Phillips J J, Bramwell R K, Graham J K
Department of Physiology, Colorado State University, Fort Collins 80523, USA.
Poult Sci. 1996 Jul;75(7):915-23. doi: 10.3382/ps.0750915.
Experiments were designed to determine when, during the cryopreservation process, sperm lose fertilizing capacity and whether the cryoprotectant, methyl cellulose (MC), could be used in combination with glycerol to cryopreserve sperm and remain in the inseminate without reducing fertility. Semen diluted in Minnesota Avian extender (MNA) and inseminated immediately had greater fertility (75%) than semen processed for cryopreservation (12 to 60%). The largest decreases in fertility were due to addition of glycerol to sperm and to cryopreservation. In another experiment, fertility of inseminates containing 0, 1, and 2% glycerol were 82, 29, and 21%, respectively, for eggs collected 2 to 5 d after insemination. When 0.5% MC was added to the same three treatments, fertility rates were 88, 63, and 69%, respectively. Semen cryopreserved in MNA containing 9% glycerol; MC + 3% glycerol; MC + 4% glycerol; MC + 9% glycerol; or 9% glycerol with the cryoprotectant removed post-thaw by dilution and subsequent centrifugation exhibited 59, 30, 35, 60, and 69% viable cells, respectively; and 65, 38, 46, 69, and 65% motile sperm, respectively. Sperm cryopreserved with MC and either 4 or 9% glycerol exhibited similar numbers of sperm binding to chicken perivitelline layers in vitro as did fresh sperm, whereas sperm frozen with MC and 3% glycerol bound oocytes with only 31% efficiency (P < 0.05). The extent to which cryopreserved sperm penetrated the perivitelline layer in vitro was independent of glycerol concentration, but was four times more efficient than that of fresh sperm (P < 0.05). The fertility rates of fresh semen, semen frozen in 9% glycerol with the cryoprotectant removed after thawing, and semen frozen in MC with either 3 or 4% glycerol were 87.4, 27.6, 0.8, and 0.5%, respectively (P < 0.05). The MC reduces the contraceptive effects of glycerol when inseminated with fresh sperm, but does not maintain fertilizing capacity in frozen-thawed sperm when used in combination with 3 or 4% glycerol.
实验旨在确定在冷冻保存过程中精子何时丧失受精能力，以及冷冻保护剂甲基纤维素（MC）是否可与甘油联合使用来冷冻保存精子，并保留在授精液中而不降低生育力。用明尼苏达禽类稀释液（MNA）稀释并立即授精的精液生育力更高（75%），高于经冷冻保存处理的精液（12%至60%）。生育力下降幅度最大是由于向精子中添加甘油以及冷冻保存。在另一项实验中，对于授精后2至5天收集的卵子，含有0%、1%和2%甘油的授精液生育力分别为82%、29%和21%。当向相同的三种处理中添加0.5% MC时，生育率分别为88%、63%和69%。在含有9%甘油的MNA中冷冻保存的精液；MC + 3%甘油；MC + 4%甘油；MC + 9%甘油；或解冻后通过稀释和随后离心去除冷冻保护剂的9%甘油，分别显示出59%、30%、35%、60%和69%的活细胞；以及65%、38%、46%、69%和65%的活动精子。用MC和4%或9%甘油冷冻保存的精子在体外与鸡卵黄膜结合的精子数量与新鲜精子相似，而用MC和3%甘油冷冻的精子结合卵母细胞的效率仅为31%（P < 0.05）。冷冻保存的精子在体外穿透卵黄膜的程度与甘油浓度无关，但比新鲜精子高四倍（P < 0.05）。新鲜精液、解冻后去除冷冻保护剂的9%甘油冷冻精液以及用3%或4%甘油的MC冷冻精液的生育率分别为87.4%、27.6%、0.8%和0.5%（P < 0.05）。当与新鲜精子一起授精时，MC可降低甘油的避孕效果，但与3%或4%甘油联合使用时，不能维持冻融精子的受精能力。
