Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand.
Department of Animal Science, Faculty of Agriculture, Rajamangala University of Technology Srivijaya, Nakhon Sri Thammarat 80110, Thailand.
Poult Sci. 2019 Sep 1;98(9):4161-4171. doi: 10.3382/ps/pez196.
Chicken semen conservation is an important tool for programs of genetic diversity management and of endangered breeds' conservation. However, the method still needs to be improved in order to be applied in a wide variety of environments and breeds. Our objective was to compare the effects of 2 external cryoprotectants saccharides (sucrose and raffinose) on the sperm freezability of a Thai local breed, Pradu Hang Dum, in which semen was frozen with a simple freezing method using nitrogen vapors and dimethyl formamide (DMF). Thirty-six males were selected on their motility vigor score for the experiments. In a first experiment, a large range of sucrose and raffinose doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the saline Blumberger Hahnen Sperma Verdünner diluent + DMF (6% v/v) with or without sucrose/raffinose. The best targeted doses of sucrose and raffinose were then kept for experiment 2 that was focused on cryopreserved semen. In this experiment, semen quality was measured on frozen-thawed sperm: different objective motility data evaluated by computer-assisted sperm analysis (CASA), membrane integrity, acrosome integrity, mitochondria function evaluated using flow cytometry, lipid peroxide production assessed by the thiobarbituric acid test. Fertility obtained with frozen-thawed semen supplemented or not with sucrose or raffinose was also evaluated after artificial insemination of laying hens. The presence of sucrose at the osmotically inactive dose 1 mmol significantly increased the vigor motility, membrane integrity, acrosome integrity, and mitochondrial functions of frozen-thawed sperm (P < 0.05), and showed the highest levels of fertility after sperm cryopreservation (91% vs. control 86%, P < 0.001). Raffinose showed negative effects on in vitro semen quality from 1 to 100 mmol. Fertility was also negatively (P < 0.001) affected by raffinose (fertility rate 66 to 70%). We thus showed in the present study the high success of a simple chicken sperm cryopreservation method with an external cryoprotectant easily available and cheap, the sucrose, used at an osmotically inactive low concentration.
鸡精液保存是遗传多样性管理和濒危品种保护计划的重要工具。然而，为了能够在各种不同的环境和品种中应用，该方法仍需改进。我们的目的是比较 2 种外部冷冻保护剂（蔗糖和棉子糖）对泰国本地品种 Pradu Hang Dum 精子冷冻能力的影响，该品种的精液是用氮气蒸气和二甲基甲酰胺（DMF）的简单冷冻方法冷冻的。选择 36 只精子活力评分较高的雄性进行实验。在第一个实验中，测试了蔗糖和棉子糖的大范围剂量。在 5°C 孵育后或在含有蔗糖/棉子糖的盐水 Blumberger Hahnen Sperma Verdünner 稀释剂+DMF（6%v/v）中用 straw 冷冻保存后，评估精液质量。然后，将最佳的蔗糖和棉子糖靶剂量保留用于专注于冷冻保存精液的实验 2。在这个实验中，通过计算机辅助精子分析（CASA）评估不同的目标运动数据、膜完整性、顶体完整性、使用流式细胞术评估线粒体功能、通过硫代巴比妥酸试验评估脂质过氧化产物，来测量冷冻解冻精子的质量。还评估了用蔗糖或棉子糖补充或不补充冷冻解冻精液的人工授精母鸡的受精能力。在 1 mmol 等渗非活性剂量下添加蔗糖可显著提高冷冻解冻精子的活力、膜完整性、顶体完整性和线粒体功能（P < 0.05），并在精子冷冻保存后显示出最高的受精率（91% vs. 对照组 86%，P < 0.001）。棉子糖在 1 至 100 mmol 范围内对体外精液质量表现出负面影响。棉子糖也对受精率产生负面影响（P < 0.001）（受精率 66%至 70%）。因此，我们在本研究中展示了一种简单的鸡精子冷冻保存方法的高成功率，该方法使用一种容易获得且廉价的外部冷冻保护剂蔗糖，以等渗非活性的低浓度使用。
