Raushan K. Singh

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.

Sperm functional attributes and oviduct explant binding capacity differs between bulls with different fertility ratings in the water buffalo (Bubalus bubalis)

We report here the differences in sperm functional attributes and sperm-oviduct binding index in bulls with different field fertility ratings. Cryopreserved spermatozoa from Murrah buffalo bulls (n=9) with different fertility ratings were evaluated for membrane integrity, capacitation status, acrosome intactness and protein tyrosine phosphorylation status. Frozen--thawed spermatozoa were incubated with oviduct explants for 1h under 5% CO2, 38.5°C with 95% relative humidity and the number of spermatozoa bound to the unit area of oviduct explants (binding index; BI) was assessed using 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescent staining. The proportion of membrane-intact and acrosome-intact spermatozoa was significantly (P<0.05) higher and the proportion of capacitated spermatozoa was significantly (P<0.05) lower in high-fertile bulls compared with medium- and low-fertile bulls. The relationship between BI and bull fertility was significant and positive (r=0.69; P=0.04). BI was negatively and significantly (r=-0.83; P=0.01) related to membrane-compromised spermatozoa. It was concluded that the sperm-oviduct explant binding index was positively related to (1) the proportion of membrane-intact spermatozoa in a given semen sample and (2) invivo fertility of the buffalo bull, indicating the possibility of developing a fertility prediction tool using a sperm-oviduct explant binding model, once validated on a greater number of bulls.

Age-related changes in transcriptional abundance and circulating levels of anti-Mullerian hormone and Sertoli cell count in crossbred and Zebu bovine males

Age-related changes in peripheral anti-Mullerian hormone (AMH) concentrations and transcriptional abundance of AMH gene in testicular tissue were studied in crossbred (Holstein Friesian × Tharparkar) and Zebu (Tharparkar) males. In both the breeds, basal AMH concentrations were estimated using ELISA method in blood plasma obtained from six males each at 1, 6, 12, 18, and 24xa0months age. After blood collection at respective ages, all the males were castrated and expression and immunolocalization of AMH was performed in the testicular tissue. The concentration of AMH in blood plasma was found to be highest at 1xa0month of age in both crossbred and Zebu males, which subsequently decreased with advancing age. Significantly (Pxa0<xa00.05) lower concentration of AMH was observed in crossbred as compared with Zebu males at 24xa0months of age. In line with peripheral AMH concentrations, the expression of AMH gene was also higher (Pxa0<xa00.05) at 1xa0month of age, which thereafter declined significantly with advancement of age in crossbred males. Furthermore, the expression of AMH gene differed significantly between Zebu and crossbred males at all the age groups studied. Immunolocalization of AMH in testicular tissue also revealed a stronger expression at 1xa0month age, which gradually decreased till 24xa0months of age. The true Sertoli cell count was significantly higher in Zebu compared with crossbred males at all age groups studied except at 6xa0months age. The relationship between Sertoli cell count and circulating AMH concentrations was negative and significant (r = -0.81; P = 0.004). In conclusion, expression of AMH gene in testicular tissue and peripheral blood concentrations of AMH were higher in young compared with adults in both crossbred and Zebu males; however, the transcriptional abundance and circulating levels of AMH were higher in Zebu compared with crossbred males.