Effect of cholesterol loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa

- First study to show the effects of CLC on DNA integrity of bull semen.
- Unique study to report the role of CLC on tyrosine phosphorylation of 32 kDa protein.
- First study in Bos indicus (Hariana bull) to report the beneficial role of CLC on frozen thawed semen.

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 × 106 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 × 106 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.