PMSG剂量对中国荷斯坦奶牛繁殖性能的影响以及抗PMSG抗体制备和应用研究
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摘要
孕马血清促性腺激素(PMSG)是雌性马属动物胎盘的尿囊绒毛膜子宫内膜杯细胞分泌的一种促性腺糖蛋白激素,也是胚胎的一种代谢产物,兼具促卵泡素和黄体生成激素的双重活性,能够促进卵泡的发育和排卵。相对于其它激素类药物,PMSG具有生产成本低,超数排卵效果好等优势,被广泛的应用于各种动物的试验研究和实际生产中。
本试验首先探索了PMSG剂量对中国荷斯坦奶牛发情、发情间隔和受胎率等繁殖性能的影响,进而探讨了PMSG剂量对卵泡发育和雌激素分泌的影响,另外,还对应用PMSG后对奶牛后期繁殖性能的影响进行了跟踪研究,为PMSG的研究和应用推广提供理论依据；根据以上研究提供的依据,同时对PMSG剂量对中国荷斯坦育成牛的繁殖性能的影响进行了研究,并与研究中经产牛的试验数据进行对比分析,分析育成牛和经产牛对不同剂量PMSG反应敏感性的差异,为PMSG在育成牛上的应用提供参考。由于PMSG存在半衰期过长等不可控性因素,在实际应用中会产生一些负面影响,为了消除PMSG应用后的不利影响,本研究还进行了抗PMSG的生产和应用方面的探索。
1. PMSG剂量对中国荷斯坦奶牛繁殖性能的影响
(1) PMSG剂量对中国荷斯坦经产牛繁殖性能的影响
选取无任何繁殖障碍和生理疾病的中国荷斯坦经产牛100头,随机分为5组,每组20头,并将每头牛进行称重。5组奶牛分别肌肉注射2、2.5、3、3.5和4IU/kgBW (BW:体重),并在48h后肌肉注射PGF2a。每天对试验奶牛的发情进行监测,在发情后8～12h进行配种,配种后30天利用B超对奶牛进行早孕检测。结果显示,在PMSG和PGF2α的共同作用下,促进了奶牛的发情,缩短了发情间隔,各试验组的发情率和发情间隔差异不显著。奶牛的受胎率随着PMSG剂量的升高而降低,其中最高剂量组(4IU/kg BW)的受胎率显著高于两个低剂量试验组(2和2.5IU/kg BW),但是各个试验组的产犊率相似。PMSG剂量显著影响试验奶牛的多胎率,其中最高剂量试验组(4IU/kg BW)的多胎率达到50%。通过对试验中发情未孕的奶牛进行跟踪观察,发现应用高剂量PMSG试验组(3、3.5和4IU/kg BW)的第一次自然返情间隔显著长于最低剂量组(2IU/kg BW),但PMSG剂量对返情后的受胎率无显著影响。
(2) PMSG剂量对中国荷斯坦经产牛卵泡数目和雌激素分泌的影响
在研究PMSG剂量对中国荷斯坦经产牛繁殖性能影响的同时,本试验还对PMSG剂量对卵泡数目和雌激素分泌的影响进行了观察和测定。在上述试验的过程中分别在注射PMSG、PGF2α、发情和配种四个时间点采血,收集血清,并通过放射性免疫(RIA)方法测定雌激素水平,并在发情时利用B超对奶牛卵巢上卵泡的发育情况进行观察,分析统计大卵泡(>10mm)和中等卵泡(5～10mm)的数量。结果表明,PMSG剂量对大卵泡的数量无显著影响,但是中等卵泡的数量随着PMSG剂量的升高显著增加。雌激素浓度的变化也受到了PMSG剂量的影响,尤其在发情到配种的这段时间,随着PMSG剂量的升高,雌激素下降的幅度明显减小。
(3) PMSG剂量对中国荷斯坦育成牛繁殖性能的影响
上述试验结果发现高剂量PMSG试验组(3.5和4IU/kg BW)的多胎率过高,为了避免育成牛产多胎而导致流产或者早产,本试验选择了3个较低PMSG剂量(2、2.5和3IU/kg BW)对育成牛进行处理。为了与以上试验中经产牛的研究数据进行对比,本试验选择在与以上试验相同的季节,天气情况、饲养方法和药物处理等条件下进行。经研究发现,育成牛的发情率和发情间隔都未受到PMSG剂量的显著影响,并且在相同PMSG剂量下,育成牛和经产牛的发情率和发情间隔无显著差异。育成牛的受胎率随着剂量的增加而降低,而且在相同PMSG剂量下,育成牛的受胎率低于经产牛。PMSG剂量对育成牛的产犊率无显著影响,并且各试验组的产犊率与经产牛相比较无差异。随着PMSG剂量的升高,育成牛的多胎率增加,在相同剂量的情况下青年牛的多胎率高于经产牛,尤其是在低剂量组(2和2.5IU/kg BW)中,本研究中经产牛没有出现多胎情况,而育成牛的多胎率为25%。与经产牛不同的是,PMSG剂量对育成牛第一次自然返情间隔的影响较小,并且在高剂量组(2.5和3IU/kg BW)中,育成牛的返情间隔显著短于经产牛。
2.抗PMSG抗体的制备和应用研究
(1)抗PMSG抗体的制备
通过碳二亚胺法将PMSG与牛血清白蛋白(BSA)偶联,增加PMSG的免疫原性。经紫外分光光度计对偶联后的产物进行鉴定分析后发现,成功获得PMSG-BSA人工抗原,并且经过动物试验测定证明,合成的人工抗原具有PMSG的生物学活性。为了制备大量抗PMSG抗体,将合成的人工抗原免疫公牛。经过多次免疫后成功获得高效价抗PMSG抗体,并利用饱和硫酸铵法对获得的抗体进行了提取和纯化。
(2)抗PMSG抗体的应用研究
选取中国荷斯坦经产牛225头,随机分为3个试验组,每组75头,再将每个试验组的75头试验奶牛随机分为5个小组,每小组15头。3个试验组中的5个小组分别肌肉注射2、2.5、3、3.5和4IU/kg BW PMSG,48h后再肌注PGF2a。每天对试验奶牛的发情进行监测,在发情后8-12h进行配种,并在配种后30天利用B超对奶牛进行早孕检测,而且其中两个试验组分别在发情时和配种时肌肉注射相应中和剂量的抗PMSG抗体作为试验组,另外一组不做抗体注射处理作为对照组。试验结果表明,抗PMSG抗体的应用提高了高剂量PMSG试验组奶牛的受胎率,并改善了PMSG对奶牛后期繁殖性能的不利影响,但是在发情时和配种时两个时间点应用抗体,对其效用的发挥无显著影响。
Pregnant mare serum gonadotropin (PMSG) is a glycoprotein hormone, produced by trophoblast cells of endometrial cups in pregnant mares. PMSG exhibits mainly FSH like activity but also have some LH like activity and its parenteral administration stimulates follicular growth and ovulation. PMSG is widely used in small and large ruminants industry as it is cost-effective and less labor involved.
The present study was conducted to evaluate effects of different doses of PMSG on estrus induction, estrus interval, and conception rate in synchronized Chinese Holstein dairy cows and compared with heifers. Furthermore, follicular population, plasma estradiol concentrations and the interval from PMSG administration to first natural estrus were also determined. Because of prolonged half-life of PMSG we also studied the production and application of PMSG antibodies.
1. Effects of PMSG Doses on Reproductive Performance in Chinese Holstein Dairy Cows
(1) Effects of PMSG Doses on Reproductive Performance in Chinese Holstein Dairy Cows
The study was conducted on one hundred cyclic dairy cows. Cows were equally divided into five treatment groups. Each group (n=20) was administered with an injection of PMSG at dose rate of2,2.5,3,3.5, or4IU/kg BW, followed by an injection of PGF2α48h later. Estrus was detected every day. The cows were artificially inseminated8-12h after standing heat. On day30after artificial insemination, pregnancy rate was confirmed by transrectal ultrasonography while calving rate and multiple calvings were added up after successful parturition. There was no significant difference in estrus response among all treatment groups. Similarly, interval from PMSG administration to estrus was decreased non-significantly with the different doses of PMSG. Pregnancy rate was significantly lower in the highest treatment group (4IU/kg BW) than in low treatment groups (2and2.5IU/kg BW). Calving rate was non-significant among different regimens. The effects of PMSG dose on multiple calving was found to be statistically significant and administration of4IU/kg BW PMSG resulted in50%multiple births. The cows failed to get pregnant returned to estrus naturally. The interval from PMSG administration to first natural estrus was significantly higher in high treatment groups (>2.5IU/kg BW) than in the lowest treatment group (2IU/kg BW), however, later pregnancy rate was non-significant in all groups.
(2) Influence of PMSG doses on follicular population and plasma estradiol concentrations in Chinese Holstein dairy cows
We studied the influence of different doses of PMSG on follicular population and plasma estradiol concentrations in Chinese Holstein dairy cows. Ovarian scanning was performed by transrectal Beta-ultrasonography around estrus while plasma estradiol concentration was measured by Radioimmunoassay at the time of administration of PMSG, PGF2a, estrus and insemination. The results showed that medium follicles (5-10mm) population varied among all treatment groups with increasing trend however, there were no significant difference on large follicles (>10mm) population.
(3) Effects of PMSG Doses on Reproductive Performance in Chinese Holstein Dairy heifers
According to the results of the last experiment where higher multiple birth rate was observed in high dose group (3.5and4IU/kg BW), we selected three low PMSG doses (2,2.5and3IU/kg BW) in order to avoid abortion in first trimester. In order to compare the response of PMSG in heifers and cows we conducted this experiment under similar managemental conditions. The response was same as found in cows, where there was no significant difference in estrus response and interval from PMSG administration to estrus among all treatment groups. Pregnancy rate was significantly lower in the highest treatment group (3IU/kg BW) than in low treatment groups (2and2.5IU/kg BW) and the conception rate of heifers was lower than cows under the similar PMSG dose. Calving rate was non-significant among different regimens, and was non-significant in each PMSG group too. Multiple birth rate increased with increase in PMSG dose and was higher in low dose group (2and2.5IU/kg BWkg) heifers (25%) than in cows (0%) The interval from PMSG administration to first natural estrus increased non-significantly among different doses of PMSG. In high dose groups (2.5and3IU/kg BW), the interval in heifers was shorter than cows.
2Preparation and Application of Antibodies Against-PMSG
(1) Preparation of Antibodies Against-PMSG
PMSG was successful coupled with bovine serum albumin (BSA) by carbodi-imide method, and identified by ultraviolet spectrophotometry. PMSG-BSA was injected in bull for possible immunization. The results revealed high antibodies titer against-PMSG and later purified by saturated ammonium sulfate (SAS) methods.
(2) Applied Research of Antibodies Against-PMSG
For this study,225cows were divided into three treatment groups (n=75). And each treatment group was further divided into five subgroups (n=15). Each subgroup was administered with an injection of PMSG at dose rate of2,2.5,3,3.5, or4IU/kg BW respectively, followed by administration of PGF2α48h later. Cows were artificially inseminated8-12h after standing heat. On day30after insemination, pregnancy rate was confirmed by transrectal beta-ultrasonography. Cows were administered with an injection of antibodies against-PMSG at the time of estrus and inseminated respectively. The third group served as control group without antibodies against-PMSG administration. The results demonstrated that antibodies against-PMSG increased the conception rate of cows in high PMSG dose groups and improved the negative impact of PMSG on consequent reproduction of cows compared with control group.
本试验首先探索了PMSG剂量对中国荷斯坦奶牛发情、发情间隔和受胎率等繁殖性能的影响,进而探讨了PMSG剂量对卵泡发育和雌激素分泌的影响,另外,还对应用PMSG后对奶牛后期繁殖性能的影响进行了跟踪研究,为PMSG的研究和应用推广提供理论依据；根据以上研究提供的依据,同时对PMSG剂量对中国荷斯坦育成牛的繁殖性能的影响进行了研究,并与研究中经产牛的试验数据进行对比分析,分析育成牛和经产牛对不同剂量PMSG反应敏感性的差异,为PMSG在育成牛上的应用提供参考。由于PMSG存在半衰期过长等不可控性因素,在实际应用中会产生一些负面影响,为了消除PMSG应用后的不利影响,本研究还进行了抗PMSG的生产和应用方面的探索。
1. PMSG剂量对中国荷斯坦奶牛繁殖性能的影响
(1) PMSG剂量对中国荷斯坦经产牛繁殖性能的影响
选取无任何繁殖障碍和生理疾病的中国荷斯坦经产牛100头,随机分为5组,每组20头,并将每头牛进行称重。5组奶牛分别肌肉注射2、2.5、3、3.5和4IU/kgBW (BW:体重),并在48h后肌肉注射PGF2a。每天对试验奶牛的发情进行监测,在发情后8～12h进行配种,配种后30天利用B超对奶牛进行早孕检测。结果显示,在PMSG和PGF2α的共同作用下,促进了奶牛的发情,缩短了发情间隔,各试验组的发情率和发情间隔差异不显著。奶牛的受胎率随着PMSG剂量的升高而降低,其中最高剂量组(4IU/kg BW)的受胎率显著高于两个低剂量试验组(2和2.5IU/kg BW),但是各个试验组的产犊率相似。PMSG剂量显著影响试验奶牛的多胎率,其中最高剂量试验组(4IU/kg BW)的多胎率达到50%。通过对试验中发情未孕的奶牛进行跟踪观察,发现应用高剂量PMSG试验组(3、3.5和4IU/kg BW)的第一次自然返情间隔显著长于最低剂量组(2IU/kg BW),但PMSG剂量对返情后的受胎率无显著影响。
(2) PMSG剂量对中国荷斯坦经产牛卵泡数目和雌激素分泌的影响
在研究PMSG剂量对中国荷斯坦经产牛繁殖性能影响的同时,本试验还对PMSG剂量对卵泡数目和雌激素分泌的影响进行了观察和测定。在上述试验的过程中分别在注射PMSG、PGF2α、发情和配种四个时间点采血,收集血清,并通过放射性免疫(RIA)方法测定雌激素水平,并在发情时利用B超对奶牛卵巢上卵泡的发育情况进行观察,分析统计大卵泡(>10mm)和中等卵泡(5～10mm)的数量。结果表明,PMSG剂量对大卵泡的数量无显著影响,但是中等卵泡的数量随着PMSG剂量的升高显著增加。雌激素浓度的变化也受到了PMSG剂量的影响,尤其在发情到配种的这段时间,随着PMSG剂量的升高,雌激素下降的幅度明显减小。
(3) PMSG剂量对中国荷斯坦育成牛繁殖性能的影响
上述试验结果发现高剂量PMSG试验组(3.5和4IU/kg BW)的多胎率过高,为了避免育成牛产多胎而导致流产或者早产,本试验选择了3个较低PMSG剂量(2、2.5和3IU/kg BW)对育成牛进行处理。为了与以上试验中经产牛的研究数据进行对比,本试验选择在与以上试验相同的季节,天气情况、饲养方法和药物处理等条件下进行。经研究发现,育成牛的发情率和发情间隔都未受到PMSG剂量的显著影响,并且在相同PMSG剂量下,育成牛和经产牛的发情率和发情间隔无显著差异。育成牛的受胎率随着剂量的增加而降低,而且在相同PMSG剂量下,育成牛的受胎率低于经产牛。PMSG剂量对育成牛的产犊率无显著影响,并且各试验组的产犊率与经产牛相比较无差异。随着PMSG剂量的升高,育成牛的多胎率增加,在相同剂量的情况下青年牛的多胎率高于经产牛,尤其是在低剂量组(2和2.5IU/kg BW)中,本研究中经产牛没有出现多胎情况,而育成牛的多胎率为25%。与经产牛不同的是,PMSG剂量对育成牛第一次自然返情间隔的影响较小,并且在高剂量组(2.5和3IU/kg BW)中,育成牛的返情间隔显著短于经产牛。
2.抗PMSG抗体的制备和应用研究
(1)抗PMSG抗体的制备
通过碳二亚胺法将PMSG与牛血清白蛋白(BSA)偶联,增加PMSG的免疫原性。经紫外分光光度计对偶联后的产物进行鉴定分析后发现,成功获得PMSG-BSA人工抗原,并且经过动物试验测定证明,合成的人工抗原具有PMSG的生物学活性。为了制备大量抗PMSG抗体,将合成的人工抗原免疫公牛。经过多次免疫后成功获得高效价抗PMSG抗体,并利用饱和硫酸铵法对获得的抗体进行了提取和纯化。
(2)抗PMSG抗体的应用研究
选取中国荷斯坦经产牛225头,随机分为3个试验组,每组75头,再将每个试验组的75头试验奶牛随机分为5个小组,每小组15头。3个试验组中的5个小组分别肌肉注射2、2.5、3、3.5和4IU/kg BW PMSG,48h后再肌注PGF2a。每天对试验奶牛的发情进行监测,在发情后8-12h进行配种,并在配种后30天利用B超对奶牛进行早孕检测,而且其中两个试验组分别在发情时和配种时肌肉注射相应中和剂量的抗PMSG抗体作为试验组,另外一组不做抗体注射处理作为对照组。试验结果表明,抗PMSG抗体的应用提高了高剂量PMSG试验组奶牛的受胎率,并改善了PMSG对奶牛后期繁殖性能的不利影响,但是在发情时和配种时两个时间点应用抗体,对其效用的发挥无显著影响。
Pregnant mare serum gonadotropin (PMSG) is a glycoprotein hormone, produced by trophoblast cells of endometrial cups in pregnant mares. PMSG exhibits mainly FSH like activity but also have some LH like activity and its parenteral administration stimulates follicular growth and ovulation. PMSG is widely used in small and large ruminants industry as it is cost-effective and less labor involved.
The present study was conducted to evaluate effects of different doses of PMSG on estrus induction, estrus interval, and conception rate in synchronized Chinese Holstein dairy cows and compared with heifers. Furthermore, follicular population, plasma estradiol concentrations and the interval from PMSG administration to first natural estrus were also determined. Because of prolonged half-life of PMSG we also studied the production and application of PMSG antibodies.
1. Effects of PMSG Doses on Reproductive Performance in Chinese Holstein Dairy Cows
(1) Effects of PMSG Doses on Reproductive Performance in Chinese Holstein Dairy Cows
The study was conducted on one hundred cyclic dairy cows. Cows were equally divided into five treatment groups. Each group (n=20) was administered with an injection of PMSG at dose rate of2,2.5,3,3.5, or4IU/kg BW, followed by an injection of PGF2α48h later. Estrus was detected every day. The cows were artificially inseminated8-12h after standing heat. On day30after artificial insemination, pregnancy rate was confirmed by transrectal ultrasonography while calving rate and multiple calvings were added up after successful parturition. There was no significant difference in estrus response among all treatment groups. Similarly, interval from PMSG administration to estrus was decreased non-significantly with the different doses of PMSG. Pregnancy rate was significantly lower in the highest treatment group (4IU/kg BW) than in low treatment groups (2and2.5IU/kg BW). Calving rate was non-significant among different regimens. The effects of PMSG dose on multiple calving was found to be statistically significant and administration of4IU/kg BW PMSG resulted in50%multiple births. The cows failed to get pregnant returned to estrus naturally. The interval from PMSG administration to first natural estrus was significantly higher in high treatment groups (>2.5IU/kg BW) than in the lowest treatment group (2IU/kg BW), however, later pregnancy rate was non-significant in all groups.
(2) Influence of PMSG doses on follicular population and plasma estradiol concentrations in Chinese Holstein dairy cows
We studied the influence of different doses of PMSG on follicular population and plasma estradiol concentrations in Chinese Holstein dairy cows. Ovarian scanning was performed by transrectal Beta-ultrasonography around estrus while plasma estradiol concentration was measured by Radioimmunoassay at the time of administration of PMSG, PGF2a, estrus and insemination. The results showed that medium follicles (5-10mm) population varied among all treatment groups with increasing trend however, there were no significant difference on large follicles (>10mm) population.
(3) Effects of PMSG Doses on Reproductive Performance in Chinese Holstein Dairy heifers
According to the results of the last experiment where higher multiple birth rate was observed in high dose group (3.5and4IU/kg BW), we selected three low PMSG doses (2,2.5and3IU/kg BW) in order to avoid abortion in first trimester. In order to compare the response of PMSG in heifers and cows we conducted this experiment under similar managemental conditions. The response was same as found in cows, where there was no significant difference in estrus response and interval from PMSG administration to estrus among all treatment groups. Pregnancy rate was significantly lower in the highest treatment group (3IU/kg BW) than in low treatment groups (2and2.5IU/kg BW) and the conception rate of heifers was lower than cows under the similar PMSG dose. Calving rate was non-significant among different regimens, and was non-significant in each PMSG group too. Multiple birth rate increased with increase in PMSG dose and was higher in low dose group (2and2.5IU/kg BWkg) heifers (25%) than in cows (0%) The interval from PMSG administration to first natural estrus increased non-significantly among different doses of PMSG. In high dose groups (2.5and3IU/kg BW), the interval in heifers was shorter than cows.
2Preparation and Application of Antibodies Against-PMSG
(1) Preparation of Antibodies Against-PMSG
PMSG was successful coupled with bovine serum albumin (BSA) by carbodi-imide method, and identified by ultraviolet spectrophotometry. PMSG-BSA was injected in bull for possible immunization. The results revealed high antibodies titer against-PMSG and later purified by saturated ammonium sulfate (SAS) methods.
(2) Applied Research of Antibodies Against-PMSG
For this study,225cows were divided into three treatment groups (n=75). And each treatment group was further divided into five subgroups (n=15). Each subgroup was administered with an injection of PMSG at dose rate of2,2.5,3,3.5, or4IU/kg BW respectively, followed by administration of PGF2α48h later. Cows were artificially inseminated8-12h after standing heat. On day30after insemination, pregnancy rate was confirmed by transrectal beta-ultrasonography. Cows were administered with an injection of antibodies against-PMSG at the time of estrus and inseminated respectively. The third group served as control group without antibodies against-PMSG administration. The results demonstrated that antibodies against-PMSG increased the conception rate of cows in high PMSG dose groups and improved the negative impact of PMSG on consequent reproduction of cows compared with control group.
引文
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