JPWO2020116361A1 - Sperm function improving agent in livestock - Google Patents

Abstract

An object of the present invention is to provide a means for improving sperm function in male livestock and improving fertilization rate of livestock. When 5-aminolevulinic acid is added to the feed and administered to male domestic animals, the malformation rate of sperm derived from male domestic animals is reduced.

Description

The present invention relates to a sperm function improving agent in livestock, and more particularly to a sperm function improving agent in livestock, which comprises 5-aminolevulinic acid (5-ALA) or a derivative thereof or a salt thereof.

In order to produce excellent meat, calves with excellent meat quality, healthy and well-weighted pedigree are bred as beef cattle. However, recently in Japan, it has been reported that the conception rate has decreased by about 10% in 20 years for beef cattle and about 20% in 20 years for dairy cows (see, for example, Non-Patent Document 1). ..

Semen for artificial insemination derived from cattle with a specific pedigree is used for breeding on a nationwide scale, and for cattle used for mating and artificial insemination, livestock improvement is performed to confirm the presence or absence of diseases and the presence or absence of impaired reproductive function. Breeding tests are required by the breeding method, and blood and semen of the target cows are collected. The collected sperm are tested for sperm malformation rate, etc., but even if the sperm is a bovine sperm with an excellent pedigree and there is no problem with the amount of semen, the rate of sperm malformation increases. It is said that there is.

The cause of teratogenic azoospermia in cattle has not yet been clarified. For example, in the case of human males, administration of a compound having an estradiol action such as beta-estradiol 3-benzoate causes malformation of sperm cells and detachment from the epithelium (see, for example, Non-Patent Document 2), and testicular toxicity. Although cases of disability due to illness have been reported, little studies have been made on whether the same reason is found in bulls.

On the other hand, in chickens as well, since the 1960s, there has been increasing interest in artificial insemination in studies aimed at improving feed efficiency (see, for example, Non-Patent Document 3). Recently, the production and production of brand chickens characterized by richness and umami have been promoted, and studies have been conducted on efficient methods of artificial insemination. Etc. have been reported (see, for example, Non-Patent Document 4).

On the other hand, 5-aminolevulinic acid (5-ALA) is known as an intermediate in the tetrapyrrole biosynthetic pathway that is widely present in animals, plants and fungi. In animals, it is biosynthesized from succinyl-CoA and glycine by aminolevulinic acid synthase. It is synthesized and biosynthesized from glutamate in plants by a three-step enzymatic reaction. The reaction from ALA synthesis to protoporphyrin IX is shared with the heme synthesis system, that is, protoporphyrin IX is a common substrate for chlorophyll and heme synthesis, while coordination of ^{Fe 2+ produces heme.} When Mg ²⁺ is coordinated, Mg-ProtoIX is synthesized and then proceeds to the chlorophyll synthesis pathway.

It has been reported that 5-ALA promotes the growth of pigs (see, for example, Patent Document 1). In addition, there is a report on a male infertility treatment agent for mammals (see, for example, Patent Document 2), but such a report is caused by male infertility and erectile dysfunction caused by oligospermia and asthenozoospermia. This is a report on the effect on male infertility, and concrete studies have been made on the actual weight and weight ratio of rat sperm sac, the effect on human erectile dysfunction, and the motility and survival rate of human sperm. , The relationship with the improvement of sperm function in domestic animals has not been investigated.

Japanese Unexamined Patent Publication No. 2003-40770 International Publication WO2009 / 139156 Pamphlet

LIAJ News No. 147, 2-5, 2014 Mol Cell Endocrinol 2001; 178: 161-168 Yoshimi Nakagawa, based on recent test results, Okayama Prefectural Poultry Farm, Okayama Livestock News 1960.09 Establishment of optimal freezing and thawing method for new brand chicken "Fuji Kogun chicken" and improvement of artificial insemination method, new agricultural technology No. 616, 2016

An object of the present invention is to provide a means for improving sperm function such as malformed sperm disease in male livestock and a means for improving the fertilization rate of livestock.

The present inventors have repeated various trials and errors to improve the fertilization rate of livestock, but when an improved diet containing 5-aminolevulinic acid is administered to bulls, the sperm malformation rate decreases. I found that. In addition, when an improved sample containing 10 ppm of 5-aminolevulinic acid in the feed was prepared and the improved sample was ingested at 150 g / day for the rooster, the sperm malformation rate of the rooster decreased and the sperm density increased. Was confirmed, and the present invention was completed.

（式中、Ｒ^１は、水素原子又はアシル基を表し、Ｒ^２は、水素原子、直鎖若しくは分岐状アルキル基、シクロアルキル基、シクロアルケニル基、アリール基又はアラルキル基を表す）
［２］奇形精子率が減少することを特徴とする、上記［１］記載の精子機能改善剤。
［３］家畜が雄牛又は雄鶏であることを特徴とする、上記［１］又は［２］記載の精子機能改善剤。
［４］ 精子機能の改善が雄牛における精子の前進運動率の向上であることを特徴とする、上記［３］記載の精子機能改善剤。
［５］精子の形態の正常率が５％未満の雄牛に投与することにより、精子の形態の正常率の数値が５倍以上となる改善が得られることを特徴とする上記［３］記載の精子機能改善剤。
［６］精子の形態の正常率の数値が５倍以上となる改善が、投与開始後５０日以内に得られることを特徴とする上記［５］記載の精子機能改善剤。
［７］精子の形態の正常率が６５％以上８０％未満の雄牛に投与することにより、精子の形態の正常率が５％以上増加する改善が得られることを特徴とする上記［３］記載の精子機能改善剤。
［８］雄鶏における精液中の精子の密度が増加することを特徴とする上記［３］記載の精子機能改善剤。
［９］精子の密度の増加が、投与開始後３カ月以内に得られることを特徴とする上記［８］記載の精子機能改善剤。
［１０］上記［１］〜［９］のいずれか記載の精子機能改善剤を添加した家畜用飼料。
［１１］上記［１］〜［９］のいずれか記載の精子機能改善剤を、家畜の雄に経口投与することを特徴とする精子の機能改善方法。
［１２］上記［１］〜［９］のいずれか記載の精子機能改善剤を、体重１ｋｇあたり、アミノレブリン酸換算で０．０１〜１．５ｍｇ／日を経口投与することを特徴とする上記［１１］記載の精子の機能改善方法。
［１３］上記［１］〜［９］のいずれか記載の精子機能改善剤を投与した家畜から採取した精子を用いて、家畜の受精率を向上させる方法。That is, the present invention is specified by the following matters.
[1] A sperm function improving agent in livestock containing the compound represented by the following formula (I) or a salt thereof.

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group or an aralkyl group).
[2] The sperm function improving agent according to the above [1], which is characterized by a decrease in the malformed sperm rate.
[3] The sperm function improving agent according to the above [1] or [2], wherein the livestock is a bull or a rooster.
[4] The sperm function improving agent according to the above [3], wherein the improvement of sperm function is an improvement in the forward motility of sperm in a bull.
[5] The above description [3], wherein administration to a bull having a normal rate of sperm morphology of less than 5% can improve the value of the normal rate of sperm morphology by 5 times or more. Sperm function improving agent.
[6] The sperm function improving agent according to the above [5], wherein an improvement in which the value of the normal rate of sperm morphology is 5 times or more is obtained within 50 days after the start of administration.
[7] The above-mentioned [3], which is characterized in that administration to a bull having a normal rate of sperm morphology of 65% or more and less than 80% can improve the normal rate of sperm morphology by 5% or more. The sperm function improving agent described.
[8] The sperm function improving agent according to the above [3], wherein the density of sperm in semen in a rooster is increased.
[9] The sperm function improving agent according to the above [8], wherein an increase in sperm density is obtained within 3 months after the start of administration.
[10] A livestock feed to which the sperm function improving agent according to any one of the above [1] to [9] is added.
[11] A method for improving sperm function, which comprises orally administering the sperm function improving agent according to any one of the above [1] to [9] to male domestic animals.
[12] The sperm function improving agent according to any one of the above [1] to [9] is orally administered at 0.01 to 1.5 mg / day in terms of aminolevulinic acid per 1 kg of body weight [12]. 11] The method for improving sperm function according to the above method.
[13] A method for improving the fertilization rate of livestock by using sperms collected from livestock to which the sperm function improving agent according to any one of the above [1] to [9] has been administered.

Furthermore, the present invention includes the following aspects.
(1) The ALA or a derivative thereof according to any one of the above [1] to [9] used for improving sperm function, or a pharmacologically acceptable salt thereof.
(2) Use of ALA or a derivative thereof according to any one of the above [1] to [9] or a pharmacologically acceptable salt thereof for improving sperm function.
(3) Use of ALA or a derivative thereof according to any one of the above [1] to [9] or a pharmacologically acceptable salt thereof in the production of a sperm function improving agent.
(4) A method for improving sperm function by administering to domestic animals a therapeutically effective amount of ALA or a derivative thereof or a pharmacologically acceptable salt thereof according to any one of the above [1] to [9].

By administering the sperm function improving agent of the present invention to livestock, it is possible to improve the sperm function of livestock, for example, to reduce the sperm malformation rate, and in particular, to prevent and treat malformation sperm disease. The effect of improving the fertilization rate of livestock can be obtained. In the present invention, the fertilization rate means the rate at which fertilization is actually established when an egg and a sperm are present and an attempt is made to fertilize by fusing the sperm nucleus and the egg nucleus.

The livestock sperm function improving agent of the present invention includes a compound represented by the above formula (I) or a salt thereof (hereinafter, these may be collectively referred to as "ALAs") as an active ingredient. The livestock in the present invention is not particularly limited, and examples thereof include cows, pigs, sheep, and horses raised by humans to use meat and milk, and further to use meat and eggs. Human-reared poultry such as chickens, ducks, quail, turkeys, and ducks can be included.

The above-mentioned cattle are not particularly limited as long as they are domesticated cattle belonging to the subfamily Bovinae and Bos, and the domesticated cattle include meat cattle, dairy cattle, and dairy cattle. Cattle can be exemplified, specifically, Aberdeen Angus, Hereford, Shorthorn, Charolais, Limousine, Japanese Black, Japanese Brown, Japanese Short Angle, Japanese Black, etc. Beef cows; dairy cows such as Brown Swiss, Gandhi, Holstein, Jersey, Kelly, Milking Devon, Norwegian Red; cows of these hybrids; but pure Japanese Black cattle, Japanese brown cattle, Japanese short-horned cattle, and non-horned Japanese cattle, which are classified as Japanese black cattle, are preferable, and Japanese Black cattle can be preferably mentioned. The age at which the sperm function improving agent of the present invention is applied is not particularly limited as long as the cow is of an age at which sperm can be used for reproduction and needs to improve sperm function. Examples of cattle that need to improve their function include bulls that have sperm malformations and are suffering from or may be suffering from bovine malformation sperm. Cattle that need to be prevented or treated can be mentioned.

The above chickens are not particularly limited as long as they are domesticated chickens belonging to the subfamily Kiji and the genus Yakei (Gallus), and the domesticated chickens are roughly classified into egg meat combined breeds, egg breeds, and meat breeds. Examples of the egg-combined species include horizontal spot Plymouth Rock species, Rhode Island Red species, and New Hampshire species, but in reality, strains specialized for eggs or meat are used. Examples of egg species include white Leghorn species, black Minorca species, and their hybrids, and examples of meat species include Cornish species and white Plymouth Rock species produced by mutation of horizontal plymouth rock species. A broiler or the like, which is a hybrid first generation (F1) obtained by crossing a white Cornish male and a white Plymouth Rock female, can be exemplified. In addition, as Japanese chickens, Aomori Shamo Rock, Okuku Jisha, pure Nagoya Cochin, Awao chicken, Satsumadori chicken, etc., which have been improved and crossed from Japanese native species in order to pursue richness and taste, etc. It can include many native breeds such as domestic brand chickens, Shamo chickens, bantams, and silkie chickens. The age at which the sperm function improving agent of the present invention is applied is not particularly limited as long as the chicken is of an age at which sperm can be used for reproduction and needs to improve sperm function. Examples thereof include chickens with malformations and chickens with low sperm density in semen.

Improvements in sperm function in the present invention include a decrease in malformed sperm rate, prevention / treatment of malformed sperm disease, improvement in sperm forward motility, and increase in sperm concentration (sperm density) in semen. Therefore, the sperm function improving agent of the present invention is not particularly limited as long as it is an improving agent capable of improving the sperm function of domestic animals, but is used for the prevention and treatment of malformed sperm reducing agents and malformed sperm disease. A prophylactic / therapeutic agent for malformed sperm disease, a sperm motility improving function improving agent capable of increasing the proportion of sperm moving forward, and an increase in sperm concentration capable of increasing the concentration of sperm in semen. Agents can be included.

The above-mentioned malformation sperm disease is one of the sperm-forming disorders related to the function of producing sperm in the testis (testis), and the proportion of sperm in normal form (normal rate of sperm morphology) in domestic sperm is less than 80%. That is, the pathological condition when the sperm malformation rate is 20% or more can be mentioned.

As for the structure of sperm, the head having nuclear DNA which is genetic information from the tip; the neck of the connecting part between the head and the tail; mitochondria surround the outside of microtubules and peripheral fibers in a spiral shape to form a mitochondrial sheath. A structure can be mentioned that includes a middle piece of mitochondria; a flagellar main part consisting of an axoneme extending from a centriole; a short end part and a terminal part without a tail sheath.

Examples of the site for determining whether or not the sperm morphology is normal include the head, neck, middle piece, flagellar main part, and / or terminal part. Regarding the sperm head, it is pointed, spherical, distorted, small, huge, dwarfed, deformed, round, with respect to the rounded outer shape called normal egg-shaped or flagellar-shaped. In the case of two heads, etc .; for the neck, small, short, deformed, cut (posterior defect in the middle piece part), flexion, etc .; for the middle piece, short, thin, deformed, etc. In the case of short / small, long, bent, curved, 2 tails, etc .; for the main part of the flagella, short, thin, 2 or more, bent, short / small, long, bent, 2 tails, etc .; for the terminal part, In the case of roundness, defect, etc .; it can be judged that the sperm morphology is not normal and is malformed. In particular, the malformation of the sperm head may be emphasized because it is more closely associated with the decline in sperm fertilization function. In addition, in the case of a complex malformation in which malformations are observed in a plurality of sites, it can be classified as a malformation in a site closer to the head.

As a method for determining whether or not a sperm is malformed, a method of observing under a microscope to determine whether or not the sperm is malformed is common, and the presence or absence of malformation in each part of the sperm can be easily determined under a microscope. A method of making an observation and making a visual judgment can be preferably mentioned.

When observing under the microscope, it is preferable to perform hematoxylin / eosin staining, Giemsa staining and the like before observing. In order to prevent the occurrence of secondary malformations, a method of smearing sperm on a slide glass after fixing glutaraldehyde and performing Giemsa staining can be preferably mentioned.

Examples of the method for collecting bovine sperm include semen collection by artificial vaginal method, intratesticular sperm collection method, microscopic epididymal sperm collection method, percutaneous epididymal sperm collection method, and sperm concentration washing method. can. As a method of collecting sperm from chickens, a method of massaging the abdomen or back of the test chicken, pinching while lightly pressing both sides of the cloaca, and collecting semen can be mentioned.

It is possible to observe the sperm morphology and the like and calculate the (advanced) motility of the collected semen, and also to calculate the normal rate of the morphology of the sperm and the like thawed after the sperm is frozen and stored. The time to determine the sperm morphology is not particularly limited, but for example, immediately after sperm collection; after transportation to the inspection site; after addition of phosphate buffered saline, centrifugation at 500 to 1000 g for 3 to 7 minutes. 3 times; immediately after thawing; can be illustrated, but in consideration of economic cost and the like, it is preferable to observe under a microscope immediately after collecting sperm to determine the morphology of sperm, and exercise. It is desirable to perform freezing treatment only for those that do not have problems such as rate and malformation rate.

In the present invention, when it is determined that malformed sperm (sperm malformation rate) decreases, the proportion of malformed sperm decreases and the proportion of normal sperm morphology (normal rate of sperm morphology) increases. There are cases. For example, the malformation rate may be reduced by 1%, preferably 3% within 3 months after the start of administration of the sperm function improving agent of the present invention as compared with that before the start of administration.

In particular, when the sperm malformation rate is reduced to the extent that azoospermia in cattle is treated, the following aspects can be mentioned. That is, when the normal rate of sperm morphology increases, the sperm function improving agent (cow) of the present invention is applied to a bull whose sperm morphology normal rate is less than 5% (sperm malformation rate is 95% or more). When a feed containing a preventive or therapeutic agent for sperm malformation) is administered, the value of the normal rate of sperm morphology collected after administration is 5 times or more, preferably 7 times or more, more preferably 10 times or more, and further. It can be preferably 15 times or more, and specifically, when a feed containing a sperm function improving agent is administered to a bull having a normal rate of sperm morphology of 1%, it is collected after administration. It can be mentioned that the value of the normal rate of sperm morphology is 5% or more, preferably 7% or more, more preferably 10% or more, still more preferably 15% or more. The improvement due to such an increase should be a value obtained on any day within 50 days, preferably within 40 days, and more preferably within 30 days after the start of continuous administration of the feed containing the sperm function improving agent of the present invention. Is desirable.

As another aspect of increasing the normal rate of sperm morphology, the sperm function improving agent of the present invention (prevention of bovine malformation sperm disease) is used in bulls whose normal rate of sperm morphology is 65% or more and less than 80%. Alternatively, when a therapeutic agent) or feed is administered, the normal rate of sperm morphology collected after administration is increased by 5% or more, preferably 7% or more, more preferably 10% before administration. .. Such an increase may be achieved on any day after 50 days, preferably after 55 days, and more preferably after 60 days after the start of continuous administration of the sperm function improving agent or feed of the present invention. desirable.

As a method for determining the sperm motility, a conventionally known method can be mentioned, but a visual determination using a microscope can be preferably mentioned. Specifically, a method of determining whether or not each sperm is stationary and determining that the sperm is a moving sperm when it can be confirmed that the sperm is moving can be mentioned.

The presence or absence of the above malformation and the forward motility rate can be determined by using an automatic analyzer such as a semen motility analyzer (Computer Assisted Sperm Analysis). It can also be measured and quantified.

Examples of the case where the forward movement rate is improved include a case where the probability of sperm actively moving forward is increased by 45 μm or more per second in the observed sperm group. can.

The method for measuring the sperm density in semen is not particularly limited as long as it is a known method, but a method of calculating the number of sperms in semen using a hemocytometer and a microscope and calculating the number of sperms / mL. Can be exemplified.

Examples of the increase in sperm density include cases where the density of sperm in the observed semen before administration is 1.3 or more, and 1.5 months after the start of administration. It is preferable to increase to 1.35 or more within.

Among the above ALAs, 5-ALA or a salt thereof in which both ^{R 1} and R ² of the formula (I) are hydrogen atoms can be preferably exemplified. 5-ALA is a type of amino acid also called δ-aminolevulinic acid. As the 5-ALA derivative, R ¹ of the formula (I) is a hydrogen atom or an acyl group, and R ² of the formula (I) is a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, or a cycloalkenyl. Compounds other than 5-ALA, which are groups, aryl groups or aralkyl groups, can be mentioned.

Examples of the acyl group in the formula (I) include linear or branched alkanoyl groups having 1 to 8 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl and benzylcarbonyl groups. , Benzoyl, 1-naphthoyl, 2-naphthoyl group and other aroyl groups having 7 to 14 carbon atoms can be mentioned.

Examples of the alkyl group in the formula (I) include linear or branched groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl and octyl groups. Alkyl groups having 1 to 8 carbon atoms can be mentioned.

Examples of the cycloalkyl group in the formula (I) include cycloalkyl groups having 3 to 12 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl groups.

Examples of the cycloalkenyl group in the formula (I) include cyclopropenyl (eg, 1-cyclopropenyl), cyclobutenyl (eg, 1-cyclobutenyl), cyclopentenyl (eg, 1-cyclopentenyl), cyclohexenyl (eg, 1-cyclo). Cycloalkenyl groups having 3 to 12 carbon atoms such as hexenyl), cycloheptenyl (eg, 1-cycloheptenyl), cyclooctenyl (eg, 1-cyclooctenyl), cyclodecenyl (eg, 1-cyclodecenyl), cyclododecenyl (eg, 1-cyclododecenyl) group. Can be mentioned.

Aryl groups in formula (I) include phenyl, naphthyl (eg, 1-naphthyl), anthryl (eg, 1-anthryl), phenanthryl (eg, 1-phenanthryl), pyrenyl (eg, 1-pyrenyl) groups and the like. Aryl groups having 6 to 18 carbon atoms can be mentioned.

As the aralkyl group in the formula (I), the aryl moiety can be exemplified in the same manner as the above aryl group, and the alkyl moiety can be exemplified in the same manner as the above alkyl group. , Trityl, naphthylmethyl, naphthylethyl group and other aralkyl groups having 7 to 15 carbon atoms can be mentioned.

As the 5-ALA derivative ^{, a compound in which R 1} is a formyl, acetyl, propionyl, butyryl group or the like, or a compound in which R ² is a methyl, ethyl, propyl, butyl, pentyl group or the like is preferable, and the above R ^{The combination of 1} and R ² can preferably exemplify a combination of formyl and methyl, acetyl and methyl, propionyl and methyl, butyryl and methyl, formyl and ethyl, acetyl and ethyl, propionyl and ethyl, butyryl and ethyl, and the like. ..

ALAs may act as an active ingredient in the state of 5-ALA of formula (I) or a derivative thereof in vivo, and may be administered in various salt forms in order to increase solubility depending on the form of administration. In addition, a prodrug (precursor) that acts after being metabolized by an enzyme in the body in order to improve gastrointestinal absorption, tissue migration, tissue selectivity, chemical stability, etc., or to reduce side effects. ) (For example, ester). For example, examples of the salt of 5-ALA and its derivative include a pharmacologically acceptable acid addition salt, metal salt, ammonium salt, organic amine addition salt and the like. Examples of the acid addition salt include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate and other inorganic acid salts, formate, acetate, propionate and toluenesulfonic acid. Salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Organic acid addition salts can be exemplified. Examples of the metal salt include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium and calcium salt, and metal salts such as aluminum and zinc. Examples of the ammonium salt include alkylammonium salts such as ammonium salt and tetramethylammonium salt. Examples of the organic amine salt include each salt such as triethylamine salt, piperidine salt, morpholine salt, and toluidine salt. These salts can also be used as a solution at the time of use.

Among the above ALAs, desirable ones are 5-ALA and various esters such as 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester and 5-ALA pentyl ester. , And these hydrochlorides, phosphates, sulfates, and 5-ALA hydrochlorides and 5-ALA phosphates can be particularly preferably exemplified.

The above ALAs can be produced by any known method of chemical synthesis, production by microorganisms, and production by enzymes. In addition, the above ALAs may form a hydrate or a solvate, and either one may be used alone or two or more thereof may be used in combination as appropriate.

As a mode of use of the sperm function improving agent of the present invention, it may be made into granules, granules, powders, or solutions and mixed with a feed normally given to cows, but powders, granules, etc. Orally-administered preparations such as fine granules and tablets; they can also be used in the form of preparations such as liquid preparations and injection preparations such as dissolution-type powders at the time of use. Examples of the administration method of the above-mentioned preparation include oral administration, intravenous administration, intramuscular administration, local administration, peritoneal administration, transdermal administration, transrectal administration and the like.

The dose of the sperm function improving agent containing ALAs of the present invention in the case of oral administration is not particularly limited as long as the effects of the present invention are exhibited, but in the case of bulls, in terms of aminolevulinic acid per 1 kg of body weight, 0.01 to 1.5 mg / day, preferably 0.05 to 1 mg / day, more preferably 0.075 to 0.5 mg / day, still more preferably 0.08 to 0.3 mg / day. In the case of roosters, an amount containing 0.2 to 25 ppm, preferably 2 to 15 ppm, more preferably 8 to 12 ppm in terms of aminolevulinic acid can be mentioned in the sample to be ingested at 150 g / day. That is, the intake in a rooster with an average body weight of about 2500 g to 3000 g (2.5 kg to 3 kg) is 0.03 mg / day to 3.75 mg / day, preferably 0.3 mg / day to 2.25 mg / day. More preferably, 1.2 mg / day to 1.8 mg / day can be mentioned, and such a value is 0.01 mg / day to 1.5 mg / day, preferably 0. It corresponds to 1 mg / day to 0.9 mg / day, more preferably 0.4 mg / day to 0.72 mg / day.

When the sperm function improving agent of the present invention is added to the feed and used, the feed feeding method includes a separate feeding method in which the feed is fed twice or more a day, or the feed is freely ingested without a specific feeding time. Although feeding methods such as continuous feeding can be mentioned, a separate feeding method in which feeding is performed twice a day in the morning and evening is preferable, and the feeding time in the morning is from 6:00 to 11:00, preferably 8:00. From 10:00 to 10:00 can be exemplified, and as the feeding time in the evening, 15:00 to 20:00, preferably 16:00 to 18:00 can be exemplified. The feed to which ALA is added is not particularly limited as long as it is a feed generally used for feeding cows and chickens, and corn, soybean meal, alfalfa, rice husk, wheat bran, rice bran, etc. Examples thereof include feeds for cattle and chickens prepared from conventionally known feed raw materials such as oats hay, cotton seed oil meal, bone meal, lime, dicalcium phosphate, sodium chloride, urea and molasses.

The sperm in the normal form obtained by the present invention can be subjected to fertilization in which a sperm nucleus and an egg nucleus are fused, and examples of the fertilization method include artificial insemination and in vitro fertilization. As the in vitro fertilization, the above fertilization can be artificially performed outside the body, and as a method of in vitro fertilization, eggs and sperms are fertilized in a culture solution to create a state close to natural fertilization outside the body. So-called conventional IVF, a method performed by co-culturing eggs and sperm taken out of the body, a transparent band opening method, an intraepithelial sperm injection method, an intracytoplasmic sperm injection method (ICSI), etc. Although the micro-fertilization method can be mentioned, the conventional IVF is preferable in terms of cost. In the case of chickens, a method of injecting the stock solution or dilution of the collected semen or the stock solution or dilution of the semen thawed after cryopreservation into the vagina of the hen can be exemplified.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

[Reference example 1]
A1 (1 year and August at the start of the experiment, weight about 490 kg), A2 (1 year and June at the start of the experiment, weight about 490 kg), A3 (1 year and August at the start of the experiment, weight about 530 kg), and A4 (start of the experiment) We examined four male Japanese black-haired cows weighing about 490 kg at the age of 1 year and June. Before administration of the feed containing the therapeutic agent of the present invention (ALA-containing feed), an ALA-free feed for breeding Japanese beef (manufactured by Nishinihon Kumiai Feed Co., Ltd.) and a roughage such as Otsuhei were allowed to be freely ingested. The average feed intake was 3 to 3.5 kg for Wagyu breeding feed and 4.0 kg to 5.5 kg for roughage such as Otsuhei per day.

Sperm morphology was observed twice for each of the above four bulls immediately before administration of the ALA-containing feed. The sperms collected from each bull by the artificial vaginal method were transported to the laboratory, and then washed 3 times with phosphate buffered saline (700 g, 3 times centrifugal washing for 5 minutes). In this test, in order to prevent the occurrence of secondary malformations, sperm were smeared on a slide glass after glutaraldehyde was fixed, and Giemsa staining was performed.
Observation of stained specimens was carried out on 100 or more sperms per specimen. In addition, photography for observation was carried out on 100 or more sperms per sample. Photography (CCD camera DP73, Olympus) was performed at 1000 times, which is the maximum magnification of the microscope, and sperm contours were sharpened using a phase inspector. The malformation rate was accurately determined by copying a magnified image of the photograph taken on a PC. The results are shown in Table 1 below.
Regarding the classification of malformations at the time of observation, if complex malformations are found, the malformation rate is calculated as a higher-level site. For example, when malformations were found in the head and the middle piece, it was regarded as a head malformation.

(result)
For A2 cattle, the normal rate of sperm morphology was as low as 1% on December 20, 2017, before administration of the therapeutic agent of the present invention. The malformation rate of the head was 94%. For other A1, A3, and A4 cows, the normal rate of sperm morphology was 72-79%, less than 80%. The head malformation rate was 14% to 22%. In addition, cervical malformations, middle piece malformations, flagellar main / terminal malformations were 0 to 4% in all A1 to A4 bulls, and sperm malformations were mainly in the form of the head. There were many.

[Example 1]
When feeding the above-mentioned 5-ALA-free Japanese beef breeding feed and roughage such as oatshay to each of the four cows of A1, A2, A3, and A4 having an average weight of 500 kg twice a day in the morning and evening, respectively. A feed containing 1% of aminolevulinic acid phosphate was sprinkled on the feed in a top dress at a rate of 5 g / dose and 10 g per day. The average intake of feed was 3 to 3.5 kg for Wagyu breeding feed and 4.0 kg to 5.5 kg for roughage such as Otsuhei, which were the same as before the test.

For each cow that continued to take ALA-containing feed, 16 days (January 5, 2018), 28 days (January 17, 2018), and 49 days (January 17, 2018) have passed since the start of intake of ALA-containing feed. Each bull at 63 days (February 21, 2018), 76 days (March 6, 2018), 90 days (March 20, 2018) After collecting sperm from the above, the presence or absence of head malformation, neck malformation, middle piece malformation, and main / terminal malformation of the whip was confirmed for about 100 sperm for each bull according to the procedure in Reference Example 1. The results of A2 cows are shown in Table 2 below.

(result)
As is clear from Table 2, the normal rate of sperm morphology before administration of the ALA-containing feed was 1%, and the values of the normal rate of A2 were 7% 16 days after the start of administration and 22% 28 days after the start of administration. After 49 days, it became 25%, confirming an increase of 24%. By continuing the oral intake of the ALA-containing feed, the normal rate of sperm morphology increased up to 25 times. The head malformation rate was 86% 16 days after the start of administration, 69% 28 days later, and 67% 49 days later, which were significantly lower than the 94% head malformation rate before administration.

[Example 2]
The results of A1 cows are shown in Table 3 below.

(result)
As is clear from Table 3, the normal rate of sperm morphology before administration of the ALA-containing feed was 78%, and the normal rate of A1 was 89% 16 days after the start of administration, 87% after 63 days, and 76 days. After the lapse of time, it became 89%, and after 90 days, it became 85%. By continuing the oral intake of the ALA-containing feed, the normal rate of sperm morphology collected after administration increased, and it increased from 78% of the value before administration to at least 78%. It increased by more than 7%.

[Example 3]
The results of A3 cows are shown in Table 4.

(result)
As is clear from Table 4, the normal rate of sperm morphology before administration of the ALA-containing feed was 79%, and the normal rate of A3 was 81% 16 days after the start of administration, 83% after 28 days, and 63 days. It became 84% after the lapse of time, 84% after the lapse of 76 days, and 84% after the lapse of 90 days. After the course, it increased by 5% or more from 79% of the value before administration.

[Example 4]
The results of A4 cows are shown in Table 5.

(result)
As is clear from Table 5, the normal rate of sperm morphology before administration of the ALA-containing feed was 72%, and the normal rate of A4 was 91% 16 days after the start of administration, 92% after 49 days, and 63 days. After the lapse, it became 85%, after 76 days, 89%, and after 90 days, it became 77%. By continuing the oral intake of the ALA-containing feed, the normal rate of sperm morphology collected after administration increased, and before administration. It increased by at least 5% or more from 72% of the value of.

[Example 5]
For A2, A3, and A4 cows, the sperm forward motility was calculated before and after administration of the ALA-containing feed. In each case, sperm were collected from each bull by the artificial vaginal method, washed 3 times with phosphate buffered saline (3 times centrifugal washing for 700 g for 5 minutes), fixed with glutaraldehyde, and observed after staining by Giemsa staining. Was done.
The results are shown in Table 6.

(result)
As is clear from Table 6, for A2 cattle, the value of sperm advance motility was 0% on December 20, 2017, before the feed containing the prophylactic or therapeutic agent of the present invention was administered. However, it became 20% after 16 days from the start of administration, 30% after 28 days, 20% after 49 days, and 20% after 63 days, and by continuing oral intake of ALA-containing feed, sperm advance movement of sperm The rate numbers increased from 0 up to 30%. In addition, for A3 cattle, the value of sperm advance motility was 40% on December 20, 2017, before the feed containing the preventive or therapeutic agent of the present invention was administered, but 49 days have passed. After 60%, after 63 days, it became 60%, and after 76 days, it became 50%. By continuing oral intake of ALA-containing feed, the sperm forward motility rate 28 to 76 days after the start of administration was the value before administration. Compared with, it increased by 12% to 50%, which was a remarkable increase. For A4 cattle, the sperm advance motility rate was 30% on December 20, 2017, before administration of the feed containing the preventive or therapeutic agent of the present invention, but 16 days after the start of administration. 60%, 65% after 28 days, 40% after 49 days, 40% after 63 days, and by continuing oral intake of ALA-containing feed, sperm forward movement 28 to 76 days after the start of administration Compared with the numerical value of the rate, it increased by 16% to 116% before administration, which was a remarkable increase.

[Example 6]
[Examination of chickens]
(Test chicken)
A male Plymouth Rock chicken with lateral spots about 500 days old was used as the test chicken. Eight males were used in the experiment and divided into two groups of four each, and a control group: (5-ALA-free) 4 birds and a treatment group: (5-ALA-added) 4 birds were provided.

As the 5-ALA-free basic feed used for the lateral spot Plymouth Rock, crude protein: 17% or more, crude fat: 3% or more, crude fiber: 6% or less, crude ash content: 14% or less, calcium: A compound feed for adult chicken breeding (manufactured by Chubu Feed Co., Ltd.) containing 3.5% or more, phosphorus: 0.4% or more, and ME: 2850 kcal / kg or more was used.

5-ALA was added to the above-mentioned compound feed for raising adult chickens at a concentration of 10 ppm to prepare an ALA-containing chicken feed, and 150 g of each chicken was ingested daily. That is, each chicken in the treatment group ingested 0.0015 g / day of 5-ALA.

In order to verify the effect of addition of 5-ALA to the feed on the fertility of roosters, eight test chickens were fed before the start of feeding the ALA-containing feed (before administration of ALA) (July 1-5, 2019). Sun); After 1.5 months of ALA-containing feed (1.5 months later) (August 19-26, 2019); After 3 months of ALA-containing feed (3 months) (October 1-7, 2019) The semen was collected in three periods of (day); Specifically, semen is collected from the cloaca by massaging the abdomen of the test chickens, as described in Bogdonoff et al., Poult. Sci., 33: 665-669, 1954, three times each period. Was done. That is, in the period before ALA administration, semen was collected on July 1, 3, and 5. For the period 1.5 months later, semen was collected on August 19, 22, and 26. In the period after 3 months, semen was collected on October 1, 4, and 7. Of the 8 male plymouth rock chickens, 4 in the "control group" (individual number 1-4) were fed an ALA-free basic diet for the entire period, and 4 in the "treatment group" (individual number). In 5-8), the basic diet containing no ALA was given during the period before the administration of ALA, but after July 6, the feed containing ALA was ingested. For each period, sperm motility, density, and malformation rates in semen collected from each chicken, as well as plasma testosterone levels were measured.

Examples of the measurement of sperm motility in semen include measurement of two items, the motility sperm rate of sperm in semen and the sperm rate of active forward movement. Using Computer Assisted Sperm Analysis: CASA, manufactured by HAMILTON, the motile sperm rate (Motility (Mol.)) And the sperm rate of active forward movement (Progressive Motility (Pro.M.)) were calculated. Regarding the motile sperm rate, it was determined whether or not each sperm was stationary, and the sperm whose movement could be confirmed was determined to be motile sperm. Regarding the active forward movement, sperms traveling 45 μm or more per second were determined to be active forward movements.

Similar to the above cow example, the sperm malformation rate in the semen is the highest magnification of the microscope after fixing the sperm with glutaraldehyde and smearing the sperm on a slide glass and performing Giemsa staining in order to prevent the occurrence of secondary malformation. It was carried out at 1000 times, and the outline of the sperm was made clear using a phase inspection device. The malformation rate was accurately determined by copying a magnified image of the photograph taken on a PC. If there was a malformation in the head, neck, middle piece, flagellar main part, and / or terminal part, it was determined that there was a malformation.

The sperm density (concentration) in the semen was calculated as the sperm count / mL by calculating the sperm count using a blood cell counter and a microscope.

During each period before, 1.5 months, and 3 months after the administration of ALA, blood was collected from the test chickens three times in the same manner as described above, and the testosterone concentration in plasma was measured. A testosterone EIA kit (manufactured by Funakoshi Co., Ltd.) was used to measure the testosterone concentration in plasma, and the concentration was measured according to the procedure manual of the kit.

Table 7 below shows the motile sperm rate, forward motile sperm rate, concentration, and malformation rate of sperm in semen on each collection day in each rooster.

Based on the results in Table 7 above, three data of motile sperm rate, forward motile sperm rate, density, and malformation rate of sperm obtained in each period before, 1.5 months, and 3 months after administration. The average value was calculated, and the effect of adding 5-ALA to the feed (effect of administration to roosters) is shown in Table 8 below.

(result)
As shown in Table 8 above, in all of the items of sperm motility, density and malformation rate, there were many individuals in the treated group that were found to be effective by administration as compared with the control group.

Next, in order to clarify the effect of administration, the ratio of the value after 1.5 months or 3 months to the value before administration (the value before administration is 1) in each individual is the above-mentioned motile sperm rate. , Forward-moving sperm rate, sperm density and sperm malformation rate were calculated. The results are shown in Table 9 below.

Furthermore, statistical processing was performed on the above-mentioned motile sperm rate, forward motile sperm rate, sperm density and sperm malformation rate in the control group (individual numbers 1 to 4) and the treatment group (individual numbers 5 to 8), and the administration of ALA was performed. A significant difference test was performed on the effects of sperm motility (motile sperm rate and forward motile sperm rate), concentration and malformation rate (significant difference test by Student's t-test after ANOVA analysis). The results are shown in Table 10 below.

(result)
As is clear from Table 10, the average values of motile sperm rate and forward motile sperm rate increased with the addition (administration) of 5-ALA to the feed for 3 months, but between the control group and the treatment group. No significant difference was observed in.

On the other hand, the sperm density (concentration) was higher after administration than before administration, and a significant difference was observed between the two sections, especially after 3 months (P <0.01). In addition, the sperm malformation rate was lower after administration than before administration, and a significant difference was observed between the two sections, especially after 1.5 months (P <0.05). From these results, the addition of 5-ALA to the feed (concentration in the feed: 10 ppm) does not show significant effect on sperm motility such as sperm motility and forward motility of roosters, but sperm density. And it was confirmed that it was effective with a significant difference in the sperm malformation rate.

[Reference example]
The measured values of the testosterone concentration in the plasma of each individual collected before, 1.5 months, and 3 months after the administration are shown in Table 11 below.

Table 12 below shows the results of statistical processing (ANOVA analysis) of each numerical value in Table 11 above and a significant difference test (Student's t-test) on the effect of ALA administration on plasma testosterone concentration.

(result)
In the chickens in the control group, the testosterone concentration was low in 1 of 4 chickens after administration (3 months later). In addition, in the chickens in the treatment group, the values were lower in 3 out of 4 chickens as compared with those before administration. However, as a result of statistically processing the values before and after administration in each group (ANOVA analysis), no significant difference was observed. In addition, as a result of comparing the values of the control group and the treatment group in each period (Student's t-test), the P values before, during the intermediate period (1.5 months later) and after administration (3 months) were 0, respectively. The values were .61, 0.49, and 0.47, and no significant difference was observed between the control group and the treatment group at any of the time periods (Table 12). From the above results, it was considered that the addition (administration) of 5-ALA to the feed for 3 months did not affect the plasma testosterone concentration.

(Summary about chicken)
Addition of 5-ALA to poultry feed (10 ppm) shows no significant effect on sperm motility and plasma testosterone concentration in roosters, but is considered to be effective on sperm density and sperm malformation rate. Was done.

Claims (13)

A sperm function improving agent in livestock containing a compound represented by the following formula (I) or a salt thereof.

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group or an aralkyl group). The sperm function improving agent according to claim 1, wherein the malformed sperm rate is reduced. The sperm function improving agent according to claim 1 or 2, wherein the livestock is a bull or a rooster. The sperm function improving agent according to claim 3, wherein the improvement of sperm function is an improvement in the forward motility of sperm in a bull. The improvement in sperm function according to claim 3, wherein the administration to a bull having a normal rate of sperm morphology of less than 5% can improve the value of the normal rate of sperm morphology by 5 times or more. Agent. The sperm function improving agent according to claim 5, wherein an improvement in which the numerical value of the normal rate of sperm morphology is 5 times or more is obtained within 50 days after the start of administration. The sperm function according to claim 3, wherein when administered to a bull having a normal rate of sperm morphology of 65% or more and less than 80%, an improvement in increasing the normal rate of sperm morphology by 5% or more can be obtained. Improving agent. The sperm function improving agent according to claim 3, wherein the density of sperm in semen in a rooster is increased. The sperm function improving agent according to claim 8, wherein an increase in sperm density is obtained within 3 months after the start of administration. A livestock feed to which the sperm function improving agent according to any one of claims 1 to 9 is added. A method for improving sperm function, which comprises orally administering the sperm function improving agent according to any one of claims 1 to 9 to male domestic animals. The sperm function according to claim 11, wherein the sperm function improving agent according to any one of claims 1 to 9 is orally administered at 0.01 to 1.5 mg / day in terms of aminolevulinic acid per 1 kg of body weight. How to improve. A method for improving the fertilization rate of livestock by using sperms collected from livestock to which the sperm function improving agent according to any one of claims 1 to 9 has been administered.