CN111956366A - Method for inducing simultaneous estrus and superovulation of female cats - Google Patents
Method for inducing simultaneous estrus and superovulation of female cats Download PDFInfo
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- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A61D17/00—Devices for indicating trouble during labour of animals ; Methods or instruments for detecting pregnancy-related states of animals
- A61D17/002—Devices for indicating trouble during labour of animals ; Methods or instruments for detecting pregnancy-related states of animals for detecting period of heat of animals, i.e. for detecting oestrus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D19/00—Instruments or methods for reproduction or fertilisation
- A61D19/02—Instruments or methods for reproduction or fertilisation for artificial insemination
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Abstract
A method for inducing oestrus and ovulation of a female cat, which belongs to the technical field of animal artificial propagation. The invention provides a method for inducing the simultaneous estrus and superovulation of a female cat, aiming at the problem that the estrus and the ovulation are difficult to synchronize in the artificial reproduction of the female cat. The specific scheme is that the adult healthy female cats are continuously fed with the altrenogest with the dose of 0.01-20 mg/cat/day for 10-20 days, after the altrenogest is stopped for 2-3 days, the female cats are given with 10-1000IU of pregnant mare serum hormone through intramuscular injection, and after 72-96 hours, the female cats are given with 10-1000IU of human chorionic gonadotropin through intramuscular injection, so that the female cats can estrus and superovulate at the same time within a preset time.
Description
Technical Field
The invention relates to the field of animal induced estrus, superovulation, artificial insemination and in vitro fertilization, in particular to a treatment method for inducing synchronous estrus and superovulation of domestic cats, belonging to the field of Assisted Reproduction Technology (ART).
Background
The synchronous estrus technology is characterized in that the ovary of an animal is stimulated by exogenous hormone, so that a group of animals can be in concentrated estrus within a certain time (24-48 hours).
The oestrus cycle of female animals can be divided into two phases, the follicular phase and the luteal phase, in terms of the change of ovarian function and morphology. The follicular phase is the period after the deterioration of the corpus luteum periodically followed by a decrease in the level of progesterone (P4) in the blood, when the follicle rapidly matures in the ovary and eventually ovulates, and sometimes dam changes are observed in behavior. The follicular phase is followed by the luteal phase, in which the follicle is ruptured to form and continue with a luteal phase, the release of pituitary Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) is inhibited by the progestogen secreted by the corpus luteum, i.e. the follicle is inhibited from developing to maturity, the dam is not in heat, the corpus luteum is degenerated by the action of Prostacyclin (PG) (without insemination) and subsequently enters another follicular phase. The precondition for the arrival of the follicular phase is that the luteal phase is ended, and after the luteal phase is ended, the level of the progestational hormone is reduced to a low limit, so that the inhibition on the development of the follicular is released, the follicular starts to grow again, and the female animals show estrus. The estrus synchronization technology is beneficial to the centralized management of animals, can save labor force, time and cost, and can solve the problem of estrus synchronization of embryo transfer donors and acceptors, and the closer the estrus starting time of the acceptors and donors is, the higher the conception rate of transfer is. The technology of estrus synchronization becomes an indispensable technology in the work of animal husbandry artificial insemination, semen freezing, superovulation, embryo transplantation, fertilized egg deep-cold preservation and the like.
But the prior art for simultaneously estrusing the female cats has a series of problems.
First, studies have shown that felines respond inconsistently to exogenous gonadotropins, and following induction with gonadotropins, ovarian hyperstimulation may occur, leading to excessive follicular development, high levels of estrogen, premature or excessive progestin release, and sometimes premature luteinization.
II, La Polt and other researches show that the ovary of the cat is very sensitive to PMSG and leads to increase of ovarian LH receptor after administration. Other studies have shown that PMSG leads to follicular cysts, premature luteinization of the ovaxanthin, a high proportion of unovulated follicles or ovulation before estrus, etc. after use.
Thirdly, although there are a variety of oestrus induction methods for felines, the success rates of oestrus induction, ovulation, pregnancy and offspring delivery vary between different regimes and internally.
Disclosure of Invention
Aiming at the problem that excellent cats are difficult to estre synchronously, the invention provides a method for inducing the cats to estre synchronously.
The female cats treated by the method are required to be healthy and disease-free, especially can not carry diseases related to reproduction, the female cats are 6-9 months old, the female cats are over 2kg in weight and have moderate physique. The method uses three medicines: altrenogest, hCG, and PMSG. Altrenogest is a progesterone analogue with the functions of prolonging luteal phase and inhibiting growth, development and oestrus expression of follicles. After a certain period of treatment, the medicine is stopped at the same time, and because the ovary loses the control of exogenous progestogen, the cycle corpus luteum on the ovary is degenerated, and then follicle development occurs at the same time, thereby causing the heat of the female animals. The hCG injection is a hormone medicine, and can promote the anterior pituitary of animals to release follicle stimulating hormone FSH and LH after injection, further cause the obvious rise of plasma LH and the slight rise of FSH, and promote the mature ovulation of oocytes of ovaries of female animals. PMSG for injection has functions of follitropin and luteinizing hormone, and can promote follicle maturation, ovulation and corpus luteum generation of female livestock, and stimulate corpus luteum to secrete progestational hormone.
According to the method for inducing the co-estrus of the queen cats, each cat is fed with the altrenogest at a fixed point every day, after the cats are continuously fed for 10-20 days and after 2-3 days, each cat is injected with the equine chorionic gonadotropin PMSG, and after 72-96 hours, each queen is injected with the human chorionic gonadotropin hCG, and after 25-30 hours, the queen cats are estrus. The sign of estrusing is with stably receiving public cat mating as the main characteristic of estrusing, also includes some actions such as excitation, appetite show decline, constantly howl, the time of rising is crouched, raise the tail, raise buttockss. When the female cat is in heat, the vulva part can be flushed and swollen, and viscous fluid with blood can flow out of the vagina. Most importantly, the ovaries of the heat-inducing queen were ovulated and red bodies appeared as confirmed by surgical procedures.
Technical advantages of the invention
The medicine used in the experiment has wide source and convenient use, and is beneficial to large-scale popularization and use.
The liquid medicine of the altrenogest in the method is colorless and tasteless, and can be directly taken by a female cat by using a feeding gun, so that the waste of medicine administration and the loss of dosage during administration are avoided.
And 3, all the treated queens are in estrus on the same day after the hCG treatment, and the estrus induction efficiency is high.
4. According to other studies, it has been shown that pretreatment of domestic cats with exogenous progestagen prior to gonadotropin-induced estrus administration can protect the queen ovaries from producing excessive estrogen and producing a uniform physiological response.
5. The invention can improve the mating rate and conception rate of recipient female cats, shorten the mating period, reduce infertility, improve the reproduction rate and reduce the consumption of manpower and material resources, is beneficial to the development of artificial insemination technology on cats, and is convenient for large-scale tissue production and timed large-scale experiments; secondly, the induction of the estrus in the same period of the female cat can reduce the abnormal phenomenon of the female cat in the breeding process, bring great economic benefit, be beneficial to saving labor force, organizing large-scale production, really realizing full-in and full-out and being beneficial to leading the domestic cat to reach the same breeding state, health state and immune state.
6. The invention is beneficial to the accurate and timed estrus of embryo transplantation acceptors and is beneficial to improving the embryo transplantation efficiency and success rate.
The invention is beneficial to the synchronous estrus and superovulation of the domestic cat as the embryo receptor model of various small wild feline endangered animals, and the endangered felines can be rescued by researching the in-vitro embryo development of the domestic cat.
Drawings
FIG. 1 is a flow chart of the present invention.
Detailed Description
The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
1 laboratory animal
Randomly selecting 20 common domestic cats with the age of 6-9 months and the weight of more than 2kg as animals for experiments, and inducing and feeding the animals for 4-5 days by using fruit juice before the experiments are carried out so that the animals for experiments can adapt to the drug feeder.
2 test grouping and procedure
(1) Grouping: firstly, adopting the existing superovulation method for the female cats, namely injecting 100IU PMSG intramuscularly, injecting 75IU hCG intramuscularly after 80 hours, and performing an ovariectomy operation after 24 hours; ② experiment group 1, adopting a mode of adding altrenogest in the feed every day, feeding continuously for 15 days, injecting 100IU PMSG after 3 days, injecting 75IU hCG after 80 hours.
(2) The process comprises the following steps: control group (n = 6): 6 female cats were randomly selected and treated with the existing superovulation method, and were treated with intramuscular injection of 100IU PMSG,80 hours later with intramuscular injection of 75IU hCG, and 25 hours later with ovariectomy. Test group (n ═ 6): feeding altrenogest at 9 am every day, and recording day1 of feeding altrenogest as day1 venous blood for detecting estradiol and progesterone content in blood serum; the ovaries, oviducts and uteri are collected in two groups, and the red body number, ovarian cyst number, ovarian diameter, oviduct length and uterine horn length of each female cat are counted. And the collected cat oocytes are subjected to in vitro fertilization and continued culture, and the blastocyst number and the blastocyst rate of each queen are observed. The specific experimental procedure is shown in FIG. 1.
3 collecting and detecting samples
(1) Content detection of estradiol (E2) and progesterone (P4) in serum: collecting blood (5 ml/head) from anterior vena cava of each group of female cats at 4 time points of the final day of allylogest feeding (day15), the day of PMSG intramuscular injection (day 18), the day of hCG intramuscular injection (day21) and the day of surgery; the blood sample is kept at 4 ℃ overnight, centrifuged at 4000r/min at 4 ℃ for 15min, the serum is separated and stored at-20 ℃. Serum samples (1 ml/head) were tested in unison and groups were analyzed for changes in the reproductive hormones E2 and P4.
(2) And (3) analyzing the ovary state: the size of the ovary on both sides is measured by observing and counting each group of red bodies (the number of ovulation points) and calculating according to the length of the maximum contact surface multiplied by the width.
(3) Oocyte recovery: oocytes of domestic cats were cultured with MEM + FSH + LH +3 mg ml BSA +1% FBS, and most of the oocytes exhibited first polar bodies after 48 hours of culture.
(4) In vitro fertilization: following In Vitro Maturation (IVM) of oocytes, the granulosa cells (COC) were washed in TALP medium supplemented with 6 mg/mL BSA, 0.36 mM sodium pyruvate, 1 mM glutamine, 2.2 mM calcium lactate, 1% non-essential MEM, amino acids (NEAA), 0.01 mg/mL heparin sodium salt and 50 mg/mL gentamicin (mTALP). Finally, 20 to 30 COCs were placed in a four-well plate containing 500 mL of supplemental mTALP, CO-incubated with 1.5-2.5X 106 sperm/mL at 5% CO2 +38.5 ℃.
(5) Embryo in vitro culture: MEM is supplemented with Essential (EAA) and non-essential amino acids (NEAA), and BSA or FBS using modified Tyrode's solution (mTy). The embryos are firstly cultured in mTy + BSA + NEAA for 2 days, then transferred into mTy + 10% FBS + NEAA EAA culture solution and cultured in a system containing 5% CO2, and the embryos reach blastocysts after 7 days.
(6) And (3) counting embryos: when in vitro culture is carried out for 7 days, early-stage development blastula in each group are randomly selected for embryo counting, and the main operation steps are as follows: ensuring that the number of the effective blastula in each group is more than 5; and continuously culturing the rest embryos, observing the development state of each group of blastocysts, and counting the development rate of the blastocysts.
In the existing superovulation method, PMSG is often used as an inducer for superovulation, but researches show that when PMSG is used for superovulation stimulation, adverse effects such as ovarian cyst triggering, normal ovulation time interference, embryo development inhibition and drug resistance of livestock can be caused. Altrenogest has a short-term reversible inhibitory effect on ovarian activity in domestic cats and no side effects were observed. The use of progestagen to suppress the ovaries, oral administration of this steroid does not alter the already ongoing follicular activity. The method uses the altrenogest as the estrus synchronization treatment, and then uses the PMSG to carry out the superovulation treatment on the queen, and the result shows that the average red body number of an experimental group is obviously higher than that of a control group and has the advantage of being higher than 10 on average, so that the method has obvious advantage in the actual domestic cat breeding, and also reflects that the ovulation effect of the queen can be obviously improved by the method. Meanwhile, compared with a control group, the number of 6-8cell embryos in the experimental group is increased remarkably, and the ovarian cyst of the female cat in the experimental group is improved remarkably, so that the invention effectively promotes the development of the embryos and improves the conditions of the prior female cat in the same period of estrus and high ovarian cyst rate in superovulation. Therefore, compared with the prior method for overranking female cats, the method has obvious advantages.
Research shows that most follicles are finally locked in the follicle development process, and only partial dominant follicles can effectively ovulate; the level of E2 in this process reflects follicular development. Compared with the existing method for overruling female cats, the method for promoting the development of the follicles in the female cats has the advantage that the content of E2 in the blood of the female cats is obviously higher than that of the control group in the using process of the method, which shows that the method has a more effective promoting effect on the development of the follicles in the female cats, and the method corresponds to the result that the red body number of the experimental group is obviously higher than that of the control group. Meanwhile, the P4 plays an important role in establishing and maintaining the pregnancy process, the relatively high-concentration P4 is more beneficial to embryo implantation, fetal development and the like, and compared with the existing method for superovulation of the queen, the method can be used for more obviously improving the content of P4 in the blood of the queen to a certain extent, which indicates that the queen using the method has a more favorable gestational environment.
Compared with the prior method for simultaneously oestrus and superovulation of the female cats, the method can effectively improve the ovulation number of the female cats, reduce the occurrence probability of ovarian cysts and provide a superior hormone internal environment for implantation, development and pregnancy maintenance of embryos.
While the invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
Claims (7)
1. A method for inducing oestrus and superovulation in female cats at the same time is characterized in that each female cat is fed with altrenogest at a fixed point every day, after 10-20 days of continuous feeding and 2-3 days of interval, each female cat is injected with pregnant mare serum hormone (PMSG), and after 72-96 hours of interval, each female cat is injected with human chorionic gonadotropin (hCG), and after 25-30 hours of interval, the female cats are in an oestrous state.
2. The method for inducing estrus synchronization and superovulation in queen cats according to claim 1, wherein said daily regimen of altrenogest is administered to each cat in an amount of 0.01-20 mg.
3. The method for inducing estrus synchronization and superovulation in queen cats according to claim 1, wherein the amount of PMSG injected per cat is 10-1000 IU.
4. The method for inducing estrus synchronization and superovulation in a queen according to claim 1, wherein said hCG is injected in an amount of 10-1000IU per cat.
5. The method for inducing estrus synchronization and superovulation in a queen according to claim 1, wherein said queen is healthy and disease-free and has no special requirements for breed.
6. The method for inducing estrus and superovulation in a queen according to claim 1, wherein the ovaries of said queen are ovulated and red bodies appear.
7. The method for inducing estrus induction and superovulation in a queen according to claim 1, wherein said method is applied to different felines at varying dosages as required.
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