Anel-López L, García-Álvarez O, Parrilla I, Del Olmo D, Maroto-Morales A, Fernandez-Santos M R, Ortiz J A, Soler A J, Martínez E M, Vazquez J M, Garde J J
SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario s. n., 02071 Albacete, Spain.
SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario s. n., 02071 Albacete, Spain; Biomedical Center, Medical Faculty in Pilsen, Charles University in Prague, Pilsen, Czechia.
Theriogenology. 2017 Feb;89:206-213. doi: 10.1016/j.theriogenology.2016.11.010. Epub 2016 Nov 14.
This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm.
本研究首次调查了性别分选和冷冻保存对马鹿（Cervus elaphus）精子解冻后特征及生育力的影响。在繁殖季节，通过电刺激采精法从10头成年雄鹿采集精液，每份射精样本分为四个实验组：大量分选精子、分选但未区分性别（BSS）；高纯度分选X精子（XSS）；高纯度分选Y精子（YSS）；以及对照未分选精子（NS）。随后，所有样本在液氮中冷冻。每头雄鹿每种样本类型取两根细管，在解冻后立即以及在37℃孵育2小时后进行分析。通过计算机辅助精子分析（CASA）评估，解冻后NS、BSS和YSS精子的总活力（TM）无差异（P<0.05）。对于XSS，解冻后TM低于NS（54%）或BSS（50%）（39%，P<0.05），但与YSS（47%）精子相似（P>0.05）。通过碘化丙啶/YO-PRO-1和流式细胞术分析评估，XSS精子的凋亡精子百分比高于NS（12%）、BSS（13%）和YSS（14%）精子（17%，P≤0.05）。孵育后，各处理组之间的TM或凋亡百分比无差异（P>0.05）。以DNA碎片化指数（%DFI）计算的解冻后染色质稳定性在各处理组之间相似；孵育后，除YSS外，所有组%DFI均增加，YSS组显示最低值（P<0.05）。对同步发情的母鹿进行人工授精，YSS、NS和标准冻融精子的分娩率分别为44%、52%和62%（P<0.05）。值得注意的是，YSS和NS精子所产小鹿分别有93%和55%为雄性（P<0.05）。总之，Y分选精子显示出可接受的解冻后精子评估参数和预期的后代性别比例。需要更多研究来了解可能损害Y分选马鹿精子生育力的精子损伤来源。
