Negative pressure in the pre-freezing of ram semen

ABSTRACT

Background: The effect of controlled stress on cells has been widely studied and there is strong evidence that positivepressure improves the survival of cryopreserved gametes and embryos. Positive pressure resulted in higher motility offrozen boar semen, and increased bull semen cryotolerance. Looking for similar effects, we developed a piece of equipment (Nitrocooler) to apply negative pressure. The “Nitrocooler” increased the cryotolerance in bovine IVP blastocysts.Nevertheless, until now, negative pressure has not been tested in sperm cells. This study aimed to determine the effectsand the best negative pressure intensity to apply in pre-freezing ram semen.Material, Methods & Results: The ram semen obtained with artificial vagina was immediately diluted (1 + 1.5) in Trisegg yolk, and fractioned into 4 experimental groups, Control Without treatment; P200 Submission to 200 mBar negativepressure; P500 Submission 500 mBar negative pressure; P800 Submission to 800 mBar negative pressure. Then, Tris-yolkglycerol was added to all groups to obtain a dilution of 1 + 3, and a sperm concentration of 180 million per straw. Thecooling and freezing procedure was identical for all groups. After thaw, progressive motility (PM) was assessed at zero,1, 2 and 3 h of TRT and after Percoll selection (PMPP). Acrosome integrity (ACI) was assessed by FITC PNA staining;plasmatic membrane integrity (PMI) by hypo-osmotic test, acrosome integrity post Percoll (ACIPP), membrane integritypost Percoll (PMIPP), and cleavage rate after heterologous IVF with bovine oocytes. Data were analyzed by t test (P <0.05). Just after thawing, control group showed higher PM (49%) than P200 (40.9%), P500 (38.9%) and P800 (38.9%),without differences among them. There were no differences between PM in the Control, P200, P500 and P800 groupsat 1 h, 2 h and 3 h of TRT, and in ACI...