Garner D L, Thomas C A, Gravance C G, Marshall C E, DeJarnette J M, Allen C H
School of Veterinary Medicine, University of Nevada, Reno 89503, USA.
Theriogenology. 2001 Jul 1;56(1):31-40. doi: 10.1016/s0093-691x(01)00540-4.
Dilution of semen to low cell numbers/dose can result in a bull-dependent reduction in the post-thaw viability of cryopreserved bovine spermatozoa. It is possible that essential seminal plasma components are lacking at the greater dilution rates, thereby contributing to the deleterious effects of semen dilution. Ejaculates of 6 Holstein bulls were diluted to 120 x 10(6) sperm/mL in an egg yolk citrate extender (EYC). Split samples were further diluted to 80, 40, 20 and 4 x 10(6) sperm/mL in EYC extender with (+SP) and without (-SP) the addition of frozen/thawed seminal plasma previously obtained from a vasectomized bull. Serial dilutions for the +SP treatments were calculated and performed such that each dilution contained a volume of seminal plasma equal to the original 120 x 10(6) sperm/mL dilution. Samples were then loaded into 0.5-mL French straws yielding final sperm concentrations of 30, 20, 10, 5 and 1 x 10(6)/dose. Straws from each dilution were analyzed using 2 stain combinations: the sperm viability stain, SYBR-14 and propidium iodide (PI); or the mitochondrial-specific, membrane potential-dependent stain JC-1 along with PI. Split-plot analysis of variance indicated that within bulls, there were greater proportions of viable spermatozoa in aliquots containing added seminal plasma than in aliquots without added seminal plasma (P < 0.05). Contrast analyses showed that sperm viability significantly decreased as sperm concentration decreased in the -SP samples. Although the dilution effect was also observed in the +SP samples, the magnitude of the effect was less than in the -SP samples. At most sperm concentrations, the proportions of spermatozoa that stained with JC-1 were correlated (r > 0.84; P < 0.05) with the percentages of SYBR- 14 stained spermatozoa. Furthermore, the proportions of JC-1-stained spermatozoa were greater in the +SP aliquots than in the -SP samples at a concentration of 10 x 10(6) sperm/0.5 mL. These results suggest that the addition of seminal plasma can be beneficial to sperm viability when semen is diluted to low cell numbers/dose.
将精液稀释至低细胞数/剂量可能导致冷冻保存的牛精子解冻后活力出现公牛个体依赖性降低。在更高的稀释率下，精液中可能缺乏必需的精浆成分，从而导致精液稀释产生有害影响。将6头荷斯坦公牛的射精精液在蛋黄柠檬酸盐稀释液（EYC）中稀释至120×10⁶个精子/毫升。将分割后的样本在添加（+SP）和不添加（-SP）先前从输精管结扎公牛获得的冻融精浆的EYC稀释液中进一步稀释至80、40、20和4×10⁶个精子/毫升。对+SP处理进行系列稀释计算并操作，使每次稀释所含精浆体积等于最初120×10⁶个精子/毫升的稀释液。然后将样本装入0.5毫升法式细管中，最终精子浓度为30、20、10、5和1×10⁶个/剂量。对每个稀释度的细管使用两种染色组合进行分析：精子活力染色剂SYBR-14和碘化丙啶（PI）；或线粒体特异性、膜电位依赖性染色剂JC-1以及PI。裂区方差分析表明，在公牛个体内，添加精浆的等分试样中活精子的比例高于未添加精浆的等分试样（P<0.05）。对比分析表明，在-SP样本中，精子活力随精子浓度降低而显著下降。虽然在+SP样本中也观察到了稀释效应，但其效应程度小于-SP样本。在大多数精子浓度下，用JC-1染色的精子比例与用SYBR-14染色的精子百分比相关（r>0.84；P<0.05）。此外，在浓度为10×10⁶个精子/0.5毫升时，+SP等分试样中JC-1染色的精子比例高于-SP样本。这些结果表明，当精液稀释至低细胞数/剂量时，添加精浆对精子活力可能有益。
