Effect of semen washing on thawed ram spermatozoa subjected to a four hour post-thawing thermal evaluation test

This study was conducted to evaluate sperm motility and qualitative characteristics of ram spermatozoa cryopreserved in the presence or absence of seminal plasma under controlled condition and subjected to a 4 h post-thawing thermal evaluation test. The aim being that exposing these frozen-thawed sperm to thermal resistance test will provide useful information on their survivability in the female genital tract towards fertilizing the ovum. Briefly, ejaculates from 5 males (5years) were collected by artificial vagina, and split into two aliquots. One aliquot was diluted (1:5) in Tris-citric acid-glucose (TCG) solution and washed twice by centrifugation at 600×g for 10 min, while the other aliquot was kept unwashed. Thereafter, washed and unwashed sperm were extended in a TCG-based media containing 5% glycerol and 15% powdered egg yolk (PEY) and frozen. Frozen-thawed sperm were thawed and incubated at 37 °C for 4 h. Sperm motility characteristics was evaluated by CASA while plasma membrane integrity (SYBR-14/PI), acrosome integrity (PE-PNA), mitochondria activity (Mitotracker deep red) and reactive oxygen species (H2DCFDA) were analysed by flow cytometry. Sperm nucleoprotein integrity assessment was done by the determination of overall levels of sperm-head disulfide bonds via spectrophotometric analysis of free-cysteine radicals' levels. Unwashed samples generally showed better results than washed samples as regards to sperm motility characteristics irrespective of incubation. The post-thawing incubation had a significant (P < 0.05) effect on acrosome integrity and mitochondria functionality irrespective of sperm treatment. This study demonstrates that the presence of seminal plasma prior to cryopreservation was beneficial in maintaining post thawed sperm motility, and as such, could be useful for ex situ ram sperm preservation towards its use for artificial insemination.