Effect of over dilution of semen with tris extender on motion and functional attributes of bull spermatozoa during cryopreservation.

The present study was aimed to evaluate the effect of over dilution of semen with tris extender on motion and functional attributes of bull sperm post-thaw. Ejaculates (n = 24; mass motility ≥3+) were collected from bulls (n = 4) using artificial vagina, diluted to 20, 15, 10 and 5 million spermatozoa/0.25 ml, and cryopreserved. The results revealed that total motility (%), progressive motility (%) and rapid motility (%), straight linear velocity (µm/s), straightness (%) reduced significantly (p < 0.05) when semen was diluted to 5 million sperm concentration. Among the various sperm function attributes, proportions of live spermatozoa, acrosome intact spermatozoa, hypo-osmotic swelling responsive spermatozoa and non-capacitated spermatozoa reduced (p < 0.05) in 5 million spermatozoa, and the proportions of moribund spermatozoa, dead spermatozoa, live acrosome reacted spermatozoa, dead acrosome intact spermatozoa, capacitated spermatozoa and spermatozoa with lipid peroxidation increased significantly (p < 0.05) when semen was diluted from 20 to 5 million. However, the over-dilution of semen did not affect slow motility, dead acrosome reacted spermatozoa, sperm protamine deficiency and spermatozoa with lipid peroxidation. In conclusion, the over dilution of semen affected sperm motion and functional attributes of frozen-thawed bull semen.

Spermatozoa produced during winter are superior in terms of phenotypic characteristics and oviduct explants binding ability in the water buffalo (Bubalus bubalis).

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants.

Supplementing extender with anandamide enhances quality of low sperm doses during cryopreservation in bulls.

The present study explored the effect of anandamide supplementation in the extender on quality of low sperm doses during cryopreservation in Sahiwal bulls. Each fresh semen sample was split into eight aliquots (I, II, III, IV, V, VI, VII and VIII). The aliquots I, II, III and IV were taken as control and diluted to 20, 15, 10 and 5 million spermatozoa/0.25 ml respectively. The aliquots V, VI, VII and VIII were diluted with extender (supplemented with anandamide at 1 µM/ml of extender) to 20, 15, 10 and 5 million spermatozoa/0.25 ml respectively. This was followed by filling of diluted semen into French mini straws, equilibrated at 4°C of 4 hr and cryopreserved. The results revealed that the proportions of motile spermatozoa, live spermatozoa and live acrosome intact spermatozoa were significantly (p < .05) higher in all anandamide-treated sperm doses compared to control. The proportions of moribund spermatozoa, dead acrosome intact spermatozoa and capacitated spermatozoa were significantly (p < .05) reduced in all anandamide-treated sperm doses compared to control, with no difference in proportion of dead acrosome-reacted spermatozoa. In conclusion, anandamide supplementation in the extender increases the post-thaw quality of low sperm doses during cryopreservation in bulls.