CN110604754B

CN110604754B - Pheromone composition capable of improving estrus mating rate of sows

Info

Publication number: CN110604754B
Application number: CN201910972187.4A
Authority: CN
Inventors: 李伟; 岑桂英; 钱星宇; 吴春山; 原敏; 何玲飞
Original assignee: Ningbo second hormone factory
Current assignee: Ningbo second hormone factory
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2021-11-26
Anticipated expiration: 2039-10-14
Also published as: CN110604754A

Abstract

The invention relates to a pheromone composition, in particular to a pheromone composition for improving the configurable estrus rate of sows by adopting traditional Chinese medicine extracts to enhance pheromone. A synergistic pheromone composition, comprising: the component A comprises: one or more combinations of androgenic steroid pheromones; and B component: one or more of herba Epimedii extract, Cistanchis herba extract, herba Cynomorii extract, semen Cuscutae extract, radix Dipsaci extract, fructus Ligustri Lucidi extract, Achyranthis radix extract, radix Ophiopogonis extract, and Corni fructus extract. The composition is safer and more reliable, and can reliably improve the reproductive performance (namely higher matchable estrus rate) of the sow, improve the utilization rate of the sow and improve the fertility of the sow.

Description

Pheromone composition capable of improving estrus mating rate of sows

Technical Field

The invention relates to a pheromone composition, in particular to a pheromone composition for improving the configurable estrus rate of sows by adopting traditional Chinese medicine extracts to enhance pheromone.

Background

The term "Pheromone" was proposed by researchers in the fifties of the last century and was synthesized by the two ancient greek words Pherein and hormone. It is considered to be a chemical species capable of causing an allogenic animal sexual response, and is therefore also known as pheromone, playing an important role in some behavioral and sexual interactions, as well as in the control of reproduction. Pheromones are chemical substances secreted from the exocrine glands of insects to the outside of the body, and act on other individuals of the same species through contact of individual examination or air transmission to cause specific behaviors or physiological responses. The insect sex pheromone can be mainly used for predicting and forecasting pests, directly trapping and killing males, cutting off the relation among pests and the like. The artificially synthesized sex pheromone is called 'sex attractant', and when the sex pheromone is scattered in the field in a large amount, the male insects can get lost and can not mate, thereby achieving the purpose of prevention and control.

At present, there are more than 18 kinds of pheromones and analogues thereof, the chemical structures of which are known and known. However, the pheromones ' androstenone (androst-16-en-3-one) and ' androstenol (androst-16-en-3-ol) ' secreted and released in the body of the boar are found to play an important role in the reproductive behavior of the sow. However, boar androstenone and androstenol have not only been chemically defined, but also have been produced as sprays and powders for use in swine production and for scientific research. According to the past accumulated knowledge, boars sexually stimulate sows before mating. Pheromone, auditory, visual and tactile stimuli are involved in this process, all of which affect the pituitary oxytocin release in sows and replacement gilts (Madej et al, 2005). Langendijk et al (2003) reported that the presence of boars induced oxytocin release in sows and significantly increased the activity of the myometrium in sows. The effects of boar exposure also include stimulation of follicle growth, resulting in oestrus and ovulation in more gilts (Langendijk et al, 2000). The salivary pheromone released by submaxillary gland of 10-month-old boar can stimulate oestrus and oestrus behavior of sow. The pharmaceutical product containing the pig pheromone can be used for enhancing the oestrus expression of sows and detecting the oestrus behavior change of the sows. In practical application, the boar pheromone is found to be used for sows, so that the arrival of the initial period of the sows can be promoted. In addition, studies have shown that boar pheromones can also affect the rhythm and intensity of oestrus in sows and affect the interval between weaning and oestrus. In weaned sows, when they encounter a princess or other form of pheromone stimulation, they develop certain behavioural changes, such as lordosis or orthoreflexes, which signal them to target male proximity, which for the breeder means that they can be bred or inseminated (a.i.).

Estrus refers to the phenomenon of the reproductive cycle of a sexually mature female mammal that manifests itself during a particular season, physiologically as ovulation, ready for fertilization and pregnancy, and behaviorally as attraction and admission of heterosis. Firstly, the ovarian follicle on the ovary is maturing and then ovulatory; female hormones secreted by follicles cause libido and are subject to crawling by males; at the same time, physiological changes such as mucosal congestion, edema, mucus increase, cervix release and the like of the genital catheter occur. The duration of the oestrus behavior and reproductive physiological changes is the oestrus period. The oestrus expression of animals is an important prerequisite for the production of reproductive results. However, in actual production, some hormones, such as estrogen, can be used to induce heat in female animals, but the heat generated in this case is mostly only "false heat", i.e. simple heat, and cannot be bred (artificial insemination) and normal breeding results cannot be generated. When the female animals induced by methods such as estrogen and the like are in estrus, the activity or development state of the ovaries of the female animals is not in a state due to the estrus, or no follicles are developed, or the ovaries do not have normal ovulation function, even corpus luteum exists on the ovaries, and other reproductive organs are not prepared in all aspects of mating and breeding. The action of inducing or promoting the oestrus of the female animals by the pheromone can ensure that the female animals are fertilized, pregnant and farrowing, and the oestrus of the female animals has a normal ovulation function.

Then, factors influencing the breeding of the sows are various, such as oestrus, oestrus identification, semen quality, mating technology, nutritional status, environmental factors and the like, the oestrus identification of the sows is one of important technical links in the breeding work, and the mating period can be correctly determined through the oestrus identification so as to improve the conception rate. The existing estrus identification mostly adopts external observation, and combines methods such as a back pressing test, boar estrus test and the like. However, in actual production, part of sows have normal ovarian function, normal follicular development, normal reproductive system and normal ovulation, but have no oestrus (no oestrus expression or behavior), i.e. recessive oestrus or no obvious oestrus, difficult oestrus identification, mismatch, missed mating and low loss in production. Although external observation is simple, it is difficult to determine the recessive or inconspicuous sow, and the mating period is easily missed. In large-scale production, one sow may not see a few boars from birth to sexual maturity and then mate, and the sow does not contact the boars, which is also a factor for recessive oestrus of the sow. It is also believed that the cause of recessive estrus is due to insufficient estrogen secretion caused by external factors interfering with the normal function of the pituitary.

At present, most pig farms use boars to check whether weaned sows or multiparous sows are in oestrus before sow mating or artificial insemination in order to improve the mating rate; however, even if boars are used for the heat-check method, there is no response in about 20% of sows. In addition, the method has the main problems that a separate colony house is needed for feeding the boars, the boars play a single role in the daily production of a pig farm, and the feeding cost is high. In addition, the disease infection risk is increased by boar inspection, and the boar inspection is a large leak in epidemic prevention links, for example, the epidemic of african pigs spreading China at present, a large part of epidemic situations are caused by that boars randomly pass in and out of a sow colony house, and the sow colony house which is not infected originally is also infected, so that destructive attack is caused to part of pig farms. According to incomplete statistics, about 1/3 sows in China die (fatality of disease + slaughter) due to African swine fever in 2018-2019. The boar oestrus test method is not only complicated, but also not beneficial to the epidemic prevention of the pig farm.

In foreign countries, a bionic method, namely simulating the sound and smell of breeding boars, is adopted to test whether sows are oestrous.

In 1968, UK patent GB1267514A mentions that pheromone has the function of improving artificial insemination of sows, and the product of the pheromone uses diethyl ether as a solvent of androstenone, wherein the diethyl ether has a low boiling point and a flash point of about 34.6 ℃, and is easy to explode when being used and stored at a higher temperature; in addition, the packaging is a pressure bottle, a large amount of alkanes such as propane and butane are filled in the pressure bottle to serve as propellants, the propane and the butane belong to flammable and explosive dangerous chemicals, and particularly, the product has great risks in storage and transportation in hot summer; daily preservation needs to be performed in a shady and cool place or in a refrigerator; in the using process, the paint is volatile and has certain irritant toxicity to respiratory tracts and the like of operators. Therefore, the products are very inconvenient to use and store and have potential safety hazards. The actual effectiveness of the product is low, namely only 55 percent (22/40), and the effect is not ideal, mainly because the product has too fast volatility, the active ingredients are not contacted by the sows, and the active ingredients are volatilized into the ambient air, and the concentration is diluted, so the actual use effect is poor.

Melrose et al, BRITISH VETERINARY JOURNAL 127(1971)497-502, compared the oestrus of sows when different pheromones were used, even after the two steroid hormones 5 α -androst-16-en-3-one and 3 α -hydroxy-5 α -androst-16-ene, the rate of the quiescence reaction (oestrus expression, mating) of sows was only 56% at the highest; C.B. Breeding et al, BRITISH VETERINARY JOURNAL (British VETERINARY science) 130(1974)61-67, studied the use of different androgenic steroid hormones in combination for oestrus detection of sows, when the pheromone is used alone, the proportion of the oestrus expression (mating) of the sows is only 58%.

The bionic method adopted by researchers in China, Heying Jun, oestrus identification of sows [ J ]. Zhejiang animal husbandry and veterinarian, 1995(4) reports that oestrus identification rate is basically the same (276vs 284) with external observation method, but conception rate is obviously higher than that of other methods. Mainly because the sow has the coupling response to the heat identification of the bionic method only when coupling is carried out (namely the full-time estrus), and the sow at the initial stage, the final stage or other sows which do not have heat has no coupling response, therefore, the sow with the coupling response basically has the right mating period, at the moment, the mating has higher conception rate, so the bionic method is not only an effective heat identification method.

A pheromone medicine combination and a method are mentioned in the US9480689B1, which can stimulate the reproductive behavior and the reproductive success rate of sows. The combination comprises at least one steroid hormone and one heterocyclic aromatic compound, after the product is used, the rate of the sow having a standing reaction is 88%, although the clinical effect is obviously improved, the steroid hormone is various, and a plurality of steroid hormones are assimilated and can not be used at will, otherwise the food safety can be influenced, such as estrogen (not beneficial to embryo implantation), androgen (inhibiting follicular development) and the like, if the use time is improper or inaccurate, the reproductive behavior of the sow can not be stimulated, but the reproductive behavior of the female livestock can be inhibited. In addition to this, the present invention is,the heterocyclic aromatic compound is also special, has certain odor and inhalation toxicity, and is an aromatic compound which is flammable when exposed to open fire and high heat. Andoxidizing agentThe reaction may take place. Toxic nitric oxide fume is released after heated and decomposed. When the quinoline vapor is used by a user, the quinoline vapor is irritant to the nose and the throat. It can cause headache, dizziness, nausea, and irritation to eyes and skin. In addition, quinoline has certain ecotoxicity, for example, can kill aquatic daphnia, algae and other organisms, and influences the ecological stability of water. The half-life period of biodegradation can reach 960h, and the half-life period of photooxidation in air can reach 99 h. Therefore, if the quinoline is improperly used, the health of users is affected, and certain influence is also caused to the ecological environment. In addition, some studies find that the heterocyclic aromatic compound may have a certain growth promoting effect, the growth promoting effect interferes with the growth and development of female animals and is not beneficial to the development of embryos, the food safety is influenced, and substances or medicines with the growth promoting effect are strictly prohibited for food animals in many national regions. Therefore, the product combination is not perfect enough, and great potential safety hazard also exists.

Therefore, the existing method cannot meet the requirements of the batch production technology of sows in the aspects of effectiveness, safety and the like, namely higher configurable estrus rate.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a synergistic pheromone composition which is safer and more reliable, and which reliably improves the reproductive performance (i.e., higher fertile oestrus rate) of sows, increases the availability of sows, and increases the fertility of sows.

In order to achieve the above object, the present application adopts the following technical solutions:

a synergistic pheromone composition, comprising:

the component A comprises: one or more combinations of androgenic steroid pheromones;

and B component: one or more of herba Epimedii extract, Cistanchis herba extract, herba Cynomorii extract, semen Cuscutae extract, radix Dipsaci extract, fructus Ligustri Lucidi extract, Achyranthis radix extract, radix Ophiopogonis extract, and Corni fructus extract.

Preferably, the A component comprises one or two of androstenone and androstenol, and preferably, the A component comprises androstenone and androstenol. Of course, component a of the present application may also add or replace androstenone and androstenol with one or more of the following combinations: androsterone, androstatetraene, 3 beta-androstenol, pheromone, 4, 16-androstadiene-3 beta-ol, 5, 16-androstadiene-3 beta-ol, estratetraene, and androstane-3-one.

Of course, the B-component of the present application may also be added or replaced with one or more combinations of the following extracts: comprises fructus Cnidii, radix Morindae officinalis, rhizoma Curculiginis, Cordyceps, Eucommiae cortex, placenta hominis, Ginseng radix, rhizoma Dioscoreae, fructus Ligustri Lucidi, Sargassum, Mori fructus, fructus Alpinae Oxyphyllae, semen astragali Complanati, fructus Schisandrae chinensis, semen euryales, cortex et radix Polygalae, Achyranthis radix, semen Platycladi, radix Ophiopogonis, rhizoma Acori Graminei, Gecko, semen Allii Tuberosi, semen Trigonellae, Polygoni Multiflori radix, pericarpium Granati, fructus Lycii, rhizoma Polygonati, Corni fructus, Rubi fructus, fructus Rosae Laevigatae, herba asari and lignum Aquilariae Resinatum.

Preferably, the mass ratio of the component A to the component B is 1-100: 0.5-10; more preferably, the mass ratio of the component A to the component B is 1-10: 0.5-5.

The best technical scheme of the composition is as follows: the composition contains androstenone, androstenol and epimedium extract, and the epimedium is considered by traditional Chinese medicine to be pungent and sweet in taste and warm in nature and walk the liver and kidney meridians. Is an essential herb for nourishing life gate, benefiting vital essence and qi, strengthening bones and muscles, tonifying kidney and strengthening yang, and is clinically used for treating impotence and impotence, spermatorrhea, premature ejaculation, urinary incontinence, infertility of women and other symptoms. The herba Epimedii extract is extracted from herba Epimedii (Epimedium brevicornum Maxim.) of perennial herb, and contains icariin, herba Epimedii extract, and herba Epimedii extract,Essential oils、Wax alcohol、Plant sterol、Tannins、Vitamin EThe components belong to Chinese herbal medicine extracts, are safer and do not cause adverse stimulation to users and target animals. Icariin (icarin) is preferred, and the purity is more than or equal to 94.0 percent.

It is another object of the present application to provide a formulation of a pheromone composition, wherein the formulation is administered by inhalation, comprising the pheromone composition and a carrier. The present application may optionally contain other ingredients as necessary or desired, depending on the form of the final product and the desired use. Such optional ingredients may include, but are not limited to, carriers (such as water, alcohols, solvents, etc.), co-solvents, surfactants, preservatives, and pharmaceutically acceptable adjuvants.

The synergistic pheromone composition mainly exists in a liquid form, and the using mode is spray treatment. The preferred mode of formulation is therefore a spray formulation.

Preferably, the concentration of the component A of the preparation is 1-200 mug/ml, and the concentration of the component B is 1-50 mug/ml.

Preferably, the formulations herein have a pH in the range of 5 to 8. Between this pH range, an unexpected stabilizing effect of the composition can be obtained. Therefore, the inventor further finds that the pH value is between 5 and 8, the use requirement can be met, when the pH value is lower than 4 or higher than 8, the nasal cavity of the sow can be stimulated to a certain extent, even the nasal mucosa is damaged, the acceptance degree of the sow is poor, in this case, the stability of the medicine is poor, and when the pH value is between 5 and 8, the stability of the solution of the composition is improved and is basically close to the normal condition of the body, and when the composition is used, the acceptance degree of the sow is high.

Preferably, the carrier of the present application is selected from one or more of alcohols, ethers, chloroform, benzene, carbon disulfide and water. As a further preference, the alcohols in the present application are selected from one or more of ethanol, propanol, isopropanol, butanol, pentanol, hexanol, heptanol, octanol, ethylene glycol, propylene glycol, dipropylene glycol and phenethyl alcohol.

Preferably, the preparation of the application also comprises cosolvent, surfactant, preservative and other pharmaceutically acceptable auxiliary materials. The surfactant can improve the solubility of the steroid in the aqueous solution, so that the steroid can be uniformly dispersed in the carrier.

Preferably, the cosolvent is one or a mixture of alcohols, ketones, ethers, sulfones, esters, soybean oil, N-N-dimethylformamide and N-N-dimethylacetamide; preferably, the mass percent concentration of the cosolvent is in the range of 0.2-20%, more preferably 1-5%, and the cosolvent is most preferably dimethyl sulfoxide.

Preferably, the surfactant is selected from nonionic surfactant and ionic surfactant; preferably, the surfactant is selected from one or more of lecithin, Tween-80, cetyl alcohol, hydrogenated castor oil PEG ester, glyceryl laurate, glyceryl palmitoleate, PEG diisostearate, sodium tocopheryl phosphate, steareth, C9-C15 alkyl phosphate, and C18-C36 glycol ester; preferably, the surfactant concentration is in the range of 0.01% to 0.5%, most preferably 0.05%, most preferably tween-80.

The application also discloses the use of said pheromone composition or said pheromone composition preparation for stimulating reproductive behavior and increasing reproductive success and productivity in female suid animals. The technical scheme of the application reliably improves the reproductive performance (namely higher matchable estrus rate) of the sow, improves the utilization rate of the sow and improves the fertility of the sow.

Drawings

FIG. 1 is a different type of spray apparatus; a and B are two different mechanical spray bottles; c, D is an electric spraying device.

FIG. 2 is a graph of the actual effectiveness increase for different pheromone processing groups; remarking: androstenone, 5 α; androstenol, 3 α; androstenone + androstenol, 5 α +3 α; epimedium 1, Y-1; epimedium 2, Y-2; androstenone + epimedium 1, 5 alpha + Y-1; androstenol + epimedium 1, 3 alpha + Y-1; androstenone + androstenol + epimedium 1, 5 alpha +3 alpha + Y-1; androstenone + androstenol + epimedium 2, 5 alpha +3 alpha + Y-2.

FIG. 3 is a schematic diagram of distribution of different piggeries in the same house in the same pig farm (I-VI represents distribution of different piggeries).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is illustrated by the following specific embodiments and examples, which are intended to better explain the present invention without limiting the scope of the present invention, and all modifications and variations that are within the basic idea and principle of the present invention are included in the scope of the present invention as claimed.

Preparation examples

The following terms describe the composition of androstenone, androstenol and epimedium extract in specific mixing ratios in the examples of the invention.

Comparative example 1

20.0mg of androstenone (Sigma-Aldrich,. gtoreq.98.00%) was added to 10.0g of dimethyl sulfoxide (Sigma-Aldrich, chromatographic grade) according to the formulation amount of the pheromone-containing composition solution listed in Table 1, and the mixture was subjected to ultrasonic assisted dissolution for 20 minutes; after complete dissolution, the solution was added to 500ml of MilliQ-water, stirring was started, 0.5g of Tween-80 (Sigma-Aldrich) was added while stirring, then stirring was continued, 3g of preservative (Caron, LONZA, Switzerland) was added, then MilliQ-water was added to 1000.0g, stirring was continued for 2 hours, and finally filtration was performed using a 0.45 micron filter (Merk-Millipore) to obtain composition A solution. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 1 pheromone composition solution formulations

Components	Percent by weight	g
			Androstenone	0.002	0.002
Dimethyl sulfoxide	1.0	1.0
			Tween-80	0.05	0.05
Preservative	0.3	0.3
			Purified water	98.648	98.648
Total of	100.000	100.000

Comparative example 2

According to the formulation of the composition containing pheromone androstenol (Sigma-Aldrich,. gtoreq.98.00%) as listed in Table 2. Adding 20.0mg of androstenol into 10.0g of dimethyl sulfoxide, and performing ultrasonic assisted dissolution for 20 minutes; after the dissolution, the mixture was added to 500ml of MilliQ-water, stirring was started, 0.5g of Tween-80 was added while stirring, then stirring was continued, 3g of preservative (Kathon, LONZA, Switzerland) was added, MilliQ-water was added to 1000.0g, stirring was continued for 2 hours, and finally filtration was performed using a 0.45 micron filter (Merk-Millipore) to obtain a solution of composition B. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 2 pheromone composition solution formulations

Components	Percent by weight	g
			Androstenol	0.002	0.002
Dimethyl sulfoxide	1.0	1.0
			Tween-80	0.05	0.05
Preservative	0.3	0.3
			Purified water	98.648	98.648
Total of	100.000	100.000

Comparative example 3

The formulation of the solution containing pheromone androstenone and androstenol composition is shown in table 3. 1000ml of the C composition solution was prepared by following the solution preparation methods of examples 1 and 2. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 3 pheromone composition solution formulations

Components	Percent by weight	g
			Androstenone	0.002	0.002
Androstenol	0.002	0.002
			Dimethyl sulfoxide	1.0	1.0
Tween-80	0.05	0.05
			Preservative	0.3	0.3
Purified water	98.646	98.646
			Total of	100.000	100.000

Example 1

The formulations of the compositions containing the pheromone androstenone and epimedium extract (Icariin, Simga-Aldrich,. gtoreq.94.0%, HPLC) listed in Table 4 were used. 1000ml of the D composition solution was prepared by following the solution preparation methods of examples 1 and 2. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 4 pheromone composition solution formulations

Example 2

The formula of the composition solution containing pheromone androstenol and epimedium extract is listed in table 5. 1000ml of E composition solution was prepared by following the solution preparation methods of examples 1 and 2. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 5 pheromone composition solution formulations

Components	Percent by weight	g
			Androstenol	0.002	0.002
Epimedium extract	0.002	0.002
			Dimethyl sulfoxide	1.0	1.0
Tween-80	0.05	0.05
			Preservative	0.3	0.3
Purified water	98.646	98.646
			Total of	100.000	100.000

Example 3

The formula of the composition solution containing pheromone androstenone, androstenol and epimedium extract is listed in table 6. Accurately weighing androstenone, androstenol and herba Epimedii extract according to the formula amount in Table 6, adding into dimethyl sulfoxide, and performing ultrasonic assisted dissolution for 20 min; and after the compound is completely dissolved, adding the compound into purified water, starting stirring, adding the Tween-80 in the formula amount while stirring, continuing stirring, adding the preservative, adding the purified water in the formula amount, continuing stirring for 2 hours, and filtering by using a 0.45-micron filter membrane to obtain the F composition solution. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 6 pheromone composition solution formulations

Example 4

The solution formula of the composition containing pheromone androstenone, androstenol and epimedium extract is listed in the table 7. Accurately weighing androstenone, androstenol and herba Epimedii extract according to the formula amount in Table 7, adding into dimethyl sulfoxide, and performing ultrasonic assisted dissolution for 20 min; and after the compound is completely dissolved, adding the compound into purified water, starting stirring, adding the Tween-80 in the formula amount while stirring, continuing stirring, adding the preservative, adding the purified water in the formula amount, continuing stirring for 2 hours, and filtering by using a 0.45-micrometer filter membrane to obtain the G composition solution. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 7 pheromone composition solution formulations

Components	Percent by weight	g
			Androstenone	0.002	0.002
Androstenol	0.002	0.002
			Epimedium extract	0.004	0.004
Dimethyl sulfoxide	1.0	1.0
			Tween-80	0.05	0.05
Preservative	0.3	0.3
			Purified water	98.642	98.642
Total of	100.000	100.000

Comparative example 4

A solution containing 0.002% of Epimedium extract was prepared according to the recipe shown in Table 8. Accurately weighing herba Epimedii extract 0.002% according to formula in Table 8, adding into dimethyl sulfoxide, and ultrasonic dissolving for 20 min; and after the compound is completely dissolved, adding the compound into purified water, starting stirring, adding the Tween-80 in the formula amount while stirring, continuing stirring, adding the preservative, adding the purified water in the formula amount, continuing stirring for 2 hours, and filtering by using a 0.45-micron filter membrane to obtain the H composition solution. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 8 pheromone composition solution formulations

Components	Percent by weight	g
			Epimedium extract	0.002	0.002
Dimethyl sulfoxide	1.0	1.0
			Tween-80	0.05	0.05
Preservative	0.3	0.3
			Purified water	98.648	98.648
Total of	100.000	100.000

Comparative example 5

A solution containing 0.004% of epimedium extract was prepared according to the formulation listed in table 9. Accurately weighing herba Epimedii extract 0.004% according to the formula in Table 9, adding into dimethyl sulfoxide, and ultrasonic-assisted dissolving for 20 min; after the compound is completely dissolved, adding the compound into purified water, starting stirring, adding the Tween-80 with the prescription amount while stirring, continuing stirring, adding the preservative, adding the purified water to the prescription amount, continuing stirring for 2 hours, and filtering by using a 0.45-micron filter membrane to obtain the composition solution I. Before use, the solution was dispensed into a spray device at 100ml per bottle.

TABLE 9 pheromone composition solution formulations

Components	Percent by weight	g
			Epimedium extract	0.004	0.004
Dimethyl sulfoxide	1.0	1.0
			Tween-80	0.05	0.05
Preservative	0.3	0.3
			Purified water	98.646	98.646
Total of	100.000	100.000

Test example 1

The effect of the composition solution on the reproductive behavior and the fertility of the sows is measured.

The study evaluated the efficacy of androstenone, androstenol, and epimedium extract, alone and in combination, to determine their effect on sow reproductive behavior, fertility. The objective of this study was to determine that androstenone, androstenol, and epimedium extract combinations were more effective than either pheromone alone or epimedium extract in improving sow reproductive behavior and fertility, and table 10 is a combination of several pheromones for testing.

Table 10 protocol for the various pheromone compositions tested

Packet sequence number	Examples of the invention	Principal Components
			A	Comparative example 1	Androstenone
B	Comparative example 2	Androstenol
			C	Comparative example 3	Androstenone + androstenol
D	Example 1	Androstenone + epimedium extract
			E	Example 2	Androstenol + Epimedium extract
F	Example 3	Androstenone + androstenol + epimedium extract (0.002%)
			G	Example 4	Androstenone + androstenol + epimedium extract (0.004%)
H	Comparative example 4	Epimedium extract (0.002%)
			I	Comparative example 5	Epimedium extract (0.004%)
J		DMSO control group

The effect of androstenone, androstenol and epimedium extract combination was tested at a commercial farm. The sow after weaning usually enters into the oestrus stage 4-6 days after weaning, so spraying in front of the nostril of the weaning sow is started on the 4 th day after weaning, and performing a back pressing test (namely pressing the back of the sow) after spraying is finished, wherein if the sow has a standing reaction, the sow is oestrous and can be inseminated. If no 'standing' reaction appears on day 4, the product is continuously used the next day. The spraying time is twice in the morning and evening. After application of the pheromone composition to sows 4-6 days post weaning, a total of 168 sows showed oestrus and underwent artificial breeding (a.i.). Table 11 shows the test results.

TABLE 11 test results of different pheromone combination protocols

a, finding that the sow has a standing reaction through a backpressure test; and b, 22 oestrus times exceed a specified period (4-6 days after weaning).

TABLE 12 synergistic test results for different pheromone treatment groups

Treatment of	Proportioning	Is effective	Increase value of%
				Control group	0	43.82	——
Androstenone	1:0	60.20	16.38
				Androstenol	1:0	54.99	11.17
Androstenone + androstenol	1:1	65.00	21.18
				Epimedium extract	1:0	60.68	16.86
Epimedium extract	2:0	62.89	19.07
				Androstenone + epimedium extraction	1:1	74.74	30.92
Extraction of androstenol and epimedium	1:1	74.75	30.93
				Androstenone + androstenol +	1:1:1	89.44	45.62
Androstenone + androstenol +	1:1:2	87.00	43.18

Test example 2

The effect of the pheromone composition on the reproductive performance and fertility of sows, namely dissolving F, was measured, and boars were used as controls. The test procedure was carried out with reference to example 10, in which the weaned sows were treated with the pheromone composition solution F once in the morning and at the evening on days 4-6, i.e. sprayed 1ml each time in front of the nostrils of the sows and then subjected to a "back pressure test" (i.e. pressing the back of the sows) after spraying, and if the sows had a "standing" reaction, this would indicate that they had oestred and were ready for insemination. If no 'standing' reaction appears on day 4, the product is continuously used the next day. In the control group, 18-month-old boars walk in a colony house and are in close contact with sows to be oestred, stimulated and communicated. After applying the pheromone composition to sows 4-6 days after weaning, a total of 238 weaned sows showed oestrus expression and underwent artificial breeding (a.i.). Table 13 shows the test results.

TABLE 13 comparison of the Effect of the pheromone composition on the detection of F by boars

Grouping	Number of test heads	Mean number of births	Number of oestrus	Oestrus rate,% of	Number of pregnant women	Conception rate,%
							Test group	261	3.6±1.32	238	91.19	226	94.96
Control group	243	3.5±1.27	213	87.65	191	89.67

Test example 3

The effect of different doses of the pheromone composition solution F on the reproductive performance and fertility of sows was determined, and boars were used as controls. The test method was carried out with reference to example 11, wherein the test components were divided into 3 groups and each group was administered with a dose of 0.5ml, 1ml, 2ml, respectively; weaning sows are treated with the pheromone composition dissolved F once in the morning and at night of 4-6 days, namely spraying in front of the nostrils of the sows, carrying out a back pressing test (namely pressing the back of the sows) after spraying, and if the sows have a standing reaction, indicating that the sows are oestrous and can be inseminated. If no 'standing' reaction appears on day 4, the product is continuously used the next day. In the control group, 18-month-old boars walk in a colony house and are in close contact with sows to be oestred, stimulated and communicated. After applying the pheromone composition to sows 4-6 days after weaning, a total of 238 weaned sows showed oestrus expression and underwent artificial breeding (a.i.). Table 14 shows the test results.

TABLE 14 test results of different dosage pheromone composition solutions F test

Grouping	Number of test heads	Mean number of births	Number of oestrus	Oestrus rate,% of	Number of pregnant women	Conception rate,%
							0.5ml	91	2.6±0.72	72	79.12	65	87.14
1.0ml	102	2.5±0.63	95	93.14	89	93.68
							2.0ml	109	2.6±0.43	102	93.58	95	93.13
Control group	81	2.7±0.47	69	85.19	64	92.75

Test example 4

The effect of the pheromone composition solution F on sows with different birth times is measured, and the effect on the reproductive behavior expression and the fertility of the sows is measured. Weaned sows with different farrowing times are selected from a certain commercial pig farm, and the weaned sows with the same farrowing time are the same group, namely the weaned sows with the 2 nd farrowing time, the 3 rd farrowing time, the 4 th farrowing time and the 5 th farrowing time. Each group of sows is treated with the pheromone composition solution F once in the morning and at the evening 4-6 days after weaning, namely, the sows are sprayed with 1ml of the pheromone composition solution in front of the nostrils, and a back pressing test (namely, the backs of the sows are pressed) is carried out after spraying, if the sows have a standing reaction, the sows are in estrus and can be inseminated. If no 'standing' reaction appears on day 4, the product is continuously used the next day. Table 15 shows the test results.

TABLE 15 statistics of the effects of pheromone composition solution F on sows at different parity

Number of test births	Number of test heads	Number of oestrus	Oestrus rate,% of	Number of pregnant women	Conception rate,%
						2	51	47	92.16	44	93.62
3	50	46	92.00	42	91.30
						4	49	47	91.83	44	93.62
5	53	50	92.45	46	92.00
						Control group	41	34	82.93	29	85.29

Test example 5

The effect of pheromone composition solution F on weaned sows in different seasons, the reproductive performance of sows, and the effect on fertility were determined. The same number of weaned sows are randomly selected in a certain commercial pig farm, the effect of the pheromone composition solution F on the weaned sows is tested in the months of 4, 7, 10 and 12, and the weaned sows with the same birth times and fertility effect on the reproductive behavior of the sows are the same group. Every group of sows are treated with the pheromone composition solution F in the morning and at the evening of 4 th to 6 th day after weaning, namely, the sows are sprayed in front of nostrils, 1ml of the solution is sprayed each time, and then the sow is subjected to a back pressing test (namely, the back of the sow is pressed), if the sow has a standing reaction, the sow is estrualized and can be inseminated. If no 'standing' reaction appears on day 4, the product is continuously used the next day. Table 16 shows the test results.

TABLE 16 results of the test using the pheromone composition solution F for different seasons

Test example 6

Clinical effect study of other 8 Chinese medicinal extract + pheromone compositions

Referring to examples 1 and 2, the following 8 kinds of Chinese medicinal extract + pheromone composition solutions were prepared, each prepared in 1000ml, and then dispensed in 100ml bottles. The concentration of each Chinese medicinal extract is 0.002% (w/w), and the proportion of the other components is unchanged. The method of use was performed with reference to example 10, and the statistical results are as follows.

Test results of solutions of the composition of Table 178 Chinese medicinal extracts and pheromone

From the data, the 8 Chinese medicinal extract + pheromone compositions screened by the application can promote the oestrus rate.

Test example 7

Clinical effect study of other Chinese medicinal extract + pheromone composition

Referring to examples 1 and 2, the following 4 solutions of the herbal extract + pheromone composition were prepared, each prepared in 1000ml and then dispensed in 100ml bottles. The concentration of each Chinese medicinal extract is 0.002% (w/w), and the proportion of the other components is unchanged. The method of use was performed with reference to example 10, and the statistical results are as follows.

Test results of the solutions of the compositions of the traditional Chinese medicine extract and the pheromone in the table 184

From the above data, it can be seen that no synergistic effect occurred for the 4 herbal extract + pheromone compositions.

Test example 8

The actual effect of the quinoline + pheromone composition and the traditional Chinese medicine extract + pheromone composition in different piggeries and different pens (different batches) of sows in the same piggery is compared. FIG. 3 shows the distribution of different piggeries in the same house in the same pig farm (I-VI represents the distribution of different piggeries).

Quinoline remains in the environment for a long time, which causes the oestrus rate of sows in different pens in the same pig farm to fluctuate obviously, mainly reduces, and the traditional Chinese medicine extract has no similar phenomenon (is very stable); quinoline causes the adaptation of the sow to the quinoline for a long time, is insensitive to the quinoline and does not have the first better effect. The Chinese herbal medicine extract and pheromone are relatively stable.

TABLE 19 results of testing different pheromone composition solutions in different pens (different batches) of sows in the same house of a pig farm

Test example 9

The effect of different pheromone compositions on oestrus rate of sows was compared.

Referring to examples 1 and 2, the following 4 solutions of the herbal extract + pheromone composition were prepared, each prepared in 1000ml and then dispensed in 100ml bottles. The concentration of each Chinese medicinal extract is 0.002% (w/w), and the proportion of the other components is unchanged. The method of use was carried out with reference to example 10.

TABLE 20 comparison of the results of the solution tests of different pheromone compositions

Test example 10

Different pH values were formulated for pheromone compositions. The procedure was carried out in accordance with the preparation process described in example 7 above. Different amounts of glacial acetic acid and 10% sodium hydroxide solution are respectively added dropwise to adjust the pH value of the solution to 4.0, 5.0, 7.0, 8.0 and 10.0.

TABLE 21 preparation of pheromone composition solutions at different pH values

Test example 11

Stability test research is carried out according to the guide principle of stability test of raw material medicaments and preparations in the appendix 302 of 'Chinese veterinary pharmacopoeia' of 2015 edition. The key items include content and pH value. The test conditions are as follows: the temperature is 40 +/-2 ℃; relative humidity, 75% ± 5%; test period, 6 months; the examination time points are shown in table 10. And (3) placing the samples with different pH values in a stability test box (meeting the test conditions) for stability examination.

TABLE 22 solution stability Studies at different pH

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A formulation of a pheromone composition, wherein the formulation is for inhalation administration, the formulation being a spray formulation; comprising a pheromone composition and a carrier;

the pheromone composition comprises:

the component A comprises: androstenone and androstenol;

and B component: the epimedium extract is icariin with the purity of more than or equal to 94.0 percent;

the mass ratio of the component A to the component B is 1-10: 0.5-5; the concentration of the component A is 1-200 mug/ml, and the concentration of the component B is 1-50 mug/ml;

the pH range of the preparation is 5-8;

the preparation also comprises cosolvent, surfactant and antiseptic; the carrier is water.

2. The pheromone composition preparation according to claim 1, wherein the cosolvent is selected from one or more of alcohols, ketones, ethers, sulfones, esters, soybean oil, N-dimethylformamide and N-dimethylacetamide; the mass percentage concentration of the cosolvent is within the range of 0.2-20 percent.

3. A pheromone composition formulation according to claim 1, wherein the surfactant is tween-80; the surfactant concentration is in the range of 0.01% to 0.5%.

4. Use of a formulation of a pheromone composition according to any one of claims 1 to 3 for the preparation of a formulation for stimulating reproductive behavior, increasing reproductive success, or increasing productivity in a female suid.