NDVSU-College of Veterinary Science and Animal Husbandry, Rewa, India.
ICAR-Indian Veterinary Research Institute, Bareilly, India.
Reprod Domest Anim. 2020 Jun;55(6):726-736. doi: 10.1111/rda.13674. Epub 2020 Apr 17.
A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 10 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca , capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 10 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 10 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 10 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca ); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca , low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca , medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 10 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing-thawing-associated damages in bubaline species.
一项研究旨在确定外源性胆固醇负载环糊精（CLC）的最佳剂量，以获得水牛精子的最大冷冻保护效果。在本研究中，将 120×10 个精子分别孵育在 2、3 和 4mg 的 CLC 中，分别分组为 Gr II、III 和 IV，然后评估冷冻保护的水牛精子的精子渐进性运动、细胞内 Ca 、通过蛋白酪氨酸磷酸化（PTP）测定评估的顶体状态、透明带结合百分比（ZBP）和体外受精试验的卵裂率（CR）与未处理的对照组（Gr I）进行比较。结果表明，在 2 至 3mg 的 CLC 处理中，具有更高细胞内 Ca 和 PTP 中具有中或高顶体状态的精子群体的百分比显著（p<.05）呈线性下降，然后增加到 4mg/120×10 个精子，而精子渐进性运动、具有低顶体状态的精子群体百分比、ZBP 和 CR 在 2 至 3mg 的 CLC 处理中显著增加（p<.05），然后减少到 120×10 个精子的 4mg。该研究清楚地表明，在 3mg/120×10 个精子中，CLC 具有最大的有益效果，可保护精子渐进性运动、膜流动性（低细胞内 Ca ）；防止冷冻顶体（免疫定位中的低顶体状态）并增强体外 ZBP 和 CR。经冻融处理的 CLC 处理精子的运动与低细胞内 Ca 的精子群体、低顶体状态的精子群体、ZBP 和 CR 呈正显著相关（p<.05），而与高细胞内 Ca 的精子群体、中或高顶体状态的精子呈负相关（p<.05）。本研究首次揭示，在更高剂量（>3mg/120×10 个精子）的 CLC 孵育下，对精子冷冻保存有不利影响，尽管在处理前用 3mg/120 百万精子孵育可将冷冻解冻相关损伤最小化在 Bubalus 物种中。
