Epididymal stallion sperm quality improves with single layer centrifugation prior to cryopreservation or post-thawing

Abstract

Cryopreservation of epididymal spermatozoa is the last method for preserving gametes of valuable stallions in case of sudden death, severe injuries, or orchiectomy. Due to harvesting methods, sperm is unavoidably contaminated with epithelial and red blood cells, leukocytes, debris, and bacteria. These contaminants and dead spermatozoa can cause detrimental effects on sperm quality. Colloid centrifugation can reduce the load of these contaminants and select a subpopulation of motile spermatozoa. The objective of this experiment was to evaluate whether single layer centrifugation (SLC) prior to cryopreservation or post-thawing improves the quality of stallion epididymal sperm. Ten stallions were submitted to bilateral orchiectomy and harvesting of epididymal cauda spermatozoa (n=20) was performed by retrograde flushing with an extender. Epididymal sperm samples were subjected to conventional centrifugation (no colloid - 600xg for 20 minutes), and SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). SLC was performed at 300 x g for 10 minutes (Androcoll-Equine, Minitube, Tiefenbach, Germany). All samples were cryopreserved, thawed, and evaluated. The post-thaw group (SLC+) was thawed, centrifuged by SLC, and resuspended in freezing extender (SLC+F) or cooling extender (SLC+C). Total motility and progressive motility were evaluated with computer-assisted semen analyses (CASA). Sperm morphology was evaluated by examining a wetmount preparation of unstained samples fixed in formol saline. Mitochondrial functionality, membrane integrity, and DNA integrity were evaluated in a fluorescence microscope with specific fluorescent dyes. All variables were normally distributed according to the Shapiro–Wilk test. Data were evaluated by descriptive statistics, one-way analysis of variance, and Tukey's test. Significance was set at p <0.05. SLC-PC (24.6 ± 3.6%) and SLC+F (26.2 ± 4%) treatments yielded significantly higher total sperm motility than SLC+C (16.7 ± 1.7%) and conventional (16 ± 3.5%) treatment. SLC+F (17.5 ± 3.8%) yielded significantly higher progressive motility than the conventional (8.8 ± 2%), SLC+C (9.8 ± 1.3%), and SLC-PC (12.9 ± 4.1%) treatments. The mitochondrial functionality of all SLC groups was significantly higher (SLC-PC 72 ± 2%; SLC+C 74.6 ± 1.8%; SLC+F 82.5 ± 1.9%), than that of the conventional group (25.2 ± 2.1%). Plasma membrane integrity was higher in SLC-PC (48.6 ± 4%) than in SLC+F (30 ± 6.5%), SLC+C (36 ± 4.8%), and conventional (22.8 ± 4.5%) samples. DNA integrity and percentage of morphologically normal samples were unaffected by treatment. SLC is a simple procedure that can be performed prior to cryopreservation or post-thawing and that can improve stallion epididymal sperm quality. Epididymal spermatozoa submitted to SLC only after thawing is best extended using freezing extender than cooling extender.