Subcutaneous injection product for cattle for inducing superovulation

Abstract

The present invention provides a subcutaneous injection product for cattle for inducing superovulation characterized by comprising aluminum hydroxide gel and an efficient amount of a gonadotropin, wherein the concentration of the aluminum hydroxide is, in terms of the amount of aluminum, higher than or equal to 0.2 mg/mL and lower than 1.0 mg/mL, which is a subcutaneous injection product that is useful for producing good calves and that can induce superovulation in a cow by a single administration. The present invention also provides a method for producing good calves undergoing steps of subcutaneously injecting the aforementioned injection product into a cow to thereby induce superovulation, inducing estrus in the cow, conducting artificial insemination, and then collecting fertilized eggs.

Claims

1. A subcutaneous injection product for cattle for inducing superovulation, characterized by comprising aluminum hydroxide gel and gonadotropin present in an amount effective to induce adequate superovulation, wherein the concentration of the aluminum hydroxide is, in terms of the amount of aluminum, 0.3 to 0.6 mg/mL.

2. The subcutaneous injection product for cattle for inducing superovulation according to claim 1, wherein the total amount of the aluminum hydroxide is, in terms of the amount of aluminum, 1.5 to 3.0 mg per administration.

3. The subcutaneous injection product for cattle for inducing superovulation according to claim 1, wherein the content of the gonadotropin is 10 to 50Armour Units or an amount of any biological valence that corresponds to 10 to 50 Armour Units per administration.

4. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 1, comprising a first container in which the aluminum hydroxide gel is enclosed, a second container in which the gonadotropin is enclosed, and a third container in which a liquid for dissolving the gonadotropin is enclosed.

5. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 1, comprising a first container in which the aluminum hydroxide gel is enclosed, wherein the concentration of the aluminum hydroxide is, in terms of the amount of aluminum, 0.3 to 0.6 mg/mL; and a second container in which the gonadotropin is enclosed.

6. A method for obtaining fertilized eggs characterized by comprising a step of subcutaneously injecting into cattle the injection product according to claim 1, a step of inducing estrus and conducting artificial insemination, and a step of collecting fertilized eggs.

7. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 2, comprising a first container in which the aluminum hydroxide gel is enclosed, wherein the concentration of the aluminum hydroxide is, in terms of the amount of aluminum, 0.3 to 0.6 mg/mL, and wherein the total amount of the aluminum hydroxide is, in terms of the amount of aluminum, 1.5 to 3.0 mg per administration, and a second container in which the gonadotropin is enclosed.

8. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 3, comprising a first container in which the aluminum hydroxide gel is enclosed, wherein the concentration of the aluminum hydroxide is, in terms of the amount of aluminum, 0.3 to 0.6 mg/mL, and a second container in which the gonadotropin is enclosed, wherein the content of the gonadotropin is 10 to 50 Armour Units or an amount of any biological valence that corresponds to 10 to 50 Armour Units per administration.

9. The subcutaneous injection product for cattle for inducing superovulation according to claim 2, wherein the content of the gonadotropin is 10 to 50Armour Units or an amount of any biological valence that corresponds to 10 to 50 Armour Units per administration.

10. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 2, comprising a first container in which the aluminum hydroxide gel is enclosed, a second container in which the gonadotropin is enclosed, and a third container in which a liquid for dissolving the gonadotropin is enclosed.

11. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 3, comprising a first container in which the aluminum hydroxide gel is enclosed, a second container in which the gonadotropin is enclosed, and a third container in which a liquid for dissolving the gonadotropin is enclosed.

12. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 9, comprising a first container in which the aluminum hydroxide gel is enclosed, a second container in which the gonadotropin is enclosed, and a third container in which a liquid for dissolving the gonadotropin is enclosed.

13. A kit for preparing the subcutaneous injection product for cattle for inducing superovulation according to claim 9, comprising a first container in which the aluminum hydroxide gel is enclosed, wherein the concentration of the aluminum hydroxide is, in terms of the amount of aluminum, 0.3 to 0.6 mg/mL, and wherein the total amount of the aluminum hydroxide is, in terms of the amount of aluminum, 1.5 to 3.0 mg per administration, and a second container in which the gonadotropin is enclosed, wherein the content of the gonadotropin is 10 to 50 Armour Units or an amount of any biological valence that corresponds to 10 to 50 Armour Units per administration.

Description

EXAMPLES

(1) Hereafter, the present invention will be particularly explained with reference to examples.

Example 1

Preparation of Aluminum Hydroxide Gel (No. 1)

(2) Aluminum chloride, 14.85 g in terms of an anhydrous one, was dissolved in water to prepare an aqueous solution of 400 mL. Separately, about 5.0 g of sodium hydroxide was dissolved in water to prepare an aqueous solution having an appropriate concentration. The aqueous sodium hydroxide solution was gradually added to the aqueous aluminum chloride solution, thereby adjusting the pH to be 6.5 to 6.6. Thereafter, water was added so that the total volume became 1 L. The concentration of the aluminum hydroxide is, in terms of the amount of aluminum, 3 mg/mL (theoretical value).

(3) The aluminum hydroxide gel thus prepared, 100 mL, was put into a vial having a 100 mL-volume, and the vial was closely sealed with a rubber stopper and an aluminum cap. It was sterilized at 105 degrees Celsius for 30 minutes in an autoclave. After the sterilization, the content of the vial was homogeneously mixed. It was used as an aluminum hydroxide gel in the following experiments.

Example 2

Preparation of Aluminum Hydroxide Gel (No. 2)

(4) Potassium aluminum sulfate, 28.74 g in terms of an anhydrous one, was dissolved in water to prepare an aqueous solution of 400 mL. Separately, about 5.0 g of sodium hydroxide was dissolved in water to prepare an aqueous solution having an appropriate concentration. The aqueous sodium hydroxide solution was gradually added to the aqueous potassium aluminum sulfate solution, thereby adjusting the pH to be 6.4 to 6.6. This liquid was centrifuged (about 3,000 rpm; 5 minutes), and supernatant was removed. To the residue, water having a same volume as that of the removed supernatant was added and then the container was sufficiently shaken. The steps of centrifugation, removal of supernatant, and addition of water were repeated again. Thereafter, 9.0 g of sodium chloride was added and dissolved. The aqueous sodium hydroxide solution was added, thereby adjusting the pH to be 6.4 to 6.6. Further, water was added so that the total volume became 1 L. The concentration of the aluminum hydroxide is, in terms of aluminum, 3 mg/mL (theoretical value). The aluminum hydroxide gel thus prepared was put into a vial and sterilized in the same manner as that in Example 1.

Experiment 1

Assay to Investigate Effects of Concentration and Total Amount of Aluminum Hydroxide for Superovulation

(5) 1. Preparation of Injection Product

(6) The aluminum hydroxide gel (concentration in terms of aluminum: 3 mg/mL) prepared in Example 1 was diluted with a physiological saline to be concentrations in terms of aluminum of 0.6 mg/mL, 0.3 mg/mL, and 0.15 mg/mL. In 5 mL of each of the aluminum hydroxide gel solutions thus prepared having respective concentrations, 30 Armour unit (A.U.) of Antrin R-10 (a gonadotropin from anterior pituitary (FSH) manufactured by Kyoritsu Seiyaku Corporation) was dissolved. Thus, four injection products were prepared. Concentrations of aluminum hydroxide (concentrations in terms of aluminum) and total amounts of aluminum hydroxide (total amounts in terms of aluminum) of these injection products were as those shown in Table 1.

(7) 2. Administration of Injection Product, Artificial Insemination, and Collection of Fertilized Eggs

(8) Six Japanese Black Beef cows constituted one group. First, 5 mL of an injection product having a concentration of 3 mg/mL in terms of aluminum was subcutaneously injected into the neck region of each cow. Forty-eight hours later, prostaglandin F2.sub. was administered. After the onset of estrus, artificial insemination was conducted. After 7 days of the insemination, embryos were collected. After a lapse of 40 days or more from the collection of the embryos, the same treatment as that described above was practiced, except that another injection product having a concentration of 0.6 mg/mL in terms of aluminum was used. Thereafter, similarly, the same treatment as that described above was practiced, except that another injection product having a concentration of 0.3 mg/mL in terms of aluminum or still other injection product having a concentration of 0.15 mg/mL in terms of aluminum was used, wherein there was similarly an interval of 40 days or more between treatments.

(9) 3. Results

(10) Table 1 shows the results. As is clear from Table 1, in the case where the concentration of aluminum hydroxide (concentration in terms of aluminum) of the injection product was 0.15 mg/mL, the number of transplantable embryos was significantly small By contrast, in the cases where the concentrations were 0.30 mg/mL or more and within the range that was examined in this experiment, there were no significant difference in the number of large ovarian follicles in estrus, the number of corpora luteum, the number of residual ovarian follicles, the number of embryos collected, and the number of transplantable embryos, even if the concentration was increased.

(11) TABLE-US-00001 TABLE 1 Aluminum hydroxide in injection product Condition of cows Conc. in Total amount Number of terms of in terms of Number Number of large residual Number of Number of aluminum aluminum of cows ovarian follicles in Number of ovarian embryos transplantable (mg/mL) (mg) treated estrus corpora luteum follicles collected embryos 3.00 15.00 6 19.0 3.5 10.7 2.9 2.8 0.9 7.5 2.9 4.8 1.6 0.60 3.00 6 20.0 4.3 11.7 1.6 3.0 1.2 8.0 1.3 4.3 1.4 0.30 1.50 6 24.8 6.6 11.7 3.8 5.3 1.0 9.3 2.9 4.8 1.7 0.15 0.75 6 24.2 6.7 10.7 3.7 3.8 1.5 7.7 1.5 1.2 0.5

Experiment 2

Assay to Investigate Effect of Concentration of Aluminum Hydroxide for Superovulation

(12) The same experiment as that of Experiment 1 was practiced, expect that the concentrations of aluminum hydroxide (concentrations in terms of aluminum) in the injection products and the administered amounts were revised to be the values shown in Table 2, and that the number of Japanese Black Beef cows subjected to the experiment were revised to be the values shown in Table 2. Table 2 shows the results. When the total amounts of aluminum hydroxide (total amounts in terms of aluminum) were same to each other, the case where the concentration of aluminum hydroxide (concentration in terms of aluminum) was the highest (1.5 mg/mL) gave the fewest number of transplantable embryos.

(13) TABLE-US-00002 TABLE 2 Injection product Conc. Total amount of of aluminum aluminum Condition of cows hydroxide in hydroxide in Number of terms of Administered terms of Number Number of large residual Number of Number of aluminum amount aluminum of cows ovarian follicles Number of ovarian embryos transplantable (mg/mL) (mL) (mg) treated in estrus corpora luteum follicles collected embryos 1.5 1 1.5 6 22.2 7.3 10.5 4.1 3.3 1.4 6.0 2.7 2.8 1.3 0.5 3 1.5 6 19.0 6.3 10.7 2.9 5.2 2.3 7.5 2.9 6.2 2.9 0.3 5 1.5 4 16.5 4.7 8.5 4.9 4.5 0.5 7.5 4.4 5.8 3.5

Experiment 3

Measurement of Sizes of Swellings or Indurations

(14) In Experiment 1, the sizes of swellings and/or indurations at the sites (neck regions) where the injection products of the present invention were administered via subcutaneously injection were measured with time. Table 3 shows the results. It is clear from Table 3 that the swelling or induration comes to be large if the concentration of aluminum hydroxide (concentration in terms of aluminum) in the injection product is high and the total amount of aluminum hydroxide (total amount in terms of aluminum) is large.

(15) TABLE-US-00003 TABLE 3 Aluminum hydroxide Conc. Total amount Size of swelling or induration (mm) in terms of in terms of Number 5 days later 10 days later 20 days later aluminum aluminum of cows the administration the administration the administration (mg/mL) (mg) treated Length Width Length Width Length Width 3.00 15.00 6 84.4 9.8 32.2 8.0 73.2 7.4 30.8 6.8 64.6 5.4 17.6 2.0 0.60 3.00 6 41.3 2.8 16.5 0.3 38.8 3.0 16.5 2.7 25.3 5.4 12.8 2.5 0.30 1.50 6 36.7 7.0 21.2 1.7 42.4 3.8 12.0 2.1 29.8 4.0 11.5 2.4 0.15 0.75 6 35.2 7.8 18.0 5.3 21.8 7.4 9.2 3.2 12.0 5.4 6.2 2.6