Effects of Centrifugation Before Freezing on Boar Sperm Cryosurvival

Abstract

Current protocols for boar sperm cryopreservation require the centrifugation of semen in order to separate sperm cells from the seminal plasma. 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). In both experiments, sperm-rich fractions from 3 boars were diluted, pooled, and cooled to 17ЊC before centrifugation. In experiment 1, the g-forces tested were 400, 800, 1600, and 2400 ϫ g for 3 or 5 minutes, using the standard regime (800 ϫ g for 10 minutes) as a reference. Sperm recovery (Bü rker Chamber) and yield (triple fluorescent stain of PI/R123/FITC-PNA [DNA-specific fluorochrome propidium iodide/mitochondria-specific fluorochrome rhodamine-123/acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin]) were calculated. The highest recovery and yield (P Ͻ .05) values were achieved using 2400 ϫ g for 5 or 3 minutes and 1600 ϫ g for 5 minutes, which showed no differences (P Ͼ .05) from the reference in terms of sperm yield. In experiment 2, cooled semen was centrifuged using 3 different regimes: C1 (2400 ϫ g for 3 minutes), C2 (1600 ϫ g for 5 minutes), and C3 (800 ϫ g for 10 minutes). Pellets were diluted in lactose-egg yolk (LEY)-glycerol-Equex STM (1 ϫ 10 9 cells/mL) and frozen in 0.5-mL straws. After thawing, sperm quality was assessed after 30 and 150 minutes of incubation (37ЊC). Centrifugation regimes C1 and C2 showed significantly (P Ͻ .05) higher postthaw sperm motility (assessed with a computer-assisted semen analysis system), viability (evaluated as for experiment 1), and percentage of uncapacitated sperm (assessed with a chlortetracycline assay) than did C3. In addition, C1 had the highest (P Ͻ .05) oocyte penetrating ability (assessed with the homologous in vitro penetration test performed with immature oocytes). Malondialdehyde production, assessed with the thiobarbituric acid reactive species test, was unaffected (P Ͼ .05) by the centrifugation regime used. We conclude that high g-force (2400 ϫ 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. Therefore, we recommend the use of short-term centrifugation with a relatively high g-force (2400 ϫ g for 3 minutes) in boar sperm cryopreservation protocol.

Kinematic and spermatic recovery after selection by centrifugation in colloid solutions of ovine cryopreserved semen

“…Similar results were found by Jimenez-Rabadan et al (2012), who selected goat semen in a solution of silica coated with silane specific for bovines. The recovery rates can be altered by several factors, among them: quality of semen before centrifugation (Alvarez et al, 2010;Martinez-Alborcia et al, 2013), caution at the time of deposition of the semen sample at the top of the gradient selection column (Thys et al, 2009), TM increase after sperm selection, and time and intensity of centrifugation force (Carvajal et al, 2004). Martinez-Alborcia et al (2013) hypothesized that non-functional spermatozoa saturate the interface between the semen and the colloid, obstructing the passage of functional spermatozoa.…”

“…Martinez-Alborcia et al (2013) hypothesized that non-functional spermatozoa saturate the interface between the semen and the colloid, obstructing the passage of functional spermatozoa. Carvajal et al (2004) tested several swine semen centrifugation protocols and suggested that the intensity of the centrifugation force is more important than the centrifugation time in the sperm recovery rate. This fact would explain the higher recovery in the methods employing silica colloidal-PVP, considering that the intensity of the g force was of 900 compared to 300 in the methods using silica colloidal-silane.…”

Evaluation of Different Cryoprotectants (CPAs) in Boar Semen Cryopreservation

“…The straws were then plunged into the liquid nitrogen. After thawing the straws at 37°C for 20 sec [5], thawed semen was evaluated for sperm motility [13], morphological defect, viability by eosin-nigrosin staining [4], and plasma-and acrosomalmembrane integrities. Sperm plasma-membrane integrity was assessed using 6-carboxyfluoresceindiacetate (6-CFDA; Sigma-Aldrich)/propidium iodide (PI; Sigma-Aldrich) fluorescent staining [12] and analyzed by flow cytometry.…”

Antioxidant Effects of Seminal Plasma on Cellular Morphological Viability of Swine Semen Post-Cryopreservation

“…Indeed, our results show a positive effect of temperature on SOD and GPX1 activity that at the same time seems to have an indirect effect on sperm quality likely to maintain a constant fitness for reproductive success ( Figure 6). Addition of SP in a range of 5-50% (v/v) to the extender minimized cryopreservation-induced membrane damage [5,9,38] or early onset of acrosome responsiveness post-thaw [16,38] due to stabilizing proteins or antioxidant enzymes in SP that provide protection to the sperm cells [6,13,31]. Results in our study indicate that addition of SP to frozen-thawed semen ameliorated the negative effects of cryopreservation compared with control sperm as reflected in higher EN as well as HOST percent values (Figures 7 and 8).…”