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Query: UMLS:C0265264 (HOS)
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Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.
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PMID:Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate. 1675 87

With the aim of finding an ideal cryoprotectant (CPA) in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of the 3 most commonly used CPAs, glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3%, 6%, and 12%) of each CPA on sperm freezability. Sperm samples were diluted to a final sperm concentration of approximately 400 x 10(6) spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37 degrees C for 2 hours in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < .05). Moreover, regardless of the level of CPA, results indicated that the cryoprotective effects on red deer epididymal spermatozoa of the 3 CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < .001). Furthermore, our results also showed an improvement in sperm parameters when the TCF diluent contained 6% of GLY. In Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate the effect of 2 different temperatures of GLY addition -22 degrees C (ambient temperature) and 5 degrees C- on sperm freezability. Our results showed a differential response (P < .05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22 degrees C (81.94 +/- 2.4% vs 72.38 +/- 2.4%). Although there were no statistically significant differences (P > .05) between the 2 temperatures of GLY addition after thawing in terms of sperm quality, after 2 hours of incubation, results tended to be better when CPAs were added at 22 degrees C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22 degrees C, as an alternative to the more common 3%-4% of GLY and addition at 5 degrees C, in red deer semen freezing protocols.
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PMID:Influence of various permeating cryoprotectants on freezability of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of concentration and temperature of addition. 1677 52

The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.
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PMID:Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants. 1707 44