An optimized centrifugation protocol for ram sperm ensuring high sample yield, quality and fertility

The optimization and implementation of artificial insemination (AI) in sheep is necessary to increase the livestock productivity through enhanced control of reproductive function. Sperm centrifugation is a common procedure in the ejaculate handling in AI and other assisted reproductive technologies (ART), as part of new methods of sperm analysis, selection or preservation. However, our research group previ-ously established that this simple procedure might cause a large sperm loss and induce deleterious ef-fects on the sperm function of the ovine species when high centrifugation forces are employed. To our knowledge, there are no studies on combined effect of extender and different centrifugal forces on ram sperm yield and quality. Furthermore, evidence of in vivo fertility rate using sperm obtained with various centrifugation forces is also lacking in this species. Thus, the objective of this work was to define the ideal conditions for ram semen centrifugation that will achieve the best quantity and quality sample to ensure unaffected fertilization ability of centrifuged ram sperm. The Experiment 1 evaluated the effect of the centrifugation procedure of two extenders (INRA 96 and Tyrode's) and two cooling protocols (Rapid and Slow Refrigeration-35 degrees C to 15 degrees C-) on sperm recovery rate and quality (motility and kinetic param-eters, viability, apoptosis and mitochondrial activity). INRA 96 combined with Slow Refrigeration and Tyrode's at room temperature registered the highest sperm recovery and quality values (P <= 0.05). In Experiment 2, the influence of three centrifugal forces (600, 1200 and 6000xg for 10 min) was assessed immediately after centrifugation on the technical performance and sperm functionality in diluted samples with INRA 96 and Tyrode's at the conditions set out in Experiment 1. The lowest pellet weight (P <= 0.05) without harmful effect on sperm physiological status (P > 0.05) was achieved at 1200xg, since 6000xg induced sperm motility damage (P <= 0.05) with both extenders. Finally, to ensure the total safety of the centrifugation protocol, Experiment 3 tested in a combined in vitro and in vivo test the effect of these three centrifugal forces on ram sperm quality after dilution (INRA 96) and liquid storage (6-8 h at 15 degrees C). The damage produced by 6000xg on sperm motility (P <= 0.05) was maintained over time, coinciding with a lower fertility (P <= 0.05). In conclusion, ram sperm can be centrifuged in INRA 96 extender up to 1200xg for 10 min at 15 degrees C as secure values with high recovery rates and without detrimental effects on sperm quality and fertility. (C) 2022 Published by Elsevier Inc.