摘要
犬属单次发情动物,其间情期和乏情期较长,生殖活动和繁殖率也具有一定的不稳定性,由于犬的特殊生殖生理特性,制约了养犬业的发展及犬繁殖控制技术的发展。2005年底公布的犬全基因组图谱显示,犬与人的基因相似度比人与鼠高,犬的疾病研究发现,许多疾病与人类疾病相似,如心脏病、癌症和免疫性神经系统疾病等。因此犬作为研究人类遗传疾病的动物模型具有很大的潜力,目前是国内外学者研究的热点。
母犬的发情周期受体内生殖激素的调控,因此研究母犬体内生殖激素的相互作用情况及内分泌规律,可为提高良种犬的繁殖率及犬在生物医学领域中的应用提供理论依据。本研究选用外源激素诱导犬发情,观察犬的发情率及配种后的产仔率;并采集发情期内犬末梢血液,用放射免疫法和化学发光免疫法测定末梢血中的生殖激素雌二醇(E_2)、孕酮(P)和促黄体生成激素(LH)的浓度。同时,收集发情母犬的粪便,通过烘烤、混合及提取等步骤,用放射免疫法测定P变化规律。本研究通过对不同年龄、不同品种犬的诱导发情及发情周期内生殖激素的变化规律和不同检测方法的研究,旨在探讨有效诱导犬发情的外源激素及简单,快速的激素检测方法。
1.比格犬发情期中生殖激素变化规律研究
采集10只自然发情的成年雌性比格犬末梢血,用放射免疫法测定E_2、LH、P的分泌规律。研究发现LH在发情前期开始后8～10d达到最高浓度7.42±0.49mIU/mL;E_2在发情前期浓度为32.0±3.98pg/mL,之后逐渐升高,在发情前期开始后的7～9d达到最高值80.65±5.73pg/mL,之后迅速下降,在2～3d内降至12.12±2.86pg/mL并维持在这一浓度, 80%的母犬E_2峰值出现在LH峰值前24 h;P在发情前期浓度为3.04±1.08ng/mL,在发情前期开始后的8～12d初次升高到14.16±2.52ng/mL,并将这一浓度维持在3～5d,之后继续升高,在LH峰值之后的第17d达到最高值37.06±1.76ng/mL,其中80%的母犬在LH峰值之前P浓度开始初次升高,同时E_2浓度开始下降。所有试验犬均在E_2峰值后及P浓度逐渐升高时开始接受交配。研究表明,犬体内E_2对LH峰值的出现具有负反馈调节作用,E_2:P浓度比的下降促使LH峰值出现,并且与母犬发情的行为特征有关。
2.犬的诱导发情研究
选用PGF+PMSG+hCG外源激素,对不同年龄和不同品种犬诱导发情,并对PGF+PMSG+hCG和PGF+FSH+hCG两种不同激素组方诱导犬发情效果进行了比较。结果表明,在不同年龄犬诱导发情实验中,PGF+PMSG+hCG可诱导青年比格犬发情,经人工配种后,青年比格犬配种成功并产仔,发情率及产仔率均为100%;虽然老龄比格犬诱导后也出现发情征兆,但接受配种后并未产仔,可能是由于老龄犬体内生殖激素分泌不足,生殖器官功能下降所致。在对不同品种犬诱导发情实验中,PGF+PMSG+hCG可有效诱导青年比格犬和青年贵宾犬发情,诱导后的雌性犬发情率及产仔率均为100%。为比较不同的外源激素对犬的诱导效果,选用PGF+PMSG+hCG和PGF+FSH+hCG两种组方对青年犬诱导发情试验。显示,PGF+PMSG+hCG激素组方诱导犬发情后的2～3d,5只母犬均出现了发情现象,人工配种后产仔,发情率及产仔率均为100%; PGF+FSH+hCG激素组方诱导的5只青年贵宾犬均未出现发情现象。
3.化学发光免疫分析法检测比格犬发情期中的生殖激素
目前,测定动物体内激素时常应用放射免疫分析法,该法对操作人员具有一定放射性危害,并且对实验室防护设备要求较高。化学发光酶免疫分析法是将化学发光分析与免疫分析相结合,相比于放射免疫分析法,其具有无放射性污染、标记物容易制备、稳定性较高、检测速度快、准确度和灵敏度高等优点。本实验采用化学发光酶免疫分析法测定成年雌性比格犬发情期中血清LH、E_2和P的浓度水平,并与RIA测定结果进行比较,结果显示两者显著相关(P<0.0001),CLEIA与RIA检测血清LH、E_2和P浓度的相关系数分别为r= 0.964,0.987,0.9997,说明可以使用CLEIA代替RIA测定犬体内各生殖激素的含量,研究犬生殖激素的内分泌规律。
4.犬粪便孕酮检测方法的建立
母犬体内孕酮变化在发情鉴定、生殖力鉴定、胚胎死亡的判断、早孕诊断等方面显示了非常重要的作用。目前对犬生殖激素研究主要是采用静脉采血的方法,该方法会引起犬应激反应,影响犬体内正常的激素分泌情况,为减少外界因素对犬造成的应激反应和静脉采血对犬造成的痛苦,本实验在母犬发情期内定时收集粪便,通过烘烤、混合及有机溶剂提取等步骤从粪便中提取P,用放射免疫法测定P的含量及变化规律,结果表明,粪便中P含量在发情前期为47.88±11.93ng/mL,在发情前期开始后初次升高到139.95±25.89ng/mL,此浓度维持在3～4d,之后继续升高达到最高值(357.68±23.14ng/mL)。与末梢血中P检测方法比较得知,母犬粪便中P的浓度与血液中P的浓度显著相关(P<0.0001),两者浓度的变化趋势基本一致,说明粪便P的含量能很好地反映血液中P的含量,因此可以采用粪便样本检测母犬体内P浓度的变化情况。
Canine, a mono-estrous species, its dioestrus and anestrus are considerably long and the productive activities and productive rate of bitches are also instability. Canine special characteristics of the reproduction and physiology above have restricted the development of kennel business and canine reproductive regμLation technology. The dog genome map published in the end of year 2005 showed that the genetic similarity between canine and human was higher than that between human and mouse. Many diseases in dog are very similarly to that in human, such as heart diseases, cancer and autoimmune nervous system diseases. So dogs as the animal models of study human genetic diseases have great potential, and now it is the hot research studied by many domestic and foreign scholars.
Since the estrous cycle of the canine is regμLated by reproductive hormones in vivo, the study of canine secretion and relationships between internal reproductive hormones can improve the canine reproductive rate and can also provide the theoretical basis of canine application in the biological and medical fields. In this study, we used exogenous hormones to induce of bitches estrus and observed the oestrous and pregnant rates after mating. We collected the feces and blood samples at regμLar time during the oestrus in beagle bitches, extracted progesterone from feces samples by roasting and mixing feces with water and using organics solvent, then used RIA and CLEIA to detect the concentrations of plasma LH, E_2, P and fecal P. We aimed to investigate the effective hormone prescriptions in use of estrus induction of bitches and to find the simple and fast methods of hormone detection.
1. Levels of plasma reproductive hormones in oestrous beagle bitches
In all ten experiment bitches during the nature estrous cycle, pre-ovμLatory LH surge was observed and the plasma LH reached the peak level (7.42±0.49mIU/mL) during 8-10 days after proestrus. The concentration of estradiol in proestrus was 32.0±3.98pg/mL, then it increased gradually and got to the peak level (80.65±5.73pg/mL) during 7-9 days after the starting of proestrus, after that the concentration of estradiol dropped sharply to 12.12±2.86pg/mL in 2-3 days and maintained at this concentration. The highest concentration of plasma estradiol occurred at 24h before the LH peak in 80% experiment beagle bitches. The progesterone in proestrus was 3.04±1.08ng/mL, then it initially increased to 14.16±2.52ng/mL during 8-12 days after the starting of proestrus and kept in this concentration for 3-5 days, after that the progesterone continued to rise and reached the peak level 37.06±1.76ng/mL at day 17 after the LH peak. The initial rise of progesterone occurred before LH peak, meanwhile the concentration of estradiol began to drop in 80% experiment beagle bitches. All experiment bitches accept the mating after the peak level of estradiol and the initial rise of progesterone. It can be concluded that estradiol had a negative feedback on LH secretion, and the peak level of LH and the estrous behaviors of bitch were involved in the decrease of E_2:P ratio.
2. Induction of bitches estrus
The hormone prescriptions of PGF+PMSG+hCG have been selected in the experiment to the use of estrus induction of bitches of different ages and different species. The resμLts confirmed that the hormone prescriptions of PGF+PMSG+hCG were effective to the induction of estrus of young bitches, and the oestrous and pregnant rates were both 100%. Althoμgh old beagle bitches appeared the estrous phenomenon after the injection of hormone prescriptions of PGF+PMSG+hCG, finally they failed to whelp after mating. The reasons may be that the old dogs have low homone levels and the dysfunction of reproductive organs. The hormone prescriptions of PGF+PMSG+hCG were also effective to the induction of estrus of young beagle bitches and poodle bitches, and the oestrous and pregnant rates were also both 100%. The two hormone prescriptions of PGF+PMSG+hCG and PGF+FSH+hCG have been chosen in this experiment to induce bitches to begin estrus. The resμLts show that they all began to proestrus during 2-3 days after injection of the PGF+PMSG+hCG hormone prescriptions and the oestrous and pregnant rates were both 100%. Moreover the hormone prescriptions of PGF+FSH+hCG were ineffective to the induction of estrus.
3. Detection of plasma reproductive hormones in oestrous beagle bitches used CLEIA
The radioimmunoassay (RIA) as the common use of detecting animal reproductive hormones has several drawbacks, such as the radiological hazards to operators, radioactive contamination of the environment and the high demand of laboratory protective equipment. The chemiluminescence enzyme immunoassay (CLEIA), combination of chemiluminescence and immunoassay, has many advantages compared with radioimmunoassay. It has no radioactive contamination, easy preparation of makers, high stability, rapid detection and high accuracy and sensitivity. This experiment used CLEIA to detect the plasma concentrations of LH, estradiol and progesterone in beagle bitches during the estrus. There was a significant positive correlation (P<0.0001) between the resμLts of CLEIA and RIA, and the correlation coefficients of the plasma concentrations of LH, estradiol and progesterone used CLEIA and RIA were 0.964,0.987,0.9997 respectively. The resμLts show that we can use CLEIA instead of RIA to detect the canine reproductive hormones in vivo and to study the secretion of reproductive hormones in the future.
4. The method of detecting canine fecal progesterone
The detection of canine progesterone is very importance in the identification of canine estrous cycle, fertility, death of embryo and early pregnancy. Measuring blood levels of sex steroid hormones is the common means of investigating the canine estrous cycle, but taking daily blood samples is not convenience and the stress caused by taking daily blood invasively can affect normal hormone levels in the blood. In order to reduce the negative impact described above, we collected the feces samples at regμLar time in this experiment during the estrus of beagle bitches, extracted progesterone from feces samples followed by roasting feces, mixing feces with water and using organics solvent to extract, then detected the level of fecal P used RIA. The resμLts showed that the concentration of fecal progesterone remained at mean 47.88±11.93ng/mL level during the proestrus, then began to initial arise after the beginning of proestrus and remained this level (139.95±25.89ng/mL) for 3-4 days, afterwards reached the peak concentration (357.68±23.14ng/mL). There was a significant positive correlation (P<0.0001) between plasma progesterone concentration and fecal progesterone concentration and the fluctuation of fecal progesterone was consistent to the plasma progesterone. The conclusion confirms that the progesterone levels in feces well reflect the progesterone levels in blood, so we can use fecal samples to investigate the changes of canine progesterone levels instead of blood samples in the further.
引文
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