Yolk Extender Research Articles

Evaluation of the use of L-carnosine in short-term storage of ram semen with spermatological and flow-cytometric analyzes

This study was conducted to investigate the effect of adding different doses of L-carnosine to semen in vitro on the short-term storage of semen. Semen taken from 6 Akkaraman rams was used in the research. Semen taken from each animal was pooled. It was diluted with tris+egg yolk extender containing L-carnosine at different doses (1, 5, 10, 20, 50, 100 and 200 mM) and stored at +4°C for 96 hours. It was determined that 1, 5, 10, 20 mM L-carnosine doses increased total, progressive and rapid motility, kinematic parameters VCL, VSL, VAP values and high mitochondrial membrane potential level during short-term storage. It was also determined that these doses protected membrane integrity, reduced the rate of abnormal spermatozoon, acrosomal damage and low mitochondrial membrane potential level. It was determined that a dose of 200 mM L-carnosine had a toxic effect and negatively affected the parameters. As a result, when ram semen is desired to be stored for a short time (for 96 hours), it may be recommended to add 1, 5, 10 and 20 mM L-carnosine amino acid to the semen extenders in order to preserve spermatological parameters.

Cryoprotective Potential of Theobromine in the Improvement of the Post-Thaw Quality of Bovine Spermatozoa.

Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [CtrlC]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (CtrlN). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity (p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to CtrlC. All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa (p < 0.001) than CtrlC. Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR (p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 (p < 0.01), while all TBR doses stabilized the levels of PKC (p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process.

The Effect of Resveratrol and Catalase on Post-Thaw Angora Buck Semen

This research aimed to examine the impact of resveratrol and catalase on the motility, plasma membrane integrity, acrosomal membrane integrity, and mitochondrial activity of Ankara buck semen following freeze-thawed process. In this study, semen samples obtained from four mature bucks were divided into four groups: control (C), resveratrol 500 µM/ml (R), catalase 50 IU/ml (CAT), and resveratrol 500 µM/ml + catalase 50 IU/ml (CATR). After dilution with Tris/egg yolk extender, the semen samples were frozen in liquid nitrogen and then thawed for assessment. The CATR group gave the highest values across all evaluated parameters (motility, plasma membrane integrity, acrosomal membrane integrity, and mitochondrial activity) compared to the other groups (61 ± 1.0%, 72.6 ± 0.70%, 70.73 ± 0.67%, 60.9 ± 0.79%, respectively) (p&lt;0.05). In conclusion, the combination of catalase and resveratrol significantly improved the quality of buck semen after freeze-thawed process, thereby contributing to enhanced reproductive outcomes and genetic preservation.

Coconut water combined with purebred chicken egg yolk as an alternative semen extender for Madura Pote bucks semen preservation

One of the many efforts to increase the quality of livestock genetics is through artificial insemination (AI). Other than increasing it, AI can be conducted to preserve semen. A successful AI is determined by seminal quality, therefore, a method to preserve semen for a longer storage time is needed. The method used is adding an extender that fulfills prerequisites for a semen extender such as coconut water combined with egg yolk citrate extender. Coconut water is rich in carbohydrates, protein, vitamins, and antioxidants while egg yolk contains lecithin. This study aims to find out the Pote buck spermatozoa quality stored in coconut water and egg yolk extender. This study uses three groups of treatments (T0: 0.1 ml semen + 0.9 ml egg yolk citrate, T1: 0.1 ml semen + 0.9 ml coconut water, and T2: 0.1 ml semen + egg yolk citrate (20%) + coconut water). All three of these are stored at 5oC and evaluated every day until day 5 of their motility, viability, intact plasma membrane, abnormality, and MDA level. Data analysis used is ANOVA and a further test called BNT is conducted if a significant difference is determined. No significant difference was found between T0 and T1 (p&gt;0.05). The highest progressive motility, viability, and intact plasma membrane (%) among the three groups of treatments happened to be from T2. Meanwhile, a low percentage of spermatozoa abnormality and MDA level were also found in T2 with its extender being coconut water combined with egg yolk citrate. To conclude, the best extender for storing Pote buck semen is stored at 5oC is coconut water combined with egg yolk citrate extender.

Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus)

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

Enhancing cryopreserved ram sperm quality at-80 °C with Spirulina platensis and Salvia verbenaca extracts.

This study was conducted in two steps to evaluate the influence of freezing methods and natural extracts on cryopreserved ram sperm quality. Initially, the research compared the effects of two freezing methods: liquid nitrogen (LN2) versus -80 °C, on post-thawed ram semen on total and progressive motilities and velocity parameters. Experiment I revealed no significant differences (P > 0.05) between the LN2 and -80 °C freezing methods, indicating similar effects on the analyzed parameters. Experiment II aimed to examine the influence of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to egg yolk extender on cryopreserved sperm quality, utilizing the -80 °C freezing method. Various concentrations (1.25, 3.75, 6.25 and 8.75 µg*mL-1) of acetone (Ac-SP and Ac-SV) and hexanoic (Hex-SP), as well as methanolic (MeOH-SV) extracts, were added into the extender. A thorough assessment of post-thawed sperm quality parameters, encompassing motility, velocity parameters, viability, membrane integrity, abnormality and lipid peroxidation was conducted. The outcomes demonstrated that 1.25 and 3.75 g*mL-1 of Ac-SP and Hex-SP and 1.25 µg*mL-1 of AC-SV and MeOH-SV increased the post-thawed ram sperm quality. In conclusion, this study emphasizes the antioxidant properties of SP and SV extracts, highlighting their potential to protect cryopreserved sperm cells from oxidative stress at -80 °C.

The effects of supplementation of vitamin D to the egg-yolk extender on cryopreservation of ram semen.

This study aimed to examine the effects of supplementation of vitamin D to the egg-yolk extender on characteristics of frozen-thawed ram semen. Semen samples obtained from adult rams were pooled and divided into five equal volumes. It was reconstituted with extenders containing different concentrations of vitamin D: 0 (control), 12.5 (VITD 12.5), 25 (VITD 25), 50 (VITD 50), and 100ng/mL (VITD 100), and then they were frozen. Sperm motility parameters, plasma membrane functional integrity, acrosomal integrity, DNA fragmentation, and mitochondrial membrane potential of the groups were evaluated after sperm thawing. Total motility and progressive motility were higher in VITD 50 than in all other groups (p<0.05). Higher sperm straightness, linearity, and wooble were higher in VITD 50 than in the control group (p<0.05). A similar pattern of VITD 50 was observed for plasma membrane integrity and mitochondrial membrane potential (p>0.05). In the study, it was observed that adding vitamin D to the extender had a beneficial effect on ram spermatological parameters. In addition, it was concluded that the use of the 50ng/mL vitamin D in the extender provided more effective protection than the other doses.

Effect of autologous platelet-rich plasma on the fertility and quality of cryopreserved buffalo bull semen: a comparative study using OptiXcell® and tris egg yolk extenders

BackgroundBuffalo spermatozoa have a distinct membrane structure that makes them more vulnerable to cryopreservation, resulting in lower-quality post-thawed sperm. This decreases the success rate of artificial insemination in buffaloes. Understanding and addressing these specific vulnerabilities are essential for improving reproductive techniques in buffalo populations. The properties of cryopreserved buffalo bull semen were examined in this study regarding the impact of adding autologous platelet-rich plasma (PRP) to OptiXcell® or Tris egg yolk-based extenders. Ten buffalo bulls were used to collect semen. Each bull’s ejaculate was separated into two main equal amounts, each of which was then diluted with either OptiXcell® or Tris egg yolk-based extender, supplemented with various PRP concentrations (5%, 10%, and 15%), and the control (0%), before being cryopreserved according to established protocols. Following equilibration and thawing, the quality and functionality of the sperm were evaluated, along with the antioxidant enzyme activities (GSH and TAC), malondialdehyde (MDA) content, and in vivo fertilization rate of the thawed semen.ResultsAll PRP concentrations in both extenders, particularly 10% PRP, improved the quality and functionality of the sperm in both equilibrated and frozen-thawed semen. Additionally, the antioxidant enzyme activities in both extenders were higher in the PRP-supplemented groups compared to the control group in thawed semen (P < 0.05). All post-thaw sperm quality, antioxidant enzyme activities, and functionality aside from DNA integrity were higher (P < 0.05) in the PRP-supplemented OptiXcell® than in the PRP-supplemented Tris egg yolk-based extender. The fertility of cryopreserved semen in the extenders supplemented with 10% and 15% PRP increased (P < 0.05) significantly more than that of the control extenders, with 10% PRP being the optimum concentration in OptiXcell® (80%) compared to that of Tris egg yolk-based extender (66.67%) and control of two extenders (53.33% and 46.67%, respectively).ConclusionsEven though autologous PRP-supplemented extenders have a protective impact on equilibrated and cryopreserved semen, 10% PRP-supplemented OptiXcell® extenders are more effective at preserving post-thaw semen quality, functionality, and antioxidant capacity, which increases the in vivo fertility of buffalo bulls.

The Quality of Cryopreserved Semen of Local Chickens Treated with Ringer’s Lactate-Egg Yolk Extender Supplemented with Glycine and Glucose

The Quality of Cryopreserved Semen of Local Chickens Treated with Ringer’s Lactate-Egg Yolk Extender Supplemented with Glycine and Glucose

A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls’ gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Quercetin in semen extender improves frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats.

This study investigated the antioxidant effect of quercetin-treated semen on frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats. In total, 32 ejaculates from four fertile bucks were diluted in Tris-based egg yolk extender with varying levels of quercetin (0, 1, 5, 10, and 15 μM). Qualified semen samples were pooled and frozen in French straws. The results revealed that the addition of quercetin in the semen extender increased (p < 0.05) frozen-thawed sperm total motility (TM), progressive motility (PM), rapid velocity (RV), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head (ALH) displacement in contrast to the control group. Quercetin supplementation had no effect on beat cross frequency (BCF), straightness (STR), and linearity (LIN) (p > 0.05). Quercetin showed significantly higher (p < 0.05) plasma membrane and acrosome integrity and viability (p < 0.05) of spermatozoa in contrast to the control group. Quercetin in the semen extender significantly increased (p < 0.05) superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and total antioxidant capacity (TAC) levels while reduced (p < 0.05) the contents of total oxidant status (TOS) and malondialdehyde (MDA), which were in contrast to the control group. Ultrasound results revealed that 24 out of 30 (80%) goats were found pregnant when semen was treated with 5 μM quercetin while the control group showed 18 out of 30 (60%) animals were pregnant. Thus, the study concluded that 5 μM quercetin-treated semen was found to be efficient, showed increased antioxidant status, and reduced oxidant production, leading to improved spermatozoa quality and in-vivo fertility in goats.

Sesame oil as a partial substitute for egg yolk in goat semen extenders

This study aimed to evaluate the effects of replacing egg yolk extender with sesame oil on the quality of sperm in goats following incubation at 37°C, chilling at 4°C, and freezing. Semen samples were collected from four intact male goats. The individual semen sample was divided into six groups consisting of a control group and five treatment groups with different egg yolk-to-sesame oil ratios. Seminal plasma was removed, and the sperm pellet was diluted with experimental semen extenders. The control group contained an extender of 10 % egg yolk (SO0), and the experimental extenders were composed of 8.75 % egg yolk and 1.25 % sesame oil (SO1.25); 7.5 % egg yolk and 2.5 % sesame oil (SO2.5); 5 % egg yolk and 5 % sesame oil (SO5); 2.5 % egg yolk and 7.5 % sesame oil (SO7.5); and 10 % sesame oil (SO10). Each group of semen was divided into three groups, incubated at 37°C for 1 h, chilled at 4°C for 4 h, or frozen for 24 h. Five replicates were performed. Sperm quality was evaluated, including motility, viability, and functional membrane integrity. The SO1.25 group achieved the highest sperm quality rate among the treatment groups, and the extender did not have a negative effect compared to the control. However, the total replacement of egg yolk with sesame oil in an extender resulted in the lowest sperm quality. In conclusion, the ratios of egg yolk and sesame oil that were acceptable for goat semen cryopreservation were 8.75 % and 1.25 %, respectively.

Apoptotic-Related MiRNAs Correlated with Functional and Flow Cytometric Parameters in Asthenozoospermic Holstein Bulls After Freeze-Thaw Process.

Many cellular processes in spermatozoa, including apoptosis and motility, are regulated by miRNA. Different miRNAs and molecular pathways are involved in asthenozoospermia (AS) conditions, which are thought to be one of the causes of infertility with reduced sperm motility. Thirty-two semen samples from four Holstein bulls with normozoospermia (NS), total motility ≥ 70%, and progressive motility ≥ 60%, and 32 semen samples from four bulls with AS, total motility ≤ 40%, and progressive motility ≤ 32% were used to investigate the function of apoptosis-related miRNAs in the AS group. Samples were then aspirated into a 0.5 mL straw after dilution with a Tris-egg yolk extender and frozen at -196°C. After freezing, semen samples were thawed for 2 weeks at 37°C and sperm kinematic parameters, plasma membrane integrity, acrosome integrity, DNA fragmentation, apoptosis status, and expression of apoptosis-related miRNAs (miR-2114, miR-296-3p, miR-455-3p, and miR345-3p) were evaluated. Our results showed that the functional and flow cytometric parameters of the NS group were significantly better than those of the AS group. In the NS group, miR-455-3pp and miR-2412 were upregulated, while miR-345-3p was downregulated compared with the AS group. In the AS group, miR-296-39, miR-2412, and miR-345-3p levels were strongly correlated with membrane integrity, DNA fragmentation, and apoptosis status. The findings demonstrated that the selected miRNAs based on bioinformatic analysis in AS and NS samples had a substantial association with functional and flow cytometry indicators and may be involved in regulating apoptosis and motility in AS samples.

Effect of the addition of soursop juice (Annona muricata L., 1753) to egg yolk citrate extender on the quality of bull semen

The present study was carried out to investigate the effect of the addition of soursop (Annona muricata L., 1753) juice to the citrate-egg yolk extender (EYC) on the quality of cryopreserved bull semen. After pre-evaluation, the semen was distributed in five extenders T1, T2, T3, T4 and T5 containing respectively 0% soursop juice (SJ) (T1), 25% SJ (T2), 50% SJ (T3), 75% SJ (T4), 100% SJ (T5) and microscopic evaluation was performed daily to assess plasma membrane integrity (PMI), progressive motility of spermatozoa (MP) and sperm pH until all sperm were dead. The results indicate a significant increase in spermatozoa PMI and MP (p&lt;0.05) in the case of treatment with 50% SJ (T3). Spermatozoa showed a motility of 50.78% 9 days later in the citrate-egg yolk extender containing 50% SJ (T3), whereas at day 9, spermatozoa in the other extenders had a progressive motility of less than 50%. Similar results were obtained for PMI with 50.78% of spermatozoa having an intact plasma membrane 9 days later in the citrate-egg yolk extender containing 50% SJ whereas the PMI of cells in the other stents was less than 50%. However, the pH of the extender containing 50% SJ was closest to 7 during the experimental period. The results of this experiment suggest that the addition of 50% soursop juice (T3) to the citrate-egg yolk extender could be adopted as an acceptable alternative to traditional egg yolk-based extenders for the cryopreservation of bull semen.

Comparison between commercial media and TRIS-egg yolk extender in the refrigerated storage of collared peccary semen at 17 °C

To assist in the conservation of collared peccary, it is important to strengthen semen processing protocols. The aim of this study was to compare the effects of different commercial extenders (BTS; NUTRIXcell+ and PRIMXcell Ultra) and TRIS + egg yolk on the functional and morphological aspects of collared peccary semen stored at 17 °C for 48 hours. Ten ejaculates obtained by electroejaculation were divided into 4 aliquots and diluted in the respective extenders, then stored in a biological incubator at 17 °C for 12, 24, 36, and 48 hours. The samples were evaluated for kinetic parameters, membrane functionality, membrane integrity, mitochondrial activity, morphology, and sperm-binding capacity. At the end of storage (48 h), promising results were found for motility parameters, with TRIS + egg yolk (71.0 ± 4.6%) being more efficient than NUTRIXcell+ (38.9 ± 10.9%) (P < 0.05) and similar to BTS (42.9 ± 11.9%) and PRIMXcell Ultra (46.8 ± 10.8%). The results for membrane integrity and mitochondrial activity were around ∼30–50%, with TRIS being the only extender to preserve both parameters (58.9 ± 5.3 and 59.2 ± 5.6%) for up to 48 hours, respectively (P < 0.05). Finally, the extenders could guarantee 60% membrane functionality and ∼ 60–70% normal sperm morphology, as well as similar binding capacity among the groups. In conclusion, TRIS + egg yolk is effective in preserving the sperm parameters of collared peccary semen at 17 °C for 48 hours, while PRIMXcell Ultra and BTS are viable alternatives for this purpose.

The kinetics of goat sperm is negatively affected after freezing in an extender including zinc oxide nanoparticles

BACKGROUND: Nanotechnology can benefit livestock industries, especially through postharvest semen manipulation. Zinc oxide nanoparticles (Np-ZnO) are potentially an example. OBJECTIVE: To investigate how the addition of zinc oxide nanoparticles (Np-ZnO) affected the characteristics of postthawed goat semen. MATERIALS AND METHODS: Seminal pools from four Saanen bucks were used. Semen was diluted in Tris-egg yolk extender, supplemented with Np-ZnO (0, 50, 100 or 200 μg/mL), frozen and stored in liquid nitrogen (−196°C), and thawed in a water bath (37°C/30s). Semen samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), and assessed for other functional properties by epifluorescence microscopy, such as plasma membrane integrity (PMi), acrosomal membrane integrity (ACi) and mitochondrial membrane potential (MMP). RESULTS: For total motility (TM), the group treated with 200 μg/mL Np-ZnO was superior to the control. In straight-line velocity (VSL), the control was better than the group containing 200 μg/mL of Np-ZnO. For average path velocity (VAP), the control was higher than with 100 μg/mL Np-ZnO. For linearity (LIN), the control was higher than with 200 μg/mL Np-ZnO. In straightness (STR), the control and 100 μg/mL Np-ZnO were higher than with 200 μg/mL Np-ZnO. In wobble (WOB), the control was better than the 50 μg/mL Np-ZnO treatment. In PMi, ACi and MMP no significant differences were found. CONCLUSION: The addition of Np-ZnO (200 μg/mL) to the goat semen freezing extender improved the total motility of cells, whilst negatively affecting sperm kinetics.

Evaluation of Metformin Supplemented Diluent on Cryopreservation of Canine Semen

Background: Cryopreservation of semen in canines is getting more advanced lately, however the fertility following the insemination of the frozen thawed semen is lower than other species. The higher content of PUFA and lipid peroxidation leading to production of reactive oxygen species is one of the reasons for the same. Antioxidants play a role in improving the post thaw semen quality in canines. Methods: Semen collected from 10 male dogs were further divided into two aliquots. The first aliquot was extended with TRIS-fructose egg yolk extender and the second one was extended with TRIS-fructose egg yolk extender supplemented with 50 µM of metformin. The extended semen was cryopreserved using conventional methods. Subsequently, both routine semen parameters and advanced semen parameters including viability, acrosomal integrity, DNA integrity and mitochondrial reactive oxygen species (ROS) were measured in the spermatozoa through the utilization of flow cytometry. Result: Present study indicated that, post thaw motility was significantly higher in the metformin supplemented group in comparison with control while, the mitochondrial ROS was significantly lower in metformin supplemented group. Our study concludes metformin could be effectively used to reduce the ROS produced during the process of cryopreservation of the canine semen.

Effects of organic and inorganic selenium supplementation on frozen-thawed ram semen at two cooling periods.

The aim of this study was to evaluate the effects of different selenium compounds on the sperm quality of cryopreserved ram semen. Ejaculates from four rams, collected using an artificial vagina heated to 38°C, were individually evaluated. The approved ejaculates were pooled and diluted (1:1 v:v) in Tris-egg yolk extender (20%, v/v) and separated into two control groups, one cooled for 2h and the other for 4h. The pooled ejaculates at the two cooling periods were supplemented with two doses (0.5 and 1μg/mL) of organic selenium (ORG), and inorganic selenium (SeNa), each. The samples were packed in 0.25ml straws, at a concentration of 400 × 106 sperms/mL and stored in liquid nitrogen. The straws were thawed in a water bath at 37°C for 20s, and the samples were subjected to sperm kinetics evaluation by Computer Assisted Semen Analysis software. Sperm membrane integrity, acrosome morphology, and mitochondrial potential were assessed. In addition, oxidative stress markers reactive oxygen species (ROS), ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive species (TBARS), and glutathione peroxidase (GPx) enzyme activity) were also evaluated. No significant improvement was observed in the ram semen quality at the two cooling times. Supplementation of the freezing extender with 0.5μg/mL ORG, subjected to 4h cooling period, increased the sperm motility when compared with the control group at the same cooling time. In addition, the 0.5μg/mL SeNa group, under the 2h cooling period, showed an increase in sperm motility when compared to the control group at the same cooling period. Considering the importance of sperm motility as a fertility parameter, our study indicates that supplementation with ORG and SeNa can help improve the total motility of the cryopreserved ram semen.

Effects of α-Tocopherol on chilled quality parameters of Tharparkar bull semen extended in Lecithin and Tris based egg yolk extender

In this experiment, four healthy Tharparkar Cattle Bulls, 4-5 years aged were used. Total 52 (n=13) ejaculates were collected, and the semen samples having motility, morphology and viability ≥70% were processed and extended into BIOXcell­­­TM and Tris Based Egg Yolk extender supplemented with and without α-tocopherol (Vitamin E) 0.02 Mm/ml. Pre-thawed assessment of motility, morphology, viability and membrane integrity with BIOXcell­­­TM + α-tocopherol (Vitamin E) (79.09±0.87, 86.13±0.79, 82.13±1.20, 81.40±0.77) showed improved quality parameters as compared to Tris+ α-tocopherol (Vitamin E) (74.69±0.94, 82.81±1.00, 77.63±1.26, 77.31±1.01), BIOXcellTM (73.55±1.20, 80.81±0.96, 76.02±1.28, 75.31±1.20) and Tris (71.72±1.28, 78.90±1.01, 74.04±1.31, 73.18±1.29) respectively. In conclusion supplementation of α-tocopherol (Vitamin E) improves the Pre thawed quality parameters i.e. Motility, Morphology, Viability and Membrane Integrity of Tharparkar Bull semen extended in BIOXcell­­­TM and Tris Based Egg Yolk extender.

Exploring enzymatic antioxidant activities in Asian Elephant (Elephas maximus) sperm and seminal plasma

This study explored the relationships between sperm quality and the activities of antioxidant enzymes (superoxide dismutase, SOD, and glutathione peroxidase, GPx) in sperm (S) and seminal plasma (SP) of Asian elephants, as well as the potential protective role of glutamine in semen extenders during chilled storage. In experiment 1, twenty ejaculates from 11 bulls were analyzed for percentages (%) of sperm motility (%MOT), viability (%VIA), and activities of SOD and GPx. A significant negative correlation was found between %VIA and SP-SOD (r = − 0.71), and positive correlations between %MOT and both S-GPx (r = 0.49) and SP-GPx (r = 0.57). Following these findings, glutamine, a precursor of glutathione and semen antioxidant was introduced in Experiment 2. Each ejaculate (in a total of 8 ejaculates obtained from four bulls) was diluted in Tris-fructose- (EYT) and Tris-glucose-egg yolk extender (TCG), respectively, supplemented with 0 (control), 20 and 40 mM glutamine and stored at 4 °C. Sperm parameters including %MOT, %VIA, functional membrane integrity (%sHOST), and DNA integrity (%DNA) were assessed after 24 and 48 h of storage. Results demonstrated that glutamine did not significantly affect %VIA or %DNA. A significant reduction in %MOT was observed at 24 h, but 20 mM glutamine added in extenders reversed this decline at 48 h. A 40 mM addition of glutamine in TCG markedly reduced %sHOST at 24 h. In conclusion, SOD and GPx activities in the sperm and seminal plasma are indicative of sperm quality. During chilling, using a higher level of glutamine may damage the spermatozoa of Asian elephants.