Effects of Extender and Packaging Devices on Ultrastructural Changes and Reproductive Index of Cryopreserved Spermatozoa of Ossimi Rams

Sperm cryopreservation is a critical technique for improving the efficacy of artificial insemination in sheep breeding programs. This study evaluated the effects of different extenders and packaging methods on post-thaw ram sperm quality, ultrastructure, and potential reproductive performance. Semen was collected from five healthy Ossimi rams and extended using three cryopreservation media: Tris-egg yolk (Tris-EY), Tris supplemented with 1% soybean lecithin (Tris-SBL), or Tris supplemented with 2 mM butylated hydroxytoluene (Tris-BHT). The extended semen was then packaged in either plastic straws or pellets and cryopreserved in liquid nitrogen. Post-thaw sperm quality was assessed by evaluating progressive motility, viability, and membrane integrity. Sperm ultrastructure was examined using transmission electron microscopy (TEM) and reproductive index. A two-way ANOVA analysis was used to study the effects of the extender and the method of packing. Cryopreservation using straws significantly improved post-thaw sperm quality compared to pellets, demonstrating higher progressive motility (p = 0.003), viability (p < 0.0001), and membrane integrity (p < 0.0001). However, neither the packaging method nor the extender type significantly affected the plasma membrane status, acrosome integrity, or mitochondrial, tail, and nuclear damage (p > 0.05). Ultrastructural analysis confirmed that straws, regardless of the extender used (Tris-EY, Tris-SBL, or Tris-BHT), effectively preserved sperm ultrastructure, including the nucleus, head, acrosome, plasma membrane, and mitochondria. The methods of packaged and extender types did not significantly affect the conception rate and litter size in sheep (p > 0.05). However, it is worth noting that Tris-SBL in straws had the highest conception rate (85.7%) and litter size (1.5 per ewe), while Tris-BHT in pellets had the lowest values (71.4% and 1.2, respectively). This study confirmed that plastic straw packaging significantly improved post-thaw sperm quality. While Tris-EY showed higher reproductive performance, further research is needed to establish statistical significance in pregnancy rates and litter size.