T. K. Mohanty

Effect of age and season on semen quality parameters in Sahiwal bulls

The objective of the study was to determine the effect of season, period, age, bull, and ejaculate on semen quality in Sahiwal bulls. Semen production records from 1996 to 2006 of 5,483 ejaculates from 46 Sahiwal bulls maintained at Artificial Breeding Complex, NDRI, Karnal, India were analyzed using least square analysis of variance by LSML software package. The overall least squares means of ejaculate volume (VOL), total volume per day (VOLD), mass activity (MA), initial motility (IM), sperm concentration per ml (SPC), and sperm concentration per ejaculate (SPCE) were 3.79u2009±u20090.02xa0ml, 5.81u2009±u20090.06xa0ml, 2.32u2009±u20090.01, 55.47u2009±u20090.001%, 766.69u2009±u20095.50u2009×u2009106/ml and 3023.25u2009±u200930.15u2009×u2009106, respectively. All semen traits (VOL, VOLD, MA, IM and SPCE) were significantly (Pu2009<u20090.01) affected by age groups, season and period, whereas season had significant effect on VOL at 5% level. During hot-humid season, highest value of VOL, VOLD, MA, IM, SPC, and SPCE were observed followed by summer and cold season. Highest value of VOL, VOLD, IM, and SPCE were observed during period-3 (2004–2006), whereas highest value of MA and SPC were observed during period-1 (1996–1999). However, lowest magnitude of MA, IM, SPC, and SPCE during period-2 (2000–2003) was observed. Ejaculate characteristics like VOL, VOLD, and SPCE increased with the increasing age of bull up to 5xa0years and then decreased. Significant (Pu2009<u20090.01) bull to bull variation was found in VOL, VOLD, MA, IM, SPC, and SPCE traits. First ejaculate had significantly (Pu2009<u20090.01) higher MA, IM, SPC, and SPCE. Hence, it could be concluded that during rainy season and period-1 and period-3 the quality of semen is quantitatively and qualitatively good. Better quality semen was obtained up to 5xa0years of age in Sahiwal bulls.

Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics

Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility.

Behavioural signs of estrus and their relationship to time of ovulation in Zebu (Sahiwal) cattle

The present study reports the behavioural signs of estrus, their temporal distribution and duration of expression and their relationship with the time of ovulation in zebu cattle in order to identify the reliable sign(s) of estrus that could fairly predict the ovulation time. The onset, intensity and expression of various signs of estrus were continuously recorded till ovulation in 60 Sahiwal cows. Time of ovulation was determined by ultrasound examinations at 2h interval. Estruses were mostly of moderate (52%) or weak (34%) intensity. Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane appeared early in relation to the ovulation time (31.27±1.97, 31.05±2.98 and 30.79±2.53h, respectively) in comparison to mounting (27.67±2.33h) and standing to be mounted (25.37±2.11h). Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane persisted significantly more duration (P<0.01) than mounting and standing to be mounted. Further these cardinal signs appeared early in relation to time of ovulation, persisted for longer duration and expressed intensely. We conclude that mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane can be good predictor ovulation in this breed of cattle.

Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders

Since the inception of bovine semen cryopreservation, egg yolk and milk based extenders have been used to protect sperm from the detrimental effects of cooling and freezing. In recent years, demand for alternatives to conventional commercial extenders has arisen as the risk of introducing exotic diseases through transporting egg yolk based products has been recognized. Egg yolk can also interfere with sperm evaluation and the presence of particulate material in the extender may reduce fertility. Soybeans contain lecithin, a phospholipid fraction that can substitute for high molecular weight lipoprotein and phospholipids from egg yolk and prevent or ameliorate damage to the sperm plasma membrane that occurs during extension, cooling, and cryopreservation. Soy lecithin based extenders have been evaluated for processing and freezing bovine semen, although extender from soybean milk has not been studied as extensively. Commercially available soy lecithin based extenders are used increasingly but remain under scrutiny and are not universally accepted. With these observations in mind, this review is intended to examine effects of conventional cryopreservation procedures, methods of assessment, and potential for developing soybean extract as an acceptable alternative to traditional egg yolk and milk based extenders for bull sperm cryopreservation.

Differential proteomic profile of spermatogenic and Sertoli cells from peri-pubertal testes of three different bovine breeds.

Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred [Holstein Friesian (HF)] and indigenous purebred [Tharparkar (TP)] bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds.

Expression of short chain fatty acid receptors and pro-inflammatory cytokines in utero-placental tissues is altered in cows developing retention of fetal membranes

INTRODUCTIONnRetention of fetal membranes (RFM) is an economically important condition in dairy cattle disturbing the health and production in affected animals. In spite of extensive research, pathogenesis for RFM and identification of a predictive marker for the condition still remains elusive. Since expulsion of fetal membranes in an inflammatory process, studying the expression of inflammatory molecules is expected to give an idea about the pathogenesis of RFM.nnnMETHODSnExpression of pro-inflammatory cytokines, short chain fatty acid (SCFA) receptors and adhesion molecules was studied using qRT-PCR in the utero-placental tissues obtained from the cows that expelled fetal membranes normally (nxa0=xa012) and from the cows that developed RFM (nxa0=xa012). Concentrations of SCFAs were estimated in the utero-placental tissues using Gas Chromatography.nnnRESULTSnThe concentration of butyrate in the utero-placental tissue extract was 3-4 times lower, and GPR41 and GPR43 mRNA expression was 7.7-10.0 and 3.4-4.4 folds lower (pxa0<xa00.05) in cows that developed RFM compared to those that expelled the fetal membranes normally. The expression of IL-1, IL-6, IL-8, TNF-α, ICAM and PECAM was higher (pxa0<xa00.05) in the cows that normally expelled the fetal membranes compared to those that developed RFM.nnnDISCUSSIONnSince a clear association of butyrate levels, expression of pro-inflammatory cytokines, adhesion molecules and SCFAs receptors was observed with expulsion of fetal membranes, modulating the levels of these molecules prior to parturition might help in reducing the incidence of RFM in cows.nnnCONCLUSIONnInflammatory response is essential for normal expulsion of fetal membranes and that inadequate or altered expression of pro-inflammatory cytokines, adhesion molecules and SCFA receptors is associated with RFM in cows.

Comparative Quality Assessment of Buffalo (Bubalus bubalis) Semen Chilled (5°C) in Egg Yolk- and Soya Milk–Based Extenders

Egg yolk-Tris is most commonly used semen extender; however, its use involves hygienic risk, interference with fertility and poor microscopic examination. Therefore, replacement of egg yolk with a plant-based component with protective effects on spermatozoa would be advantageous. In present study, we observed effect of soya milk-based extenders on dilution and liquid preservation of Murrah buffalo bull semen at 5°C up to 72 h in comparison with conventional egg yolk-Tris extender (Ext.1). In experiment one, a total of 32 buffalo semen ejaculates from four animals were extended and preserved at 5°C for 72 h in soya milk-based extender (Ext.2) with different percentages (10%, 15%, 20%, 25% and 30%) of soya milk for optimization of soya milk concentration. Semen quality was assessed for individual motility, viability, membrane integrity and acrosome integrity at 0, 24, 48 and 72 h of liquid preservation. The results of experiment one indicated that 25% soya milk is an optimum concentration for buffalo bull semen extender preparation. A modified method was used to prepare another soya milk-based extender (Ext.3). In the second experiment, two soya extenders (Ext.2 and 3) with optimized concentration (25%) of soya milk were comparatively assessed with egg yolk-Tris extender (Ext.1) for semen quality parameters at 0, 24, 48 and 72 h of liquid preservation. The individual sperm motility at 0 and 24 h following dilution were found non-significant among extenders. However, after 48 h of dilution, individual motility in Ext.3 was observed significantly (p < 0.05) higher than Ext.1. After 24, 48 and 72 h of dilution sperm membrane integrity in Ext.3 was found significantly (p < 0.05) higher than Ext.1. Overall, comparative evaluation of sperm parameters obtained revealed that Ext.3 containing 25% soya milk can be used as a substitute of egg yolk-based extender for buffalo semen liquid preservation.

Identification of suitable combinations of in vitro sperm-function test for the prediction of fertility in buffalo bull

The present study assessed sperm functional characteristics in the frozen-thawed semen of buffalo bulls and estimated their relationship with field fertility. Frozen semen samples from three different freezing operations each from nine Murrah buffalo bulls were used for the assessment of different sperm functions related to fertilizing potential. Bulls were classified into high (n = 2), medium (n = 5), and low (n = 2) fertile based on adjusted field fertility. The sperm functions estimated included membrane integrity using carboxyfluorescein diacetate-propidium iodide, acrosome reaction status using fluorescein isothiocyanate peanut agglutinine, status of apoptosis using Annexin-V, protamine deficiency using Chromomycin A3, membrane stability using Merocyanine 540 and lipid peroxidation status using 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene. The relationship between the proportion of live acrosome-intact spermatozoa and fertility was positive and significant (r = 0.59; P = 0.001). The proportion of moribund spermatozoa showed a significantly negative correlation with fertility (rxa0=xa0-0.50; Pxa0=xa00.008). Similarly, the relationship of spermatozoa with unstable membrane (rxa0=xa0-0.51; P = 0.007), necrotic (rxa0=xa0- 0.42; P = 0.028), early necrotic (rxa0=xa0-0.42; Pxa0=xa00.031), and apoptotic spermatozoa (rxa0=xa0-0.39; Pxa0=xa00.046) with bull fertility was negative and significant. The correlation between the protamine-deficient spermatozoa and fertility was negative, but not significant. Among different combinations of tests, live acrosome-intact spermatozoa and lipid peroxidation status of spermatozoa revealed high positive correlation with buffalo bull fertility (adjusted R2xa0=xa00.73, C[p]xa0=xa00.80). These preliminary findings may help in developing tools for assessing fertility of buffalo bulls, once validated in more animals.

Testicular Cell Indices and Peripheral Blood Testosterone Concentrations in Relation to Age and Semen Quality in Crossbred (Holstein Friesian×Tharparkar) Bulls

Present study analyzed the changes in peripheral blood testosterone concentrations and testicular cytogram in relation to age and semen quality in crossbred males. Three different age groups of crossbred males viz. bull calves (6 months, n = 5), young bulls (15 months, n = 5) and adult bulls (4 to 6 years, n = 8) were utilized for the study. Testicular fine needle aspiration cytology technique was used to quantify testicular cytology and their indices. Peripheral blood testosterone concentrations were measured using enzyme-linked immunosorbent assay method. Semen samples collected from adult bulls were microscopically evaluated for quality parameters. Mean peripheral blood testosterone concentrations in bull calves, young bulls and adult bulls were 2.28±0.09 ng/mL, 1.42±0.22 ng/mL and 5.66±1.08 ng/mL respectively, and that in adult bulls were significantly different (p<0.01) from young bulls and bull calves. There was no significant difference between the proportion of different testicular cells in bull calves and young bulls. Between young and adult bulls, significant differences (p<0.01) were observed in the proportion of spermatocytes, spermatozoa, and sperm: Sertoli cell ratio. The proportions of Sertoli cells showed a significant difference (p<0.01) between the three age groups. The number of primary spermatocytes had a positive correlation with peripheral blood testosterone concentrations in bull calves (r = 0.719, p<0.01). Number of Sertoli cells per 100 germ cells was negatively correlated with blood testosterone concentration in young bulls (r = −0.713, p<0.01). Among different semen parameters in adult bulls, ejaculate volume (r = 0.790, p<0.05) had positive relationship, and sperm motility had significant negative correlation (r = −0.711, p<0.05) with testosterone concentrations. The number of Sertoli cells and Sertoli cell index had a positive correlation with various semen quality parameters (p<0.001). Results of the present study conclude that number of Sertoli cells and Sertoli cell index are good indicators of semen quality, but peripheral blood testosterone concentrations may not have a direct relationship with various seminal attributes in crossbred bulls.

Alteration in peripheral blood concentration of certain pro-inflammatory cytokines in cows developing retention of fetal membranes

Retention of fetal membranes (RFM) adversely affects the production and reproduction potential of the affected cows leading to huge economic loss. Physiological separation of fetal membranes is reported to be an inflammatory process. The present study compared the concentrations of certain pro inflammatory cytokines [Interleukin 1β (IL-1), Interleukin 6 (IL-6), Interleukin 8 (IL-8) and Tumor necrosis factor α (TNF-α) between the cows that developed RFM (n=10) and the cows that expelled fetal membranes normally (n=10) to find out if they could serve as a predictive tool for RFM. Blood samples were collected from the cows from 30 days before expected parturition through day -21, day -14, day -7, day -5, day -3, day -1, on the day of parturition (day 0), day 1 postpartum and the pro-inflammatory cytokines were estimated in blood plasma by ELISA method. The IL-1β concentration was significantly lower (P<0.05) in cows that developed RFM compared to those that expelled fetal membranes normally from 3 days before calving till the day of calving. The plasma concentrations of IL-6 and IL-8 were also lower (P<0.05) in cows that developed RFM than those calved normally. On the day of calving, significantly (P<0.05) lower concentrations of TNF-α was observed in cows that developed RFM compared to those expelled fetal membranes normally. It may be inferred that the concentrations of IL-1, IL-6, IL-8 and TNF-α around parturition were altered in cows developing RFM compared to those expelled fetal membranes normally.