JP2899534B2 - Method of inducing superovulation in cattle - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for inducing superovulation in cattle. Furthermore, it relates to a method for inducing superovulation in a cow in a highly stable manner and efficiently collecting normal fertilized eggs. The present invention also provides a cow capable of recovering a significantly large number of eggs or embryos when a cow having an increased number of follicles capable of responding to the follicle-stimulating hormone administered in the process of superovulation is produced and subjected to superovulation. Also relates to the breeding method of.

[0002]

2. Description of the Related Art Animals are roughly divided into singletons (cattle, horses, humans, etc.) that give birth to only one child at one delivery, and multiple animals (pigs, dogs, etc.) that give birth to multiple children. The regulation of the number is considered to be controlled by the endocrine (hormone) from the hypothalamus-pituitary-gonad. In order to exert the effect of fertilized egg transplantation in a single animal, superovulation treatment that induces multiple ovulation is currently performed, but conventional superovulation treatment in cattle is exogenously treated with hormone agents ( It has been performed by administering porcine follicle stimulating hormone (FSH), pregnant mare serum gonadotropin). That is,
Fertilized egg (embryo) transfer technology is one of the techniques for breeding cows, and the superovulation induction technique, which is one of a series of fertilized egg transfer techniques, is a method for efficiently collecting fertilized eggs (embryos) from cows. Is widely used as In the superovulation treatment of cattle, a large number of follicles are developed by administering a follicle-stimulating hormone on the 9th to 13th day of the estrous cycle of the cattle, and prostaglandin F is produced.
The ₂ alpha induces administered luteolysis after 48-60 hours of follicle stimulating hormone administration, bring to ovulation by releasing the hypothalamic pituitary inhibiting action of progesterone. On the other hand, immunosuppression of inhibin, which suppresses the secretion of follicle-stimulating hormone from the pituitary gland in cattle, promotes the secretion of follicle-stimulating hormone from the pituitary gland, which can induce follicular development and multiple ovulation. It has been reported. By the way, inhibin is a hormone of a protein which is secreted from the testes of males or follicles of females and suppresses the production and secretion of FSH in the pituitary gland. Inhibin, the existence and structure of which had not been clarified until the 1980s, gradually became clear in its full range and action due to rapid progress in genetic engineering, and in mature cows, F from the pituitary gland was observed.
It is becoming clear that SH secretion is specifically suppressed. If inhibin can be controlled suppressively in cows, it is expected that endogenous FSH secretion is promoted and multiple ovulation can be induced in each estrous cycle. In recent years, inhibin was suppressed by immunization with inhibin, and the number of ovulation was increased. Attempts to increase the number of animals have become enthusiastic in sheep and cows.

[0003]

However, in the conventional superovulation treatment in which follicle stimulating hormone or pregnant mare serum gonadotropin is administered, the number of follicles capable of responding to the hormone varies greatly among individuals or from time to time. , Resolving this point has become a major issue. From this,
It is difficult to collect high-quality embryos with higher stability than cows with excellent qualities, and the average number of embryos collected is reduced, resulting in higher production cost per embryo.
Therefore, by elucidating the relationship between the hormone related to the state of ovarian follicles or follicular development and the reactivity of superovulation, and by performing a certain treatment, it is possible to produce a cow that responds to superovulation in a stable manner at all times. It is certain that the economic effects of bovine fertilized egg transfer technology will be considerable.

[0004]

[Means for Solving the Problems] 4 at the start of superovulation treatment
It has been reported that the number of ~6mm follicles and the number of ovulations by superovulation are positively correlated (Romero, A., J. Alber.
t, Z. brink and GE Seidel, Jr., Theriogenology,
35, 265, abstract, 1991). Superovulation treatment decreases the number of ovulations in the luteal phase when functional principal follicles were present (Guilbaut, LA, F. Grasso, JG Lussier, P. R.
ouillier and P. Matton, J. Reprod.Ferti., 91, 81-8
9, 1991 and Huhtinen, MV Rainio, J. Aalto, P.
Bredbacka and A. Maki-Tanila, Theriogenology, 37,
457-463, 1992), follicles show active inhibin secretion with development (Martin, TL, RL Fogwell a.
nd JJ Ireland, Biology of Reproduction, 44, 693
-700, 1991) has been reported. Recently, the present inventors have found that active immunization with inhibin increases the number of small and medium follicles. It has not been clarified how the dominant follicles influence the reaction of superovulation, but the above reports indicate the presence of the dominant follicle-enhanced inhibin secretion-decreased number of small and medium follicles-superovulation treatment. You can draw a diagram of decreased reactivity.

Therefore, from a new point of view, the present invention has found that the number of ovulations is significantly increased when immunization with inhibin is performed and mature cows with a large number of 4-6 mm follicles are subjected to conventional superovulation treatment. It was When this method is used, the secretion of endogenous follicle-stimulating hormone is enhanced, so that there is no production of antibodies against follicle-stimulating hormone by repetitive treatment, and there is no fear of diminished efficacy.

The present invention will be described in detail below with reference to examples.

[0007]

Example 1 The following experiment was carried out in order to examine the number of ovulations in a cow subjected to superovulation treatment while immunized with inhibin. 1. Experimental method (1) Experiments 1 and 2 were set. Experiment 1. In order to confirm the transition of the antibody titer to inhibin and the transition of the number of small and medium follicles due to inhibin active immunization, 5 heifers of the nulliparous state were provided and confirmed. Experiment 2. The effect of inhibin active immunization on the superovulation treatment results was investigated using 30 Holstein and Japanese Black cattle. After inactive immunization of 5 Holstein heifers and 9 Japanese Black heifers with inhibin, superovulation with follicle-stimulating hormone was performed according to a conventional method.
On the other hand, as a control, 5 Holstein heifers and 1
One Japanese Black cattle was subjected to superovulation without imhibin immunization, and the egg collection results were compared. (2) Antigen: Rabbit serum albumin conjugated to a synthetic peptide imitating the 1st to 26th amino acid sequence from the N-terminus of porcine inhibin α chain. (3) Test animals: heifers of the heifer and Japanese black cattle of the heifer that show a normal estrous cycle. (4) Immunization method: Active immunization was performed. For initial administration, 1 mg of the peptide (indicating a fragment of inhibin) was mixed with Freund's complete adjuvant and subcutaneously administered at 3 to 4 sites of the test animal. At 8th and 15th week of the initial administration, 0.5 mg peptide was administered as a booster administration in the same manner as the initial administration. (5) Follicle-stimulating hormone: Porcine pituitary extract (or pregnant horse serum gonadotropin (PMSG)) (6) Lutein-degrading agent: cloprostenol (7) Superovulation treatment: 10th to 12th day of estrous cycle Administration of follicle-stimulating hormone is started. In the hormone administration method, the dose is reduced intramuscularly 6 times at intervals of 12 hours. Dose 6, 6, 5,
5, 3 and 3 mg. 48 hours after the start of hormone administration, 750 μg of cloprostenol is intramuscularly administered. (8) Investigation of the number of ovulated eggs and the number of recovered eggs: On the 7th to 8th day of estrus after the superovulation treatment, eggs were non-surgically collected from the uterus, and the number of recovered eggs and the normal egg number/rate were investigated. 2. Results In this study, we could induce an increase in the antibody titer against inhibin in test cows by the technique of active immunization against inhibin. It was observed that the number of small and medium follicles (5 to 10 mm) tended to increase when the antibody titer increased. In this way, when the antibody against inhibin increased and the number of small and medium follicles was large and the superovulation treatment was performed on cows, the number of recovered eggs was increased without impairing the normal embryo rate.

[0008]

[Changes in the appearance of small and medium follicles due to inhibin active immunization]

[Table 1] From the results in the above table, it was observed that the follicles having a size of 5 to 10 mm increased after the initial administration and the booster administration.

[0009]

[Improved responsiveness to superovulation by inhibin immunity]

[Table 2] When superovulation was performed, the number of recovered eggs and the number of normal embryos were significantly increased in the inhibin immunized group as compared with the untreated group.

[Brief description of drawings]

FIG. 1 shows changes in antibody titer against inhibin due to active immunization of inhibin.

Claims (7)

(57) [Claims] 1. A method for inducing superovulation in cattle, which comprises performing active immunization against inhibin existing in the body of a cow to generate antibodies in the body of the cow, and then exogenously administering a follicle-stimulating hormone. 2. The method according to claim 1, wherein the follicle-stimulating hormone agents are follicle-stimulating hormone and pregnant mare serum gonadotropin. 3. The method according to claim 1, wherein the cows are dairy cows and beef cows having a normal sexual cycle after the onset of spring. 4. A method for active immunization with inhibin, wherein a peptide synthesized by replicating the amino acid sequence from 1 to 26th from the N-terminal of α chain of pig and bovine inhibin and albumin conjugated to the peptide is used as an antigen and mixed with an adjuvant. The method according to claim 1, wherein the cow is subcutaneously administered a plurality of times in this state. 5. The method according to claim 1, wherein the immunization with inhibin is active immunization (vaccine) in which an antigen is administered. 6. A cow that has been subjected to active immunization with inhibin,
Ordinary superovulation treatment is performed on the 9th to 13th day of the luteal phase, or when the luteinizing hormone preparation is left for 7 to 12 days, the follicle-stimulating hormone is administered, and further 2 to 3 days later, the luteinizing agent is administered or A method of inducing estrus by removing luteinizing hormone preparations and allowing a large number of ova to ovulate by a conventional method. 7. The method according to claim 6, wherein the antigens used for active immunization are bovine or porcine natural (extracted) inhibin, synthetic inhibin fragments and duplicate inhibin.