应用超数排卵与胚胎冷冻技术保护马头山羊品种的研究
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摘要
本研究的目的是通过马头山羊的超数排卵、超排胚胎的冷冻、冷冻胚胎的移植等技术的有机结合，实现马头山羊的胚胎冷冻保种。本试验围绕这个目的，对马头山羊的超数排卵、超排胚胎的冷冻、冷冻胚胎的移植分别进行了研究。
本试验首先研究了处理方法、配种方法、上胎产羔数、膘情、重复超排、基因多态性等因素对马头山羊超数排卵效果的影响，旨在充分发掘优秀马头山羊母畜的繁殖潜力。试验结果表明：(1)在CIDR+FSH+PG超排方法的基础上，同时运用LHRH-A_3与P_4进行超排可以显著提高只均获胚数(15.63vs12.75)、只均可用胚数(14.75vs11.25)和可用胚率(94.40％vs88.24％)(P＜0.05)，得到较好的超排效果；(2)超排供体羊配种时，每隔8h本交一次(公母比例1：1)或每隔8h人工授精一次均可得到较好的超排效果，其只均获胚数、只均可用胚胎数、可用胚率均显著高于每隔8h本交一次(公母比例1：2)或每隔12h本交一次(公母比例1：1)(P＜0.05)；(3)上胎产羔4只的供体羊只均获胚数(16.83vs14.17)和只均可用胚数(15.83vs13.33)显著高于上胎产羔2只以下的供体羊(P＜0.05)；(4)中等膘情的供体羊超排效果较好，其只均获胚数、只均可用胚数和可用胚率均显著高于上等膘情和下等膘情的供体羊(P＜0.05)；(5)对供体羊进行重复超排时，其只均获胚数(15.17vs15.67)、只均可用胚胎数(14.17vs14.83)和可用胚率(93.41％vs94.68％)等指标均略低于首次超排，但统计差异不显著(P＞0.05)；(6)INHA基因不同基因型对供体羊只均获胚数和只均可用胚数的影响一致，均表现为GG＞AG＞AA，但是统计差异不显著(P＞0.05)。
本试验随后研究了冷冻方法、解冻时水浴温度以及解冻方法等对马头山羊胚胎冷冻后发育效果的影响，旨在探索出适合马头山羊胚胎的稳定可靠的冷冻保存方法，提高其冷冻保存效率。试验结果表明：(1)采用细管玻璃化冷冻和OPS玻璃化冷冻对胚胎进行冷冻，比较两种方法的效果，发现解冻后胚胎形态正常率(84.09％vs81.08％)、囊胚发育率(63.51％vs60.00％)以细管玻璃化冷冻效果略好，但在方法间统计差异不显著(P＞0.05)；(2)胚胎经细管玻璃化冷冻后，分别采用25℃和37℃水浴进行解冻，发现37℃水浴解冻后的胚胎发育效果略好于25℃水浴解冻，但解冻后胚胎的形态正常率(83.78％vs81.48％)和囊胚发育率(64.52％vs59.09％)在方法间统计差异均不显著(P＞0.05)；(3)胚胎经细管玻璃化冷冻后，分别采用二步法和一步法进行解冻，解冻后胚胎的形态正常率(87.50％vs83.78％)和囊胚发育率(66.67％vs64.52％)在方法间统计差异均不显著(P＞0.05)。
本试验最后对受体的同期发情以及马头山羊冻胚移植进行了研究，旨在进一步检验其胚胎冷冻的效果及建立胚胎冷冻保种库进行马头山羊保种的可行性。试验结果表明：(1)受体羊用CIDR进行处理时同期发情率为85.71％，受体羊用孕酮海绵栓进行处理时同期发情率为78.57％，两者同期发情率在方法间统计差异不显著(P＞0.05)；(2)马头山羊超排获得的胚胎经细管玻璃化冷冻-解冻后进行不同类型受体羊移植，经产受体羊与育成受体羊的移植妊娠率分别为46.67％和37.50％，经产受体羊移植妊娠率比育成受体羊高，但统计差异不显著(P＞0.05)。
The purpose of this study was the conservation of Matou goat by use of thesuperovulation、embryo cryopreservation and frozen embryo transfer techniques. Thesuperovulation, embryo cryopreservation, frozen embryo transfer were studied in thisexperiment.
To improve the reproductive performance of the excellent female Matou goat, thefactors affecting superovulation, such as treatment methods, mating methods, litter sizes,body condition, repeated superovulation and gene polymorphism were firstly studied inthis experiment. The results indicated that: (1)The number of recovered embryos per goat(15.63vs12.75), the number of useful embryos per goat (14.75vs11.25) and the useful rateof embryos (94.40％vs88.24％) were significantly improved when superovulation by useof LHRH-A_3 and P_4, compared with the basic superovulation method of CIDR+FSH+PG;(2)The number of recovered embryos per goat, the number of useful embryos per goatand the useful rate of embryos were significantly higher than that of mating every 8 hours(male:female=1:2) or mating every 12 hours (male:female=1:1), when mating every 8hours (male:female=1:1) or artificial insemination every 8 hours (P＜0.05); (3)The numberof recovered embryos per goat (16.83vs14.17) and the number of useful embryos pergoat (15.83vs13.33) of the donors with four feta was significantly higher than the donorswith less than two feta at previous lambing (P＜0.05); (4)The number of recoveredembryos per goat, the number of useful embryos per goat and the useful rate of embryosof the donors with medium body condition were significantly higher than the donors withsuperior or inferior body condition (P＜0.05); (5)The number of recovered embryos pergoat (15.17vs15.67), the number of useful embryos per goat (14.17vs14.83) and theuseful rate of embryos (93.41％vs94.68％) had no significant difference between therepeated superovulation and the first time superovulation (P＞0.05); (6)The differentgenotype had the same effect on the number of recovered embryos per goat and thenumber of useful embryos per goat for Matou goat, it indicated that GG＞AG＞AA, butthere was no significant difference (P＞0.05).
To explore the stable and reliable method of embryo cryopreservation and improvethe cryopreservation effect, the factors affecting the development effect of the frozenembryos, such as freezing methods, thawing temperature and thawing methods weresecondly studied in this experiment. The results indicated that: (1)The normoplasia embryos rate (84.09％vs81.08％) and the developed blastocyst rate (63.51％vs60.00％)had no significant difference between the straw vitrification freezing and OPSvitrification freezing(P＞0.05), while, straw vitrification freezing was better than OPSvitrification freezing; (2)The normoplasia embryos rate (83.78％vs81.48％) and thedeveloped blastocyst rate (64.52％vs59.09％) had no significant difference on thawing in25℃and 37℃water bath, for embryos freezed by straw vitrification (P＞0.05), while,37℃was better than 25℃; (3)The normoplasia embryos rate (87.50％vs83.78％) and thedeveloped blastocyst rate (66.67％vs64.52％) had no significant difference on thawing byuse of one-step and two-step, for embryos freezed by straw vitrification (P＞0.05).
To further check the effect of embryo cryopreservation and the feasibility of theconservation of Matou goat by establishing frozen embryos bank, the synchronization ofestrus and frozen embryos transfer of Matou goat was finally studied in this experiment.The results indicated that: (1)The rate of synchronization of estrus by use of CIDR was85.71％, by use of progesterone sponge plug was 78.57%, the rate of synchronization ofestrus had no significant difference between them, when chosen healthy, with normaloestrous cycle, local Banjiao goat as recipients (P＞0.05); (2)The pregnancy rate ofmultiparity recipients was 46.67％, the pregnancy rate of virginal recipients was 37.50％,the pregnancy rate had no significant difference between them, when transferred Matougoat superovulation embryos after straw vitrification frozen-thawing, but the pregnancyrate of multiparity recipients was higher than the virginal recipients (P＞0.05).
本试验首先研究了处理方法、配种方法、上胎产羔数、膘情、重复超排、基因多态性等因素对马头山羊超数排卵效果的影响，旨在充分发掘优秀马头山羊母畜的繁殖潜力。试验结果表明：(1)在CIDR+FSH+PG超排方法的基础上，同时运用LHRH-A_3与P_4进行超排可以显著提高只均获胚数(15.63vs12.75)、只均可用胚数(14.75vs11.25)和可用胚率(94.40％vs88.24％)(P＜0.05)，得到较好的超排效果；(2)超排供体羊配种时，每隔8h本交一次(公母比例1：1)或每隔8h人工授精一次均可得到较好的超排效果，其只均获胚数、只均可用胚胎数、可用胚率均显著高于每隔8h本交一次(公母比例1：2)或每隔12h本交一次(公母比例1：1)(P＜0.05)；(3)上胎产羔4只的供体羊只均获胚数(16.83vs14.17)和只均可用胚数(15.83vs13.33)显著高于上胎产羔2只以下的供体羊(P＜0.05)；(4)中等膘情的供体羊超排效果较好，其只均获胚数、只均可用胚数和可用胚率均显著高于上等膘情和下等膘情的供体羊(P＜0.05)；(5)对供体羊进行重复超排时，其只均获胚数(15.17vs15.67)、只均可用胚胎数(14.17vs14.83)和可用胚率(93.41％vs94.68％)等指标均略低于首次超排，但统计差异不显著(P＞0.05)；(6)INHA基因不同基因型对供体羊只均获胚数和只均可用胚数的影响一致，均表现为GG＞AG＞AA，但是统计差异不显著(P＞0.05)。
本试验随后研究了冷冻方法、解冻时水浴温度以及解冻方法等对马头山羊胚胎冷冻后发育效果的影响，旨在探索出适合马头山羊胚胎的稳定可靠的冷冻保存方法，提高其冷冻保存效率。试验结果表明：(1)采用细管玻璃化冷冻和OPS玻璃化冷冻对胚胎进行冷冻，比较两种方法的效果，发现解冻后胚胎形态正常率(84.09％vs81.08％)、囊胚发育率(63.51％vs60.00％)以细管玻璃化冷冻效果略好，但在方法间统计差异不显著(P＞0.05)；(2)胚胎经细管玻璃化冷冻后，分别采用25℃和37℃水浴进行解冻，发现37℃水浴解冻后的胚胎发育效果略好于25℃水浴解冻，但解冻后胚胎的形态正常率(83.78％vs81.48％)和囊胚发育率(64.52％vs59.09％)在方法间统计差异均不显著(P＞0.05)；(3)胚胎经细管玻璃化冷冻后，分别采用二步法和一步法进行解冻，解冻后胚胎的形态正常率(87.50％vs83.78％)和囊胚发育率(66.67％vs64.52％)在方法间统计差异均不显著(P＞0.05)。
本试验最后对受体的同期发情以及马头山羊冻胚移植进行了研究，旨在进一步检验其胚胎冷冻的效果及建立胚胎冷冻保种库进行马头山羊保种的可行性。试验结果表明：(1)受体羊用CIDR进行处理时同期发情率为85.71％，受体羊用孕酮海绵栓进行处理时同期发情率为78.57％，两者同期发情率在方法间统计差异不显著(P＞0.05)；(2)马头山羊超排获得的胚胎经细管玻璃化冷冻-解冻后进行不同类型受体羊移植，经产受体羊与育成受体羊的移植妊娠率分别为46.67％和37.50％，经产受体羊移植妊娠率比育成受体羊高，但统计差异不显著(P＞0.05)。
The purpose of this study was the conservation of Matou goat by use of thesuperovulation、embryo cryopreservation and frozen embryo transfer techniques. Thesuperovulation, embryo cryopreservation, frozen embryo transfer were studied in thisexperiment.
To improve the reproductive performance of the excellent female Matou goat, thefactors affecting superovulation, such as treatment methods, mating methods, litter sizes,body condition, repeated superovulation and gene polymorphism were firstly studied inthis experiment. The results indicated that: (1)The number of recovered embryos per goat(15.63vs12.75), the number of useful embryos per goat (14.75vs11.25) and the useful rateof embryos (94.40％vs88.24％) were significantly improved when superovulation by useof LHRH-A_3 and P_4, compared with the basic superovulation method of CIDR+FSH+PG;(2)The number of recovered embryos per goat, the number of useful embryos per goatand the useful rate of embryos were significantly higher than that of mating every 8 hours(male:female=1:2) or mating every 12 hours (male:female=1:1), when mating every 8hours (male:female=1:1) or artificial insemination every 8 hours (P＜0.05); (3)The numberof recovered embryos per goat (16.83vs14.17) and the number of useful embryos pergoat (15.83vs13.33) of the donors with four feta was significantly higher than the donorswith less than two feta at previous lambing (P＜0.05); (4)The number of recoveredembryos per goat, the number of useful embryos per goat and the useful rate of embryosof the donors with medium body condition were significantly higher than the donors withsuperior or inferior body condition (P＜0.05); (5)The number of recovered embryos pergoat (15.17vs15.67), the number of useful embryos per goat (14.17vs14.83) and theuseful rate of embryos (93.41％vs94.68％) had no significant difference between therepeated superovulation and the first time superovulation (P＞0.05); (6)The differentgenotype had the same effect on the number of recovered embryos per goat and thenumber of useful embryos per goat for Matou goat, it indicated that GG＞AG＞AA, butthere was no significant difference (P＞0.05).
To explore the stable and reliable method of embryo cryopreservation and improvethe cryopreservation effect, the factors affecting the development effect of the frozenembryos, such as freezing methods, thawing temperature and thawing methods weresecondly studied in this experiment. The results indicated that: (1)The normoplasia embryos rate (84.09％vs81.08％) and the developed blastocyst rate (63.51％vs60.00％)had no significant difference between the straw vitrification freezing and OPSvitrification freezing(P＞0.05), while, straw vitrification freezing was better than OPSvitrification freezing; (2)The normoplasia embryos rate (83.78％vs81.48％) and thedeveloped blastocyst rate (64.52％vs59.09％) had no significant difference on thawing in25℃and 37℃water bath, for embryos freezed by straw vitrification (P＞0.05), while,37℃was better than 25℃; (3)The normoplasia embryos rate (87.50％vs83.78％) and thedeveloped blastocyst rate (66.67％vs64.52％) had no significant difference on thawing byuse of one-step and two-step, for embryos freezed by straw vitrification (P＞0.05).
To further check the effect of embryo cryopreservation and the feasibility of theconservation of Matou goat by establishing frozen embryos bank, the synchronization ofestrus and frozen embryos transfer of Matou goat was finally studied in this experiment.The results indicated that: (1)The rate of synchronization of estrus by use of CIDR was85.71％, by use of progesterone sponge plug was 78.57%, the rate of synchronization ofestrus had no significant difference between them, when chosen healthy, with normaloestrous cycle, local Banjiao goat as recipients (P＞0.05); (2)The pregnancy rate ofmultiparity recipients was 46.67％, the pregnancy rate of virginal recipients was 37.50％,the pregnancy rate had no significant difference between them, when transferred Matougoat superovulation embryos after straw vitrification frozen-thawing, but the pregnancyrate of multiparity recipients was higher than the virginal recipients (P＞0.05).
引文
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