Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen-thawed bull spermatozoa.
In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.
Gadea
Joaquín
J
Department of Physiology, School of Veterinary, University of Murcia, Murcia, Spain. jgadea@um.es
Gumbao
David
D
Cánovas
Sebastián
S
García-Vázquez
Francisco Alberto
FA
Grullón
Luis Alberto
LA
Gardón
Juan Carlos
JC
D000818:Animals; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002843:Chromatin / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D047108:Embryonic Development / Q000187:drug effects; D005307:Fertilization in Vitro; D005615:Freezing; D005978:Glutathione / Q000494:pharmacology*; D008297:Male; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*
D002843:Chromatin; D017382:Reactive Oxygen Species; D005978:Glutathione