S.K. Ghosh

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.

Bovine seminal PDC-109 protein: An overview of biochemical and functional properties

Although long-term storage of bovine semen is desirable for wider use, successful cryopreservation depends on several factors, including various proteins present in seminal plasma. One such group of proteins, viz. bovine seminal plasma (BSP) proteins represents the major protein fraction in bovine seminal plasma. They constitute three major heparin-binding (HB-) acidic proteins secreted by seminal vesicles, viz. BSP-A1/-A2 (PDC-109), BSP-A3 and BSP-30-kDa. By purification studies it was deduced that PDC-109 is a polypeptide of 109 amino acids and contains two tandem repeating fibronectin type-II (Fn-II) domains, preceded by a 23 residue N-terminal domain. Though BSP-A1 and BSP-A2 are biochemically similar they differ only in glycosylation and their mixture is called PDC-109 or gonadostatins. PDC-109 exists as a polydisperse, multimeric self-associated molecule and possesses multifunctional properties, viz. binding to the surface of plasma membrane of spermatozoa causing conformational change in the sperm surface proteins and enhances motility. Besides binding, PDC-109 protein provokes cholesterol efflux from sperm membrane and promotes sperm reservoir by interacting with oviductal membrane. Interaction of sperm with PDC-109 protein induces sperm capacitation and acrosome reaction. However, prolonged exposure of spermatozoa with free floating PDC-109 protein as during processing for preservation, increases cholesterol efflux from spermatozoa. The efflux of sperm membrane cholesterol and disturbance in cholesterol:phospholipids ratio causes destabilization of plasma membrane thereby inducing cryoinjury to the sperm. In this review, the biochemical, functional properties of PDC-109 protein and its role during semen cryopreservation is summarized.

Activity of antioxidative enzymes in fresh and frozen thawed buffalo (Bubalus bubalis) spermatozoa in relation to lipid peroxidation and semen quality

Abstract Objective To investigate the activity of antioxidative enzymes in fresh and frozen thawed spermatozoa in relation to lipid peroxidation and semen quality in buffalo ( Bubalus bubalis ) bulls. Methods Forty two semen ejaculates from seven buffalo bulls were collected by artificial vagina method and were used for the study. Sperm motility, livability, plasma membrane and acrosomal integrity, buffalo cervical mucous penetration test were assessed in fresh and frozen thawed semen. Intracellular antioxidative enzymatic activity such as super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) and reduced glutathione (GSH), reactive oxygen species (ROS) and lipid peroxidation (LPO) were estimated in fresh and frozen thawed semen. Results A significant ( P 9 sperm in fresh semen, but after freezing and thawing this activity was not detectable. These enzyme activities had a strong positive association with sperm motility, membrane integrity and distance traveled by vanguard spermatozoa in buffalo cervical mucus and negative correlation with LPO and ROS. However, no significant correlation with acrosomal integrity was found. Conclusion It was concluded that loss of activity of intracellular antioxidative enzymes was evident after freezing and thawing and there was a strong association between the antioxidative enzyme activities, ROS, lipid peroxidation and sperm function in buffalo semen.

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.

Gas chromatographic-mass spectrometric analysis of chemical volatiles in buffalo (Bubalus bubalis) urine

Isolation of active fraction and characterization of chemosignals from urine have been attempted in several mammalian species in the recent years. The objective of this study was to identify the urinary volatiles across various reproductive stages of buffalo cow, namely, estrus, diestrus, and pregnancy, and in bull, by chemical extraction followed by gas chromatography-linked mass spectrometry (GC-MS). Urine samples were collected from six buffalo cows at two different phases of estrous cycle, namely, estrus and diestrus. Besides, urinary samples were collected from five pregnant buffalo cows (60-75 days after artificial insemination (AI)) and six adult bulls. Thin-layer chromatography was performed as a preliminary test for qualitative comparison of different compounds extracted by organic solvents. Identification of the urinary compounds was carried out in a gas chromatograph (Perkin Elmer, Autosystem XL) linked to a mass spectrometer (Turbomass). The results of GC-MS analysis indicated the presence of 21 compounds with varying molecular weights and retention time, which were further categorized as diestrus-specific, pregnancy-specific, and bull-specific urinary compounds. No compound, however, could be identified as estrus-specific. We concluded that qualitative differences do exist in estrus, diestrus, and pregnant buffalo cow urine and in bull urine, as evidenced by GC-MS.

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.

Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium-regulating multifunctional protein

Regucalcin (RGN) is a calcium-regulating, anti-apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real-time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34-kDa, 28-kDa and 24-kDa isoforms, wherein the 34-kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium-related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.

Counting Sperm Numbers

Selection of the best ejaculate from the donor species depends greatly on precise calculation of the concentration of the live sperm cells. Different laboratories engaged in the semen biology work employ variety of instruments and procedures. These, coupled with the variability of the result induced by the objective assessment (inter-technician variation), make it difficult for an investigator to decide on the suitability of an assay for his work. This chapter addresses this dilemma by listing protocols of various assays for determination of concentration of spermatozoa and their relative merits. Thus, procedures of haemocytometer, spectrophotometer and NucleoCounter SP-100 are covered. Since the flow cytometer and CASA have been described elsewhere, their procedural details are not explained here.