Buffalo Spermatozoa Research Articles

Motion characteristics and plasma integrity evaluation of Murrah buffalo semen

Buffalo semen collected from Murrah bull were cryopreserved and evaluated for different motility parameter, kinematics and plasma membrane integrity. Buffalo bulls were maintained uniform standard management and nutritional practices. Semen was collected regularly twice a week semen collection schedule from four (04) Murrah bull. Collected semen was immediately transported to laboratory and evaluated for different macroscopic parameter (color, volume and thickness). Fresh semen was then diluted with saline solution and evaluated for sperm concentration, motility, sperm kinematics and morphology. Semen samples that fill all the standard were selected for freezing and diluted with Tris-egg yolk citrate diluter. Diluted semen was equilibrated, cryopreserved and finally evaluated for post thaw sperm quality. Different motility parameter (total, progressive, static and slow motility) varied significantly (p<0.01) irrespective of different freezing stages. Significantly higher progressive sperm motility and viability of buffalo spermatozoa were observed at fresh semen whereas lower progressive sperm motility and viability was found at post thaw stage. Total and progressive motility reduced by 2.5 and 2.12% following equilibration, whereas following cryopreservation, total and progressive motility reduced by 35.7 and 28.51% and static motility increases accordingly (35.4%). Significantly higher plasma membrane integrity of sperm was observed at fresh semen followed by pre freeze and post thaw semen. Following freezing, integrity of plasma membrane reduces at the rate of 10.81% and 26.7% at pre freezing and post thaw stages. Significantly higher average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were found for fresh semen followed by pre-freeze and post-thaw semen. Frozen buffalo semen with higher progressive motility and motion characteristics may be produced if motility losses can be reduced during freezing stage as this stage results higher motility losses. Asian Australas. J. Biosci. Biotechnol. 2022, 7(2), 75-81

Kisspeptin-10 in cryodiluent improves the post-thaw quality of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa.

Effects of kisspeptin-10 as antioxidant in cryodiluent were evaluated on post-thaw quality of buffalo spermatozoa. Qualified semen samples from five bulls were pooled, divided into five aliquots and extended in Tris-citric acid cryodiluent containing differential doses of kisspeptin-10 (5, 10, 15, and 20 μmol L-1 and negative control. Extended sperm suspension was cooled to 4°C, packaged in 0.54 ml straws and cryopreserved. At post-thawing, catalase (unit mg-1 ), peroxidase (unit mg-1 ) and reduced glutathione (μmol L-1 ) levels were highest (p < 0.05) with 20 μmol L-1 of kisspeptin-10 as compared to negative control. Moreover, lipid peroxidation (nmol L-1 min-1 mg protein-1 ) level was lowest (p < 0.05) with 20 μmol L-1 of kisspeptin-10. Sperm progressive motility (%), rapid velocity (%) and kinematics were higher (p < 0.05) with 15 and 20 μmol L-1 of kisspeptin-10 as compared to negative control. Supra-vital plasma membrane integrity (%), viable sperm with intact acrosome (%) and DNA integrity (%) were improved (p < 0.05) with all doses of kisspeptin-10 as compared to negative control. It was concluded that the addition of 15 and 20 μmol L-1 kisspeptin-10 in cryodiluent ameliorated the overall frozen-thawed quality parameters of Nili-Ravi buffalo spermatozoa.

Development of homologous hemizona assay as a potential predictor of fertility in buffalo bulls: A preliminary approach

Although, myriads of tests are routinely used, no single test can accurately predict fertilization potential of semen. The hemizona assay (HZA) has advantages in two ways: (a) it determines multitude traits of sperm and (b) it is a controlled sperm function test. In the present study, we developed homologous HZA in buffaloes to predict bull fertility. In this experiment, bulls with fertility rate 53.3% and 48.5% were used as control, whereas bulls with fertility rate 32.6% and 32.2% were used as test semen samples. For HZA, matching buffalo hemizonae were co-incubated with processed buffalo sperm for 4 h. The number of sperms bound to the outer surface of hemizona was determined. No significant difference was observed in sperm binding for co-incubation of same bull sperm with matching hemizona (p < .05). Significant difference in sperm binding to matching hemizona was seen, while two halves were incubated with control and test semen, respectively (p < .05). Hemizona assay index (HZAI) of test bull semen has been determined from percentage of test-sperm bound to the matching hemizona in comparison to control-sperm. For finding relation, HZAI was correlated with respective fertility rates of semen samples, and it was found that a significant positive correlation was present with r=0.83, p < .1. A regression equation of Y=1.39X - 55.8 (where Y=pregnancy rate of test semen sample and X=HZAI of test semen sample) was presented to predict fertility rates of unknown semen samples. Thus, HZA can be used as a potential predictor of buffalo bull fertility.

X chromosome-linked genes in the mature sperm influence semen quality and fertility of breeding bulls

The role of sperm expressed X-linked genes on bull fertility has not been studied in detail. The objective of the present study was to assess the influence of X-linked genes on the sperm functional parameters and field fertility rate in the Holstein Friesian cattle (n = 12) and Murrah buffalo (n = 7) bulls. The enrichment analysis (cattle = 8; buffalo = 8) of the X-linked genes was carried out using retrospective RNA-seq data and mRNA expression levels of functionally relevant genes were validated using the RT-qPCR. The mRNA expression levels of these genes were functionally associated with sperm attributes and field fertility rate. The sperm transcriptome studies revealed that the total number of expressed genes and the transcript content of the X-linked genes in the mature sperm were very low in both species, and only 23.31% of these genes were commonly expressed between them. The transcript pool corresponding to the X-linked genes represents embryonic organ development (p = 0.03) and reproduction (p = 0.02) processes in cattle and buffalo sperm, respectively. The mRNA expression levels of X-linked genes, RPL10 and ZCCHC13 in cattle; AKAP4, TSPAN6, RPL10 and RPS4X in buffalo were significantly (p < 0.05) correlated with sperm kinematics. Importantly, the mRNA expression levels of the genes RPL10 (r = −0.68) and RPS4X (r = 0.81) had a significant correlation with the field fertility rate in cattle and buffalo, respectively. Multivariate regression models and receiver operating curve analysis suggest that the mRNA expression levels of X-linked genes may be useful in predicting bull fertility. The study indicates that sperm-expressed X-linked genes influence semen quality and field fertility rate in both cattle and buffalo.

Comparative Proteomic Analyses of Poorly Motile Swamp Buffalo Spermatozoa Reveal Low Energy Metabolism and Deficiencies in Motility-Related Proteins.

Simple SummaryProteomic analysis of normal and poorly motile buffalo sperm indicates that the formation of poorly motile buffalo sperm is associated with inefficient energy metabolism, decreased sperm protamine proteins, a lack of motility-related proteins, and changes in tail structural proteins. The results provide clues for finding molecular mechanisms for poor motility of buffalo sperm.The acquisition of mammalian sperm motility is a main indicator of epididymal sperm maturation and helps ensure fertilization. Poor sperm motility will prevent sperm cells from reaching the fertilization site, resulting in fertilization failure. To investigate the proteomic profiling of normal and poorly motile buffalo spermatozoa, a strategy applying liquid chromatography tandem mass spectrometry combined with tandem mass targeting was used. As a result, 145 differentially expressed proteins (DEPs) were identified in poorly motile spermatozoa (fold change > 1.5), including 52 upregulated and 93 downregulated proteins. The upregulated DEPs were mainly involved in morphogenesis and regulation of cell differentiation. The downregulated DEPs were involved with transport, oxidation-reduction, sperm motility, regulation of cAMP metabolism and regulation of DNA methylation. The mRNA and protein levels of PRM1 and AKAP3 were lower in poorly motile spermatozoa, while the expressions of SDC2, TEKT3 and IDH1 were not correlated with motility, indicating that their protein changes were affected by transcription or translation. Such changes in the expression of these proteins suggest that the formation of poorly motile buffalo spermatozoa reflects a low efficiency of energy metabolism, decreases in sperm protamine proteins, deficiencies in motility-related proteins, and variations in tail structural proteins. Such proteins could be biomarkers of poorly motile spermatozoa. These results illustrate some of the molecular mechanisms associated with poorly motile spermatozoa and provide clues for finding molecular markers of these pathways.

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.

The ameliorating effects of crocetin on frozen-thawed quality, and fertility via attenuating oxidative status of bubaline spermatozoa

Current study was conducted to appraise the cryoprotective influence of crocetin on quality, oxidative status, and fertility potential of bubaline spermatozoa. Collected semen from four bulls was diluted in five aliquots with (10 μM, 5 μM, 2 μM, 1 μM, and control [0 μM] supplementation of crocetin). After gentle dilution (37 °C), cooling (4 °C, in 2 h), equilibration (4 °C, for 4 h) and packaging of samples was done in straws (polyvinyl French, 0.5 ml), and then frozen (programmable cell freezer). This study established that crocetin supplementation significantly (p < 0.05) improves CASA (Computer Assisted Sperm motion Analyzer) total motility (%), rapid velocity (%), average-path, and curved-line velocities (μm/sec, 10 μM vs. control), and progressive motility (%), straight-line velocity (μm/sec), total antioxidant capacity (TAC, μMol/l), ATP concentrations (nmol/million), and fertility potential (%) (10 μM vs. control, and 1 μM), and mitochondrial potential (%) of buffalo spermatozoa (5, and 10 μM vs. control). Crocetin supplementation significantly (p < 0.05) alleviates DNA fragmentation, seminal plasma ROS (104 RLU/20/25 million, RLU = Relative light unit) levels, and lipid peroxidation (LPO, μMol/ml) in buffalo spermatozoa (10 μM vs. control). In a nutshell, crocetin supplementation improves post-thaw quality by means of motility parameters, motion kinematics, TAC, and ATP concentrations, and fertility potential, and abolished DNA fragmentation parameters, seminal plasma ROS, and LPO concentrations of buffalo spermatozoa. The exact mechanism by which crocetin acts are not fully elucidated; however, it is probable to speculate that the reduction in ROS, and LPO recorded in this study may be related to scavenging ability of this antioxidant during cryopreservation.

Correlation Between Dissolved Oxygen Level, Antioxidants and Oxidants in Semen Diluted with Partially Deoxygenated Extender at Various Stages of Cryopreservation

BACKGROUND: Dissolved oxygen (DO) in semen dilutor may lead to the production of reactive oxygen species (ROS) and buffalo sperm may become more prone to deleterious effects of ROS due to the presence of high amounts of polyunsaturated fatty acids (PUFAs) in their membranes. OBJECTIVE: To study the correlation between dissolved oxygen level, antioxidants and oxidants in semen diluted with partially deoxygenated extender at various stages of cryopreservation. MATERIALS AND METHODS: Each semen sample was split into two aliquots viz., Aliquot I [diluted with Extender I (control: without deoxygenation)] and Aliquot II [diluted with Extender II: partially deoxygenated by liquid nitrogen (LN) flushing], which were diluted, filled in straws, cryopreserved and evaluated post-thaw. RESULTS: The DO levels ( P &lt; 0.05) decreased significantly after LN flushing of the extender and they increased significantly ( P &lt; 0.05) in post-thaw semen. The progressive motility, viability, hypo-osmotic swelling response, acrosomal integrity, glutathione peroxidase (GPx), total antioxidant capacity (TAC) and superoxide dismutase (SOD) decreased significantly ( P &lt; 0.05) in both control and treated semen after thawing. SOD and TAC were positively correlated in semen treated with normal extender at the pre-freeze stage; however, in semen treated with partially deoxygenated extender, no correlation was found between SOD and TAC at the pre-freeze stage. ROS and total TAC were negatively correlated in semen treated with partially deoxygenated extender at the pre-freeze stage; however, no correlation was found between ROS and TAC in control semen. CONCLUSION: The partial deoxygenation of extender affects the correlation between sperm quality parameters, antioxidants, and oxidants during different stages of semen cryopreservation.

Freezability biomarkers in the epididymal spermatozoa of swamp buffalo

As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES.

Effect of cholesterol-loaded cyclodextrin enriched extenders on the quality of prefrozen and frozen buffalo semen

Objective: To investigate the effects of non-permeable cryoprotectant, cholesterol-loaded cyclodextrin, when added at different concentrations into cooled and frozen-thawed semen extended with Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders. Methods: A total of 40 ejaculates from four buffalo bulls were collected using artificial vagina. Ejaculates were extended with one of Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders which contained different concentrations [0 (control), 0.75, 1.50, 2.25 and 3.00 mg/mL] of cholesterol-loaded cyclodextrin. The extended semen samples were cooled to 5 °C and then frozen slowly to -196 °C in 0.25 mL ministraws before being stored in liquid nitrogen pending its evaluation. Sperm motility, live sperm, normal sperm morphology, sperm membrane integrity and acrosome morphology were measured. Results: Supplementation of cholesterol-loaded cyclodextrin improved progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 1.50-2.25 mg/mL depending upon types of used extenders and stages of pre- and post-freezing process (P&lt;0.01). The best concentration was 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post- freezing. However, greater concentration (3.00 mg/mL) of cholesterol-loaded cyclodextrin had a detrimental effect compared to the control group with the two evaluated extenders (P&lt;0.01). Conclusions: Cholesterol-loaded cyclodextrin supplementation at 1.50-2.25 mg/mL concentration could improve pre-frozen and post-thawed buffalo sperm quality. The most suitable concentration is 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.

Effect of platelet-rich plasma (PRP) on post-thaw quality, kinematics and in vivo fertility of fertile and subfertile buffalo (Bubalus bubalis) spermatozoa.

This study investigated the effect of adding platelet-rich plasma (PRP) in semen extender prior cryopreservation on post-thaw quality, kinematics, and in vivo fertility of fertile and subfertile buffalo spermatozoa. Eleven buffalo bulls were classified based on their conception rate (CR) into fertile (n = 8, CR > 55%) and subfertile (n = 3, CR < 35%) groups. Ejaculates were collected with artificial vagina, pooled, and dispensed into 6 aliquots, diluted with Tris-egg yolk-glycerol extender supplemented with different proportions of PRP [0% (control), 5%, 10%, 15%, 20%, and 25%] followed by cryopreservation using standard procedures. Post-thaw sperm quality, kinematics, antioxidant activity, cryosurvival rate, and in vivo fertility were compared between fertile and subfertile groups and among proportions of PRP within each group. The results showed that 15% PRP greatly (P < 0.001) improved sperm characteristics, average path velocity, and curvilinear velocity of the subfertile group. Interestingly, 5%, 10%, and 15% PRP greatly (P < 0.001) reduced malondialdehyde content and improved enzymatic (glutathione peroxidase and superoxide dismutase) and total antioxidant capacity in fertile and subfertile groups. However, these three proportions of PRP significantly (P < 0.001) improved the cryosurvival rate of the subfertile group; only 15% PRP greatly improved CR of subfertile (60.83% vs. 34.17%) animals to be comparable with that of fertile ones treated with 5 (59.17%) and 10% (60.83%) PRP. In conclusion, adding 15% PRP to semen extender before cryopreservation is recommended to improve post-thaw quality, antioxidant activity, and in vivo fertility of buffalo semen particularly of the subfertile animals.

The Evaluation of Rosemary (Rosmarinus Officinalis) Leaf Extract Inclusion in Freezing Medium on Quality Parameters of Buffalo Bull Spermatozoa

BACKGROUND:The discrepancy between the endogenous antioxidants concentrations and free radicals results in oxidative stress and cellular injury.OBJECTIVE:To appraise the usefulness ofRosemarinus officinalis(RO) aqueous extract in protecting buffalo spermatozoa during freezing / thawing process.MATERIALS AND METHODS:Qualifying ejaculates from four well-restrained bulls were evaluated initially and then diluted in a freezing medium supplemented with RO-0.00, RO0.50%, RO-1.00%, RO-2.00%, and RO-4.00%, cooled to 4ºC in 2 h, equilibrated for 4 h at 4ºC, packed in straws, and cryopreserved, and thawed at 37ºC for 30 s followed by evaluation.RESULTS:We found that freezing medium supplemented with RO-2.00% improves progressive motility (%) compared to the control. Similarly, a lower rate of apoptosis-like changes (%) was recorded with RO4.00% than the control, RO-0.50% and RO-1.00%. This response was accompanied by an increment in viable spermatozoa. Semen samples supplemented with RO-2.00% and RO-4.00% displayed higher TAC (total antioxidant capacity, μM/L) and ATP (nmol/million) content than the control. In addition, semen samples supplemented with RO-2.00% displayed lower concentrations of ROS (reactive oxygen species, 10 4 RLU/20 min/25 million) than the control and RO-0.05%. Also LPO (lipid peroxidation, μM/L) with RO-2.00% and RO-4.00% was lower than the control.CONCLUSION:The inclusion of rosemary aqueous extract ameliorates motility features, structural and functional parameters, viability, TAC and ATP content of bull sperm. Conversely, the inclusion of rosemary aqueous extract alleviates apoptosis-like changes, ROS and LPO in comparison to the control. Further studies are required to determine the mechanism of action of rosemary aqueous extract in ameliorating semen quality and fertility of buffalo spermatozoa.

The Quality of Buffalo Sperm Following Preservation Using Different Diluents and Sperm Concentrations

Artificial Insemination (AI) success depends on the quality of the frozen semen. The quality of the frozen semen of swamp buffalo in Indonesia is still low. The study was conducted to determine the quality of buffalo sperm following freezing using three different diluents and three different doses. The study used buffalo semen from the Tuah Sakato Artificial Insemination Center, Payakumbuh (n = 3). The semen collecting was carried out once a week for 10 weeks (replication). The research method used was 3x3 factorial randomized block design. The first factor was diluent (Triladyl®, Andromed® and Tris egg- yolk) and the second factor was the dose of spermatozoa (10 and 15 and 20 x 106 sperm/ml). Data were analyzed using variant analysis, while the differences between treatments were tested by Duncan Multiple Range Test. The results showed that the plasma membrane integrity of buffalo sperm was found in Andromed® diluent, while tris egg-yolk diluent gave better motility, viability, plasma membrane integrity and recovery rate at a sperm concentration of 20 x106 sperm/mL compared to triladyl® diluent and a sperm concentration of 10 and 15 x106 sperm/mL. It was concluded that andromed® diluent and tris egg-yolk gave better motility, viability, plasma membrane integrity and recovery rate at a sperm concentration of 20 x106 sperm/mL compared to triladyl® diluent and a sperm concentration of 10 and 15 x106 sperm/mL. of Buffalo of sperm abnormalities not sicnificantly by the type of diluent but are influenced by sperm concentration.

Flow Cytometric Evaluation of Acrosomal integrity of Buffalo Spermatozoa at Different Stage of Cryopreservation using Different Extenders

Flow Cytometric Evaluation of Acrosomal integrity of Buffalo Spermatozoa at Different Stage of Cryopreservation using Different Extenders

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. For the experiment, semen extender was supplemented with Mito TEMPO (50 μM), acetovanillone (50 μM), and a combination of Mito TEMPO + acetovanillone (50 μM+ 50 μM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. The addition of Mito TEMPO (50 μM) and acetovanillone (50 μM) individually and in combination significantly (p<0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p<0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 μM supplemented group. The improvement observed in acetovanillone (50 μM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

Cryopreservation-induced changes in buffalo spermatozoa with special reference to their DNA, membrane, acrosomal and mitochondrial integrities

Cryopreservation-induced changes in buffalo spermatozoa with special reference to their DNA, membrane, acrosomal and mitochondrial integrities

Cryoprotectant With A Mitochondrial Derived Peptide, Humanin, Improves Post-Thaw Quality Of Buffalo Spermatozoa

BACKGROUND:Semen cryopreservation results in deleterious effects on spermatozoa, including lipid peroxidation and a reduction in the total antioxidant components of seminal plasma. The ultimate outcome of these changes is a reduction in post-thaw semen quality. A mitochondrial derived peptide, humanin, a potent cytoprotective and antioxidant agent was used in the present study.OBJECTIVE:To evaluate the efficacy of a mitochondrial-derived peptide, humanin to improve the post-thaw quality of buffalo spermatozoa.MATERIALS AND METHODS:A total of 18 ejaculates from three Murrah buffalo bulls (n=6 each) were collected. Each ejaculate was divided into four aliquots. The first aliquot was diluted with standard EYTG dilutor (Group I, control), whereas the other three aliquots were diluted with EYTG supplemented with 2 μM (Group II), 5 μM (Group III) and 10 μM humanin (Group IV), respectively. Semen was evaluated for physico-morphological and functional attributes such as progressive motility, viability, abnormality, acrosome integrity, plasmamembrane integrity of fresh samples, pre-freeze and post-thaw stages. Oxidative stress parameters [lipid peroxidation (LPO) and total antioxidant capacity (TAC)] were also measured at the pre-freeze and post-thaw stages.RESULTS:Humanin s upplementation resulted in significantly higher (p≤0.05) post- thaw motility in all treatment groups and, higher (p≤0.05) viability in Groups III and IV in comparison to the control at the post-thaw stage. Spermatozoa with intact acrosome and plasma membran e were higher (p≤0.05) in Groups III and IV as compared to Group s I and II. The LPO levels at the post- thaw stage were found to be lower (p≤0.05) in all treatment groups versus the control group, whereas, higher (p≤0.05) TAC value s were recorded in Groups III and IV in comparison to the control and Group II.CONCLUSION:Humanin supplementation in the extender improved the freezabilty of buffalo spermatozoa.

Effect of Milk Type Subjected to Different Heat Treatments on Cryo-Survivability and In Vivo Fertility of Buffalo (Bubalus bubalis) Spermatozoa in a Milk-Based Extender.

Oxidative stress is a major contributory factor to cellular damage during semen cryopreservation and results in a decreased fertilizing capacity of cryopreserved bull sperm. The inclusion of exogenous antioxidants sometimes exerts deleterious effects on sperm quality. Thus, enhancing the endogenous production of antioxidants is a requirement. This study aimed to investigate the effect of milk type heated at different temperatures on the antioxidant potential of extenders, and the subsequent post-thaw quality parameters and in vivo fertility of buffalo bull semen. Cow (C) and buffalo whole milk (B) were used separately for semen extender preparation, heated at five different temperatures (T1 = 90°C, T2 = 100°C, T3 = 110°C, T4 = 120°C, T5 = 130°C) for 10 minutes. Reactive sulfhydryl groups were measured in each subgroup by Ellman's reagents as CT1 = 143.2 μM, CT2 = 147.4 μM, CT3 = 151.5 μM, CT4 = 157.2 μM, CT5 = 161.8 μM, BT1 = 168.3 μM, BT2 = 172.5 μM, BT3 = 176.7 μM, BT4 = 196.3 μM, and BT5 = 205.7 μM. All semen samples were cryopreserved in milk-based extenders by using standard procedures. Post-thaw quality parameters including total and progressive motility, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity were found to be higher (p < 0.05) in the group (BT3) containing buffalo milk heated at 110°C, whereas in the same group, lipid peroxidation was found to be lower (p < 0.05) as compared with other treatment groups and control group. In vivo fertility of cryopreserved buffalo sperm was compared among BT3, CT1 (conventionally used milk extender), and a Tris egg yolk extender group. The fertility rates [47% (54/114), 30% (33/108), and 36% (37/103)] were higher (p < 0.05) in BT3 as compared with other groups. This study suggests that buffalo milk heated at 110°C has high antioxidant potential and improves post-thaw quality and in vivo fertility of cryopreserved buffalo bull semen.

Recombinant expression and molecular characterization of buffalo sperm lysozyme-like protein 1

Several sperm lysozyme-like genes evolved from lysozyme by successive duplications and mutations; however their functional role in the reproduction of farm animals is not well understood. To understand the function and molecular properties of buffalo sperm lysozyme-like protein 1 (buSLLP1), it was expressed in E. coli; however, it partitioned to inclusion bodies. Lowering of temperature and inducer concentration did not help in the recovery of the expressed protein in the biologically active form. Therefore, buSLLP1 was cloned and expressed in Pichiapink system based on auxotrophic Pichia pastoris in a labscale fermenter. The expressed protein was obtained in flow-through by using a 30 kDa ultrafiltration membrane followed by MonoQ anion exchange chromatography, resulting in a homogenous preparation of 40 mg recombinant buSLLP1 per liter of initial spent culture-supernatant. Circular dichroism spectroscopy showed that recombinant buSLLP1 possessed a native-like secondary structure. The recombinant buSLLP1 also showed thermal denaturation profile typical of folded globular proteins; however, the thermal stability was lower than the hen egg white lysozyme. Binding of buSLLP1 to chitin and zona pellucida of buffalo oocytes showed that the recombinant buSLLP1 possessed a competent binding pocket, therefore, the produced protein could be used to study its functional role in the reproduction of farm animals.

L-Carnitine Improves Cytoprotection during Cryopreservation: A case study on Nili-Ravi Buffalo Sperm

The current study was aimed to evaluate the antioxidative effect of L-Carnitine at post thawing following cryopreservation of Nili-Ravi buffalo sperm. For this purpose, semen from three buffalo bulls were collected for 3 weeks using an artificial vagina (N=18; replicates). The qualified ejaculates were diluted employing tris-citric acid extender i.e., control did not receive any L-Carnitine and experimental groups having 0.5, 1.0, and 1.5 ng/mL of L-carnitine at 37 C with approximately 50 x 106 sperm/mL. The semen was cooled at 4 C and then equilibrated (4 hours), filled in straws (0.5 mL) at4 C, placed on LN2 vapours for 10 min, and kept into an LN2 container. The thawed semen was evaluated for post-thaw quality. The integrity of the sperm plasma membrane and motility (P?0.05) was highest in the extenders having 1.0 ng/mL of L-carnitine as compared to the control(received no L-Carnitine). However, sperm chromatin integrity and viability(live sperm with intact acrosome) remained similar. It was concluded that supplementing 1.0 ng/mL L-Carnitine of extender can improve the post-thaw quality of cryopreserved sperm. Based on the results of the current experiments it is recommended to include L-carnitine extender to improve post-thaw quality of buffalo sperm in terms of its motility and integrity ofits plasma membrane. Keywords: Buffalo, Sperm, Cryopreservation, Extender, L-Carnitine, Artificial insemination.