Cryopreservation of Balinese Bull Semen Using Three Different Types of Extenders and Equilibration Times

Abstract

Bali cattle (bos javanicus) is considered as the most preferable breed in the small holding systems in Indonesia because of their high resistance against diseases, their remarkable ability to grow on low quality fodders and their high quality meat. However there is a decrease in the total population of the Bali cattle which could be because of the excessive slaughtering such a problem can be overcome by applying strict management planning. The quality of the insemination dose plays an important role in the success of the artificial insemination process however in compare with fresh semen; the post-thawed semen quality is generally lower because of the cryo-injury which could lead to the decrease in sperm motility, viability and membrane integrity. Another problem is regarding the needed equilibration time. Generally four hours equilibration time is applied before semen freezing which is considered as a time consuming step, moreover there are a desire for the reduction of such time. The objectives of the present study were to compare the post-thawed quality of Balinese bull sperm cryopreserved in three different extenders; Tris-clarified egg yolk (Tris-cEY), Bioxcell® and Optixcell® extenders in combination with three different equilibration times (30min, 2hrs and 4hrs). Thirty six ejaculates were collected from six Balinese bulls and were frozen in three different extenders (Tris-cEY, Bioxcell®and Optixcell® extenders) after equilibration in three different equilibration times (30 min, 2hrs and 4hrs) by following the routine freezing protocol. The post-thawed semen parameters were evaluated for motility using computer-assisted sperm analysis (CASA), membrane integrity using hypo-osmotic swelling test (HOST) and for viability using eosin nigrosin staining. There were significant interactions between equilibration time and extender type for sperm motility, viability and membrane integrity. Thirty minutes equilibration time had the lowest values (P<0.05) for total and progressive motilities, percentages of sperm with intact plasma membrane and viability independent of extender type. Sperm velocity parameters and membrane integrity were better in semen frozen with Optixcell® than commercial lecithin based and Tris-cEY extenders. Overall, semen extended in Tris-cEY, Optixcell® and Bioxcell®were better equilibrated at 4 hrs however, commercial Liposome based extender equilibrated at 2 hrs showed good motility and viability which were not significantly different from 4hrs equilibration (P>0.05). In conclusion, based on the evaluations of sperm motility, viability and functional membrane integrity, thirty minutes and two hrs equilibration were not sufficient for maintaining the post-thawed semen quality. Semen equilibration at 4 hrs. yielded the greatest sperm survival, independent of the used extender type. Semen which was equilibrated with Optixcell® extender for 2 hrs showed good motility and viability however more evaluations regarding the acrosome status and DNA fragmentation are needed. Either Bioxcell® or Optixcell® could be used for Balinese bull semen cryopreservation.