Past Issue

Volume 16, Number 3, Autumn 2014, Serial Number: 63, Pages: 279-288

Effect of Extender and Equilibration Time on Post Thaw Motility and Chromatin Structure of Buffalo Bull (Bubalus Bubalis) Spermatozoa


Abdolhossain Shahverdi, Ph.D., 1, Abdolreza Rastegarnia, D.V.M., Ph.D., 2, *, Tohid Rezaei Topraggaleh, D.V.M., 3,
Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Department of Clinical Science, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran
Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran
*Corresponding Address: P.O.Box: 969 Department of Clinical Science Faculty of Veterinary Medicine Islamic Azad University Urmia Branch Urmia Iran Email:a.rastegar@iaurmia.ac.ir

Abstract

Objective

The aim of the present study was to investigate the effects of four equilibration times (2, 4, 8 and 16 hours) and two extenders (tris or Bioxcell®) on cryopreservation of buffalo semen.

Materials and Methods

In this experimental study, split pooled ejaculates (n=4), possessing more than 70% visual sperm motility were divided in two aliquots and diluted in Bioxcell® and tris-citric egg yolk (TCE) extenders. Semen was cooled to 4°C within 2 hours, equilibrated at 4°C for 2, 4, 8 and 16 hours, then transferred into 0.5 ml French straws, and frozen in a programmable cell freezer before being plunged into liquid nitrogen. Postthaw motility characteristics, plasma membrane integrity, acrosome morphology and DNA integrity of the buffalo sperm were studied after thawing.

Results

There were significant interactions between equilibration times and extenders for sperm motility and membrane integrity. Post thaw sperm motility (PMOT), progressive motile spermatozoa (PROG), plasma membrane integrity (PMI) and normal apical ridge (NAR) measures were lower for sperm equilibrated for 2 hours in both TCE and Bioxcell® extender compared to others equilibration times. PMOT, PMI and NAR for sperm equilibrated for 4, 8 and 16 hours showed no significant differences in either extender, although PROG measures were superior in Bioxcell®compared to TCE at all equilibration times (p<0.05). Kinematic parameters such as average path velocity, curvilinear velocity and linearity in the Bioxcell®extender were superior to those in the TCE extender studied. In contrast to motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected by different equilibration times.

Conclusion

Equilibration time is necessary for preservation of the motility and integrity of buffalo sperm membranes. Equilibration times of over than 2 hours resulted in the greatest preservation of total semen parameters during cryopreservation. There were no significant interactions between equilibration times over 4 hours and type of extender which lead to greater post thaw sperm survival.