Abstract
Eleven semen samples from Santa Ines breed rams were cryopreserved with the objective of studying the effect of the dimethyl acetamide (DMA, 3%) or glycerol (GL, 6%) in an extender (Tris) with or without trehalose (TRE, 100 mOsmol) on post-thaw sperm viability and fertility. After the evaluation, the semen was diluted in extenders (TRIS+GL6%; TRIS+GL6%+TRE; TRIS+DMA3%; Tris+DMA3%+TRE) and then frozen. The post-thaw kinetic sperm parameters evaluated were the percentage of total motility (TM) and rapid spermatozoa (RAP), track speed (VCL), lateral amplitude of head (ALH), and linearity (LIN). The sperm viability was verified using the fluorescence markers propidium iodide (PMI, plasmatic membrane integrity), JC-1 (MMP, mitochondrial membrane potential), and FITC-PSA (ACI, acrosomal integrity). The spectrum of DNA sperm damage was assessed by single cell gel electrophoresis (Comet assay), and the measurements performed were tail moment (TMOM) and tail intensity (TINT; Comet Assay II software, Perceptive Instruments, Haverhill, UK). The sheep estrus synchronization protocol (n = 170) was 12 days of sponge with 60 mg of medroxyprogesterone acetate and 300 UI of eCG i.m. at sponge withdrawal. The ewes were inseminated on 57.5 h after sponge removal with 150 × 106 of live spermatozoa. The conception rates (CR) were verified for ultrasonography 45 days after insemination. The statistical analysis was performed using SAS software (SAS Institute Inc., Cary, NC, USA). The means (±SEM) of in vitro post-thaw sperm parameters and fertility rates are shown in Table 1. The greatest (P < 0.05) rates of TM and RAP for the GLY groups were observed in relation to the DMA ones. It was also verified the inferiority (P < 0.05) in the VCL and ALH for the trehalose extenders in both groups with GLY and DMA. The values (%) of TINT and TMOM were 14.9 and 03.9, 15.8 and 03.8, 14.7 and 02.9, and 16.6 and 04.4, respectively, for TRIS+GL6%, TRIS+GL6%+TRE, TRIS+DMA3%, and TRIS+DMA3%+TRE (P > 0.05). The integrity rates of all sperm membranes were superior for TRIS+GL6% in relation to TRIS+DMA3%. In conclusion, the DMA had apoor cryoprotective effect for ram semen. The addition of trehalose promoted negative effect on sperm kinetics, although it improved levels of PMM post-thawing in the DMA extender. The conception rate did not present a difference (P > 0.05) among the groups. Table 1.
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