Our objectives were to ascertain the comparison of Tris egg yolk-based, Triladyl and Optixell extender on postthaw quality, CASA parameters and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 35) from five bulls were diluted in Tris egg yolk-based, Triladyl , Optixell extender and frozen in 0.50 ml French straws. Postthaw sperm CASA motility (%) was higher (p < 0.05) in Optixell extender as compared to Triladyl and Tris egg yolk-based extender. Although sperm progressive motility (%), morphology (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), amplitude of lateral head displacement (μm), beat cross-frequency (Hz), straightness (%), length of curvilinear path (μm), length of average path (μm), intact plasma and acrosome membrane (%), and DNA integrity (%) were higher (p < 0.05) in spermatozoa cryopreserved in Optixell extender as compared to Tris egg yolk-based and Triladyl extender. The fertility rates (68.18%, 45.45%, 55.4%) were higher (p < 0.05) in buffaloes inseminated with semen doses frozen in Optixell extender than the Tris egg yolk-based and Triladyl extender respectively. It is concluded that Optixell extender improves postthaw semen quality and fertility in Nili Ravi buffaloes.

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Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca 2+ ] i and plasma membrane fluidity in viable, acrosomeintact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5 C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n = 17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca 2+ ] i decreased over time (1 h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca 2+ ] i in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1 h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5 C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions.

Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prepTM solution (20–60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prepTM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (n = 3) with high sperm DFI (>20%). The optimal Opti-prepTM solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prepTM as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased (p < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prepTM cushion and a 20% top layer.

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