Spermatozoa vitrification of sex-reversed rainbow trout (Oncorhynchus mykiss): Effect of seminal plasma on physiological parameters

This study was designed to test a vitrification method on spermatozoa of sex-reversed rainbow trout and determine the ability of sucrose, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA) and seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium comprised a standard buffer for fish spermatozoa (Cortland (R) medium) + 10% DMSO + 2% BSA + 0.13 M sucrose + SP at concentrations of 30% (group 1), 40% (group 2) or 50% (group 3). Fresh sperm (F) was used as a control. For cooling, 30 mu L suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. After cooling, the spheres were placed in cryotubes for storage in liquid nitrogen. For thawing, cryotubes with the vitrified spermatozoa were placed in a water bath at 37 degrees C for 60 s. After warming, the following sperm quality parameters were determined by flow cytometry: viability and plasma membrane integrity (SYBR-14/PI, staining technique); mitochondrial membrane integrity (JC-1 staining); and DNA fragmentation (TUNEL). Sperm function was assessed by fertility trials. Spermatozoa quality variables were best preserved when the highest SP concentration (50%) was used (viability 97.3%, plasma membrane integrity 98.4%, mitochondrial membrane integrity 36.2%, DNA fragmentation 11.1%). Similarly, fertilization rates were higher with sperm that were vitrified in medium with 50% SP. This is the first report on the physiological parameters of the cryopreservation of spermatozoa from sex-reversed rainbow trout (Oncorhynchus mykiss) by direct plunging into liquid nitrogen (vitrification). (C) 2012 Elsevier B. V. All rights reserved.