Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura 281001, Uttar Pradesh, India.
College of Biotechnology, College of Veterinary Science and Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura 281001, Uttar Pradesh, India.
Theriogenology. 2017 Jul 1;96:164-171. doi: 10.1016/j.theriogenology.2017.04.016. Epub 2017 Apr 10.
The beneficial effects of cholesterol loaded cyclodextrin (CLC) addition were evaluated in cryopreserved bull semen. Forty ejaculates were collected from Hariana bulls (n = 4), pooled and divided into 4 aliquots. All the aliquots were initially diluted in to egg yolk tris citrate and supplemented with CLC @ 0.5 mg (Group-II), 1.0 mg (G-III) and 2.0 mg (G-IV) CLC/120 × 10 spermatozoa or without CLC (G-I) that served as control. Extended semen was cryopreserved at -196 °C for 24 h. Seminal attributes like motility, viability, cryocapacitation like changes, tyrosine phosphorylation, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA integrity were evaluated after equilibration and thawing. Results showed a significant increase in the motility, viability and acrosome intact spermatozoa in Group II as compared to other three groups. Further, the proportion of spermatozoa showing capacitation and acrosome reaction was also decreased (P < 0.05) significantly in Group II as compared to Group I, III, and IV. Immunoblot demonstrated a 32 kDa (p32) protein showing differential variation in the band intensity in all the four groups being lower in Group II. Further, the immunolocalization study revealed positive immune reactivity for tyrosine phosphorylated proteins at middle piece and neck (high fluorescence), post-acrosomal region (medium fluorescence), and principal piece (low fluorescence) of spermatozoa. Addition of CLC significantly increased (P < 0.05) the percentage of spermatozoa showing high transmembrane mitochondrial potential, and also, CLC @ 0.5 mg/120 × 10 in semen extender significantly decreased (P < 0.05) spermatozoa showing fragmented DNA after thawing as compared to control. Results of the present study indicate beneficial effects of CLC supplementation on cryodamage of spermatozoa by reducing the cryocapacitation and apoptosis like changes.
研究了胆固醇包合环糊精（CLC）添加物对冷冻牛精液的有益作用。从哈里亚纳公牛（n=4）采集了 40 份精液，混合并分为 4 份。所有等分试样最初均在卵黄三柠檬酸中稀释，并添加 0.5mg（第 II 组）、1.0mg（第 III 组）和 2.0mg（第 IV 组）CLC/120×10 个精子的 CLC，或不添加 CLC（第 I 组）作为对照。扩展精液在-196°C下冷冻 24 小时。在平衡和解冻后评估了运动性、活力、冷冻致精子质膜通透性和 DNA 完整性改变、酪氨酸磷酸化、凋亡等方面的变化。结果表明，与其他三组相比，第 II 组的运动性、活力和顶体完整精子显著增加。此外，与第 I 组、第 III 组和第 IV 组相比，第 II 组显示顶体反应和获能的精子比例也显著降低（P<0.05）。免疫印迹显示 32kDa（p32）蛋白在所有四组中的条带强度存在差异，第 II 组的条带强度较低。此外，免疫定位研究显示，精子中段和颈部（高荧光）、顶体后区（中荧光）和主段（低荧光）有酪氨酸磷酸化蛋白的阳性免疫反应。CLC 的添加显著增加（P<0.05）了显示高跨膜线粒体潜能的精子百分比，并且在精液稀释液中添加 0.5mg/120×10 的 CLC 也显著降低（P<0.05）了与对照组相比解冻后显示碎片化 DNA 的精子数量。本研究结果表明，CLC 补充剂通过减少冷冻致精子质膜通透性和凋亡样变化，对精子的冷冻损伤具有有益作用。
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