J. K. Prasad

Sequestration of PDC-109 protein improves freezability of crossbred bull spermatozoa

A study was carried out to assess the effect of sequestration of PDC-109 protein, a majority constituent of heparin binding proteins (HBP) of seminal plasma, on freezability and in vitro fertilizing ability of crossbred bull spermatozoa after cryopreservation. The study consisted of isolation and characterization of PDC-109 protein to raise anti-sera against it in rabbits. Following which, raised antibodies against PDC-109 protein was quantitated and coated in tubes used for collection of ejaculates. Semen ejaculates thus collected were cryopreserved using EYTG extender. Physico-morphological characteristics, viz. motility, viability, acrosomal integrity and HOS response as an indicator of freezability of cryopreserved spermatozoa were determined at pre freeze as well as post thaw stage. At pre freeze stage, a significant (p<0.05) improvement in viability (83.83 ± 2.18 vs 75.17 ± 2.42) and acrosome integrity (81.33 ± 2.38 vs 72.83 ± 2.39) in antibodies treated group than control was observed. Similarly, increase in HOS responsive spermatozoa was highly significant (p<0.01) than control (78.83 ± 1.69 vs 67.5 ± 1.75). At post thaw stage, significant (p<0.05) improvement in viability (69.50 ± 2.16 vs 60.33 ± 2.19) and HOS responsive spermatozoa (68.67 ± 1.62 vs 58.50 ± 1.32) and highly significant (p<0.01) increase in individual motility (56.17 ± 1.83 vs 47.00 ± 1.86) and acrosome integrity (75.17 ± 2.38 vs 61.83 ± 2.1) was observed in antibodies treated group when compared to control was observed. The results from the study revealed that sequestration of PDC-109 protein from semen samples leads to significant improvement in pre-freeze and post-thaw values of above parameters in cryopreserved spermatozoa. It is thus concluded that sequestration of PDC-109 protein from ejaculates improves freezability of crossbred bull spermatozoa.

Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo Spermatozoa

Advancements in reproductive technologies have shown seminal plasma (SP) as a nutritive-protective medium for spermatozoa metabolism, function and transport. At the same time quality variables and thus freezability of spermatozoa are influenced by SP proteins originating from male reproductive tract. One such protein, viz. PDC-109 is reported to influence freezability of spermatozoa in cattle. Thus the present investigation was designed to evaluate effect of seminal PDC-109 protein concentration on post-thaw cholesterol content and semen quality variables (SQP) as an indicator of membrane integrity and freezability, respectively of buffalo spermatozoa. Ejaculates (n=42) selected on the basis of mass activity and individual motility were divided into three parts, first part for SP proteins isolation, second for cholesterol estimation and third part was cryo-preserved to evaluate freezability based on post-thaw SQP, viz. individual progressive motility, viability and acrosome integrity of spermatozoa. A total of 28 (66.7%) and 14 (33.3%) ejaculates from four bulls were found as freezable or non-freezable, respectively. Though total seminal plasma protein (TSPP) concentration was found similar in freezable and non-freezable ejaculates, the heparin binding proteins (HBP) content in non-freezable semen was greater (P<0.01) than freezable ejaculates. There was a similar trend for the PDC-109 protein content in respective ejaculates. Cholesterol content of spermatozoa and SQP were greater (P<0.05 and 0.01, respectively) in freezable as compared to non-freezable ejaculates of each bull at post-thaw stage. This study showed that concentrations of HBP and PDC-109 in non-freezable semen might be responsible for greater cryo-damage reflecting in poor freezability of buffalo spermatozoa.

Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa

Buffalo spermatozoa are comparatively more susceptible to freezing hazards than cattle spermatozoa. In recent times incubation of spermatozoa with cholesterol-loaded-cyclodextrins (CLC) has shown improvements in semen quality in several species. Therefore, this study was undertaken to evaluate the incubation level of CLC at which maximum benefit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa were incubated in 2, 3 and 4 mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phospholipids content, their ratio, flow cytometric evaluation of plasma membrane integrity (PMI), plasma membrane fluidity and extent of cryoinjury (Chlortetracycline, CTC assay) were compared with an untreated control (Gr I). Additionally the ability of cholesterol-loaded-spermatozoa to undergo induced acrosome reaction (IAR) using ionophore calcium (A23187) was evaluated in frozen-thaw samples. Data show a significant and linear increase (CV=0.88) in cholesterol content of spermatozoa in Gr II, III and IV and a significant decrease in phospholipids content at frozen-thaw stage in Gr IV than Gr III spermatozoa. The study revealed a significant improvement in PMI and significant reduction in plasma membrane fluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groups compared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly less responsive to ionophore calcium (A23187) than Gr I. This study shows for the first time that incubation of buffalo bull spermatozoa with CLC (3mg/120×10(6)) prior to processing permits greater numbers of sperm to survive cryopreservation while allowing spermatozoa to capacitate and the acrosome to react to AR inducer ionophore calcium (A23187).

Effects of seasons on enzymatic changes and cholesterol efflux in relation to freezability in Tharparkar bull semen

Abstract Objective To assess the effect of different seasons on sperm motility, viability, total sperm abnormality, acrosomal and plasma membrane integrity, antioxidant profiles such as superoxide dismutase (SOD) and catalase (CAT), enzymatic profiles such as aspartate amino transaminase (AST), lactic acid dehydrogenase (LDH) and biochemical profiles such as total cholesterol in Indian breed, Tharparkar bull. Methods Total numbers of 60 ejaculates from 3 bulls were collected through artificial vagina method twice a week during summer and winter season (30 ejaculates from each season). The semen samples were pooled and diluted with the standard TEYC extender and these seminal, biochemical parameters were studied at fresh, pre- freeze and post thaw stage of semen cryopreservation. Results Seminal parameters such as volume, plasma membrane integrity and biochemical parameters such as SOD at fresh semen, acrosomal integrity, LDH, AST and total cholesterol at pre–freeze level and acrosomal integrity, SOD at post thaw stage of cryopreservation were differed significantly ( P P P P P P P Conclusion It can be concluded that cryopreservation of Tharparkar bull semen in summer and winter season do not show any definite pattern in relation to seminal and biochemical profile changes and the semen can be cryopreserved in both seasons throughout the year in this prestigious Indian breed of cattle.

Uterine Infection Influences Size and Follicular Fluid Composition of the Largest Follicle in Buffalo (Bubalus bubalis)

The effect of uterine infection on size and follicular fluid composition of the largest follicle was studied in buffalo. Reproductive tracts were collected from 102 graded Murrah buffaloes at an abattoir. Uterine infection was diagnosed by physical examination of uterine mucus, white side test and uterine cytology. Samples with pus-containing mucus, positive reaction on white side test and/or >5% neutrophils were considered to be positive for uterine infection. Diameter of the largest follicle was measured, and follicular fluid was aspirated and assayed for nitric oxide (NO), ascorbic acid (AA), cholesterol, oestradiol (E(2)) and progesterone (P(4)). Infected buffaloes had smaller-sized (p < 0.0001) largest follicles than non-infected buffaloes. Follicular fluid collected from the largest follicle in infected buffaloes had greater (p < 0.0001) NO and P(4) concentrations coincident with lesser AA (p < 0.001), cholesterol (p < 0.0001) and E(2) (p < 0.0001) concentrations. Results indicated that uterine infection has an inhibitory effect on growth of the largest follicle in buffalo. The changes in follicular fluid composition in infected buffaloes suggest that the direct effect of uterine infection on ovarian function may be mediated through an alteration in the follicular microenvironment. Greater NO and lesser AA concentrations in the follicular fluid of infected animals are novel findings.

Evaluation of follicular oxidant-antioxidant balance and oxidative damage during reproductive acyclicity in water buffalo (Bubalus bubalis)

Abstract Objective To investigate changes in follicular fluid concentrations of reactive oxygen species (ROS) and total antioxidant capacity (TAC) and degree of oxidative damage to follicular cells, using protein carbonyl (PC) as marker of oxidative stress, were investigated during reproductive acyclicity in buffalo. Methods Follicular fluid was aspirated from follicles grouped into three classes depending upon their diameter [small (5.0–7.0 mm), medium (7.1–10.0 mm), and large (>10.0 mm)]. Progesterone and estradiol were estimated to determine functional status (P:E ratio) of the follicles. Results Acyclic buffaloes had greater concentrations of ROS ( P P =0.0412) and lower concentrations of TAC ( P =0.0280) than cyclic buffaloes. An interesting novel finding was the complete absence of low P:E functionally active follicles in acyclic buffaloes. Results indicated a pronounced follicular fluid oxidant-antioxidant imbalance and oxidative damage to follicular cells during acyclicity in buffalo. Conclusion In conclusion, this study provided evidence about role of oxidative stress in pathogenesis of reproductive acyclicity.

Partial deoxygenation of extender improves sperm quality, reduces lipid peroxidation and reactive oxygen species during cryopreservation of buffalo (Bubalus bubalis) semen

The present study was designed to investigate the effect of partial deoxygenation of extender on sperm quality, lipid peroxidation (LPO) and reactive oxygen species (ROS) in buffalo (Bubalus bubalis) during cryopreservation of semen. Semen extender was prepared freshly and split into three sub-extenders [Extender I: control (non-deoxygenated), Extender II (partially deoxygenated by using LN2 flushing) and Extender III (partially deoxygenated mechanically by vacuum pump)]. Amounts of dissolved oxygen (DO) were determined in all the three extenders and also in post-thaw semen. Ejaculates with mass motility of  ≥3+ and individual progressive motility of 70% or greater were collected from Murrah buffalo bulls and utilized in the study. Each semen sample was divided into Groups I (diluted with Extender I), II (diluted with Extender II) and III (diluted Extender III) with a maximum of 60 × 106 sperm/mL. French mini straws (0.25 mL) were filled with the extended semen samples, sealed with polyvinyl alcohol powder, kept for 3 h at 5 °C for equilibration and then stored in an automatic programmable freezer until the temperature of straws reached -145 °C followed by plunging the straws into liquid nitrogen (-196 °C). Semen samples were evaluated at pre-freeze and post-thaw stages for various variables [sperm motility, live sperm count, acrosomal integrity, hypo-osmotic swelling (HOS) response, LPO and ROS concentrations]. The mean DO was less (P < 0.05) in Extender II as compared to I and III. The DO was less (P < 0.05) in Group II (semen extended with Extender II) as compared with III (semen extended with Extender III) and I (semen extended with Extender I). The percentages for sperm motility, viability and intact acrosomes (PIA) were greater (P < 0.05) in Groups II and III as compared to the control group at the pre-freeze stage, while at the post-thaw stage, percentages of sperm motility, viability, PIA and HOS response were greater (P < 0.05) in Group II as compared with the control group and Group III. Pre-freeze HOS response (%) was greater (P < 0.05) in Group II as compared with the control and Group III. At the pre-freeze stage, sperm LPO and ROS were less (P < 0.05) in Groups II and III as compared with the control and at post-thaw stage, spermatic LPO and ROS concentrations were less (P < 0.05) in Group II than in the control group and Group III. In conclusion, partial deoxygenation of extender improves sperm quality, reduces sperm LPO and ROS concentrations in buffalo during cryopreservation. Partial deoxygenation of the extender with LN2 flushing may be one of the ways for improving quality and fertility of frozen-thawed buffalo sperm.

Effect of incubation on freezability of cholesterol-loaded cyclodextrin treated buffalo (Bubalus bubalis) spermatozoa

Aim: The aim of this study was to investigate the effect of incubation on freezability of cholesterol loaded cyclodextrin (CLC) treated buffalo spermatozoa. Materials and Methods: Semen samples with mass motility of 3+ and greater, collected from Murrah buffalo bulls were utilized. Immediately after collection, four equal groups of semen sample were made. Group I was kept as control and diluted with Tris upto concentration of 60×106 sperm/ml, where as Groups II, III, and IV were treated with CLC at 3 mg/120× 106 spermatozoa, incubated at 37°C for action of CLC for 10, 15 and 20 min, respectively, and diluted with tris upto concentration of 60×106 sperm/ml. All groups were subjected to equilibration and freezing. The evaluation of semen samples from all groups was carried out at fresh, pre-freeze and post-thaw stage for progressive motility, viability and hypo-osmotic swelling response (HOS response). Results: At the pre-freeze stage, significantly (p<0.05) higher percentage of progressive motility and viability was observed in treatment groups as compared to control with no significant difference among treatment groups. HOS response was significantly (p<0.05) higher in treatment groups as compared to control at pre-freeze stage. At post-thaw stage, significantly (p<0.05) higher percentage of progressive motility, viability and HOS response was recorded in Group II as compared to control and other treatment groups (III and IV). Group II retained significant post-thaw motility and viability at various post-thaw incubation periods. Conclusion: Incubation period of 10 min for CLC treated buffalo spermatozoa yielded significantly higher results in terms of freezability as compared to incubation for 15 and 20 min.

Studies on effect of different seasons on expression of HSP70 and HSP90 gene in sperm of Tharparkar bull semen

Abstract Objective To assess the effect of different seasons on expression of HSP70 and HSP90 genes of spermatozoa in Indian breed, Tharparkar bull. Methods Total numbers of 60 ejaculates from 3 bulls were collected through artificial vagina method twice a week during summer and winter season (30 ejaculates from each season). The semen samples were pooled and diluted with the standard TEYC extender and these semen samples were allowed to study the expression of HSP 70 and HSP 90 genes of spermatozoa with commercially available kit. Results No significant difference was observed in spermatozoal mRNA expression of HSP 70 and HSP 90 during winter and summer season in this bull semen. But the mRNA expression of both HSP 70 and HSP 90 during summer season was found non-significantly higher in comparison to winter season. Conclusion It was concluded from the present study that there was no significant difference in the mRNA expression of HSP 70 and HSP 90 between the winter and summer season, presence of similar type of stress resistant spermatozoa in Tharparkar bull semen and the semen can be cryopreserved throughout the year in this prestigious Indian breed.

Reduction of dissolved oxygen in semen extender with nitrogen gassing reduces oxidative stress and improves post-thaw semen quality of bulls

The objective of the present study was to investigate the effects of two different concentrations of dissolved oxygen (DO, 4 and 8) ppm in the extender on oxidative stress affecting plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and deoxyribonucleic acid (DNA) damage of bull spermatozoa following cryopreservation. For the experiment, nitrogen (N2) gassing of the extender for varied time intervals yielded extender with DO concentration of 4 ppm and 8 ppm (Groups II and III, respectively). For the Control (Group I) without N2 gassing, a DO concentration of 11.7 ppm was recorded. Following sample selection, ejaculates were divided into three aliquots and were extended to have 80 × 106 spermatozoa/mL of extender in the three groups. Semen samples were evaluated for reactive oxygen species (ROS), lipid peroxidation (LPO), total antioxidant capacity (TAC) and superoxide dismutase (SOD) at the fresh, pre-freeze, and post-thaw stages. Evaluation of PMI, MMP, and DNA damage were conducted on frozen-thawed samples. There were greater (P < 0.05) increase in ROS and LPO and decrease in TAC concentrations in Group I than Groups II and III. Mean values of SOD at the post-thaw stage was greater (P < 0.05) in Group II than Group I. There was a similar trend in the PMI in Groups II and III; MMP and DNA integrity in Group II was greater compared with Group I. In conclusion, results indicate there was a beneficial effect of maintaining DO concentrations at 4 rather than of 8 or 11.7 ppm in extender for sustaining post-thaw semen quality.