Composition for chemical castration of pig comprising estradiol benzoate as active ingredient

Abstract

The present invention relates to a composition for chemical castration of pigs comprising estradiol benzoate as an active ingredient. Specifically, according to the present invention, when estradiol benzoate is injected into the subcutaneous fat of the muscles of pigs, the size of the testes is significantly reduced compared to a control group of pigs. The invention provides a composition for chemical castration of pigs capable of eliminating the characteristic boar odor of male pigs by causing the testicular function of pigs using estradiol benzoate.

Description

Technology Field

[0001] The present invention relates to a method for chemically neutralizing piglets by treatment with estradiol benzoate. Background Technology

[0003] In the case of boars (male pigs), their commercial value is generally assessed as low due to a distinctive boar taint. Boar taint refers to an unpleasant smell or taste that appears when pork or pork products are cooked or chewed; while it is difficult to detect in fresh meat, it develops when the meat is heated.

[0004] For this reason, although boar taint is not a substance harmful to the human body in terms of hygiene, it adversely affects the taste, aroma, and flavor of pork, negatively impacting consumer preference and causing consumers to avoid purchasing it. Therefore, from the perspective of pig farmers, the removal of boar taint from boars is very important for improving pork quality.

[0005] There are various steroid compounds that cause boar taint in pork, but the representative causative substances are known to be androstenone, skatole, and indole, and among them, androstenone and skatole are particularly the main causes of boar taint.

[0006] Androsterone is a type of male hormone that generates blood testosterone. Produced in the testes, it travels through the bloodstream and is either broken down in the liver or converted into sex hormones. Any excess that is not broken down or converted is characterized by its accumulation in fat in an oxidized form. The amount of androsterone accumulated in fat varies significantly depending on the location of the fat.

[0007] Skateol is a chemical naturally produced from the breakdown of bacterial proteins in the large intestine, and is known to be primarily generated during the breakdown of tryptophan, an amino acid. Skateol is also found in human feces. This generated skateol is broken down in the liver, but androsterone plays a role in inhibiting its breakdown. Undigested skateol is absorbed in the intestines and accumulates in lean meat and fat, with particularly high accumulation occurring in back fat.

[0008] Skateol production is found in castrated and uncastrated pigs, boars, and sows, but skateol accumulation is significantly observed in the fat of boars.

[0009] Androsterone and skatole accumulated in the fat in this way evaporate into the air and disappear during the cooking process of pork, and the odor left by the vaporized molecules is the boar odor.

[0010] For centuries, pig farms at home and abroad have been castrating male pigs at about 2 to 3 weeks of age to prevent this boar taint.

[0011] Castration of boars is highly advantageous for farmers seeking to supply high-quality pork, as it not only prevents boar taint but also reduces the pig's aggression, thereby decreasing the risk of harm to humans and other animals; prevents the risk of unwanted hybridization; increases individual growth rates and feed efficiency; and thins muscle fibers to improve pork tenderness, which can increase the proportion of optimal meat quality. For these reasons, most domestic farms have been performing castration on fattening pigs.

[0012] Surgical castration methods include severance, severance, severance, severance, partial castration, and bloodless castration; in addition, there is also a method of rendering the gonads incapable by irradiating them with a certain amount of radiation sensitive to cause a loss of function.

[0013] Surgical castration is the most preferred method by farmers because it is relatively simple to perform and can effectively remove boar taint at a low cost compared to other castration methods (such as chemical castration).

[0014] However, surgical castration has the following disadvantages.

[0015] It is known that the secretion of androgens, which increase red meat production, decreases, resulting in lower productivity in castrated pigs compared to uncastrated pigs; consequently, castrated pigs show a growth rate of about 10%, feed efficiency of about 12%, and red meat production of about 6.5% lower than uncastrated pigs ((Jensen, M. et al. 1995. Appl. Environ. Microbiol.61: 3180-3184).

[0016] Furthermore, the physical surgical castration currently performed on farms is carried out without anesthesia, causing excessive stress and suffering to boars due to the pain of the procedure (Prunier et al., 2005). This stress and suffering lead to a decrease in the pig's immunity and a consequent weakening of disease resistance, which actually contributes to an increase in mortality rates (English, P. et al. 1975. Vet Ann 15:95-104; English, P. et al. 1984. Pig News and Information 5: 369-376). As corroborating evidence for this, there are research results indicating that non-surgical castration can increase the profitability of lean meat production by approximately 30% (Lange, C. et al. 1995. Ontario Swine Research Review: 41-44).

[0017] Moreover, with the recent rise in global interest in animal welfare, surgical castration—particularly methods performed without anesthesia—is becoming a major point of contention regarding livestock management practices. While some countries, such as the Netherlands, Norway, and Switzerland, use anesthetics to alleviate stress and pain caused by the physical castration of boars, public opinion is increasingly calling for the restriction of such practices for the sake of animal welfare, regardless of whether anesthesia is used.

[0018] According to the European Union agreement published in 2010, for the sake of animal welfare, anesthesia or analgesics must be administered when performing surgical castration starting from January 2012, and surgical castration must be phased out starting from 2018 (De Briyne et al., 2016). Although Korea does not yet have such restrictions, it is considering similar measures in the near future.

[0019] Accordingly, as a method to eliminate boar odor, instead of surgical castration to remove the testicles, research is actively being conducted on methods to specifically remove only androstenone and skateol, the main substances causing boar odor.

[0020] The most traditional alternative is genetic breeding. This is a research method that analyzes the correlation between genetic variation occurring while raising boars and the amount of boar odor substances accumulated in vivo, but it has the disadvantage of taking a long time to research and requiring a large amount of research funding for long-term studies.

[0021] Another alternative is immuno-castration using an immuno-castration vaccine. Immuno-castration vaccines are one of the safe and efficient methods for suppressing boar taint by utilizing the pig's immune system. By generating specific antibodies against porcine gonadotropin-releasing hormone (GnRH), immuno-castration vaccines suppress testicular function, thereby inhibiting the production and accumulation of androsterone and skatole. In this regard, Pfizer's immuno-castration vaccine, 'Improvac,' has been commercialized in many countries worldwide and obtained product approval in Korea in 2006. However, in Korea, immuno-castration alone does not fully meet the castration criteria. Furthermore, globally, castration using immuno-castration vaccines costs nearly 50 times more than surgical castration. Consequently, this technology has effectively become obsolete as it has failed to serve as a realistic alternative for pig farmers seeking economic benefits.

[0022] Another method for removing boar taint from boars is chemical castration.

[0023] Chemical castration methods began in 1960 with research into substances that inject sclerosing agents directly into the testicles to induce the loss of testicular function.

[0024] Substances known to date to induce testicular failure include ferrous chloride (Kar et al. 1965), danazol (Dixit et al., 1975), BCG (Das et al. 1982), zinc tannin (Fahim et al., 1982), glycerol (Weinbauer et al., 1985, Immegart 2000), glucose, NaCl (Heath et al., 1987, Russell et al. 1987), DBCP (Shemi et al. 1988), lactic acid (Fordyce et al. 1989), zinc arginine (Fahim et al. 1993), sodium fluoride (Sprando et al. 1996), formalin (Balar et al. 2002), and calcium chloride (Samanta 1998, Jana et al. 2002), as well as glacial acetic acid. Potassium permanganate (Giri et al. 2002), lactic acid (Hill et al. 1985), tannic acid, zinc sulfate (Feher et al. 1985), alpha-hydroxypropionic acid (Cohen et al. 1995), formalin (Ijaz et al. 2000), and CastrateQuin 14 (Soerensen et al. 2001) are known, and their subjects are monkeys, hamsters, rabbits, mice, and dogs.

[0025] Estradiol benzoate, an estrogenic substance distinct from the aforementioned substance, has been reported to induce testicular insufficiency primarily in rats, a type of rodent (Korean Patent Publication 10-2021-0114399, ROUGE, Marion, et al. Ex vivo effects of 17β-estradiol on the prepubertal rat testis. Reproductive Toxicology, 2023, 118: 108363.).

[0026] However, there were no studies utilizing estradiol benzoate for the castration of male piglets. Accordingly, the inventors conducted research to find a method to eliminate boar taint without castrating boars, not only for the improvement of productivity and feed efficiency and disease resistance of pigs, but also for animal welfare. As a result, they confirmed that when boar piglets were consistently treated with estradiol benzoate for 3 weeks, the size of the testes was significantly reduced and the function of Leydig cells was impaired, thereby completing the present invention. The problem to be solved

[0028] The object of the present invention is to provide a composition for chemical castration of pigs comprising estradiol benzoate as an active ingredient.

[0029] In addition, the objective of the present invention is to provide a method for chemically castrating pigs using a composition for chemically castrating pigs containing estradiol benzoate as an active ingredient.

[0030] In addition, the objective of the present invention is to provide a pig that has been castrated by a method of chemical castration of a pig using a composition for chemical castration of a pig containing estradiol benzoate as an active ingredient. means of solving the problem

[0032] In order to achieve the above objective,

[0033] The present invention provides a composition for chemical castration of pigs comprising estradiol benzoate as an active ingredient.

[0034] The composition according to the present invention can be locally injected into male pigs.

[0035] According to the present invention, the estradiol benzoate can be injected into male pigs at a dose of 0.6 mg / kg to 2.4 mg / kg.

[0036] In addition, the composition according to the present invention can be injected starting from day 1 and for at least 21 days.

[0037] In addition, the present invention provides a pig that has been chemically castrated by the chemical castration method of the pig.

[0038] In addition, the present invention provides a pig that has been chemically castrated by the above-described chemical castration method, and has a sperm concentration in the testes that is 30 times lower than that of a pig that has not been chemically castrated. Effects of the invention

[0040] The present invention relates to a composition for chemical castration of pigs containing estradiol benzoate as an active ingredient. Specifically, according to the present invention, when estradiol benzoate is injected into male piglets for about 3 weeks, the size of the testes is significantly reduced compared to the control group, and the secretion of sex hormones and testicular production are inhibited, whereas there is no significant difference in mortality rate, change in body weight, blood analysis, and carcass yield compared to the control group. It has been confirmed that by using the composition of the present invention, the testicular function of male pigs can be selectively inhibited without side effects, thereby eliminating boar odor. Brief explanation of the drawing

[0042] Figure 1 is a diagram showing the size of testes by treatment group observed visually. Figure 2 is a figure showing the ratio of testicular weight to body weight by treatment group. Figure 3 is a figure showing testicular tissues by treatment group stained with H&E. Figure 4 shows the expression of cytochrome P450, a marker protein of Leydig cells in testicular tissues, according to treatment group. Figure 5 is a figure showing the ratio of testicular weight to body weight of the treatment group by treatment period. Figure 6 is a figure showing the size of the testes observed visually over the course of the treatment period for each treatment group. Figure 7 shows the number of sperm per unit area of ​​testicular tissue in the treatment group according to the treatment period. Figure 8 is a figure showing the expression levels of LH and GNRH of the treatment group according to the treatment period. Figure 9 shows the expression levels of FSH and Testosterone in the treatment group according to the treatment period. Specific details for implementing the invention

[0043] The present invention will be described in detail below.

[0044] The present invention provides a composition for chemical castration of pigs comprising estradiol benzoate (EB) as an active ingredient.

[0045] The above estradiol benzoate is (17β)-17-hydroxyestra-1,3,5(10)-trien-3-yl benzoate according to IUPAC (International Union of Pure and Applied Chemistry) nomenclature.

[0046] The above estradiol benzoate has the structure of the following chemical formula I, and the chemical formula is C 25 H 28 It is a compound having O3 and a molecular weight of 376.488, and derivatives thereof are also included as long as they have the effect of reducing or eliminating boar odor in pigs.

[0047] Chemical Formula I

[0048]

[0050] The above estradiol benzoate is an analog of estradiol that binds to estrogen receptors.

[0051] The above active ingredient refers to an amount (effective amount) sufficient to reduce or eliminate boar taint in pigs. The effective amount of estradiol benzoate for reducing or eliminating boar taint in pigs may vary depending on the age (years) and body weight of the pigs, and may be in the range of 0.6 mg / kg to 2.4 mg / kg, preferably 1.2 mg / kg to 2.2 mg / kg, most preferably 1.0 mg / kg to 2.0 mg / kg.

[0052] The above composition may further comprise suitable pharmaceutically acceptable carriers, excipients, and diluents. The composition of the present invention may further comprise carriers, excipients, and diluents commonly used when preparing pharmaceutical compositions containing steroid derivatives, and preferably further comprise corn oil, but is not limited thereto.

[0053] In addition, the present invention provides a method for chemically castrating a pig by injecting the above composition into a male pig.

[0054] The method of administering the above-described composition for chemical castration of pigs may be administered through a general route capable of reaching the target tissue. Depending on the purpose, the composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, or orally, and preferably intramuscularly.

[0055] In addition, the present invention provides a method for chemically castrating pigs by injecting the above composition into male pigs, preferably male pigs that are not adult pigs, most preferably male pigs that are one day old.

[0056] In addition, the present invention provides a method for chemically castrating a pig by injecting the above composition continuously for 7 days or more, preferably 14 days or more, most preferably 21 days or more.

[0057] In addition, according to the present invention, in order to maximize castration efficiency and boar odor removal effect while minimizing side effects, it may be advantageous to inject estradiol benzoate at a dose of 0.6 mg / kg body weight to 1.4 mg / kg body weight, preferably 0.8 mg / kg body weight to 1.2 mg / kg body weight, most preferably 1.0 mg / kg body weight for 1 to 14 days after the start of the composition injection, and from the 15th day after the start of the injection at a dose of 1.6 mg / kg body weight to 2.4 mg / kg body weight, preferably 1.8 mg / kg body weight to 2.2 mg / kg body weight, most preferably 2.0 mg / kg body weight.

[0058] In addition, the present invention provides a pig that has been chemically castrated by the above-described method for chemical castration of pigs. The chemically castrated pig is characterized by having a sperm concentration in the testes that is at least 10 times lower than that of a normal male pig.

[0060] In specific embodiments and experimental examples of the present invention, the inventors treated 1-day-old male pigs with estradiol benzoate (EB) at different concentrations [control group, test group-1 (low dose administration, 1 mg / kg), test group-2 (high dose administration, 2 mg / kg)] and investigated body weight, mortality rate, and changes in testicular tissue.

[0061] As a result, there were no significant differences in mortality rate and changes in body weight among the treatment groups. On the other hand, it was confirmed that the testicular weight of the low-dose and high-dose treatment groups was significantly reduced compared to the control group (Figs. 1 and 2).

[0062] In addition, H&E staining of testicular tissue revealed that the spacing between seminiferous tubules in the testicular tissue of the low-dose and high-dose treatment groups decreased compared to the control group (Fig. 3). Furthermore, verification of the function of Leydig cells in the control and high-dose treatment groups confirmed that the expression of cytochrome P450, a marker protein of Leydig cells, was significantly reduced in the high-dose treatment group (Fig. 4).

[0063] To verify whether there were any side effects other than the inhibition of testicular tissue function by EB, the blood count (CBC) of each treatment group at 4 weeks of age was analyzed, and there was no significant difference compared to the control group.

[0064] Thus, it was confirmed that there were no side effects associated with low and high doses, and the EB injection amount for piglets was determined to be only high dose. Short-term treatment (1 day) and long-term treatment (continuous treatment for 21 days) were selected, and the long-term (7 months) chemical neutralization effect was verified. The treatment groups were divided into a control group, test group-3 (short-term treatment, high dose injection for 1 day), and test group-4 (continuous high dose injection for 1-21 days).

[0065] Although there were no significant differences in mortality rates and changes in body weight among the treatment groups, only in test group-4 did testicular size and the testicular-to-body weight ratio decrease at months 1, 2, 3, and 7 (Figs. 5 and 6). In addition, the sperm concentration in the testes in test group-4 (high-dose continuous infusion from days 1-21) was approximately 30 times lower compared to the control group (Fig. 7). Furthermore, it was confirmed that LH and GNRH hormones significantly decreased in the blood of test group-4 (low-dose continuous infusion from days 1-14 / high-dose continuous infusion from days 15-21) (Fig. 9). On the other hand, by confirming that there were no significant differences in blood analysis, carcass yield, large cut meat production, carcass yield relative to fasting body weight, and large cut meat yield relative to fasting body weight between the control and treatment groups, it was found that male pigs can be chemically castrated without side effects by treating with EB for 3 consecutive weeks.

[0067] The present invention will be explained in detail below through examples and experimental examples.

[0068] However, the following examples and experimental examples are merely illustrative of the present invention in one aspect, and the present invention is not limited to the following examples and experimental examples.

[0070] <Example 1> Setting the Optimal Estradiol benzoate Concentration for Deriving Neutralization Effect

[0071] To determine the optimal concentration of Estradiol benzoate (EB) that does not affect growth and mortality in piglets, piglets were treated with Estradiol benzoate at different concentrations, and changes in body weight, mortality rate, and testicular tissue were investigated.

[0072] Specifically, 27 one-day-old male piglets (among male piglets born from artificial insemination of sows on the same date at the National Institute of Animal Science, those born on the same date were used as the same treatment group, with a one-day difference in birth dates between treatment groups. Pig rearing management was conducted in compliance with the National Institute of Animal Science management manual) were randomly divided into three treatment groups [control group, test group-1 (low dose, 1 mg / kg), test group-2 (high dose, 2 mg / kg)]. Neutralization reagents (Estradiol benzoate and Corn oil) were purchased from Sigma, and other reagents and glassware were general laboratory grade products.

[0073] To treat 1-day-old piglets with estradiol benzoate, estradiol benzoate was dissolved in corn oil. The control group used only corn oil, while test group-1 (low dose, 1 mg / kg) and test group-2 (high dose, 2 mg / kg) were each injected into the hip muscles of the piglets at concentrations of 1 mg / kg or 2 mg / kg, adjusted to individual body weight, for 21 consecutive days. The amount of estradiol benzoate injected was also increased in accordance with body weight gain (maintaining the injection amount of estradiol benzoate relative to body weight at 1 mg / kg for test group-1 and 2 mg / kg for test group-2). The volume of corn oil or estradiol benzoate injected into the pigs was 1 ml during weeks 1 to 2, and increased to 2 ml during week 3. Estradiol benzoate was infused continuously for 3 weeks, and Estradiol benzoate was not infused during the last 4 weeks.

[0074] After 4 weeks, all pigs were subjected to a test slaughter (blood collection and tissue sampling including testes). The sampled testes were weighed and measured, washed with physiological saline for tissue fixation, and immersed in a fixative for tissue staining. The testicular tissues were prepared into paraffin blocks, sectioned, and subjected to tissue H&E and immunohistochemistry. Immediately before the test slaughter, approximately 5 ml of blood was collected via jugular vein using a Vacutanier EDTA (K2) tube for whole blood and a vacutainer SST II plus plastic serum tube for serum. The whole blood collected was immediately analyzed for general blood properties (such as RBC, HCT, HGB, NEU, EOS, and BASO) using ProCyte Dx reagent (99-26306-00) and a ProCyte Dx Hematology Analyzer. For serum biochemical analysis, the collected blood was left to stand at room temperature for 1 hour, then centrifuged (4℃, 3000xg, 20 min) to separate the serum, and physicochemical analysis was performed using an automated analyzer with the IDEXX Catalyst Chem 17 CLIP (IDEXX) kit.

[0075] division month fury number neck gold saturday Day date 3 / 21 22 23 24 25 26 27 Control / Low / High Dose Week 1 EB Infusion (1ml) EB injection (1ml) EB injection (1ml) EB injection (1ml) First 1 2 3 date 28 29 30 31 4 / 1 2 3 Control / Low / High Dose EB injection (1ml) EB injection (1ml) EB injection (1ml) EB injection (1ml) Week 2 EB Infusion (1ml) EB injection (1ml) EB injection (1ml) First 4 5 6 7 8 9 10 date 4 5 6 7 8 9 10 Control / Low / High Dose EB injection (1ml) EB injection (1ml) EB injection (1ml) EB injection (1ml) Week 3 EB Infusion (2ml) EB injection (2ml) EB injection (2ml) First 11 12 13 14 15 16 17 date 11 12 13 14 15 16 17 Control / Low / High Dose EB injection (2ml) EB injection (2ml) EB injection (2ml) First 18 19 20 21 22 23 24 date 18 19 20 21 22 23 24 Control / Low / High Dose blood draw Sampling First 25 26 27 28

[0076] Body weight and mortality rates by treatment group over 4 weeks

[0077] Mortality rate : 1 out of 9 pigs in test group-1 died (1 / 9). No deaths occurred in the 9 control pigs and 9 test group-2. This confirmed that low dose (1 mg / kg) and high dose (2 mg / kg) of Estradiol benzoate administered did not induce mass mortality in pigs.

[0078] weight There was no significant difference in the pattern of body weight gain among the treatment groups following Estradiol benzoate treatment.

[0080] Weight gain pattern over 4 weeks Life Week 1 Week 2 Week 3 Week 4 Control group 1.45 2.56 4.74 7.08 8.77 High capacity 1.40 2.52 4.71 7.02 8.56 low dose 1.46 2.60 4.81 7.43 8.67

[0081] Comparison of Week 4 weights by treatment group weight Average (kg) Standard deviation Control group (n=7) 8.77 0.72 Low dose (n=8) 8.56 0.84 High capacity (n=9) 8.34 0.94

[0083] Thus, statistical analysis results confirmed that there was no significant difference in body weight between the low-dose and high-dose Estradiol benzoate injection treatment groups in 4-week-old pigs.

[0085] Investigation of testicular weight by treatment group at 4 weeks of age

[0086] The testicular size of the 4-week-old treatment group was observed, and it was confirmed that testicular growth was inhibited in both the low-dose and high-dose treatment groups compared to the control group (Fig. 1).

[0087] To go beyond visual observation and for accurate analysis, the ratio of testicular weight to body weight was investigated for each individual in the treatment group, and it was confirmed that the ratio of testicular weight to body weight was significantly reduced in both the low-dose and high-dose treatment groups (Fig. 2).

[0089] Changes in internal testicular tissue of pigs by treatment group at 4 weeks of age

[0090] To identify the cause of inhibition of testicular size growth in the low-dose and high-dose treatment groups compared to the control group, testicular tissues from the control group and the low-dose / high-dose treatment groups at week 4 were stained with H&E, and the number of seminiferous tubules and the diameter of the seminiferous tubules within the stained testes were examined.

[0091] H&E staining results showed that the number of seminiferous tubules per unit area in the testicular tissue of the low-dose / high-dose treatment groups significantly increased compared to the control group, and the spacing between seminiferous tubules in the testicular tissue of the low-dose / high-dose treatment groups decreased compared to the control group (Fig. 3).

[0092] On the other hand, there was no significant difference in the diameter of the seminiferous tubules between the control group and the low / high dose treatment groups.

[0093] Number of cleaning pipes per treatment district Cleaning water (mm) 2 ) SE Control group 282.25 29.56 low dose 431.13 29.56 High capacity 419.63 29.56

[0094] Cleaning pipe diameters by treatment zone Diameter of the drainage tube (um) SE Control group 46.67 2.03 low dose 49.39 2.03 High capacity 48.41 2.03

[0095] In order to determine the cause of the decrease in the spacing between seminiferous tubules in the testicular tissue compared to the control group in the low-dose / high-dose treatment groups, the testicular tissues of the control and high-dose treatment groups at week 4 were immunostained and the function of Leydig cells was verified.

[0096] As a result, it was confirmed that the expression of cytochrome P450, a marker protein of Leydig cells, was significantly reduced in the high-dose treatment group (Fig. 4).

[0097] As is known, the primary function of Leydig cells is their involvement in the secretion of male hormones; therefore, it could be inferred that the decline in Leydig cell function observed in the testicular tissue of the high-dose treatment group resulted in a decrease in testicular function, namely sperm production capacity (as confirmed in the long-term effect verification experiment following Estradiol benzoate treatment described later, the sperm production capacity of the long-term Estradiol benzoate treatment group was reduced).

[0099] Biochemical and hemocytic analysis of pig blood by treatment group at 4 weeks of age

[0100] The blood cell count (CBC) of the blood of each treatment group at 4 weeks of age was analyzed, and the results are shown in Table 6 below.

[0101]

[0102] The analysis results showed no significant differences in major blood indicators between the control group and the treatment group.

[0103] Furthermore, the biochemical characteristics of the blood were analyzed for each treatment group at 4 weeks of age, and the results are shown in Table 7 below.

[0104]

[0105] As a result of analyzing the blood cell and biochemical characteristics of the 4-week-old treatment groups, neither the low-dose nor the high-dose treatment groups showed significant differences compared to the control group, which confirmed that there were no harmful side effects in the body other than in the testes, even with high-dose treatment (2 mg / kg) as well as low-dose treatment.

[0107] <Example 2> Verification of Long-term Effects of Chemical Neutralization of Estradiol benzoate in Piglets

[0108] It was confirmed that there were no harmful side effects in the body other than in the testes even with high-dose treatment (2 mg / kg), so piglets were treated with 2 mg / kg of Estradiol benzoate and the resulting long-term (7 months) chemical neutralization effect was verified.

[0109] Specifically, 30 one-day-old male piglets (among male piglets born from artificial insemination of sows on the same date at the National Institute of Animal Science, those born on the same date were used as the same treatment group, with a difference of 3 days in birth dates between treatment groups. Pig rearing management was conducted in compliance with the National Institute of Animal Science management manual) were randomly divided into three treatment groups [control group, test group-3 (short-term treatment group: high dose for 1 day), test group-4 (long-term treatment group: high dose for 21 consecutive days)]. Neutralization reagents (Estradiol benzoate and Corn oil) were purchased from Sigma and used, while other reagents and glassware were general laboratory grade products.

[0110] For 1-day-old piglets, the control group was injected daily with only corn oil for 21 consecutive days, while Test Group-3 (injection daily for 1 day) and Test Group-4 (injection daily for 21 consecutive days) were each individually injected with estradiol benzoate dissolved in corn oil at a dose of 2 mg / kg into the hip muscles of the piglets for 1 or 21 consecutive days. Additionally, the amount of estradiol benzoate injected was increased in accordance with the body weight gain of the pigs as they grew (ensuring that the injection amount of estradiol benzoate relative to body weight was maintained at 2 mg / kg).

[0111] The volume of corn oil or estradiol benzoate injected into pigs was 1 ml during weeks 1 to 2, and 2 ml during week 3. Estradiol benzoate was injected for a maximum of 3 weeks, after which no treatment was performed. Additionally, two pigs per treatment group were test-slaughtered each month at 1, 2, and 3 months from the time of the initial estradiol benzoate treatment, and four pigs per treatment group were test-slaughtered in the final 7th month.

[0112] division month fury number neck gold saturday Day date 8 / 29 30 31 9 / 1 2 3 4 Control group Corn oil Week 1 Corn oil Week 1 Corn oil Week 1 Test Zone-3 EB (subcutaneous) once Test Zone-4 EB (Muscle) Week 1 work date 5 6 7 8 9 10 11 Control group Corn oil Week 1 Corn oil Week 1 Corn oil Week 1 Corn oil Week 1 Corn oil week 2 Corn oil week 2 Corn oil week 2 Test Zone-3 Test Zone-4 Week 1 of EB Week 1 of EB Week 1 of EB Week 1 of EB Week 1 of EB Week 1 of EB Week 2 of EB work date 12 13 14 15 16 17 18 Control group Corn oil week 2 Corn oil week 2 Corn oil week 2 Corn oil week 2 Corn oil week 3 Corn oil week 3 Corn oil week 3 Test Zone-3 Test Zone-4 Week 2 of EB Week 2 of EB Week 2 of EB Week 2 of EB Week 2 of EB Week 2 of EB Week 3 of EB work date 19 20 21 22 23 24 25 Control group Corn oil week 3 Corn oil week 3 Corn oil week 3 Corn oil week 3 Test Zone-3 Test Zone-4 Week 3 of EB Week 3 of EB Week 3 of EB Week 3 of EB Week 3 of EB Week 3 of EB work

[0113] Sampling and treatment of blood (blood collection and hemocytic biochemical analysis before test slaughter) and testes (weighing and immersion in fixative after test slaughter) were carried out in the same manner as described in Example 1, according to the test slaughter time (1, 2, 3, and 7 months). For the analysis of sperm in pig semen, semen was collected twice from 3 pigs in each treatment group out of the 4 surviving 6-month-old pigs in each treatment group, and the sperm concentration in the semen was measured using a hemocytometer. For the analysis of sex hormones in the blood of 4 7-month-old pigs in each treatment group, an ELISA kit for analyzing porcine sex hormones (LH, FSH, GNRH, Testosterone) was used. Additionally, carcass yield (the ratio of meat, fat, bone, etc. that can be obtained from one pig) was investigated to compare the meat production of the 7-month-old pigs (4 pigs / per treatment group) that were test-slaughtered.

[0115] Pig survival rate and body weight change by treatment group

[0116] No mortality occurred across all treatment groups. The change in body weight by treatment group was statistically analyzed using the body weight (kg) of pigs aged 0 to 7 months in each treatment group (Table 9), and no significant difference was found between treatment groups.

[0117]

[0119] Average weight by treatment group at month 7 Average (kg) SD Contrast (n=4) 72.13 10.62 Short term (n=4) 77.00 10.19 Long-term (n=4) 82.63 9.04

[0120] Survey of pig testicular size by treatment group

[0121] After treatment with estradiol benzoate, the size of pig testes and the ratio of testes to body weight per unit body weight were measured for each treatment group (Figs. 5 and 6).

[0122] Testis / body weight ratio by treatment group and treatment duration Testis / Weight Ratio (%) 1 month (n=2) 2 months (n=2) 3 months (n=2) 7 months (n=4) Control group 0.051 0.037 0.038 0.222 Short-term (1 time) 0.058 0.045 0.049 0.188 long time 0.026 0.026 0.024 0.165

[0123] Analysis results showed that only in test group-4 (low dose administered from days 1-14; high dose administered from days 15-21) a decreasing trend in the testis-to-body weight ratio was observed at 1, 2, 3, and 7 months. Statistical analysis was not possible for the treatment groups at months 1, 2, and 3 as there were only 2 pigs in each group.

[0124] It was found that Estradiol benzoate inhibited testicular growth through the reduced testicular-to-body weight ratio in test group-4.

[0126] Investigation of pig sperm production capacity by treatment group

[0127] Sperm production capacity was assessed by analyzing sperm concentration in the testes, and the results showed that the sperm concentration in testes in test group-4 was about 30 times lower than that in the control group. This indicates that the reproductive capacity of males was reduced by Estradiol benzoate (Fig. 7).

[0128] Sperm count by treatment group Average ( x 10 5 / ml) SD Contrast (n=6) 545 232.8 Short term (n=6) 535 163.2 Long-term (n=6) 18 18.9

[0129] Investigation of pig sex hormone levels by treatment group

[0130] The levels of sex hormones (LH, FSH, GNRH, Testosterone) in the blood of 7-month-old pigs (4 pigs / treatment group) in the control and treatment groups were analyzed. As a result, it was confirmed that the LH and GNRH hormones in the blood of test group-4 were significantly reduced (Fig. 8).

[0131] On the other hand, FSH and Testosterone hormones showed a decreasing trend in treatment group-4, but since the number of individuals per treatment group was 4, the variation between individuals was large, so it was found to be statistically insignificant (Fig. 9).

[0133] Analysis of porcine blood cells (CBC) by treatment group

[0134] The blood counts (CBC) of 7-month-old pigs (4 pigs per treatment group) in the control and treatment groups were analyzed. As a result, the WBC and NEU indicators were found to be slightly higher in test group-3 compared to the control group, but excluding these, no significant difference was observed between the control and treatment groups for the remaining indicators.

[0135]

[0137] Biochemical analysis of pig blood by treatment group

[0138] The biochemical characteristics in the blood of 7-month-old pigs (4 pigs per treatment group) in the control and treatment groups were analyzed. As a result, no significant difference was found between the control and treatment groups.

[0139]

[0141] Pig slaughter by treatment district Comparison of production volume (kg) before and after

[0142] The carcass yield of 7-month-old pigs (4 pigs / group) was investigated for each control group and treatment group, and as a result, there was no significant difference in the comparison of production volume (kg) before and after slaughter between treatment groups.

[0143]

[0144] (p<0.05)

[0146] Analysis of Pork Major Meat Production by Treatment Group

[0147] In addition to the comparison of production volume (kg) before and after pig slaughter by treatment group above, the production volume of 7 types of large cut meat was compared in detail.

[0148] As a result, there was no significant difference in the production (kg) of large cut meat (7 types) between the control group and the short / long-term treatment groups of 7-month-old pigs, similar to the production volume.

[0149]

[0150] Cold carcass weight: Weight including feet, kidneys, and kidney fat

[0151] Meat Production: Meat yield with only body fat removed during large cut meat trimming.

[0152] Traded Meat: Meat production volume with surface fat removed to a minimum level (0mm) during large cut trimming.

[0153] ** No significant difference between treatment groups (p<0.05)

[0155] Comparison of Carcass Yield (%) Relative to Fasting Weight by Treatment Group

[0156] The carcass yield relative to fasting body weight (body weight measured immediately before slaughter after stopping feed for 1 day prior to slaughter) of 7-month-old pigs (4 pigs / group) in the control group and short / long-term treatment groups was investigated. As a result, there was no significant difference in carcass yield (%) relative to fasting body weight between the control group and the short / long-term treatment groups.

[0157]

[0158] (p<0.05)

[0160] Comparison of Large Broth Yield (%) Relative to Fasting Body Weight by Treatment Group

[0161] As a result of investigating the large meat yield of 7-month-old pigs (4 pigs / per treatment group) in the control group and short / long-term treatment groups, no significant difference was found in the large meat yield (%) relative to fasting body weight between the control group and the treatment groups.

[0162]

[0163] * Cold Carcass Rate: Ratio of cold carcass weight to fasting body weight

[0164] Meat Yield and Trade Meat Yield: Ratio of large cut meat production to fasting body weight

[0165] ** No significant difference between treatment groups (p<0.05)

Claims

Claim 1 A method for chemically castrating pigs, wherein estradiol benzoate is administered daily by intramuscular injection to 1-day-old male pigs, at a dose of 0.6 mg / kg to 1.4 mg / kg body weight for 1 to 14 days from the start of administration, and at a dose of 1.6 mg / kg to 2.4 mg / kg body weight from the 15th day onwards, wherein the sperm concentration in the testes of the male pigs according to the above method is at least 10 times lower than that of untreated male pigs, and LH and GnRH expression is reduced compared to untreated male pigs. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 In claim 1, the method wherein the injection is performed daily for 21 days or more. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 A pig that has been chemically castrated by the method of paragraph 1 or 6. Claim 11 delete

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