Abstract
individual variation of samples following cryopreservation. Cholesterol is known to play an important role as cryoprotectant in semen freezing. Egg yolk of greater rhea (Rhea americana) is known to have 4.54 times more cholesterol than the chicken egg yolk. The aim of this work was to compare the use of two commercial extenders using chicken egg yolk and substituting Rhea egg yolk to chicken egg yolk, in order to maximize the cryoprotectant effect of cholesterol. Semen was collected six times from each of six Criollo breed stallions during the non-breeding season using a Hannover model artificial vagina and an estrous mare. The ejaculate was evaluated for total and progressive motility; concentration was determined with a Neubauer counting chamber. Sperm morphology slides were stained with Cerovsky stain. After evaluation, semen samples were divided in two aliquots and diluted 1:1 in one of the centrifugation extenders: A (extender with glycerol) or B (extender with methylformamide). The semen samples were centrifuged at 400 g for 10min and 90–95% of the supernatant (extender plus seminal plasma) was removed. All samples were re-suspended to a final concentration of 100 106 sperm/ml either in extender A with or without rhea egg yolk or extender B with or without rhea egg yolk. Extended semen were packaged into 0.5-ml straws and frozen 3 cm above liquid nitrogen level for 20 min. Samples were then plunged into liquid nitrogen and stored at 196C. Semen samples were examined earliest one week after freezing for total and progressive motility and for physical and functional membrane integrity (HOST and CFDA-PI fluorescence). Differences between treatments were computed using Prism5 (GraphPad, USA) and an analysis of the 2x2 factorial design was done using the Bonferroni test as post-test; the significance was set at 5%. Both extenders, with or without Rhea egg yolk, showed no significant difference in total or progressive motility, although there was a difference (P 1⁄4 0.05) between extenders when different extenders were compared, no matter if they were supplemented with Rhea egg yolk or not. Membrane integrity was not significantly different. The present results show that Rhea americana egg yolk might be an alternative supplement to stallion semen extenders, although there is no improvement of semen quality in comparison to egg yolk.
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