Pakistan is one of the most important agricultural country. Livestock as a subsector of agriculture contributes 11.4% in the national economy and 56.3% in agriculture. Buffalo is honored as the black gold of Pakistan and Nili-Ravi is the most important animal in this regard. Approximately 60% of the total milk produced within a country comes from buffalo. Artificial insemination is considered to be the most important tool for the prompt genetic improvement of livestock. Mammalian spermatozoa undergo many structural and biochemical changes during cryopreservation which may leads to an impaired fertility. Buffalo bull spermatozoa are sensitive and more prone to damage than cattle. Various experiments have been conducted in order to improve the post thaw semen quality which includes supplementation of egg yolk from different bird species that tends to work as a cryoprotectent against cold shock. In the present study it is assumed that replacement of chicken egg yolk with quail egg yolk in cryodiluents improves the freezability of Nili-Ravi buffalo bull spermatozoa.
Three adult Nili-Ravi buffalo bulls without any clinical reproductive anomaly and kept at Semen Production Unit (SPU), Qadirabad District Sahiwal were used in this study. Semen from these three experimental bulls was collected twice a day twice a week by using an artificial vagina already maintained at 42 ºC. Each bull was subjected to a minimum of seven replicas. Both semen samples were collected on each collection day from each of the three experimental bulls with an interval of about ten minutes between the ejaculates. After initial evaluation for sperm motility and concentration, both ejaculates collected from the same bull at the same collection day were pooled and again evaluated for sperm cell concentration. Every pooled semen sample was then divided into five aliquots and extended with one of the five experimental extenders namely A, B, C, D, and E in order to maintain a final concentration of 40 million spermatozoa per 0.54 ml of diluted semen. Instantly after dilution motility percentile for each semen sample was recorded. After filling, open ends of straws were sealed with polyvinyl pyrolidine powder and allowed to cool and then it was stored at 4-5 ºC for equilibration. Finally the samples were frozen and stored in liquid nitrogen. On post thaw evaluation, motility, live/dead count, plasma membrane integrity, acrosomal integrity, and DNA integrity along with CASA evaluation parameters for motility characteristics were performed. Data were analyzed by means of two way ANOVAand comparison between means was done by using Duncan’s multiple range test. Results showed that post-extension motility and DNA integrity did not differ significantly between extender A and E. While post-thaw motility, plasmolemma intactness, acrosomal integrity, and viable sperm count was found to be higher in extender E compared to extender A. Similarly, CASA evaluation factors like progressive sperm cells motility, straight line velocity, straightness, linearity index, and the percentile of rapidly moving spermatozoa were also significantly different among extender E and A. While curvilinear velocity, and amplitude of lateral head displacement was higher in extender B that contained 5% QEY. So it was concluded that substitution of 20% CEY with 20% QEY in cryodiluents resulted in an improved post-thaw motility, viable sperm count, plasmolemma intactness, and sperm kinematics. Furthermore, it was seen that reduction in the concentration of QEY from 20% to 5% resulted in decreased sperm motility characteristics.