Induction Of Bitches Estrus And Detection Of Reproductive Hormones In Bitches During Estrus

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, E₂, 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 bitchesIn 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 E₂:P ratio.2. Induction of bitches estrusThe 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 CLEIAThe 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 progesteroneThe 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.