![]() ![]() However, the development and implementation of new assisted reproductive technologies, alternative preservation models based on washing sperm from a cellular ageing-accelerating substance such as the seminal plasma, and basic studies in spermatology is associated with the use of centrifugation. In conclusion, ram sperm can be centrifuged in INRA 96 extender up to 1200 X g for ten minutes at 15 ☌ as secure values with high recovery rates and without detrimental effects on sperm quality and fertility.Ĭentrifugation is routinely employed in handling the ejaculates of some species, but it is not part of the commonly used protocols in ram. The damage produced by 6000 X g on sperm motility (P ≤ 0.05) was maintained over time, coinciding with a lower fertility (P ≤ 0.05). ![]() 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 (six to eight hours at 15 ☌). The lowest pellet weight (P ≤ 0.05) without harmful effect on sperm physiological status (P > 0.05) was achieved at 1200 X g, since 6000 X g induced sperm motility damage (P ≤ 0.05) with both extenders. In Experiment 2, the influence of three centrifugal forces (600, 12 X g for ten minutes) 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. INRA 96 combined with Slow Refrigeration and Tyrode's at room temperature registered the highest sperm recovery and quality values (P ≤ 0.05). 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 ☌ to 15 ✬–) on sperm recovery rate and quality (motility, viability, apoptosis and mitochondrial activity). 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. Furthermore, evidence of in vivo fertility rate using sperm obtained with various centrifugation forces is also lacking in this species. To our knowledge, there are no studies on combined effect of extender and different centrifugal forces on ram sperm yield and quality. ![]() However, our research group previously established that this simple procedure might cause a large sperm loss and induce deleterious effects on the sperm function of the ovine species when high centrifugation forces are employed. 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. The optimization and implementation of artificial insemination (AI) in sheep is necessary to increase the livestock productivity through enhanced control of reproductive function. Therefore, we recommend the use of short-term centrifugation with a relatively high g-force (2400 x g for 3 minutes) in boar sperm cryopreservation protocol. We conclude that high g-force (2400 x g) and short centrifugation time (3 minutes) do not affect sperm recovery and yield and that, moreover, they have a positive effect on the cryosurvival of boar sperm. Centrifugation regimes C1 and C2 showed significantly (P. After thawing, sperm quality was assessed after 30 and 150 minutes of incubation (37 degrees C). Pellets were diluted in lactose-egg yolk (LEY)-glycerol-Equex STM (1 x 10(9) cells/mL) and frozen in 0.5-mL straws. In experiment 2, cooled semen was centrifuged using 3 different regimes: C1 (2400 x g for 3 minutes), C2 (1600 x g for 5 minutes), and C3 (800 x g for 10 minutes). 05) from the reference in terms of sperm yield. Sperm recovery (Bürker Chamber) and yield (triple fluorescent stain of PI/R123/FITC-PNA ) were calculated. In experiment 1, the g-forces tested were 400, 800, 1600, and 2400 x g for 3 or 5 minutes, using the standard regime (800 x g for 10 minutes) as a reference. In both experiments, sperm-rich fractions from 3 boars were diluted, pooled, and cooled to 17 degrees C before centrifugation. This study evaluated the influence of different centrifugation regimes on both sperm recovery and yield (percentage of viable sperm with an intact acrosome relative to the initial sperm population) after centrifugation (experiment 1) as well as the influence of different centrifugation regimes on boar sperm cryosurvival (experiment 2). Current protocols for boar sperm cryopreservation require the centrifugation of semen in order to separate sperm cells from the seminal plasma. ![]()
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