JP4537663B2 - Relaxin-like protein secretagogue - Google Patents

Description

数値：平均±標準誤差 単位：μｇ／ｍｌ
【００２９】
表１によると、精漿中のリラキシン様蛋白質の濃度は、試験後半の８月以降、添加区が対照区を上回る数値を示した。
上記の結果から、本分泌促進剤は、雄豚の精漿中のリラキシン様蛋白質濃度を増加させることが解る。
また、上記の試験データについて、元々リラキシン様蛋白質の濃度が低い各豚５頭のデータを示すと、表２の結果となる。
【００３０】
【表２】

数値：平均±標準誤差 単位：μｇ／ｍｌ
【００３１】
表２の結果から、添加区では３００μｇ／ｍｌまで回復しており、添加区と対照区を比較すると、特に７〜１０月での差が大きく、本分泌促進剤は、元々リラキシン様蛋白質の濃度が低い豚に特に有効であることが解る。
以上の結果、本発明の分泌促進剤は、雄豚の精漿中のリラキシン様蛋白質濃度を増加させ高めることが確認された。
【００３２】
【実施例２】
１１頭の種豚から精漿を集め、リラキシン様蛋白質濃度を測定した。平均リラキシン様蛋白質濃度は、３００μｇ／ｍｌであった。平均濃度を基準にして高リラキシン様蛋白質濃度区（８頭）と低リラキシン様蛋白質濃度区（３頭）の二つのグループに分けた。高リラキシン様蛋白質濃度区のリラキシン様蛋白質の平均濃度は３４６μｇ／ｍｌであり、低濃度区リラキシン様蛋白質平均濃度は１７４μｇ／ｍｌであった。各濃度区の種豚の精子の性状並びに対外受精率を測定した。結果は、表３に示す。
【００３３】
【表３】

ＲＬＰ：リラキシン様蛋白質 数値：平均±標準偏差
【００３４】
表３の結果では、顕微鏡下で評価する運動精子率、生存精子率、未成熟精子率に有意差は見られなかった。
それに対して、実際に、卵子を使用している体外受精率測定では、高リラキシン様蛋白質濃度区と低リラキシン様蛋白質濃度区で、明確な差が認められた。
このように、従来行われていた顕微鏡下で観察する方法による評価では、正常精液の判別が難しく、精漿中のリラキシン様蛋白質濃度を測定することで初めて判別することができることが解った。
【００３５】
以上の実施例１及び実施例２の結果から、オリゴ糖は、リラキシン様蛋白質の分泌を促進させ、雄豚の生殖能力を向上させることが解る。
そして、従来の技術では、精子の運動能で問題のない精液を用いて人工授精しても十分な繁殖成績が得られないことがあったが、精漿中のリラキシン様蛋白質濃度を測定することで、生殖能力の高い精液を判別出来ることが可能となった。
【００３６】
【発明の効果】
（１）本発明によれば、リラキシン様蛋白質の分泌が促進される結果、精漿中のリラキシン様蛋白質の濃度低下が防止されるので、牛、豚、鶏などの雄性動物においても暑熱ストレスなどの環境変化による精子活力の低下を引起こすことがなく、農場成績を向上・安定させるという、格別の効果が達成される。また、イヌ、ネコなどの愛玩動物では、精漿中のリラキシン様蛋白質を増加させ、円滑な繁殖が可能となる。
【００３７】
（２）従来の顕微鏡を用いた判定では発見できなかった雄豚等の精液異常を精漿中のリラキシン様蛋白質の濃度を測定することにより、早期に発見できるので、経済的な損失は最小限で食い止めることが出来る。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for improving the fertility of a male animal with a relaxin-like protein secretion promoter.
[0002]
[Prior art]
Sexual reproduction performed by many life forms requires two sexes, male and female, and forms a new generation of life forms by fertilization of the gametes presented by each. Needless to say, reproduction is a vital activity of life.
However, in recent years, there has been a change in fertility due to a decrease in the number of human spermatozoa and seafood due to the effects of environmental hormone contamination, and environmental pollution surrounding reproduction has become a social problem.
[0003]
And in order to pursue production efficiency, industrial animals such as cattle, pigs, and chickens are often bred intensively in a small barn, and improvements are being promoted in terms of breeding management and sanitary environment. But still not enough. Such various stresses on livestock cause deterioration of the reproductive performance and hinder production efficiency.
In particular, in the field of pig farming, in addition to the intensive production, in addition to the import pressure of cheap livestock products, the new burden of manure processing due to the regulation of livestock excrement has been added. The surrounding environment is severe, and the reduction of production costs is a serious problem for producers.
[0004]
The most important thing for reducing production costs is to carry out efficient breeding and smooth livestock production. However, in industrial animals, sweat glands are degenerated, and pigs and the like are particularly covered with thick subcutaneous fat, and it is said that some kind of reproductive disorder is caused by heat stress exceeding 25 ° C. In particular, semen properties such as a decrease in the number of sperm in bred animals and a decrease in sperm vitality (decreased motility) have been caused, often causing reproductive disorders.
As a method for solving such a problem, for example, a method for improving the reproductive performance of a boar characterized by using a feed for seed pigs containing oligosaccharides, particularly saccharides mainly containing fructooligosaccharides, is known. However, it is difficult to say that it is sufficient (see, for example, Patent Document 1).
[0005]
In addition, in vitro fertilization, since sperm fertility is insufficient, it is known to add caffeine or β-cyclodextrin to enhance motility. However, these methods have a low probability of obtaining a normal fertilized egg such as the occurrence of multisperm entry. Thus, a method for improving the fertilization rate by adding a specific peptide to sperm to enhance motility has been developed.
As such a method, for example, a testis-specific insulin homolog peptide is added to sperm before fertilization to enhance motility (for example, refer to Patent Document 2), or buffalo semen / plasma-derived sperm movement. An ability promoting polymer protein (for example, see Patent Document 3) is known.
However, in order to make these peptides, it is indispensable to have a living body for obtaining a genetic recombination technique or protein, it must be purified to increase purity, and it is necessary to perform in vitro fertilization, It was an extremely complicated and expensive method.
[0006]
Relaxin is one of the pregnancy hormones found in the genitals during pregnancy.
On the other hand, relaxin-like protein is a peptide hormone homologous to relaxin and is contained in the seminal plasma of male animals. Relaxin-like proteins are thought to bind to sperm receptors and increase their motility. That is, it mainly binds to the sperm acrosome at the time of ejaculation, and if it is exposed to epididymis tail sperm at a concentration of 150 to 200 μg / ml, it is said to significantly induce fertility and acrosome reaction (for example, non (See Patent Document 1 and Non-Patent Document 2).
As such relaxin-like protein, for example, there is a secretory protein having a molecular weight of about 13 kDa isolated from porcine seminal gland. As a result of examining the concentration of seminal plasma relaxin-like protein in 10 breed pigs, 117-700 μg / ml (average 300 ± 170 μg / ml) (see, for example, Non-Patent Document 3).
[0007]
[Patent Document 1]
JP-A-8-205789 [Patent Document 2]
JP 2002-204698 A [Patent Document 3]
JP-A-11-279075 [Non-patent Document 1]
Journal of Reproduction and Development, Vol.47, No.4, 2001, 197-204
[Non-Patent Document 2]
“Abstracts of the 98th Annual Meeting of the Japanese Society of Livestock Science 2001”, Japan Society of Animal Science, p.109
[Non-Patent Document 3]
"Abstracts of the 94th Annual Meeting of the Japanese Reproductive Biology Society" 2001, Japanese Reproductive Biology Society, p.88
[0008]
Recently, it has been known that low fertility semen having a low fertility with an egg is known even when the number of sperm and vitality (motility) of sperm are normal by microscopic examination. Although this cause has not been clearly clarified yet, it is considered that the resistance to stress of the sperm nucleus is low and the swing amplitude width of the sperm head is small.
In the management of breeding animals, the characteristics of semen require skillful and time-consuming microscopic observation, and in fact, female animals are examined for the presence or absence of estrus, and expensive electronic equipment such as ultrasonic examination equipment is required. Here, the determination in female animals is very difficult to find even if there is a problem with semen, such as the conception rate or the number of offspring, because the influence of the individual is great.
Therefore, since a sire with abnormal semen properties is used for a long time, economic loss is great. For this reason, if a semen abnormality of a breeding sire that cannot be found by a determination using a conventional microscope can be discovered at an early stage, economic loss can be minimized.
From the above, development of a method for improving the fertility of male animals is awaited.
[0009]
[Problems to be solved by the invention]
It is an object of the present invention to provide a method for increasing the reproductive performance of male animals by increasing the concentration of relaxin-like protein in semen.
[0010]
[Means for Solving the Problems]
The present inventors consider that relaxin-like protein is a tablet molecule that imparts motility to sperm, and as a result of intensive research to solve the above-mentioned problems, between the oligosaccharide and the secretion of relaxin-like protein, Knew that there was a relationship, and as a result of further research, the present invention was completed.
[0011]
That is, the present invention is as follows.
1. By administering 0.01-0.3 g / day / kg body weight of isomaltoligosaccharide to a boar with a relaxin-like protein of less than 300 μg / ml in seminal plasma, 300 μg / kg of relaxin-like protein in seminal plasma is administered. A method for increasing the concentration of relaxin-like protein in the seminal plasma of boar, characterized by increasing to more than ml.
2. A method for discriminating the normality of semen of a boar characterized by measuring the concentration of relaxin-like protein in seminal plasma and determining that the relaxin-like protein in seminal plasma is 300 μg / ml or more as appropriate.
[0012]
The present invention has been made on the basis of the discovery of a new fact first discovered by the present invention that oligosaccharides have an excellent relaxin-like protein secretion-promoting action. According to the present invention, the secretion of relaxin-like protein is promoted by the action of the oligosaccharide, and as a result, the concentration of relaxin-like protein in semen is prevented from decreasing, so that heat stress can also occur in male animals such as cattle, pigs and chickens. A special effect of improving and stabilizing the farm performance is achieved without causing a decrease in sperm vitality due to environmental changes.
In addition, abnormalities in semen, such as boars, that could not be detected using conventional microscopes can be detected at an early stage by measuring the concentration of relaxin-like protein in semen. I can do it.
[0013]
Hereinafter, the present invention will be described in more detail.
Examples of the oligosaccharide of the present invention include isomaltoligosaccharide, galactooligosaccharide, fructooligosaccharide, soybean oligosaccharide, raffinose, xylo-oligosaccharide, lactulose, lactosucrose and the like, in particular, isomaltose, isomaltlyose, panose, Oligosaccharides in which glucose has bonds other than α-1,4 bonds, for example, isomaltoligosaccharides, are optimal.
Since isomaltooligosaccharides are highly stable against heat and acid, there is little inactivation due to heat treatment of feed or addition of acidic agents, so the use of relaxin-like protein secretion promoters consisting of isomaltooligosaccharides is not limited This is also advantageous.
[0014]
In order to use semen with a high fertilization rate for reproduction, it is necessary to use semen that exceeds a certain standard relaxin-like protein concentration.
The standard is that you can actually measure the relaxin-like protein concentration and measure the conception rate by artificial insemination (as a supplementary data, you can also measure the sperm motility such as the rate of sperm. Good). There is actually a method to check fertility by the birth rate and birth rate, but it takes a lot of time for judgment, for example, pregnancy period is 280 days for cattle and 144 days for pigs. It needs to be bigger.
[0015]
Judgment criteria vary depending on the species, and also for animals with protected pedigree. It also varies depending on the breeding season.
In the case of pigs, the relaxin-like protein in seminal plasma is 280 μg / ml or more, preferably 300 μg / ml or more. Semen with a relaxin-like protein of 240 μg / ml or less is judged to be defective semen and should not be used.
[0016]
The relaxin-like protein secretion promoter of the present invention may contain oligosaccharides alone or other additives such as excipients, disintegrants, nutrients, herbs (herbal medicines) and the like.
Excipients include silicic acid, zeolite, calcium carbonate, calcium phosphate, sugar (lactose, sucrose, mannitol, sorbitol, etc.), starch (corn starch, wheat starch, rice starch, potato starch, etc.), cellulose derivatives (methylcellulose, hydroxy) Propylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, etc.), gelatin, tragacanth rubber, etc., disintegrating agents include cross-linked polyvinylpyrrolidone, agar, alginic acid or salts thereof, and nutrients include amino acids, vitamins, etc. .
[0017]
The relaxin-like protein secretagogue of the present invention has been found to be effective in industrial animals such as humans, pigs, cows, horses, sheep, goats and chickens, and pets such as dogs and cats. It can be used by adding to pharmaceuticals.
The effect of the relaxin-like protein secretion promoter of the present invention is about 0.01 g to 0.3 g / day / kg body weight as an active ingredient oligosaccharide, but preferably 0.02 g to 0. 2g / day / kg body weight is good.
In addition, since the function of oligosaccharides is thought to be mainly due to the improved balance of the intestinal microflora, it is desirable to continuously supply this secretagogue, but as a summer measure for pigs, etc. If it is used in a limited way, you may pay 2 to 3 months before entering the summer.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these do not limit the present invention.
1. Measurement of relaxin-like protein concentration in seminal plasma The relaxin-like protein concentration was measured according to a time-resolved fluorescence immunoassay established as a method for measuring relaxin concentration (see, for example, Non-Patent Document 4).
[0019]
[Non-Patent Document 4]
"Clinical Veterinary" special issue, Vol.20, No.3, 2002, P.151-159
[0020]
(1) Preparation of tracer Using a purified relaxin-like protein and labeling with a non-radioactive fluorescent substance europium, fractionation by gel filtration is performed to obtain a relaxin-like protein labeled with europium.
[0021]
(2) Preparation of relaxin-like protein specific antibody Relaxin-like protein specific antibody is prepared by immunizing rabbits.
The resulting antibody cross-reacts only with relaxin-like protein, but does not cross-react with other components in seminal pus extract, and does not cross-react with insulin or FSH. Confirm that it is a specific antibody.
[0022]
(3) Time-resolved fluorescence immunoassay (TR-FIA)
Using a time-resolved fluorescence immunoassay based on a competitive principle in which the second antibody was immobilized, the relaxin-like protein concentration in seminal plasma was measured by the following method.
The sample or standard and the first antibody are added to the microtiter plate on which the second antibody is immobilized, and incubated for 1.5 hours. Subsequently, europium labeled relaxin-like protein is added as a tracer and incubated for an additional 3.5 hours. After the incubation is complete, the plate is washed 6 times to stop the reaction. Thereafter, an europium ion is released from the labeled relaxin-like protein, and an enhancement reagent (Na N ₃ ) for forming a chelate compound exhibiting high fluorescence is added. After incubation for 5 minutes, the fluorescence is counted with a time-resolved fluorescence apparatus.
[0023]
[Example 1]
69 parts of syrup containing 87.5% isomaltoligosaccharide (Isomalt 900: made by Showa Sangyo Co., Ltd.) and 31 parts of silicic acid (made by United Silica Industrial Ltd.) as an excipient are spray-dried. A protein secretagogue was prepared.
The above relaxin-like protein secretion promoter was fed to a boar for artificial insemination, and the time course of the relaxin-like protein concentration in seminal plasma was investigated while comparing with the control group without addition.
[0024]
(1) Test method Twenty male boars for artificial insemination are divided into an added group and a control group, each containing 25 g / day / pig body weight as the above-mentioned secretagogue of the present invention as an isomaltooligosaccharide. About 250kg was paid, and the control ward was unpaid. The test period was 7 months from April to October, assuming summer measures for boars.
[0025]
(2) Survey items During the test period, semen is collected at a rate of about once / week, the collected semen is observed with a microscope to investigate its properties, and relaxin in the seminal plasma collected at a rate of once / month. The concentration of the like protein was measured.
[0026]
(3) Test results 1) Sperm properties In the control group, 2 out of 10 test pigs showed abnormal semen properties from September and became swine. In contrast, the test pigs in the addition group were good with no abnormal semen properties during the test period.
[0027]
2) Table 1 shows the measurement results of the concentration of relaxin-like protein.
[0028]
[Table 1]

Numerical value: Mean ± standard error Unit: μg / ml
[0029]
According to Table 1, the concentration of relaxin-like protein in seminal plasma showed a value that the added group exceeded the control group after August in the latter half of the test.
From the above results, it is understood that the present secretagogue increases the concentration of relaxin-like protein in the seminal plasma of boars.
In addition, regarding the above test data, the data of 5 pigs each originally having a low concentration of relaxin-like protein are shown in Table 2.
[0030]
[Table 2]

Numerical value: Mean ± standard error Unit: μg / ml
[0031]
From the results of Table 2, in the added group, it was recovered to 300 μg / ml. When the added group and the control group were compared, the difference was particularly large in July to October, and the secretagogue was originally a relaxin-like protein concentration. It can be seen that it is particularly effective for low pigs.
As a result, it was confirmed that the secretagogue of the present invention increases and increases the concentration of relaxin-like protein in the seminal plasma of boars.
[0032]
[Example 2]
Seminal plasma was collected from 11 sows and the relaxin-like protein concentration was measured. The average relaxin-like protein concentration was 300 μg / ml. Based on the average concentration, it was divided into two groups, a high relaxin-like protein concentration group (8 heads) and a low relaxin-like protein concentration group (3 heads). The average concentration of relaxin-like protein in the high relaxin-like protein concentration group was 346 μg / ml, and the average concentration of relaxin-like protein in the low concentration group was 174 μg / ml. The sperm properties and the external fertilization rate of the pigs in each concentration group were measured. The results are shown in Table 3.
[0033]
[Table 3]

RLP: Relaxin-like protein Value: Mean ± standard deviation
In the results of Table 3, no significant difference was observed in the motor sperm rate, viable sperm rate, and immature sperm rate evaluated under a microscope.
In contrast, in vitro fertilization rate measurement using eggs, a clear difference was observed between the high relaxin-like protein concentration group and the low relaxin-like protein concentration group.
Thus, it has been found that the conventional evaluation by the method of observing under a microscope makes it difficult to discriminate normal semen, and it can be discriminated for the first time by measuring the concentration of relaxin-like protein in seminal plasma.
[0035]
From the results of Examples 1 and 2 above, it can be seen that oligosaccharides promote the secretion of relaxin-like proteins and improve the reproductive performance of boars.
And with the conventional technology, even if artificial insemination was not possible even with artificial insemination using semen that has no problem with sperm motility, the concentration of relaxin-like protein in seminal plasma should be measured. This makes it possible to discriminate semen with high fertility.
[0036]
【The invention's effect】
(1) According to the present invention, since the secretion of relaxin-like protein is promoted, the concentration of relaxin-like protein in seminal plasma is prevented from decreasing, so that heat stress and the like can be observed in male animals such as cows, pigs and chickens. A special effect of improving and stabilizing the farm performance is achieved without causing a decrease in sperm vitality due to environmental changes. Moreover, in pet animals such as dogs and cats, relaxin-like protein in seminal plasma is increased and smooth breeding becomes possible.
[0037]
(2) Since semen abnormalities such as boars that could not be detected by conventional microscopy can be detected early by measuring the concentration of relaxin-like protein in seminal plasma, economic loss is minimal. You can stop at.

Claims (2)

  By administering 0.01-0.3 g / day / kg body weight of isomaltoligosaccharide to a boar with a relaxin-like protein in seminal plasma of less than 300 μg / ml, 300 μg / kg of relaxin-like protein in seminal plasma is administered. A method for increasing the concentration of relaxin-like protein in the seminal plasma of boar, characterized by increasing to more than ml.   A method for discriminating the normality of semen of a boar, wherein the concentration of relaxin-like protein in seminal plasma is measured and it is determined that the relaxin-like protein in seminal plasma is 300 μg / ml or more.