JP3008082B2 - Estrus and ovulation inducers - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an estrus and ovulation inducer capable of inducing estrus and ovulation by administering to females of domestic animals such as cattle, horses, pigs and the like by intrauterine administration.

[0002]

2. Description of the Related Art As is well known, the reproductive activity of females of domestic animals such as cattle, horses and pigs begins with the development of ovarian follicles, estrus, ovulation, luteal formation, embryo development, implantation, and pregnancy. Refers to a series of processes such as maintenance, parturition, regression of the corpus luteum, and rearing of calves. After parturition, livestock females repeat the reproductive activity again, with a period of reproductive activity specific to each animal species.

[0003] Such a series of breeding activities are greatly affected by environmental factors such as nutrition, weather, and breeding management, in addition to living body factors such as endocrine and nervous factors. Originally, domestic females have a function that can cope with such changes in the factors, but the reproductive activity may temporarily or temporarily not be performed normally due to various causes.

[0004] Such a condition is called a reproductive disorder, and its causes are roughly classified into intrauterine infections caused by pathogens such as bacteria, viruses, protozoa, rickettsiae, fungi, and follicular cysts, luteal cysts, and luteal dysplasia.・ It is divided into those due to genital dysfunction such as luteal residue, ovulation disorder, etc., which show no estrus, or estrus signs at the time of follicle maturation although follicle development, ovulation and luteal formation are seen periodically. It is characterized by estrous abnormalities such as no dull estrus.

[0005] As a method for treating estrus abnormalities due to the intrauterine infection, intrauterine administration of antibiotics after washing the uterus is performed. As a treatment method for estrous abnormalities due to genital dysfunction such as follicular cysts, luteal cysts, luteal abnormalities, ovulation disorders, etc., gonadotropin (GTH), gonadotropin releasing hormone (GnRH), prostaglandin F ₂ α
Administration of various hormonal agents such as (PGF ₂ α) has been performed.

[0006]

However, the use of antibiotics for intrauterine infections as described above has been limited due to the problem of persistence in livestock meat and milk for meat.

[0007] Further, regarding the use of various hormonal agents for genital dysfunction, it is difficult to set the dose because the optimal dose is different for each individual. In this case, if the dose is insufficient, there is no effect. In addition, excessive administration may cause irreversible abnormalities such as serious side effects.

In particular, as a hormonal agent that induces estrus by regressing the corpus luteum, the prostaglandin F ₂ α (P
When GF ₂ α) is used, blunt estrus is often observed, and the conception rate is not high in such cases, and even if estrus can be induced, the next estrus may fall into anovulation. In many cases, it is not yet a fundamental solution to infertility.

The present invention has been made in view of the above-mentioned circumstances and problems, has no problem of persistence, is easy to set the amount of use, and is capable of normal administration by direct administration into the uterus. It can induce normal estrus and ovulation not only in domestic animals but also in animals with genital dysfunction, and further promote the healing of genital diseases that cause reproductive disorders such as endometritis and follicular cysts. In addition, an object of the present invention is to provide an estrus and ovulation inducer capable of inducing normal estrus and ovulation in such livestock.

[0010]

Means for achieving the above object are, firstly, to use chitin as an active ingredient. Second, the chitin is in the form of a fine powder having a particle size of 500 μm or less. Third, the chitin has a β-type crystal structure.

[0011]

Embodiments of the present invention will be described below. The estrus and ovulation inducer according to this embodiment contains chitin as an active ingredient.

The chitin is a homopolysaccharide containing only N-acetyl-D-glucosamine as a constitutional unit, or a polysaccharide containing N-acetyl-D-glucosamine and D-glucosamine as a constitutional unit. The ratio (degree of deacetylation) is 50% or less. This chitin is
A naturally occurring biopolymer, such as crab
It can be obtained by purifying from the cuticle of crustaceans such as shrimp, the soft shell of squid, or the exoskeleton of insects.

As a method for purifying chitin, the above-mentioned naturally occurring chitin source is subjected to removal of protein by alkali treatment, removal of ash by acid treatment, and, if necessary, removal of pigments and the like. For example, a known purification method such as a purification method may be used.

As a method for producing the estrus and ovulation inducer, for example, chitin purified by the above-mentioned operation is dissolved in a soluble solvent such as hydrochloric acid or sulfuric acid, and then a large amount of water is added to form a colloid. Method for producing chitin (Chitin and chitosan experiment manual, page 5,
Precipitation methods such as “1.2 Preparation method of colloidal chitin”),
Alternatively, it is formed into a fine powder by a pulverizing method using a pulverizer or the like, and is manufactured by suspending in a physiologically acceptable dispersion medium such as physiological saline in advance, or when administering to a female animal. Examples of the method include a method of manufacturing by suspending in a dispersion medium. The estrus and ovulation inducer may optionally contain medically acceptable additives such as adjuvants, stabilizers, emulsifiers, pH regulators, thickeners, and dispersion aids. .

As a method for suspending the chitin formed into a fine powder in a physiologically acceptable dispersion medium, a predetermined amount of the chitin is mixed with the dispersion medium, and then, for example, ultrasonic irradiation or A method of dispersing by shaking or the like can be used.

As a method for suspending fine powdered chitin in the dispersion medium at the time of administration, a fine powdery water-soluble substance or the like is used as a dispersing aid, and the water-soluble substance and the fine powdery In a dry state, the chitin thus formed is uniformly mixed in a predetermined ratio, and the above-mentioned dispersion medium is added at the time of administration, and the mixture is shaken gently to uniformly disperse the chitin.

As the easily water-soluble substance as a dispersing aid, any substance can be used as long as it is a physiologically acceptable substance which can be formed into a fine powder in a dry state and is easily dissolved in water. Preferably, low molecular weights such as salts such as sodium chloride, sodium hydrogen carbonate, diammonium hydrogen phosphate, sodium citrate, etc., monosaccharides such as sorbitol, glucose, fructose, mannitol, and oligosaccharides such as lactose, cyclodextrin, etc. One.

Examples of the pulverizer for pulverizing chitin into fine powder include a jet mill for pulverizing chitin by colliding it with the inner wall of a mill by a jet stream, and a pulverizer by colliding chitin with a pin rotating at high speed. Pin mills, vibrating ball mills that crush by vibrating media balls, planetary mills that crush by rotating a mill that revolves largely around a revolution axis parallel to the rotation axis, or centrifugal force at the bottom of a rotating mill A dry pulverizer that can finely pulverize chitin in a dry state, such as a centrifugal fluidized mill that pulverizes a given medium by reciprocating between a stationary upper part of the mill and the like, may be used.

The particle diameter of chitin formed into a fine powder by the above-mentioned precipitation method or pulverization method is 500
μm or less, more preferably 200 μm or less. As described above, if chitin is in the form of fine powder having a particle size of 500 μm or less, chitin that is not soluble in water can be uniformly suspended in a physiologically acceptable dispersion medium, so that good fluidity is provided. Therefore, the agent for inducing estrus and ovulation can be easily administered into the uterus using an injection device such as a catheter for injecting an intrauterine drug. In addition, since it is a fine powder, the contact area between chitin and the genital organs can be increased, so that a small amount can exert a sufficient effect, and the receptivity to the enzyme can be improved, so long-term use in the uterus There is an advantage that residual can be effectively prevented.

From the crystal structure, chitin has an α-type in which polysaccharide chains are oriented in opposite directions, a β-type in which polysaccharide chains are oriented in the same direction, and a mixture of α-type and β-type. It can be divided into three types of γ-type
Chitin, Chitosan ”, Chapter 2,“ 2.1.1 Chitin ”, edited by Chitin and Chitosan Study Group, February 20, 1988.
Among them, the chitin used for the estrus and ovulation inducer is β
Those having a type crystal structure are desirable. As described above, when chitin having a β-type crystal structure is used, the crystal structure is looser than that of α-type or γ-type. The swelling property with respect to water and the acceptability with respect to a decomposition enzyme such as lysozyme can be increased.

The chitin having the β-type crystal structure is purified from squid such as squid belonging to the order Coleoptera, such as squid, red squid, watermelon, spear squid, firefly squid, mica, and squid, or squid belonging to the order Cucumber. Can be obtained by:

As a method of administering the estrous and ovulation-inducing agent constituted as described above to the uterus of a domestic animal, the estrus and ovulation-inducing agent dispersed in the dispersion medium or the like is used, for example, for injecting a drug into the uterus. A method of administration into the uterus using a catheter or the like is included.

The dose of the estrus and ovulation inducer varies depending on the type and weight of the livestock, the presence or absence of reproductive disorders, and the cause and degree thereof. It is sufficient that the amount is sufficient to induce estrus. As a guide of the dose, for example, a body weight of about 400 kg
The healthy cows can be administered in an amount containing about 300 mg of chitin, which is an active ingredient for a estrus and ovulation inducer, per cow. In this case, even if the minimum effective dose is greatly exceeded, the chitin reacts mildly to the genital organs, so that no particular side effect occurs. In addition, chitin is decomposed into N-acetyl-D-glucosamine or D-glucosamine, which is a constituent unit of the chitin, in a large amount in the living body, particularly by lysozyme which is an enzyme present in the living body such as a mucous membrane. There are no toxicity or persistence issues. Even if the chitin remains in the uterus, since this chitin is a substance safe for living organisms, which is also recognized as a food additive, the meat of livestock to which the estrus and ovulation inducers are administered is used for food. However, there is an advantage that there is no particular problem.

[0024]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 10 kg of squid soft shell was pulverized by a feather mill (passing through a 5 mm screen), and the pulverized product was poured into a 1N aqueous solution of sodium hydroxide and kept at 90 ° C. for 3 hours. The solution was washed with water until it became acidic, and then immersed in a 0.1N aqueous hydrochloric acid solution at room temperature for 2 hours.

Next, after washing with water again until the washing liquid becomes neutral, it is further kept in a 1N aqueous solution of sodium hydroxide (90 ° C.) for 1 hour, washed thoroughly with water, and dried in an oven (50 ° C.) for 10 hours. After drying for 1.8 hours, 1.8 kg of purified chitin having a β-type crystal structure derived from squid was obtained. As a result of measurement by elemental analysis,
The ratio (degree of deacetylation) of D-glucosamine in the constituent units of the purified chitin was 5%.

This purified squid chitin is pulverized to a maximum particle size of 3
Fine powdery chitin having a particle size of 2 μm and an average particle size of 8.2 μm (particle size distribution measurement: SK laser PRO-7000S, manufactured by Seishin Enterprise Co., Ltd., measured in ethanol) was obtained.

The obtained finely powdered chitin is sterilized with ethylene oxide gas, and a predetermined amount of sterile physiological saline is added.
This fine powdered chitin was uniformly dispersed by ultrasonic irradiation to produce an estrous and ovulation inducer having a chitin concentration of 6 mg / ml.

50 ml of this estrus and ovulation inducer (containing 300 mg of chitin) was administered into the uterus of a cow that had formed a corpus luteum and exhibited no estrus by using an intrauterine drug infusion catheter. On the 5th day after administration, the cervical mucus viscosity decreased, and favorable estrus signs such as leakage of clear mucus from the vulva were observed.
In addition, when the intrauterine mucus was observed on the 5th day after administration, no remaining chitin was observed.

After washing the uterus once, 50 ml of the estrus and ovulation inducer (containing 300 mg of chitin) produced above was injected into the uterus of a cow having excreted pus and developing endometritis. Was administered using an intrauterine drug infusion catheter. Before administration, the cow did not become pregnant (repeat bleeder) even after 5 artificial inseminations, and the luteal body had been formed. On the 5th day after administration of the estrus and ovulation inducer as described above, In addition, the cervical mucus viscosity decreased, and favorable estrus signs such as leakage of transparent mucus from the vulva were observed.
Observation of the uterine mucus excreted on the 5th day after the administration revealed that the administered chitin had disappeared, no accumulation of pus was observed, and the endometritis was cured.

Further, the estrus and ovulation-inducing agents produced as described above were introduced into the uterus of four normal cows in the luteal phase, each containing 90 mg and 150 mg of chitin as an active ingredient, respectively.
The dose was 300 mg and 600 mg. As a result, in cows to which 90 mg and 150 mg of chitin were respectively administered into the uterus, weak estrus signs such as uterine contraction were observed, but did not reach ovulation. In contrast, in cows to which 300 mg and 600 mg of chitin were respectively administered intrauterinely, the viscosity of cervical mucus decreased 5 days after administration, and clear mucus leaked from the vulva, and no normal side effects were observed. Sufficient signs of estrus were seen, and she was pregnant after artificial insemination.

Example 2 10 kg of cuticles of crabs were immersed in 2N hydrochloric acid at room temperature for 1 day, replaced with fresh 2N hydrochloric acid, immersed again at room temperature for 1 day to demineralize, and then sufficiently washed. This decalcified crab cuticle is placed in 1N sodium hydroxide, boiled for 6 hours with occasional stirring, replaced with new 1N sodium hydroxide, and boiled again for 24 hours to perform decalcification and deproteinization. Cuticles were prepared.

The cuticle of the crab which has been decalcified and deproteinized is refluxed for 5 hours in 95% ethanol to remove pigments and the like, and 2.9 kg of purified chitin having an α-type crystal structure derived from crab shells is obtained. Prepared. As a result of measurement by elemental analysis,
The ratio of D-glucosamine to the constituent units of the purified chitin (degree of deacetylation) was 15%.

The purified chitin is pulverized to a maximum particle size of 180.
μm, average particle size 45 μm (particle size distribution measurement: SK laser P
RO-7000S, manufactured by Seishin Enterprises Co., Ltd., measured in ethanol) to obtain finely powdered chitin.

10 g of the obtained finely powdered chitin and 15 g of lactose (manufactured by Wako Pure Chemical Industries, Ltd.) finely pulverized to a particle size of 250 μm or less as a dispersing aid were mixed for 1 minute using a tabletop powder mixer. (Weight mixing ratio: 40:60), dispensed in a predetermined amount, and sterilized with ethylene oxide gas to produce a estrus and ovulation inducer of the suspension type before use.

This estrus and ovulation inducer was administered to cows with follicular cysts. Before administration, the cow received artificial insemination one month before but did not become pregnant, and as a result of diagnosis, a follicular cyst of about 7 cm was formed. At this time, the amount of progesterone, a gonad hormone, was 0%. 0.4 ng / ml. After surgically destroying the follicular cyst, a gonadotropin (trade name: Gonatropin,
As a result of administering 15000 units of Teikoku Organs Co., Ltd., the amount of progesterone was increased to 6.2 ng / ml, and the formation of corpus luteum was observed. (Containing 300 mg of chitin)
Was added thereto, and the mixture was shaken gently to suspend the chitin, which was then administered to the uterus. As a result, weak signs of estrus such as uterine contraction were observed, but they did not reach ovulation.
mg (containing 600 mg of chitin).

As a result, on day 5 after the administration, the viscosity of the cervical mucus decreased, and normal and sufficient estrus signs such as leakage of clear mucus from the vulva were observed, and ovulation was also performed. In addition, the amount of progesterone decreased to 0.3 ng / ml,
No subsequent follicular cysts developed.

For the purpose of in utero repair and for pregnancy at an early stage after parturition, the above-mentioned suspension estrus and ovulation inducer 1500 mg (chitin 600 mg) prepared as above is used.
) Was added, and the chitin was suspended by gently shaking and administered to the uterus of a cow 30 days after parturition.

As a result, on day 6 after the administration, the cervical mucus viscosity decreased, and normal and sufficient signs of estrus such as leakage of transparent mucus from the vulva were observed. pregnant.

Further, 50 mg of physiological saline was added to 1500 mg of the oestrus and ovulation inducer (containing 600 mg of chitin) produced above, and the mixture was shaken gently to suspend chitin. Administered.

As a result, on day 5 after administration, normal and sufficient signs of estrus such as a decrease in the viscosity of cervical mucus and leakage of clear mucus from the vulva were observed. pregnant.

[0042]

As described above, according to the first aspect of the present invention, since chitin is used as an active ingredient, not only normal females such as cattle, horses and pigs but also genital dysfunctions are present. Intravenous female animals can also respond to the reproductive organs mildly when administered to the uterus, induce estrus and ovulation very similar to spontaneous estrus, and have reproductive disorders such as endometritis and follicular cysts. It can also promote the healing of livestock with reproductive illness that causes estrus, and then also induce estrus and ovulation, very similar to natural estrus.

In addition, since chitin reacts mildly with the genital organs, it does not cause any side effects even if it exceeds the effective dose, and in particular, its constituent unit, lysozyme, which is an enzyme present in the body such as mucous membrane. Since it is decomposed into N-acetyl-D-glucosamine and D-glucosamine which are present in large amounts in the living body, there is no problem of persistence. Even if the chitin remains in the uterus, the chitin is a biologically safe substance that is also recognized as a food additive, and there is no particular problem in terms of safety even if the meat of the livestock is eaten.

In addition, since estrus is induced in about 4 to 7 days after administration of the effective dose, estrus can be induced systematically, and therefore, postpartum for the purpose of intrauterine repair. Early pregnancy, shortening of the delivery interval, synchronization of the estrous cycle for artificial insemination and embryo transfer to a large number of livestock at once, improvement of insemination rate and success rate of embryo transfer by inducing good estrus, etc. Can be achieved. Therefore, a significant cost reduction can be achieved more effectively.

According to the second aspect of the present invention, the chitin is in the form of a fine powder having a particle size of 500 μm or less, and the chitin that does not dissolve in water can be uniformly suspended in a physiologically acceptable dispersion medium. Therefore, good fluidity can be imparted, so that the estrus and ovulation inducer can be easily administered into the uterus using an injection device such as an intrauterine drug injection catheter. In addition, since it is in the form of a fine powder, the contact area between chitin and the genital tract can be increased, so that a small amount can exert a sufficient effect, and the receptivity to the enzyme can be improved. The remaining period can be prevented.

According to the third aspect of the present invention, since the chitin has a relatively loose β-type crystal structure, the chitin swells upon contact with a physiologically acceptable dispersion medium or body fluid, and the chitin and the genital organs Since the contact area with the metal can be made larger, a smaller amount of the metal can exert a sufficient effect. Furthermore, since the receptivity to the enzyme can be improved, it is possible to more effectively prevent chitin from remaining in the uterus for a long period of time.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 31/715 A61K 9/14 C08B 37/08 CA (STN)

Claims (3)

(57) [Claims] 1. An estrus and ovulation inducer comprising chitin as an active ingredient. 2. The estrous and ovulation inducer according to claim 1, wherein the chitin is in the form of a fine powder having a particle size of 500 μm or less. 3. The estrous and ovulation inducer according to claim 1, wherein the chitin has a β-type crystal structure.