Optimization of conditions for long-term prefreezing storage of XXXX sperm before cryopreservation.

@entity1054 @entity16745 @entity653 @entity1319
UNASSIGNED: @entity16745 ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48  hours (1  hour vs. long-term storage [LS] to 24 and 48  hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), @entity653 concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from @entity16745 were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37    C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% @entity653 were tested: 5    C (T5), 15    C (T15), and @entity1054 ( @entity1054 ). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P  <  0.05); for 48 hours, the LS-T5 sample reached higher total and progressive motility (25.9% and 9%, respectively) and nonapoptotic values (36.5%). Recovery rates revealed susceptibility to freezing at LS-15 or LS- @entity1054 samples at 24 hours (viability) or 48 hours ( @entity1319 ). In experiment 2, samples were stored at 5    C up to 48  hours and three @entity653 concentrations were evaluated: 0% (0Gly), 3% (3Gly), and 6% (6Gly). Postthawing @entity1319 increased progressively with the percentage of @entity653 for 24 hours at 5    C; 6% @entity653 during 48-hour storage had beneficial effects on sperm cryopreservation. Besides, 6% @entity653 had a clearly superior freezability for viability (42.7% and 40.8% for 24  hours and 48  hours, respectively) and motility (24  hours: total, 44.1%; progressive, 17.1%; 48  hours: total, 38.4%; progressive, 16%). In experiment 3, samples were stored up to 48 hours at 5    C with 6% of @entity653 and two dilution methods were evaluated: dilution 1:1 (average: 1782  *  10(6) sperm/mL; low) or final dilution (100  *  10(6) sperm/mL; high). Both dilution rates showed similar postthawing and postincubation results within 24 hours of long-term storage. After 48  hours, high dilution supported better postthawing quality. Both dilutions showed similar resistance to cryopreservation, except after 48  hours, when the high dilution reached a higher percent recovery rate of viability (38.8% vs. 21.6%, P  <  0.05). In conclusion, our results suggested that the best conditions for long-term prefreezing storage (up to 48 hours) of @entity16745 electroejaculates are at 5    C, at a concentration of 100  *  10(6) sperm/mL, and with 6% @entity653 .
