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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The beneficial effects of cholesterol loaded cyclodextrin (CLC) addition were evaluated in cryopreserved bull semen. Forty ejaculates were collected from Hariana bulls (n = 4), pooled and divided into 4 aliquots. All the aliquots were initially diluted in to egg yolk tris citrate and supplemented with CLC @ 0.5 mg (Group-II), 1.0 mg (G-III) and 2.0 mg (G-IV) CLC/120 × 10 6 spermatozoa or without CLC (G-I) that served as control. Extended semen was cryopreserved at −196 °C for 24 h. Seminal attributes like motility, viability, cryocapacitation like changes, tyrosine phosphorylation, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA integrity were evaluated after equilibration and thawing. Results showed a significant increase in the motility, viability and acrosome intact spermatozoa in Group II as compared to other three groups. Further, the proportion of spermatozoa showing capacitation and acrosome reaction was also decreased (P < 0.05) significantly in Group II as compared to Group I, III, and IV. Immunoblot demonstrated a 32 kDa (p32) protein showing differential variation in the band intensity in all the four groups being lower in Group II. Further, the immunolocalization study revealed positive immune reactivity for tyrosine phosphorylated proteins at middle piece and neck (high fluorescence), post-acrosomal region (medium fluorescence), and principal piece (low fluorescence) of spermatozoa. Addition of CLC significantly increased (P < 0.05) the percentage of spermatozoa showing high transmembrane mitochondrial potential, and also, CLC @ 0.5 mg/120 × 10 6 in semen extender significantly decreased (P < 0.05) spermatozoa showing fragmented DNA after thawing as compared to control. Results of the present study indicate beneficial effects of CLC supplementation on cryodamage of spermatozoa by reducing the cryocapacitation and apoptosis like changes. [ABSTRACT FROM AUTHOR]