波尔山羊冷冻精液稀释液配方研究
摘要
本试验通过逐级筛选研制出波尔山羊冷冻精液稀释液试验配方，为了分析其优劣，与国内外报道的四个典型配方进行对比，应用超微结构分析，谷草转氨酶和乳酸脱氢酶活力测定及精子异种穿卵试验等多种检测方法，评定了试验配方在波尔山羊精液冷冻保存效果上的优劣性。结果如下：
1．筛选基础液pH。分别设计出pH为6.6、6.8、7.0、7.2的四个试验组，其中试验Ⅳ组(pH7.2)效果最好，其组成为：葡萄糖3.2g、乳糖4.8g、柠檬酸钠1.7g、Tris0.01g、柠檬酸0.03g、双蒸水100ml。用其冷冻精液，其精子冻后活率0.489±0.011，复苏率(％)67.1±0.398，冻后顶体完整率(％)69.6±1.535。冻后活率、复苏率极显著优于(p＜0.01)试验Ⅰ组(pH值6.6)；冻后活率、复苏率、冻后顶体完整率均显著高于(p＜0.05)试验Ⅱ组(pH6.8)。
2．筛选基础液渗透压。分别设计出渗透压Δ0.526℃、Δ0.682℃、Δ0.754℃、Δ0.819℃四个试验组，其中试验Ⅳ组(pH7.2，渗透压Δ0.819℃)效果最好，其组成为：葡萄糖3.2g、乳糖4.8g、柠檬酸钠1.7g、Tris0.01g、柠檬酸0.03g、双蒸水100ml。用其冷冻精液，精子冻后活率0.478±0.012、解冻后顶体完整率(％)68.2±3.129，均极显著高于(p＜0.01)试验Ⅰ组(Δ0.526℃)；显著高于(p＜0.05)试验Ⅱ组(Δ0.682℃)、试验Ⅲ组(Δ0.754℃)。
3．筛选甘油的添加量水平。用试验Ⅳ组作为基础液，在此基础上分别添加2.4％、4.0％、5.6％、7.2％、8.8％的甘油，组成五个试验组，其中试验Ⅱ组(4％)效果最好。用其冷冻精液，精子冻后活率0.495±0.011，复苏率(％)65.066±0.216，均显著高于其余四个试验组(P＜0.05)。
4．筛选最佳稀释液配方。将筛选出的最好稀释液配方作为配方Ⅴ，与国内外四个典型配方比较，用配方Ⅴ冷冻保存的精液解冻后活率、冻后存活时间、冻后8h活率、冻后顶体完整率(％)分别是0.492±0.012、11.616±0.117、0.381±0.012、69.866±1.535，均极显著高于配方Ⅰ、配方Ⅲ(p＜0.01)；除精液冻后存活时间外均极显著高于配方Ⅱ(p＜0.01)，除冻后顶体完整率外均显著高于配方Ⅳ(p＜0.05)，表明
配方V冷冻保存精液效果最好。
5.精浆中GOT和LDH活力测定。用配方V进行冷冻保存波尔山羊精液，解冻
后精浆中的GOT和LDH活力分别是268.1士1.820、85.1士0.258，均显著低于配方
I、配方11、配方m(p<0.05)。表明用配方V冷冻保存精液对精子质膜保护效果
较好。
6.精子异种穿卵试验。配方V冷冻保存后精液的精子穿卵率(%)83.194士2.164，
极显著高于配方I和配方m(p<0.01)，显著高于配方11(P<0.05)。表明本试验中
应用配方V冷冻保存精液，其解冻后的精子受精能力效果显著。
The aim of this study was to improve the quality of Boer goat frozen semen, making to arrive the level of the Boer goat fresh semen. In this study, The ingredient of diluent was finished at the base of the four finest twice diluted ingredients of Boer goat frozen semen diluted ingredient of Boer goat frozen semen diluent. The analyse of the goat spermatozoa ultrastructures, measuring the active of GOT-released and LDH-released, and the ability of penetration were investigated respectively. The results were following:
1. In treatment IV (pH7.2), after frozen-thawed, the active of spermatozoa , recovery were respectively: 0.489 0.011, 69.6 1.535. But in treatment I (pH6.6), It was: 0.411 0.006 , 59.7 0.317, there were a very significant difference (p<0.01), and had a significant difference (p<0.05) with treatment II (pH6.8) , about the rate of integral top, treatment IV (pH7.2) was 68.2 3.129, which had a significant difference (p<0.05) with treatment I (pH6.6),treament II (pH6.8). The result indicated that the effect of treatment IV (pH7.2) was the best.
2. In treatment IV ( 0.819 ), after frozen-thawed, the percentages of spermatozoa 0.478 0.012, the rate of integral top 68.2 3.129, these were all elevated very remarkably (p<0.01), comparing with treatment I ( A 0.526 ); there were a significant difference (p<0.05) between treatment IV (A 0.819 ) and treatment II (A 0.682 ), treatment III( A 0.754 ). It shown the higher infiltrate pressure was favorable of the conservation of Boer goat frozen semen.
3. In treatment II, which contains 4% glycerin, the percentages of spermatozoa motility, recovery were respectively: 0.495 0.011, 65.066 0.216., There were a significant difference with treatment 1(2.4%), treatment III (5.6%), treatment IV (7.2%) and treatment V (8.8%) (P<0.05) This explains that the fine concentration of glycerin had an effect on the quality of the goat frozen semen, also the effect of 4% is the finest in the study.
4. In the ingredient V, after frozen-thawed active , the survive time, the active of frozen-thawed conserving (8h) , the rate of integral top were consequently: 0.492
0.012, 11.616 0.117% 0.381 0.012, 69.866 1.535,these were reduced remarkably between I , II, III., IV(P<0.05). The result indicated that the comprehensive effect on using the alleviant of Tris and citrate was better.
5. Ingredient V, the leakage of GOT and LDH were reduced remarkably(P<0.05), comparing with the ingredient I , ingredient II, ingredientlll, the active of GOT and LDH were respectively:268.1 1.820 , 85.1 0.258,It is important that sugar had several functions in the diluent, as the nutrient , protection and alleviant.
6. SPA.in the ingredient V, the ability of penetration was83.194 2.164,there was a very significant difference(P < 0.01) with ingredient I , III .and had a significant difference(P<0.05). It shown ingredient V was the best of these ingredients on the ability of fertilization.
1．筛选基础液pH。分别设计出pH为6.6、6.8、7.0、7.2的四个试验组，其中试验Ⅳ组(pH7.2)效果最好，其组成为：葡萄糖3.2g、乳糖4.8g、柠檬酸钠1.7g、Tris0.01g、柠檬酸0.03g、双蒸水100ml。用其冷冻精液，其精子冻后活率0.489±0.011，复苏率(％)67.1±0.398，冻后顶体完整率(％)69.6±1.535。冻后活率、复苏率极显著优于(p＜0.01)试验Ⅰ组(pH值6.6)；冻后活率、复苏率、冻后顶体完整率均显著高于(p＜0.05)试验Ⅱ组(pH6.8)。
2．筛选基础液渗透压。分别设计出渗透压Δ0.526℃、Δ0.682℃、Δ0.754℃、Δ0.819℃四个试验组，其中试验Ⅳ组(pH7.2，渗透压Δ0.819℃)效果最好，其组成为：葡萄糖3.2g、乳糖4.8g、柠檬酸钠1.7g、Tris0.01g、柠檬酸0.03g、双蒸水100ml。用其冷冻精液，精子冻后活率0.478±0.012、解冻后顶体完整率(％)68.2±3.129，均极显著高于(p＜0.01)试验Ⅰ组(Δ0.526℃)；显著高于(p＜0.05)试验Ⅱ组(Δ0.682℃)、试验Ⅲ组(Δ0.754℃)。
3．筛选甘油的添加量水平。用试验Ⅳ组作为基础液，在此基础上分别添加2.4％、4.0％、5.6％、7.2％、8.8％的甘油，组成五个试验组，其中试验Ⅱ组(4％)效果最好。用其冷冻精液，精子冻后活率0.495±0.011，复苏率(％)65.066±0.216，均显著高于其余四个试验组(P＜0.05)。
4．筛选最佳稀释液配方。将筛选出的最好稀释液配方作为配方Ⅴ，与国内外四个典型配方比较，用配方Ⅴ冷冻保存的精液解冻后活率、冻后存活时间、冻后8h活率、冻后顶体完整率(％)分别是0.492±0.012、11.616±0.117、0.381±0.012、69.866±1.535，均极显著高于配方Ⅰ、配方Ⅲ(p＜0.01)；除精液冻后存活时间外均极显著高于配方Ⅱ(p＜0.01)，除冻后顶体完整率外均显著高于配方Ⅳ(p＜0.05)，表明
配方V冷冻保存精液效果最好。
5.精浆中GOT和LDH活力测定。用配方V进行冷冻保存波尔山羊精液，解冻
后精浆中的GOT和LDH活力分别是268.1士1.820、85.1士0.258，均显著低于配方
I、配方11、配方m(p<0.05)。表明用配方V冷冻保存精液对精子质膜保护效果
较好。
6.精子异种穿卵试验。配方V冷冻保存后精液的精子穿卵率(%)83.194士2.164，
极显著高于配方I和配方m(p<0.01)，显著高于配方11(P<0.05)。表明本试验中
应用配方V冷冻保存精液，其解冻后的精子受精能力效果显著。
The aim of this study was to improve the quality of Boer goat frozen semen, making to arrive the level of the Boer goat fresh semen. In this study, The ingredient of diluent was finished at the base of the four finest twice diluted ingredients of Boer goat frozen semen diluted ingredient of Boer goat frozen semen diluent. The analyse of the goat spermatozoa ultrastructures, measuring the active of GOT-released and LDH-released, and the ability of penetration were investigated respectively. The results were following:
1. In treatment IV (pH7.2), after frozen-thawed, the active of spermatozoa , recovery were respectively: 0.489 0.011, 69.6 1.535. But in treatment I (pH6.6), It was: 0.411 0.006 , 59.7 0.317, there were a very significant difference (p<0.01), and had a significant difference (p<0.05) with treatment II (pH6.8) , about the rate of integral top, treatment IV (pH7.2) was 68.2 3.129, which had a significant difference (p<0.05) with treatment I (pH6.6),treament II (pH6.8). The result indicated that the effect of treatment IV (pH7.2) was the best.
2. In treatment IV ( 0.819 ), after frozen-thawed, the percentages of spermatozoa 0.478 0.012, the rate of integral top 68.2 3.129, these were all elevated very remarkably (p<0.01), comparing with treatment I ( A 0.526 ); there were a significant difference (p<0.05) between treatment IV (A 0.819 ) and treatment II (A 0.682 ), treatment III( A 0.754 ). It shown the higher infiltrate pressure was favorable of the conservation of Boer goat frozen semen.
3. In treatment II, which contains 4% glycerin, the percentages of spermatozoa motility, recovery were respectively: 0.495 0.011, 65.066 0.216., There were a significant difference with treatment 1(2.4%), treatment III (5.6%), treatment IV (7.2%) and treatment V (8.8%) (P<0.05) This explains that the fine concentration of glycerin had an effect on the quality of the goat frozen semen, also the effect of 4% is the finest in the study.
4. In the ingredient V, after frozen-thawed active , the survive time, the active of frozen-thawed conserving (8h) , the rate of integral top were consequently: 0.492
0.012, 11.616 0.117% 0.381 0.012, 69.866 1.535,these were reduced remarkably between I , II, III., IV(P<0.05). The result indicated that the comprehensive effect on using the alleviant of Tris and citrate was better.
5. Ingredient V, the leakage of GOT and LDH were reduced remarkably(P<0.05), comparing with the ingredient I , ingredient II, ingredientlll, the active of GOT and LDH were respectively:268.1 1.820 , 85.1 0.258,It is important that sugar had several functions in the diluent, as the nutrient , protection and alleviant.
6. SPA.in the ingredient V, the ability of penetration was83.194 2.164,there was a very significant difference(P < 0.01) with ingredient I , III .and had a significant difference(P<0.05). It shown ingredient V was the best of these ingredients on the ability of fertilization.
引文
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