评估种马精子的顶体功能主要基于使用顶体胞吐诱导物(AE),如钙离子载体A23187或孕酮。最近,据报道,在假定的获能条件下孵化种马精子(即,用钙配制的培养基,碳酸氢盐,和牛血清白蛋白)使用仅含乳酸的培养基(Lac-MW)导致有活力的精子中自发AE的高速率(AE/Viable)。在目前的研究中,在仅以乳酸作为能量底物(Lac-MW)配制的培养基中孵育精子后,我们开发了一种替代的种马精子顶体功能测定法。在实验1中,将新鲜射精的种马精子与10μMA23187,Lac-MW,或控制,在容量化条件下长达6小时。活动精子的百分比,有活力的精子,总AE(总AE),在治疗组之间比较了活精子中的AE(AE/Viable)。在Lac-MW中孵化,但不是对照或A23187,导致AE/活菌百分比的时间依赖性增加,通过流式细胞术确定,特别是在孵育4和6小时时(P<0.05)。在实验2中,将新鲜射精的精子在Lac-MW中孵育长达6小时,并确定了蛋白酪氨酸磷酸化和AE/Viable的发生。在Lac-MW中孵育4h和6h时,40%的精子显示出蛋白质酪氨酸磷酸化免疫荧光模式,这与最近与种马精子获能相关的模式一致(即,顶体和中段的免疫荧光信号)。在实验3中,比较了新鲜射精的AE/活精子率,冷藏,和冷冻/解冻的种马精子。除了在Lac-MW中孵育2小时外,新鲜,冷藏,未观察到冷冻/解冻的精子(P>0.05)。在实验4中,总AE(A23187),或AE/可行(Lac-MW),并测定了5种马的体内繁殖力。平均AE/Viable与每周期(r=0.93;P<0.05)和季节性(r=0.66;P<0.05)之间存在线性关系。在实验5中,来自低生育能力纯种(TB)种马的冷冻/解冻精子,在Lac-MW中孵育后,评估了已知携带受损顶体胞吐症(IAE;FKBP6A/A-A/A)的易感性基因型。患有IAE的低生育能力结核种马的精子平均AE/活生生较低,在Lac-MW中孵育4h和6h时,与可育对照种马相比(P<0.05)。总的来说,本研究中验证的Lac-MW模型可能是评估种马精子生理上经历AE的能力和研究种马生育潜力的有用补充试验。这种顶体功能测定可用于评估新鲜,冷藏,或冷冻/解冻的种马精子,并描述了与人工授精计划中使用的种马的体内生育力之间的强烈线性关系。
Evaluation of acrosome function in stallion sperm is mostly based on the use of inducers of acrosomal exocytosis (AE), such as the calcium ionophore A23187 or progesterone. Recently, it has been reported that incubation of stallion sperm under presumed capacitating conditions (i.e., medium formulated with calcium, bicarbonate, and bovine serum albumin) using a lactate-only containing medium (Lac-MW) results in a high rate of spontaneous AE in viable sperm (AE/Viable). In the current study, we developed an alternative assay of acrosome function for stallion sperm following the incubation of sperm in a medium formulated only with lactate as an energy substrate (Lac-MW). In Experiment 1, freshly ejaculated stallion sperm was incubated with 10 μM A23187, Lac-MW, or Control, for up to 6 h under capacitating conditions. The percentages of motile sperm, viable sperm, total AE (Total AE), and AE in viable sperm (AE/Viable) were compared among treatment groups. Incubation in Lac-MW, but not with Control or A23187, resulted in a time-dependent increase in the percentage of AE/Viable, as determined by flow cytometry, particularly at 4 and 6 h of incubation (P < 0.05). In Experiment 2, freshly ejaculated sperm was incubated in Lac-MW for up to 6 h, and the occurrence of protein tyrosine phosphorylation and AE/Viable were determined. At 4h and 6h of incubation in Lac-MW, ∼40% of the sperm displayed a protein tyrosine phosphorylation immunofluorescence pattern that coincides with that recently associated with stallion sperm capacitation (i.e., immunofluorescence signal at the acrosome and midpiece). In Experiment 3, the rate of AE/Viable sperm was compared among freshly ejaculated, cool-stored, and frozen/thawed stallion sperm. Except at 2h incubation in Lac-MW, differences in mean AE/Viable among fresh, cool-stored, and frozen/thawed sperm were not observed (P > 0.05). In Experiment 4, the relationship between Total AE (A23187), or AE/Viable (Lac-MW), and in vivo fertility of 5 stallions was determined. A linear relationship was observed between mean AE/Viable and the per-cycle (r = 0.93; P < 0.05) and seasonal (r = 0.66; P < 0.05) pregnancy rates of five stallions used for artificial insemination with cool-stored semen. In Experiment 5, frozen/thawed sperm from subfertile Thoroughbred (TB) stallions, known to carry the susceptibility genotype for Impaired Acrosomal Exocytosis (IAE; FKBP6 A/A-A/A) was evaluated following incubation in Lac-MW. Sperm from subfertile TB stallions with IAE had lower mean AE/Viable, at both 4h and 6h incubation in Lac-MW, when compared to that of fertile control stallions (P < 0.05). Overall, the Lac-MW model validated in the current study may be a useful complementary assay to evaluate the ability of stallion sperm to physiologically undergo AE and to study stallion fertility potential. This acrosome function assay can be used to evaluate fresh, cool-stored, or frozen/thawed stallion sperm, and describes a strong linear relationship with in vivo-fertility of stallions used in artificial insemination programs.