CN112603990A - Kit for increasing number of canine ovarian cumulus-oocyte complexes and application thereof - Google Patents
Kit for increasing number of canine ovarian cumulus-oocyte complexes and application thereof Download PDFInfo
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Abstract
The invention relates to a kit for increasing the number of canine ovarian Cumulus-Oocyte Complexes (COCs) and application thereof, wherein the kit is added with three hormones, namely estradiol benzoate EB, chlorprostenol PGF2 alpha and a novel hormone preparation FG600, and a simple hormone induction method capable of enabling an episodic Beagle dog to enter the early stage of estrus in a short time is also established. The kit for increasing the number of the canine ovarian cumulus-oocyte complexes enables the intercalary dogs to enter the early estrus stage in ten days, and the number of COCs recovered from the ovaries by a puncture method is about 58 on average and is obviously higher than that of normal estrus dogs (about 33.7 on average).
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a kit for increasing the number of canine ovarian cumulus-oocyte complexes and application thereof.
Background
Beagle dogs are internationally recognized experimental animals and are widely used in various fields of scientific research. The domestic Beagle dog resource is introduced from abroad, and the control on the germplasm resource of the Beagle is strict abroad at present, so the method is particularly important for the storage and development of the domestic existing resource. Meanwhile, the Beagle dog has clear genetic background, the similarity of the genome of the Beagle dog and human is as high as 95.7%, and the construction of a disease model by taking the Beagle dog as an experimental material becomes a hotspot for the research of broad scholars. Obtaining mature oocytes is a crucial step in the canine in vitro conservation work and disease model preparation process. The assisted reproduction technology of dogs is not mature relative to other mammals due to the unique physiological characteristics of the dogs.
The bitches are in estrus intervals most of the time, most of the bitches only estrus for 1-2 times in one year, and the overlong estrus cycle restricts the production benefit and breeding progress of the bitches and also influences the preparation efficiency of disease model dogs. The oocytes are in the Germinal Vesicle (GV) phase at the time of ovulation of dogs, and need to develop further in the oviduct to mature. Taking mature oocytes through a living body by surgery consumes a lot of manpower and material resources, and the number of ovulated mother cells per bitch is limited. For example, collecting ovarian Cumulus-Oocyte Complexes (COCs) from canine ovaries and culturing in vitro to obtain mature oocytes would greatly improve the work efficiency.
The quantity of COCs provided by ovaries of dogs in different estrus stages is greatly different, and studies prove that the quantity of high-quality COCs obtained in the follicular phase is the largest (22.7), the luteal phase is the next (10.7) and the episodic phase is the smallest (7.3) (Lisanpeng, Wangxiang, Xijun, and the like. Furthermore, the ability of COCs to mature in vitro from ovaries at different stages has been controversial in numerous studies (Lopes G, Sousa M, Luvoni G C, et al recovery rate, morphological quality and nuclear quality of cancer-ova complex collected from either or both of the genetic agents [ J ]. Theriogenology,2007,68(6): 825.). It is generally believed that COCs obtained from ovarian follicles (pre-estrus/estrus) have a higher potential for nuclear maturation than COCs obtained from other reproductive stages. However, the dogs only estrus 1-2 times per year, the intermittent period is as long as 8 months, and it is not easy to obtain the female dogs with ovaries in the follicular phase. Therefore, if a method for rapidly and effectively enabling the intermittent Beagle dog to enter the prophase estrus/estrus (ovary is in follicular phase) can be found, the method has a positive promoting effect on the in vitro maturation culture of the Beagle dog oocyte and the subsequent research of an auxiliary propagation technology, and can greatly accelerate the in vitro preservation of important germplasm resources and the construction research of disease model dogs.
Since 1939, scholars at home and abroad have conducted many studies on the method for inducing estrus and ovulation in dogs, and according to the reports of relevant documents, exogenous hormones and drugs for inducing estrus in dogs generally include: gonadotropin-releasing hormone, gonadotropin, prostaglandin, estrogen, dopamine receptor agonist and the like, wherein the combined use of several hormones is superior to that of a single hormone, and dopamine receptor agonist can achieve good oestrus induction effect, but the administration time is long (Kutzler M.Industration and synchronization of estrogen in dogs [ J ]. Theriogenology,2005,64(3): 766) 775.), which limits the large-scale production application. The novel hormone preparation FG600 comprises the novel long-acting recombinant follicle stimulating hormone rFSH, has high stability, longer half-life, higher activity, low price and easy acquisition, and can be effectively applied to sows (old shish, old prediction, Yuanmeng, and the like. the influence of the novel hormone preparation FG600 on the simultaneous estrus and reproductive performance of replacement gilts [ J ]. pig industry science, 2014,31(12):102-104.), but no relevant report is found temporarily when the novel hormone preparation FG600 is applied to dogs.
The invention content is as follows:
one of the purposes of the invention is to provide a kit for increasing the number of canine ovarian cumulus-oocyte complexes.
The technical scheme for achieving the purpose is as follows.
A kit for increasing the number of canine ovarian cumulus-oocyte complexes comprises estradiol benzoate EB, chlorprostenol PGF2 alpha and a novel hormone preparation FG 600.
In some of these embodiments, the dog is in estrus.
In some of these embodiments, the dog is a Beagle dog.
In some of these embodiments, the ratio of the units of administration of the lorprostenol PGF2 α to estradiol EB benzoate is: 0.1 mg: 490-510. mu.g.
In some of these embodiments, the dosage units of the novel hormone formulation FG600 are 60, 40, 30 and 20 IU/head, respectively.
One of the purposes of the invention is also to provide application of the estradiol benzoate EB, the chlorprostenol PGF2 alpha and the novel hormone preparation FG600 in preparation of a kit for increasing the number of canine ovarian cumulus-oocyte complexes.
It is also an object of the present invention to provide a method for increasing the number of canine ovarian cumulus-oocyte complexes.
The technical scheme for achieving the purpose is as follows.
Firstly, carrying out combined treatment on a maternal dog in an episodic period by using PGF2 alpha and EB (Epstein-Barr), then using a novel hormone preparation FG600 for treatment, anesthetizing an experimental dog on the third day after the hormone treatment is finished, taking an ovary by an operation, and obtaining a cumulus-oocyte complex by a puncture method; PGF2 α was injected in combination with EB and PGF2 α was injected at 0.1 mg/head, i.e., intramuscularly. The injection amount of EB is 490-; the novel hormone formulation FG600 was injected in a dose decreasing manner (60, 40, 30 and 20 IU/head) for four consecutive days.
The invention provides a kit for increasing the number of canine ovarian cumulus-oocyte complexes and application thereof, wherein the kit comprises estradiol benzoate EB, chlorprostenol PGF2 alpha and a novel hormone preparation FG600, and a simple hormone induction method capable of enabling an episodic Beagle dog to enter the early estrus period in a short time is also established. The kit for increasing the number of the canine ovarian cumulus-oocyte complexes enables the intercalary dogs to enter the early estrus stage in ten days, and the number of COCs recovered from the ovaries by a puncture method is about 58 on average and is obviously higher than that of normal estrus dogs (about 33.7 on average).
Drawings
FIG. 1: episodic and pre-estrus entering dam vulva and vaginal smears.
FIG. 2: hormone-treated ovaries.
FIG. 3: recovered COCs form.
Detailed Description
The following examples of the present invention are experimental methods without specifying specific conditions, generally according to conventional conditions, or according to conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device that comprises a list of steps is not limited to only those steps or components listed, but may alternatively include other steps or components not listed, or inherent to such process, method, product, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The method for distinguishing the estrus and the promestrus of the dogs comprises the following steps:
selecting a Beagle dog without reproductive diseases at the age of 2-3 years, wherein the characteristics of the intermittent period can be mainly judged by vulva and vaginal smear of the dog. Dogs in estrus: the vulva is shriveled without obvious swelling and vaginal fluid secretion outflow; the vaginal mucosa has few exfoliated epithelial cells, and most of the visible epithelial cells are intermediate or accessory stromal cells. Dogs in the pre-estrus stage: swelling of vulva, stiffness in touch, and hanging red fluid droplets at the lower corner of vulva; the vaginal epithelial cells have obvious cell nucleuses, are large and clear in staining, are circular or oval, visible epithelial cells begin to increase in visual field, intermediate and accessory stromal cells gradually decrease, the vaginal epithelial cells begin to gradually keratinize, and more red blood cells exist.
Method for injection of hormones
On the first and third days, PGF2 alpha 0.1 mg/head is injected intramuscularly, EB 490-; four consecutive injections were given at decreasing doses of 60, 40, 30 and 20 IU/head for four days starting on day four.
Method for recovering COCs
The experiment dog is anesthetized on the tenth day after hormone treatment, the ovary is kept fixed, the ovary is taken out after operation and placed in normal saline at 38.5 ℃ containing 1% double antibody, the normal saline is sent back to a laboratory as soon as possible, fat and ovarian membrane are removed, the ovary is separated, the normal saline added with 1% double antibody is used for cleaning three times, then the ovary is placed in M199 ovum flushing liquid containing 10% Fetal Bovine Serum (FBS), the ovarian surface follicles are repeatedly punctured by a sterile 1mL injection needle with moderate strength on a constant temperature table, all follicles are punctured as much as possible, then the ovary is sufficiently sheared by surgical scissors, COCs are released as much as possible, and COCs with regular shapes and wrapped with more than two layers of cumulus cells and dark grey ootheca are selected under a stereo microscope.
Example 1:
the kit for increasing the number of the canine ovarian cumulus-oocyte complexes comprises estradiol benzoate EB, chlorprostenol PGF2 alpha and a novel hormone preparation FG 600.
Chloroprostenol (PGF2 α), specification: 2 ml: 4mg (Ningbo Sansheng Biotech Co., Ltd.).
Estradiol Benzoate (EB), specification: 2 ml: 4mg (Hangzhou animal pharmaceuticals factory).
FG600 (cell engineering product class FSH + hCG), specification: 1200 IU/vial (Weisheng pharmaceutical science, Guangzhou).
2-3 years old female Begle dog 5, from Guangzhou institute of medicine, Inc. (laboratory animal Production license number: SCXK (Guangdong) 2018-.
The selection requirements of experimental dogs are as follows: the vulva is shriveled without obvious swelling and vaginal fluid secretion outflow; the vaginal mucosa has few desquamated epithelial cells, and is mostly intermediate or accessory stromal cells; normal reproduction records have been made.
The processing steps are as follows: on the first and third days, PGF2 alpha 0.1 mg/head is injected intramuscularly, EB 490-; starting on day four, FG600 was injected continuously for four days at decreasing doses of 60, 40, 30 and 20 IU/head, and on day three of FG600 injection, the vulva of the experimental dog began to swell and gradually entered the prophase of estrus; and (3) anesthetizing the experimental dog on the tenth day, taking the ovary after the operation, placing the ovary into normal saline with 1% double antibody and at the temperature of 38.5 ℃, returning the ovary to the laboratory, washing the ovary, repeatedly puncturing ovarian follicles on the surface of the ovary by using a sterile 1mL injection needle with moderate strength to puncture all the ovarian follicles as much as possible, and then fully shearing the ovary by using surgical scissors to release the COCs as much as possible.
As a result: in this example, all 5 dogs successfully entered the pre-estrus stage (as shown in FIG. 1). The canine ovaries post hormone treatment were swollen (as shown in figure 2). 174 high-quality COCs (shown in fig. 3) were obtained in this example, and 58.0 COCs per dog were obtained on average.
Comparative example 1:
selecting 6 Beagle dogs which have natural oestrus in the prophase of oestrus and are anogenital diseases, taking ovaries after anaesthesia and placing the ovaries in normal saline with 1% double antibody and at the temperature of 38.5 ℃ and sending the ovaries back to a laboratory, cleaning the ovaries, repeatedly puncturing ovarian follicles on the surface of the ovaries with proper strength by using a sterile 1mL injection needle, enabling all the ovarian follicles to be punctured as much as possible, and then fully shearing the ovaries by using surgical scissors to release COCs as much as possible. On average, 48.6 COCs per dog were obtained.
Comparative example 2:
selecting 3 Beagle dogs in the anaphase and with the anogenital disease, taking ovaries after anesthesia operation, placing the ovaries into normal saline with 1% double antibody and at 38.5 ℃, sending the ovaries back to a laboratory, cleaning the ovaries, repeatedly puncturing ovarian follicles on the surface of the ovaries with a sterile 1mL injection needle with proper strength to puncture all the ovarian follicles as much as possible, and then fully shearing the ovaries with surgical scissors to release the COCs as much as possible. On average, 17.7 COCs per dog were obtained.
Comparative example 3:
6 Beagle dogs with intermittent and anogenital diseases were selected and randomly divided into two groups. The used drugs are estradiol EB benzoate, Clrostenol PGF2 alpha and a novel hormone preparation FG 600.
Chloroprostenol (PGF2 α), specification: 2 ml: 4mg (Ningbo Sansheng Biotech Co., Ltd.).
Estradiol Benzoate (EB), specification: 2 ml: 4mg (Hangzhou animal pharmaceuticals factory).
FG600 (cell engineering product class FSH + hCG), specification: 1200 IU/vial (Weisheng pharmaceutical science, Guangzhou).
Group 1 dogs were given 0.1 mg/head of intramuscular PGF2 α on the first and third days, starting on day four and FG600 was given for four consecutive days at decreasing doses of 60, 40, 30 and 20 IU/head; group 2 dogs were injected subcutaneously with EB 490-. And anaesthetizing the two groups of experimental dogs on the tenth day, taking ovaries after the operation, placing the ovaries into normal saline with the temperature of 38.5 ℃ and containing 1% double antibody, sending the ovaries back to a laboratory, cleaning the ovaries, repeatedly puncturing ovarian follicles on the surface of the ovaries by using a sterile 1mL injection needle with moderate strength to puncture all the ovarian follicles as much as possible, and then fully shearing the ovaries by using surgical scissors to release the COCs as much as possible. Group 1 gave an average of 26.7 COCs per dog, and group 2 gave an average of 33.4 COCs per dog.
It can be seen by comparing example 1 with comparative examples 1, 2 and 3 that the kit for increasing the number of canine ovarian cumulus-oocyte complexes can effectively enable an episodic Beagle dog to enter the prophase of estrus, and the number of COCs obtained from ovaries is obviously higher than that of dogs in the prophase of normal estrus and the episodic period. The kit for increasing the number of the canine ovarian cumulus-oocyte complexes can effectively increase the utilization rate of ovaries and provide more experimental materials for oocyte in-vitro culture research.
The kit for increasing the number of the canine ovarian cumulus-oocyte complexes comprises estradiol benzoate EB, chlorprostenol PGF2 alpha and a novel hormone preparation FG600, and is ready to be put on the market.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (10)
1. The kit for increasing the number of the canine ovarian cumulus-oocyte complexes is characterized by comprising estradiol benzoate EB, chlorprostenol PGF2 alpha and a novel hormone preparation FG 600.
2. The kit for increasing the number of canine ovarian cumulus-oocyte complexes according to claim 1, wherein the dog is in an episodic stage.
3. The kit for increasing the number of ovarian cumulus-oocyte complexes in a dog according to claim 1, wherein the dog is a Beagle dog.
4. The kit for increasing the number of ovarian cumulus-oocyte complexes in a dog according to any one of claims 1 to 3, wherein the ratio of the administration units of the lorprostenol PGF2 a to the estradiol EB benzoate is as follows: 0.1 mg: 490-510. mu.g.
5. The kit for increasing the number of canine ovarian cumulus-oocyte complexes according to any one of claims 1 to 3, wherein the dosage units of the novel hormone formulation FG600 are 60, 40, 30 and 20 IU/head, respectively.
6. Estradiol benzoate EB, chlorprostenol PGF2 alpha and a novel hormone preparation FG600 are applied to the preparation of a kit for increasing the number of canine ovarian cumulus-oocyte complexes.
7. The use of claim 6, wherein the ratio of the amount of the lorprostenol PGF2 α to the amount of estradiol EB benzoate is as follows: 0.1 mg: 490-510. mu.g.
8. The use of claim 6 wherein the dog is in estrus.
9. The use according to claim 6, wherein the dog is a Beagle dog.
10. A method for increasing the number of ovarian cumulus-oocyte complexes of dogs is characterized in that firstly, a maternal dog in an episodic period is treated by PGF2 alpha and EB in a combined mode, then a novel hormone preparation FG600 is used for treatment, a test dog is anesthetized on the third day after the hormone treatment is completed, ovaries are taken out through an operation, and the cumulus-oocyte complexes are obtained through a puncture method; PGF2 α was injected in combination with EB and PGF2 α was injected at 0.1 mg/head, i.e., intramuscularly. The injection amount of EB is 490-; the novel hormone formulation FG600 was injected in a dose decreasing manner (60, 40, 30 and 20 IU/head) for four consecutive days.
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