Lindsey A C, Schenk J L, Graham J K, Bruemmer J E, Squires E L
Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, USA.
Equine Vet J. 2002 Mar;34(2):121-7. doi: 10.2746/042516402776767321.
The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.
本实验的目的是确定流式细胞仪分选和冷冻对公马精子生育力的影响。采用2×2析因设计来描述流式分选和冷冻精子的效果。通过连续10天经口给予41匹母马10毫升烯丙孕素（2.2毫克/毫升），随后在第11天肌肉注射250微克氯前列醇，使发情同步（7 - 8月）。在输精前6小时（新鲜精子）或30小时（冷冻/解冻精子）静脉注射3000国际单位人绒毛膜促性腺激素诱导排卵。母马被随机分配到4个精子处理组之一。从2匹种马用人工阴道采集精液并对每种处理进行处理。处理1（n = 10个母马周期）由新鲜、未分选的精子组成，处理2（n = 16个母马周期）由新鲜、流式分选的精子组成。待分选的精子用Hoechst 33342染色，并使用SX MoFlo精子分选仪根据DNA含量分选到携带X和Y染色体的群体中。处理3（n = 16个母马周期）由冷冻/解冻的未分选精子组成（以33.5×10⁶精子/毫升的浓度冷冻于0.25毫升细管中），处理4（n = 15个母马周期）由流式分选的冷冻/解冻精子组成（以64.4×10⁶精子/毫升的浓度冷冻）。根据预先确定的平均解冻后活力调整两种冷冻处理中精子的浓度，以使每次输精包含约5×10⁶个活动精子。所有处理均采用宫腔镜输精，将5×10⁶个活动精子以230微升的体积注入。在排卵后16天通过超声检查确定妊娠情况。用新鲜未分选精子输精的母马（4/10 = 40.0%）、新鲜流式分选精子输精的母马（6/16 = 37.5%）、冷冻/解冻未分选精子输精的母马（6/16 = 37.5%）和流式分选冷冻/解冻精子输精的母马（2/15 = 13.3%）的妊娠率未发现差异（P>0.1）。冷冻/解冻流式分选精子输精后的妊娠率有降低的趋势（P = 0.12）。需要用更多数量的母马进行进一步研究，以确定流式分选冷冻/解冻精子的生育力是否可以提高。
