奶牛不孕症临床调查与益母草—丹参对CdCl_2诱导的大鼠不孕症治疗研究
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摘要
奶牛繁殖性能是影响产奶量、增加淘汰率和配种成本、降低犊牛销售利润的主要因素,产后正常受孕可实现牧场收益最大化。近几十年来,奶牛育种选育过多注重产奶量,有导致奶牛繁殖性能下降的趋势。奶牛繁殖性能低降的相关诱发因素有很多,包括遗传因素、环境因素、管理因素及其相互作用等,临床上很难确定影响奶牛繁殖性能下降的具体因素。本论文拟以奶牛场不孕母牛为研究对象,利用免疫组织化学和生物学方法,分析卵巢基质金属蛋白酶(MMP)-1,2、血清一氧化氮(NO)和生殖相关激素水平间的关系；利用氯化镉(CdCl2)中毒诱导建立雌性大鼠不孕症模型,探讨中药益母草和丹参提取物对大鼠不孕相关活性物质的影响,旨在为临床诊断和治疗奶牛不孕症提供科学依据。
1.繁殖性能低下奶牛基质金属蛋白酶、抑制素、一氧化氮和生殖激素水平间的关系研究
从奶牛场选取了40头母牛,其中21头为不孕母牛,19头为健康母牛,纳入母牛处于同一发情周期。尾静脉采血,利用ELISA方法对血清中的LH、FSH、E2、睾酮、抑制素(A和B)和NO进行测定,用免疫组化法和荧光定量PCR分别检测了卵巢组织中MMP-1和MMP-2的分布及其表达水平。结果表明,与健康母牛比较不孕母牛血清中LH(17.60±0.65vs.12.25±0.32；P<0.05)、E2(82.53±6.49vs.49.70±8.08；P<0.05)、NO(15.53±1.42vs.9.76±1.10；P<0.05)、抑制素A(12.25±0.32vs.17.60±0.65；P<0.05)和抑制素B(49.70±8.08vs.82.53±6.49；P<0.05)呈显著下降趋势,而FSH则呈升高趋势(P<0.05),睾酮水平各组间无显著差异(P>0.05)；PCR结果表明,不孕奶牛卵巢组织中MMP-1和MMP-2的表达水平显著降低于健康奶牛(P<0.05)；免疫组化分析结果表明,MMP-1和MMP-2分布在卵巢组织的不同部位或细胞中,主要包括排卵前卵泡的粒层和卵泡膜细胞、小毛囊、基质细胞和血管内皮细胞的上皮滤泡细胞。上述结果提示,不孕症奶牛的发生可能与血清中LH、E2、NO、抑制素A和抑制素B下降,FSH水平升高,以及卵巢组织中MMP-1和MMP-2表达水平下降有关。
2.益母草和丹参对CdCl2诱导的不孕大鼠的治疗作用研究
以CdCl2中毒诱导建立Wister雌性大鼠不孕模型,探讨了益母草提取液(HL)、丹参提取液(RSM)以及益母草+丹参(HL+RSM)混合提取液(每1mL药液中含原生药0.5g)对大鼠不孕相关因子的影响作用。取72只雌性Wister大鼠,随机分为6组,正常对照组、丹参组、益母草组、益母草+丹参组、阳性对照(+)组以及空白对照(-)组,每组12只,除正常对照组外,其余各组在2个情期内,进行4次腹腔注射CdCl2。与注射CdCl2前正常状态下的大鼠比较,注射CdCl2后,受试大鼠的LH (173.93±1.77vs.236.97±2.48; P<0.05). E2(0.87±0.0209vs.1.46±0.29; P<0.05)、INH-A (82.33±2.02vs.125.71±0.88; P<0.05)、INH-B (29.43±2.07vs.50.15±2.08; P<0.05)及卵巢组织中MMP-1(0.28±0.04vs.0.89±0.037; P<0.05)、MMP-2(0.29±0.03vs.1.07±0.081;P<0.05)和Smad3(0.27±0.045vs.0.85±0.046; P<0.05)基因表达水平显著降低,表明不孕症大鼠模型建立成功；药物处理后第4天,与正常对照组相比,阳性对照(+)组、治疗组(LH、RSM、LH+RSM)及空白(-)组中大鼠血清LH、E2、INH-A和INH-B水平有显著增高,其中LH+RSM组中大鼠血清激素水平最高,但均显著低于正常对照组中大鼠血清激素水平(P<0.05)；治疗组大鼠的血清FSH水平均有降低现象,其中LH+RSM组的大鼠血清的中FSH水平下降幅度最大,但仍显著高于正常对照组(P<0.05)；治疗组大鼠卵巢组织中MMP-1,2和Smad3基因有上调趋势,其中LH+RSM组上升幅度最大高,仍代于正常对照组(P<0.05)。药物处理后第8天,阳性对照(+)组、治疗组(LH、RSM)及空白组(-)中大鼠血清LH、E2、INH-A、INH-B及卵巢组织中MMP-1,2和Smad3基因表达水平均有显著增加趋势,但仍显著低于正常对照组(P<0.05)；LH+RSM治疗组大鼠血清激素水平及卵巢组织中MMP-1,2和Smad3表达水平增幅最大,与正常对照组相比呈不显著性差异(P>0.05)；LH+RSM组大鼠血清FSH接近正常组大鼠血清FSH水平,两组间呈差异不显著(P>0.05)。结果表明,LH. RSM和LH+RSM治疗可不同程度地减轻CdCl2对雌性大鼠生殖功能的损伤,其中LH+RSM效果优于二者单独使用,对不孕症大鼠血清中LH, E2、INH-A、INH-B及卵巢组织中MMP-1,2和Smad3基因表达水平有较好调节作用。
综上所述,不孕症奶牛血清中LH、E2、抑制素A、抑制素B和总NO水平会明显下降,FSH水平升高,卵巢组织中MMP-1和MMP-2表达水平下降,表明奶牛繁殖性能低下与卵巢及卵泡发育异常有关,且与机体E2、INH-A、INH-B及血清中总NO水平呈现正相关性；CdCl2中毒可诱导雌性大鼠生殖功能紊乱,是建立不孕症动物模型的有效方法；中药RSM和LH+RSM对CdCl2中毒引进的雌性大鼠生殖功能损伤有治疗作用,LH+RSM组成的复方制剂效果优于两种中药单独使用,治疗后血清中LH、E2、INH-A、INH-B及卵巢组织中MMP-1,2和Smad3基因表达水平均趋向正常水平,LH+RSM合剂有望开发为防治生殖功能低下的中药组合物,其对奶牛临床不孕症的治疗作用有待进一步研究。
Reproductive performance is one of the most important factor affecting dairy farm profitability, because it directly or indirectly influences the yield of milk, reproductive culling rate and the cost for breeding and calf sales. Dairy reproductive function had declined obviously in the past twenty years. Cows that have been highly selected for milk production in recent decades have suffered a decline in cow fertility, fertility is a multi-factorial trait and its deterioration has been caused by a network of genetic, environmental and managerial factors and their complex interactions make it difficult to determine the exact reason for this decline. For this reasons, we carried out two studies were conducted during2012,2013and2014.
1. Immunohistochemical and biological investigations ovarian Matrix Metalloproteinase (MMP)1,2and nitric oxide (NO) and hormones profile in infertile dairy cows
The objective is to investigate several hormones correlated to the reproduction, inhibin A, inhibin B and NO levels and demonstrate the ovarian MMP1,2to illustrate the physiological causes of dairy cows infertility.40Holstein cows included21infertile cows and19normal cows during their estrous phase to limit as possible as the hormones fluctuation. Blood samples used for measurement of LH, FSH, E2, inhibins (A and B), testosterone and NO. The ovarian tissues used for demostrate of MMP-1and MMP-2with Real-Time PCR and Immunohistochemistry. LH, E2and NO results observed significant decreased in the infertile cows P<0.05compared to normal cows, in spite of decreasing levels in testosterone of the infertile cows but not significantly (P>0.05). Inhibins A and B showed significant decrease levels (P<0.05) in the infertile cows compared to their levels obtained from normal cows. FSH illustrated significantly higher levels in the infertile cows than normal cows (P<0.05). Real time-PCR results of MMP-1and MMP-2expressions in the ovarian tissue were lower in infertile cows compared with normal cows (P<0.05), the immunohistochemical localizations (in situ) showed decrease expression of MMP-1and MMP-2of ovaries of infertile cows in different parts of ovarian tissues and granulosa and theca cells of preovulatory follicle, epithelial follicular cells of primary and secondary follicles, stromal ovarian cells and endothelial cells of ovarian blood vessels.
2. Herbaleonuri (HL) and Radix salviae miltiorrhizae (RSM) for treatment the induced infertility by cadmium chloride (CdC12) of female Wister rats
The objectives of the present work to evaluate the treatment of induced infertility of female Wister rats by CdC12using two kinds of Chinese herbs (herba leonuri and Radix salviae meltiorrhizae) through recover the ovarian functions and reproduction disturbance and repair the expression of MMP-1,2, and Smad3genes in ovaries.72females of Wistar rats were participated in this work, compromised6groups, Normal group (n=12) and the other60females rats were undergo of infertility induced by intraperitoneal (IP) injection of CdCl2for4times within2estrous cycles, these rats were randomly divided into5groups (n=12of each) according to kinds of treatment:RSM group; HL group; Mix herbs (1:1), Vit. E group as control (+) group; and infertile group (control-ve). The results of LH, E2, INH-A and INH-B (ng/L) at the normal state before CdC12injection were236.97±2.48,1.46±0.29,125.71±0.88and50.15±2.08 respectively, while after CdC12injections showed sharply decrease (P<0.05) recorded173.93±1.77,0.87±0.0209,82.33±2.02and29.43±2.07respectively. FSH recorded216.36±2.93and304.23±6.6for normal and infertile rats respectively, statistically this increase was significant (P<0.05). The results after4days of treatment with different kinds of treatments include (Vit. E, HL, RSM, mixed herbs, and control±ve) showed increased in the levels of LH, E2, INH-A, and INH-B, the animals group of treated with mixed herbs (HL and RSM) showed the higher levels compared to others but all of groups showed significant decrease to the normal state P<0.05. FSH after4days of treatment showed decrease in levels in all groups recorded a higher decreasing in mixed herbs groups but also statistically was significant comparing to normal values P<0.05. The genes expression of MMP-1,2and Smad3in the ovarian tissue decreased significantly P<0.05in infertile rats after CdCl2injections. After4days of treatments showed slightly increase in the expression of these genes with different kinds of treatments, the animals treated with mixed herbs showed higher expression than others but all groups were significantly P<0.05compared to the normal state.
Treatment after8days of showed more increase in the levels of LH, E2, INH-A and INH-B and expression of MMP-1,2and Smad3genes in ovarian tissues in groups treated with Vit. E, HL, RSM and control (-ve) but this increase statistically still showing significant different P<0.05compared to normal state, except the animals treated with mixed herbs (HL and RSM) showed higher increased levels of the hormones and genes expression than other groups also showed no significant difference to normal state P>0.05. The FSH levels decrease in all groups but only the mixed herbs group showed no significant difference P>0.05compared with normal values, but other groups revealed a significant difference P<0.05compared with normal values.
Conclusions:the study of the first part confirmed that the infertile dairy cows have a poor ovarian activity and poorly follicular development, affecting the numbers of developing follicles and abnormal follicular development due to the adverse effect of hormones, inhibins and NO. A decrease of ovarian MMP-1and MMP-2expressions and the total nitric oxide of infertile cows indicated the improper development of follicles and the reproduction.The second experiment approved using a combination of Herba leonuri and Radix salviae miltiorrhizae provided optimum treatment of female infertility through repairing the ovarian function and adjustment the levels of hormones to the normal values. Mixed herbs (HL and RSM) repair and maintain the endocrine mechanism related to the reproduction and the ovarian functions by increase the quality and numbers of developing follicles due to augmentation of the molecules referring to increasing the numbers and maturation of the follicles which are (Inhibins, hormones, MMP1,2and the Newly aspect approves through positive effects on expression of Smad3gene in the ovarian tissue.
1.繁殖性能低下奶牛基质金属蛋白酶、抑制素、一氧化氮和生殖激素水平间的关系研究
从奶牛场选取了40头母牛,其中21头为不孕母牛,19头为健康母牛,纳入母牛处于同一发情周期。尾静脉采血,利用ELISA方法对血清中的LH、FSH、E2、睾酮、抑制素(A和B)和NO进行测定,用免疫组化法和荧光定量PCR分别检测了卵巢组织中MMP-1和MMP-2的分布及其表达水平。结果表明,与健康母牛比较不孕母牛血清中LH(17.60±0.65vs.12.25±0.32；P<0.05)、E2(82.53±6.49vs.49.70±8.08；P<0.05)、NO(15.53±1.42vs.9.76±1.10；P<0.05)、抑制素A(12.25±0.32vs.17.60±0.65；P<0.05)和抑制素B(49.70±8.08vs.82.53±6.49；P<0.05)呈显著下降趋势,而FSH则呈升高趋势(P<0.05),睾酮水平各组间无显著差异(P>0.05)；PCR结果表明,不孕奶牛卵巢组织中MMP-1和MMP-2的表达水平显著降低于健康奶牛(P<0.05)；免疫组化分析结果表明,MMP-1和MMP-2分布在卵巢组织的不同部位或细胞中,主要包括排卵前卵泡的粒层和卵泡膜细胞、小毛囊、基质细胞和血管内皮细胞的上皮滤泡细胞。上述结果提示,不孕症奶牛的发生可能与血清中LH、E2、NO、抑制素A和抑制素B下降,FSH水平升高,以及卵巢组织中MMP-1和MMP-2表达水平下降有关。
2.益母草和丹参对CdCl2诱导的不孕大鼠的治疗作用研究
以CdCl2中毒诱导建立Wister雌性大鼠不孕模型,探讨了益母草提取液(HL)、丹参提取液(RSM)以及益母草+丹参(HL+RSM)混合提取液(每1mL药液中含原生药0.5g)对大鼠不孕相关因子的影响作用。取72只雌性Wister大鼠,随机分为6组,正常对照组、丹参组、益母草组、益母草+丹参组、阳性对照(+)组以及空白对照(-)组,每组12只,除正常对照组外,其余各组在2个情期内,进行4次腹腔注射CdCl2。与注射CdCl2前正常状态下的大鼠比较,注射CdCl2后,受试大鼠的LH (173.93±1.77vs.236.97±2.48; P<0.05). E2(0.87±0.0209vs.1.46±0.29; P<0.05)、INH-A (82.33±2.02vs.125.71±0.88; P<0.05)、INH-B (29.43±2.07vs.50.15±2.08; P<0.05)及卵巢组织中MMP-1(0.28±0.04vs.0.89±0.037; P<0.05)、MMP-2(0.29±0.03vs.1.07±0.081;P<0.05)和Smad3(0.27±0.045vs.0.85±0.046; P<0.05)基因表达水平显著降低,表明不孕症大鼠模型建立成功；药物处理后第4天,与正常对照组相比,阳性对照(+)组、治疗组(LH、RSM、LH+RSM)及空白(-)组中大鼠血清LH、E2、INH-A和INH-B水平有显著增高,其中LH+RSM组中大鼠血清激素水平最高,但均显著低于正常对照组中大鼠血清激素水平(P<0.05)；治疗组大鼠的血清FSH水平均有降低现象,其中LH+RSM组的大鼠血清的中FSH水平下降幅度最大,但仍显著高于正常对照组(P<0.05)；治疗组大鼠卵巢组织中MMP-1,2和Smad3基因有上调趋势,其中LH+RSM组上升幅度最大高,仍代于正常对照组(P<0.05)。药物处理后第8天,阳性对照(+)组、治疗组(LH、RSM)及空白组(-)中大鼠血清LH、E2、INH-A、INH-B及卵巢组织中MMP-1,2和Smad3基因表达水平均有显著增加趋势,但仍显著低于正常对照组(P<0.05)；LH+RSM治疗组大鼠血清激素水平及卵巢组织中MMP-1,2和Smad3表达水平增幅最大,与正常对照组相比呈不显著性差异(P>0.05)；LH+RSM组大鼠血清FSH接近正常组大鼠血清FSH水平,两组间呈差异不显著(P>0.05)。结果表明,LH. RSM和LH+RSM治疗可不同程度地减轻CdCl2对雌性大鼠生殖功能的损伤,其中LH+RSM效果优于二者单独使用,对不孕症大鼠血清中LH, E2、INH-A、INH-B及卵巢组织中MMP-1,2和Smad3基因表达水平有较好调节作用。
综上所述,不孕症奶牛血清中LH、E2、抑制素A、抑制素B和总NO水平会明显下降,FSH水平升高,卵巢组织中MMP-1和MMP-2表达水平下降,表明奶牛繁殖性能低下与卵巢及卵泡发育异常有关,且与机体E2、INH-A、INH-B及血清中总NO水平呈现正相关性；CdCl2中毒可诱导雌性大鼠生殖功能紊乱,是建立不孕症动物模型的有效方法；中药RSM和LH+RSM对CdCl2中毒引进的雌性大鼠生殖功能损伤有治疗作用,LH+RSM组成的复方制剂效果优于两种中药单独使用,治疗后血清中LH、E2、INH-A、INH-B及卵巢组织中MMP-1,2和Smad3基因表达水平均趋向正常水平,LH+RSM合剂有望开发为防治生殖功能低下的中药组合物,其对奶牛临床不孕症的治疗作用有待进一步研究。
Reproductive performance is one of the most important factor affecting dairy farm profitability, because it directly or indirectly influences the yield of milk, reproductive culling rate and the cost for breeding and calf sales. Dairy reproductive function had declined obviously in the past twenty years. Cows that have been highly selected for milk production in recent decades have suffered a decline in cow fertility, fertility is a multi-factorial trait and its deterioration has been caused by a network of genetic, environmental and managerial factors and their complex interactions make it difficult to determine the exact reason for this decline. For this reasons, we carried out two studies were conducted during2012,2013and2014.
1. Immunohistochemical and biological investigations ovarian Matrix Metalloproteinase (MMP)1,2and nitric oxide (NO) and hormones profile in infertile dairy cows
The objective is to investigate several hormones correlated to the reproduction, inhibin A, inhibin B and NO levels and demonstrate the ovarian MMP1,2to illustrate the physiological causes of dairy cows infertility.40Holstein cows included21infertile cows and19normal cows during their estrous phase to limit as possible as the hormones fluctuation. Blood samples used for measurement of LH, FSH, E2, inhibins (A and B), testosterone and NO. The ovarian tissues used for demostrate of MMP-1and MMP-2with Real-Time PCR and Immunohistochemistry. LH, E2and NO results observed significant decreased in the infertile cows P<0.05compared to normal cows, in spite of decreasing levels in testosterone of the infertile cows but not significantly (P>0.05). Inhibins A and B showed significant decrease levels (P<0.05) in the infertile cows compared to their levels obtained from normal cows. FSH illustrated significantly higher levels in the infertile cows than normal cows (P<0.05). Real time-PCR results of MMP-1and MMP-2expressions in the ovarian tissue were lower in infertile cows compared with normal cows (P<0.05), the immunohistochemical localizations (in situ) showed decrease expression of MMP-1and MMP-2of ovaries of infertile cows in different parts of ovarian tissues and granulosa and theca cells of preovulatory follicle, epithelial follicular cells of primary and secondary follicles, stromal ovarian cells and endothelial cells of ovarian blood vessels.
2. Herbaleonuri (HL) and Radix salviae miltiorrhizae (RSM) for treatment the induced infertility by cadmium chloride (CdC12) of female Wister rats
The objectives of the present work to evaluate the treatment of induced infertility of female Wister rats by CdC12using two kinds of Chinese herbs (herba leonuri and Radix salviae meltiorrhizae) through recover the ovarian functions and reproduction disturbance and repair the expression of MMP-1,2, and Smad3genes in ovaries.72females of Wistar rats were participated in this work, compromised6groups, Normal group (n=12) and the other60females rats were undergo of infertility induced by intraperitoneal (IP) injection of CdCl2for4times within2estrous cycles, these rats were randomly divided into5groups (n=12of each) according to kinds of treatment:RSM group; HL group; Mix herbs (1:1), Vit. E group as control (+) group; and infertile group (control-ve). The results of LH, E2, INH-A and INH-B (ng/L) at the normal state before CdC12injection were236.97±2.48,1.46±0.29,125.71±0.88and50.15±2.08 respectively, while after CdC12injections showed sharply decrease (P<0.05) recorded173.93±1.77,0.87±0.0209,82.33±2.02and29.43±2.07respectively. FSH recorded216.36±2.93and304.23±6.6for normal and infertile rats respectively, statistically this increase was significant (P<0.05). The results after4days of treatment with different kinds of treatments include (Vit. E, HL, RSM, mixed herbs, and control±ve) showed increased in the levels of LH, E2, INH-A, and INH-B, the animals group of treated with mixed herbs (HL and RSM) showed the higher levels compared to others but all of groups showed significant decrease to the normal state P<0.05. FSH after4days of treatment showed decrease in levels in all groups recorded a higher decreasing in mixed herbs groups but also statistically was significant comparing to normal values P<0.05. The genes expression of MMP-1,2and Smad3in the ovarian tissue decreased significantly P<0.05in infertile rats after CdCl2injections. After4days of treatments showed slightly increase in the expression of these genes with different kinds of treatments, the animals treated with mixed herbs showed higher expression than others but all groups were significantly P<0.05compared to the normal state.
Treatment after8days of showed more increase in the levels of LH, E2, INH-A and INH-B and expression of MMP-1,2and Smad3genes in ovarian tissues in groups treated with Vit. E, HL, RSM and control (-ve) but this increase statistically still showing significant different P<0.05compared to normal state, except the animals treated with mixed herbs (HL and RSM) showed higher increased levels of the hormones and genes expression than other groups also showed no significant difference to normal state P>0.05. The FSH levels decrease in all groups but only the mixed herbs group showed no significant difference P>0.05compared with normal values, but other groups revealed a significant difference P<0.05compared with normal values.
Conclusions:the study of the first part confirmed that the infertile dairy cows have a poor ovarian activity and poorly follicular development, affecting the numbers of developing follicles and abnormal follicular development due to the adverse effect of hormones, inhibins and NO. A decrease of ovarian MMP-1and MMP-2expressions and the total nitric oxide of infertile cows indicated the improper development of follicles and the reproduction.The second experiment approved using a combination of Herba leonuri and Radix salviae miltiorrhizae provided optimum treatment of female infertility through repairing the ovarian function and adjustment the levels of hormones to the normal values. Mixed herbs (HL and RSM) repair and maintain the endocrine mechanism related to the reproduction and the ovarian functions by increase the quality and numbers of developing follicles due to augmentation of the molecules referring to increasing the numbers and maturation of the follicles which are (Inhibins, hormones, MMP1,2and the Newly aspect approves through positive effects on expression of Smad3gene in the ovarian tissue.
引文
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