猪精液冷冻与常温保存技术的研究

Cryopreservation and normal temperature preservation of porcine spermatozoa

为研究冷冻保存和常温((17±0.5)℃)保存后猪精子活率、活力和顶体完整率,比较了猪精液冷冻稀释液中2种甘油含量细管法的快速冷冻效果,以及不同解冻温度对冷冻精液的影响,采用细胞计数法、FDA-PI双重染色法与Giemsa染色方法检测精子的活率、活力和顶体完整率。结果显示:3%(体积分数,下同)甘油冷冻解冻后精液的活率、活力(38.54%、52.24%)显著(P<0.05)高于2%甘油组(15.63%、28.22%),但3%甘油组顶体完整率(11.55%)显著(P<0.05)低于2%甘油组(21.71%)。38℃水浴解冻后的精子在39℃培养箱中4 h内活率高于0.3,而50℃水浴解冻后1 h后的活率低于0.3。精液稀释液Anderohep和BTS在常温((17±0.5)℃)保存新鲜猪精子的活率与顶体完整率上效果相似,保存3 d内精子活率达0.6以上,保存6 d内精子活率为0.3以上。结果表明,使用3%甘油冷冻保存及2种常温保存稀释液均可以较好地保存精子。

The effect of cryopreservation and normal temperature preservation on porcine spermatozoa was investigated by comparing with the sperm motility, survival and acrosome-intact rates, The effects of thawing temperature after cryopreservation were also compared, The porcine liquid and frozen sperm were studied in order to improve the effect of preserved spermatozoa. The methods of cytometry, double staining of FDA-PI and Giemsa staining were used to test the motility, survival and acrosome-intact rates of preserved spermatozoa. The motility and survival rates of the group with 3 % glycerin concentrations（38.54 % and 52.24 %, respectively） were significantly（ P 〈 0.05） higher than that of the group with 2 % glycerin concentrations（15.63 % and 28.22 %, respectively）. However, acrosome-intact rate of the group with 2% glycerin concentrations （21.71%）was significantly（ P 〈 0.05）higher than that of the group with 3% glycerin concentrations （11.55%）. The result of different thawing temperatures indicated that the spermatozoa thawn in 38 ℃ water showed a better survival rate than those thawn in 50 ℃ water; the duration of preservation on above 30% survival rate were 4 and 1 h, respectively. In the experiment of normal temperature（（17 ± 0.5）℃）preservation, the effects in Anderohep and BTSdilution had no difference on the motility and acrosome-intact rates of spermatozoa. The motility of spermatozoa was above 60% in 3 days and the motility was not less than 30% until 6 days after preservation, Cryopreservation using 3% glycerin and the normal temperature preservation are feasible and can meet practical need.