viability of thawed sperm was assessed by using nigrosin-eosin staining and sperm motility was determined by using computer-assisted sperm analysis. Fertility was assessed by inseminating 144 laying hens. The TRJFE +6% DMF combination significantly improved post-thaw viability (64.88 ± 0.51%) and motility (68.58 ± 1.13%) of Thai red junglefowl sperm, and the semen had the highest fertility (60.97 ± 0.72%). The findings suggest that TRJFE +6% DMF is a suitable freezing medium to conserve Thai red jungle fowl genetic resources.

Preconditioning of bull semen with sub-lethal oxidative stress before cryopreservation: Possible mechanism of mitochondrial uncoupling protein 2.

Preconditioning of sperm using sub-lethal oxidative stress before cryopreservation is an innovative approach that can improve sperm cryo-survival. Mitochondrial uncoupling proteins (UCPs) are critical in reducing ROS level during stress conditions. The aim of the current study was to investigate whether mild sub-lethal stress induced by low concentrations of nitric oxide and hydrogen peroxide has a protective effect on quality parameters of post-thaw bull semen through modulations of mitochondrial uncoupling protein 2 (UCP2) expression. Semen samples were collected from 6 mature Holstein bulls, then mixed and divided into 8 aliquots: fresh, frozen control and frozen groups treated with NO: 0.1 (NO-0.1), 1(NO-1), 10 µM (NO-10), and H2O2: 0.1(H2O2-0.1), 1(H2O2-1) and 10µM (H2O2-10). A significantly higher percentage of total motility, progressive motility and viability was observed in NO-1 and H2O2-10 compared to the other frozen groups (P < 0.05). Sperm exposed to 1 µM NO and 10µM H2O2 showed significantly increased percentages of mitochondria activity and membrane integrity (P < 0.05). Moreover, the lowest percentage of apoptotic percentage was observed in the NO-1 and H2O2-10 in comparison to the other frozen groups. In addition, the expression level of UCP2 was higher in the NO-1 and H2O2-10 compared to the other groups (P < 0.05). It can be concluded that stress preconditioning of bull sperm before cryopreservation can increase UCP2 expression of sperm, that can play a protective role against cryoinjury after thawing.

Relationship between sperm ubiquitination and equine semen freezability.

This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very low ubiquitin intensity and IV = very high ubiquitin intensity) and location of ubiquitin staining (classification 2). Statistical analyses were performed using SAS software (version 9.4), and p ≤ .05 was considered significant. We observed that PF stallions showed higher percentages (p < .05) of sperm cells with high ubiquitination (11.82% of ubiquitin intensity grade I, 39.13% of ubiquitin intensity grade II, 27.25% of ubiquitin intensity grade III, and 20.67% of grade IV), while GF stallions showed higher percentages (p < .05) of sperm cells with lower staining intensity (28.52% grade I, 59.83% grade II, 7.92% grade III, and 7.02% grade IV). Furthermore, for PF stallions, 23 significant correlations were detected (p < .05) between sperm abnormalities and ubiquitin intensity in different sperm regions. Increased ubiquitination of the sperm head, midpiece, and tail was positively correlated with their respective morphological defects. We concluded that high sperm ubiquitin levels are observed in ejaculates from stallions with poor semen quality (poor freezability), and ubiquitin marking in specific cellular locations can identify sperm morphological defects.

Sperm cryopreservation during the SARS-CoV-2 pandemic.

PURPOSE: Sperm cryopreservation is fundamental in the management of patients undergoing gonadotoxic treatments. Concerns have risen in relation to SARS-CoV-2 and its potential for testicular involvement, since SARS-CoV-2-positive cryopreserved samples may have unknown effects on fertilization and embryo safety. This study therefore aimed to analyze the safety of sperm cryopreservation for cancer patients after the onset of the pandemic in Italy, through assessment of the risk of SARS-CoV-2 exposure and viral RNA testing of semen samples. METHODS: We recruited 10 cancer patients (mean age 30.5 ± 9.6 years) referred to our Sperm Bank during the Italian lockdown (from March 11th to May 4th 2020) who had not undergone a nasopharyngeal swab for SARS-CoV-2 testing. Patients were administered a questionnaire on their exposure to COVID-19, and semen samples were taken. Before cryopreservation, SARS-CoV-2 RNA was extracted from a 150 µl aliquot of seminal fluid in toto using QIAamp viral RNA kit (Qiagen) and amplified by a real time RT PCR system (RealStar SARS-CoV2 RT PCR, Altona Diagnostics) targeting the E and S genes. RESULTS: The questionnaire and medical interview revealed that all patients were asymptomatic and had had no previous contact with COVID-19 infected patients. All semen samples were negative for SARS-CoV-2 RNA. CONCLUSION: This preliminary assessment suggests that a thorough evaluation (especially in the setting of a multidisciplinary team) and molecular confirmation of the absence of SARS-CoV-2 in seminal fluid from asymptomatic cancer patients may assist in ensuring the safety of sperm cryopreservation.

Effects of Spermine and Spermidine supplemented extenders on post-thaw Spermatological Parameters in Stallion Semen Cryopreservation.

In this study, the effects of polyamines, Spermine and Spermidine, on long-term preservation and post-thaw spermatological parameters were evaluated. Moreover, determination of the most suitable polyamine and its dose that can be added to standard extenders were aimed. Four adult Arabian stallions were used in the study. Five ejaculates were collected from each of four stallions via artificial vagina two days interval. Each ejaculate was divided into 13 aliquots. INRA96 (95,5%), egg yolk (2%), and glycerol (2,5%) were used as a control extender. Extenders of experimental groups were prepared with different doses of Spermine and Spermidine (0,1 mg/ml; 0,2 mg/ml; 0,4 mg/ml; 1 mg/ml; 2 mg/ml; 4 mg/ml). Stallion semen that were cryopreserved with Control and experimental extenders were evaluated in terms of Total Motility, Progressive Motility, Plasma Membrane Integrity, Capacitation Index, Acrosome Integrity and DNA Fragmentation Index. At the end of the evaluations, it was determined that 0,2 mg/ml Spermine and 0,4 mg/ml Spermidine showed better Total Motility and Progressive Motility, numerically. On the other hand, it was observed that 4 mg/ml Spermine and Spermidine had the lowest statistically significant values (p < 0,001). While statistically similar differences were obtained between groups in terms of the Plasma Membrane and Acrosome Integrity, it was determined that all experimental groups had lower and statistically significant values in terms of Capacitation and DNA Fragmentation Index (p < 0,001). As result, it was observed that the stallion semen cryopreservation success can be increased by the addition of 1-2 mg/ml Spermine that had effective protection on Capacitation and DNA Fragmentation Index without damaging other spermatological properties.

Comparative evidence support better antioxidant efficacy of mitochondrial-targeted (Mitoquinone) than cytosolic (Resveratrol) antioxidant in improving in-vitro sperm functions of cryopreserved buffalo (Bubalus bubalis) semen.

The present study compared the effect of mitochondria-targeted (Mitoquinone, MitoQ) and untargeted cytosolic antioxidant (Resveratrol, RESV) supplementation on lipid peroxidation (LPO) and in-vitro sperm functions of cryopreserved buffalo bull semen. To optimize additive's concentration, sperm pellet obtained from twenty-four ejaculates was supplemented with different concentrations of MitoQ (20 nM, 100 nM, 200 nM); and RESV (10 µM, 25 µM, 50 µM) against control in the extender. The post-thaw sperm motility, livability, and membrane integrity were higher (P < 0.05) in 200 nM MitoQ and 50 µM RESV than other concentrations used. In another experiment, sperm pellet from thirty-two ejaculates was supplemented with 200 nM MitoQ and 50 µM RESV in the extender. Pre-freeze and post-thaw progressive motility and livability were higher (P < 0.05) in MitoQ (200 nM) than RESV (50 µM) treatment. MitoQ supplementation improved post-thaw membrane integrity (CFDA-PI) higher (P < 0.05) than RESV, however, hypo-osmotic swelling response observed no improvement with RESV treatment. Post-thaw LPO rate was lower (P < 0.05) and Bovine cervical mucus penetration was higher (P < 0.05) in MitoQ than RESV treatment. In post-thaw semen, MitoQ showed higher (P < 0.05) proportion of acrosome intact (FITC-PNA), live non-apoptotic (P < 0.01) sperm with a higher reduction (P < 0.05) in membrane scrambling. MitoQ improved (P < 0.01) proportion of sperm with high Mitochondrial Membrane Potential and low LPO (P < 0.01) than RESV treatment. In conclusion, improvement in post-thaw in-vitro sperm functions and cryo-tolerance was more evident in MitoQ than RESV supplemented buffalo bull semen. Our study provides a better strategy to mitigate oxidative stress by enhancing mitochondrial antioxidant system with targeted antioxidants than cytosolic antioxidant supplementation.

SARS-CoV-2 infection, male fertility and sperm cryopreservation: a position statement of the Italian Society of Andrology and Sexual Medicine (SIAMS) (Società Italiana di Andrologia e Medicina della Sessualità).

PURPOSE: The recent pandemic of severe acute respiratory syndrome (SARS) due to coronavirus (CoV) 2 (SARS-CoV-2) has raised several concerns in reproductive medicine. The aim of this review is to summarize available evidence providing an official position statement of the Italian Society of Andrology and Sexual Medicine (SIAMS) METHODS: A comprehensive Pubmed, Web of Science, Embase, Medline and Cochrane library search was performed. Due to the limited evidence and the lack of studies, it was not possible to formulate recommendations according to the Oxford 2011 Levels of Evidence criteria. RESULTS: Several molecular characteristics of the SARS-CoV-2 can justify the presence of virus within the testis and possible alterations of spermatogenesis and endocrine function. Orchitis has been reported as a possible complication of SARS-CoV infection, but similar findings have not been reported for SARS-CoV-2. Alternatively, the orchitis could be the result of a vasculitis as COVID-19 has been associated with abnormalities in coagulation and the segmental vascularization of the testis could account for an orchitis-like syndrome. Finally, available data do not support the presence of SARS-CoV-2 in plasma seminal fluid of infected subjects. CONCLUSION: Data derived from other SARS-CoV infections suggest that in patients recovered from COVID-19, especially for those in reproductive age, andrological consultation and evaluation of gonadal function including semen analysis should be suggested. Studies in larger cohorts of currently infected subjects are warranted to confirm (or exclude) the presence of risks for male gametes that are destined either for cryopreservation in liquid nitrogen or for assisted reproduction techniques.

Effects of extender type on the quality of post-thaw giant panda (Ailuropoda melanoleuca) semen.

Sperm cryopreservation is an essential approach for assisted reproduction and genetic resources conservation in captive giant pandas. Cryopreservation, however, leads to a significant decrease in sperm quality and, consequently, a low fertilization rate. Therefore, it is mandatory to disclose more suitable and efficient freezing strategies for sperm cryopreservation. In the present study, we compared for the first time the performance of two commercial freeze extender (INRA96 versus TEST) freezing methods on post-thawed semen quality. Semen cryopreserved with the INRA96 showed better total motility (73.00 ± 4.84% vs 57.56 ± 3.60%, P < 0.001), membrane integrity (60.92 ± 2.27% vs 40.53 ± 2.97%, P < 0.001) and acrosome integrity (90.39 ± 2.74% vs 84.26 ± 4.27%, P < 0.05) than stored with TEST. There was no significant difference in DNA integrity after thawing between the two extenders (95.69 ± 3.60% vs 94.26 ± 4.84%). In conclusion, the INRA96 method showed to be better for giant panda sperm cryopreservation and should therefore be recommended for use in order to increase success of artificial insemination.

Effect of relaxin on cryopreserved beef bull semen characteristics.

This study aimed to improve the quality of cryopreserved beef bull (Piedmontese) semen by incorporation of relaxin in diluted semen before cryopreservation procedures. Semen samples were collected from 4 proven fertile bulls, using artificial vagina, once per week for 8 consecutive weeks and pooled together then diluted with Bullxcell® extender, and supplemented with different concentrations of relaxin (0 (control), 25, 50 and 100 ng/ml) before cooling, equilibration and freezing procedures. Frozen semen was thawed and assessed for motility by Computer-Assisted Sperm Analysis and vitality parameters such as acrosome, plasma membrane and DNA integrities, apoptosis, mitochondrial membrane potential, mucus penetration and SOD activity. The developmental potential of bovine embryos produced in vitro by using relaxin-treated was also investigated. In the present study, 50 and 100 ng/ml relaxin incorporation in extended bull semen before cryopreservation induced a reduction of sperm motility immediately after thawing (0h), whereas, during long incubation periods (1-2 h), relaxin showed a significant positive effect on sperm quality by improving the sperm motility and velocity parameters. Interestingly, sperm vitality was improved by 25 and 100 ng/ml relaxin and the blastocyst developmental rate was significantly increased in the 25 ng/ml relaxin group compared with controls (52/118, 44.0% vs. 32/116, 27.6%, respectively). These findings suggest a potential use of relaxin at the doses tested in the present study as an additive in the cryopreservation media of bull semen to improve sperm quality.

Cryopreservation of Ghagus chicken semen: Effect of cryoprotectants, diluents and thawing temperature.

The present study evaluated the effects of cryoprotectants, semen diluents and thawing temperature during Ghagus chicken semen cryopreservation. Four different experiments were conducted; Experiment 1-semen was cryopreserved using 6% dimethylacetamide (DMA) and 2% dimethylsulphoxide (DMSO) in Sasaki diluent (SD) and Lake and Ravie diluent (LR), Experiment 2 and 3-semen was cryopreserved using 8% ethylene glycol (EG) in SD, LRD and Red Fowl Extender (RFE), Experiment 4-semen was cryopreserved using 6% dimethylformamide (DMF) in SD, LR and Beltsville poultry semen extender (BPSE). Semen was cryopreserved in 0.5 ml French straws. Thawing was done at 5°C for 100 s in ice water in Experiments 1, 2 and 4, whereas in Experiment 3 thawing was done at 37°C for 30 s. The post-thaw sperm motility, viable sperm and acrosome-intact sperm were significantly (p < .05) lower in cryopreserved samples in all the experiments. No fertile eggs were obtained from cryopreserved samples in Experiments 1 and 2, except for 8% EG RFE treatment where the fertility was 0.83%. In Experiments 3 and 4, highest fertility was obtained in LR treatment 48.12 and 30.89%, respectively. In conclusion, using cryoprotectant EG (8%) and thawing at 37°C for 30 s, and DMF(6%) resulted in acceptable level of fertility in Ghagus chicken. Though the diluents influenced post-thaw in vitro semen parameters, the fertility was not affected. In addition, results indicated that thawing temperature may be a critical stage in the cryopreservation protocol.

Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols.

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to -10 °C (5 °C/min), and then from -10 °C to -130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.

Animal protein-free OptiXcell and shortened equilibration periods can replace egg yolk-based extender and slow cooling for rhinoceros semen cryopreservation.

OptiXcell (OP) was tested as an animal protein-free alternative to an egg yolk-based extender for rhinoceros semen cryopreservation and shorter chilling/equilibration periods were evaluated. Semen was collected from three rhinoceros species: black (Diceros bicornis; n = 2), white (Ceratotherium simum; n = 2), and greater one-horned (GOH; Rhinoceros unicornis; n = 3). Controls were diluted with equine extender (EQ) or OP and equilibrated for 1 h. Treatments were diluted with extender and cooled for 15 min (fast: FEQ; FOP) or not cooled (immediate: IEQ; IOP), prior to cryopreservation. Motility decreased post-thaw (EQ: 50.7 ±â€¯5.2%; OP: 52.9 ±â€¯3.4%) from fresh (82.9 ±â€¯2.9%), was higher in OP than IOP (38.6 ±â€¯4.9%; P ≤ 0.05) and decreased over time (P ≤ 0.05). Post-thaw acrosomal integrity was lower in EQ, FEQ, and IEQ (56.9 ±â€¯0.7; 56.6 ±â€¯4.5; 54.9 ±â€¯2.9%) than OP, FOP, IOP (71.8 ±â€¯4.7; 71.9 ±â€¯3.8; 69.9 ±â€¯4.5%) and fresh (72.6 ±â€¯1.4%; P ≤ 0.05). Progression and viability were lower in EQ (2.8 ±â€¯0.2; 61.9 ±â€¯7.4%) and OP (3.1 ±â€¯0.2; 53.4 ±â€¯6.9%) than fresh (3.7 ±â€¯0.2; 87.2 ±â€¯1.3%), decreased over time (P ≤ 0.05) but not different among treatments (P > 0.05). Morphology did not differ between fresh (75.0 ±â€¯4.9% normal) and any treatment group (70.0-77.8%) or over time (P > 0.05). OptiXcell is comparable to egg yolk-based EQ when used for rhinoceros semen cryopreservation. Furthermore, chilling/equilibration can be reduced with little impact on sperm characteristics.