Application of the combination of tomato extract and eugenol in the preparation of feed additives for improving the reproductive performance of boars

Abstract

The present invention provides the application of the combination of tomato extract and eugenol in the preparation of a feed additive for improving the reproductive performance of boars, belonging to the technical field of efficient livestock and poultry breeding. First, the tomato extract is prepared and then mixed with eugenol. The mixture is added to the complete feed for boars at a ratio of 1.5‰ to feed the boars. The results show that by combining tomato extract and eugenol in a specific ratio, the sperm motility, sperm density and ejaculate volume of boars can be improved, and the sperm malformation rate of boars can be reduced, achieving the purpose of improving the quality of pig semen and maintaining the stable reproductive performance of boars.

Description

Technical Field

[0001] The present invention belongs to the technical field of efficient livestock and poultry breeding, and mainly relates to the application of the combination of tomato extract and eugenol in improving the reproductive performance of boars. Background Art

[0002] With the development of modern pig farming, intensive farming has become increasingly common. In large-scale production, the reproductive efficiency of boars is very crucial and is an important factor affecting production efficiency. Artificial insemination (AI) technology has been widely used in animal production and breeding work. At present, in production, the breeding tasks of boars are arranged tightly, which puts high requirements on the physical fitness and reproductive performance of boars. The daily metabolic needs of boars are generally met by feed. In production practice, proteins, carbohydrates, etc. are often added additionally to the complete feed for boars to strengthen nutrition, or green forage is supplemented to promote digestion and absorption. However, there is still a large gap in the reproductive efficiency of domestic boars compared with that of foreign countries, and the passing rates of phenotypic indicators such as sperm motility, malformation rate, and density of boar fertility still need to be improved.

[0003] Nutrition is an important factor affecting the reproductive performance of boars. Different amino acid ratios in the diet can affect the reproductive performance of boars (Ren, B et al., 2015. Effect of different amino acid patterns on semen quality of boars fed with low-protein diets. Anim. Reprod. Sci. 161, 96-103). Research has shown that increasing dietary lysine from 0.86% to 1.03% or supplementing threonine can improve the semen quality of boars (Rupanova, M., 2006. Influence of different lysine's levels in the compound feeds for boars on quantity and quality of the semen; Close, W.H et al., 1993. Nutrition of the working boar. Recent Dev. Pig Nutr. 2, 347-368). A lack of tryptophan can lead to a decrease in sperm count (Castrogiovanni, P et al., 2014. Effects of high-tryptophan diet on pre-and postnatal development in rats: a morphological study. Eur. J. Nutr. 53, 297-308). In addition, supplementing selenium and vitamin E can improve the sperm quality of boars (Marin-Guzman et al., Effect of dietary selenium and vitamin E on spermatogenic development in boars. J. Anim. Sci. 78: 1537–1543, 2000). In addition to regulating the fertility of boars through conventional nutrients such as mineral elements, amino acids, vitamins (or complexes of the above nutrients), in recent years, researchers have also paid more and more attention to the application of functional feed additives such as plant extracts and fatty acids in improving the reproductive performance of boars (Wei Hongkui, Peng Jian. Research progress on nutritional regulation of boar semen quality. Acta Zoonutrimenta Sinica, 2020, 32(10): 4540-4549). With the promotion of the action to reduce and replace antibiotics in feed, the effects of plant extracts on animal growth and reproduction have gradually been discovered and applied.

[0004] Oxidative stress is one of the main causes of male factor infertility, and the use of antioxidants to improve male reproductive performance is a research hotspot. Many studies have shown that antioxidants such as vitamin E and C can reduce oxidative stress in male animals (Agarwal A, et al., Role of antioxidants in treatment of male infertility: an overview of the literature. Reprod Biomed Online. 2004;8:616–27). Lycopene is a natural pigment in plants, a tomato extract made from tomatoes, and belongs to the carotenoid family. It contains 11 conjugated double bonds, can transfer electrons to free radicals, and then neutralize free radicals. It is one of the strongest antioxidants found in plants in nature. There is a relatively high concentration of lycopene in the testis, indicating that lycopene may play an antioxidant role in the process of spermatogenesis (Zini A, et al., Lycopene supplementation in vitro can protect human sperm deoxyribonucleic acid from oxidative damage. Fertil Steril. 2010;94:1033–6). At the same time, studies have shown that the concentration of lycopene in the seminal plasma of infertile men is significantly lower than that of normal men (Palan P, Naz R. Changes in various antioxidant levels in human seminal plasma related to immunoinfertility. Arch Androl. 1996;36:139–43), indicating that lycopene plays an important role in sperm function. In addition, eugenol is a natural spice extracted from cloves, has good antioxidant activity and antibacterial effect, and is often used in combination with other antioxidant substances to promote animal growth and regulate intestinal health. At present, the development of plant-derived substances that promote animal production performance and the application of nutritional means to improve animal productivity have become research hotspots in disciplines such as animal husbandry. Goyal et al. demonstrated that the concentration of lycopene in seminal plasma increases with the supplementation of lycopene in the diet (Goyal A et al., The effects of dietary lycopene supplementation on human seminal plasma. BJU Int. 2007; 99:1456–60), which provides a new idea for us to add tomato extract to the diets of livestock and poultry to improve the antioxidant capacity of animals.

[0005] At present, there is no relevant report on adding tomato extract and eugenol to boar diets to promote boar semen quality. Developing related substances as feed additives and applying them to livestock and poultry breeding is of great significance for promoting animal growth and improving animal reproductive performance. Summary of the Invention

[0006] In order to solve the problem of unstable boar semen quality in current production management, the first object of the present invention is to provide the application of the combination of tomato extract and eugenol in the preparation of feed additives for improving boar reproductive performance. The second object is to provide a mixture additive for improving boar semen quality. The third object is to provide a feeding method for improving boar semen quality. By combining tomato extract and eugenol in a specific ratio, the present invention improves boar sperm motility, sperm density and ejaculation volume, reduces the sperm malformation rate of boars, and achieves the purpose of improving pig semen quality and maintaining stable boar reproductive performance.

[0007] In order to achieve the above object, the specific solutions adopted by the present invention are as follows:

[0008] In the first aspect, the present invention provides the application of the combination of tomato extract and eugenol in the preparation of feed additives for improving boar reproductive performance.

[0009] In the second aspect, the present invention provides a mixture additive for improving boar semen quality, which is prepared by mixing tomato extract and eugenol.

[0010] As a further optimization of the above mixture additive, the tomato extract is prepared by the following method:

[0011] Step 1: Remove tomato residue leaves, wash, remove surface dirt, then crush and homogenize, and dilute with 2 times the weight of 0.25M acetic acid concentration to obtain a homogenate;

[0012] Step 2: Add 200 ppm of β-glucanase, 500 ppm of cellulase, 300 ppm of pectinase and 350 ppm of xylanase to the homogenate, mix well, and perform enzymatic hydrolysis treatment in a 55°C constant temperature water bath for 2 h;

[0013] Step 3: After enzymatic hydrolysis is completed, use ultrasonic-assisted extraction on the enzymatic hydrolysate under the conditions of ultrasonic power of 600 W, temperature of 50°C and time of 1 h; after ultrasonic treatment, centrifuge the product at 4°C and 3000 g for 20 min, and take the supernatant for freeze-drying to obtain tomato extract dry powder.

[0014] As a further optimization of the above mixture additive, the mass ratio of the tomato extract to eugenol is 1:1 to 3:1, preferably 2:1.

[0015] Thirdly, the present invention provides a feeding method for improving the semen quality of boars, which is to add the above mixture additive to the basal diet at a ratio of 1.5‰ for feeding.

[0016] As a further optimization of the above feeding method, by mass percentage, the basal diet includes: 63% corn, 16% soybean meal, 9% wheat bran, 4% fat powder, 4% fish meal, and 4% premix; the premix provides per kilogram of diet: 20000 IU of vitamin A, 10 mg of vitamin B1, 80 mg of vitamin B2, 30 mg of vitamin B6, 25 μg of vitamin B12, 15 mg of vitamin K3, 250000 IU of vitamin D3, 100 mg of vitamin E, 31.96 mg of vitamin K, 20.00 mg of pantothenic acid, 1100 mg of niacin, 1.3 mg of biotin, 9.0 g of choline, 19 mg of folic acid, 20 mg of manganese, 100 mg of zinc, 80 mg of iron, 50 mg of copper, 0.15 mg of iodine, 0.15 mg of selenium, and 30 mg of cobalt.

[0017] Beneficial effects: The present invention uses the combination of tomato extract with good antioxidant effect and eugenol, which not only supplements nutrients such as carotene for boars and improves the physical condition of boars, but also can effectively increase the ejaculation volume, sperm density and motility of boars, and enhance the reproductive performance of breeding boars. The action mechanism of the present invention is clear, the effect is reliable, and it is easy to promote. Description of the Drawings

[0018] Figure 1 It is a comparison chart of the antioxidant effect of pig semen after feeding with the mixture additive; among them, CN is the measurement result of the antioxidant capacity index of sperm of the control group pigs, and Additive1, 2, 3 are the measurement results of the antioxidant capacity index of sperm of experimental groups 1, 2, and 3 respectively. Detailed Embodiments

[0019] Currently in production management, due to individual factors, feeding factors and other reasons, the semen quality of boars is unstable, such as less ejaculation volume, high sperm malformation rate, low motility, etc., which reduces the fertilization ability of sperm and greatly affects the reproductive efficiency and economic benefits of pig farms. The purpose of the present invention is to provide a tomato extract added to the compound feed for boars, and through nutritional regulation, to improve the sperm motility, sperm density and ejaculation volume of boars, reduce the sperm malformation rate of boars, and achieve the purpose of improving the pig semen quality and maintaining the stable reproductive performance of boars.

[0020] The technical solution is to first prepare the tomato extract, then mix it with eugenol, add this mixture to the complete feed for boars at a ratio of 1.5‰ to feed the boars, detect the relevant indicators of the boar semen quality, and evaluate the influence effect of the tomato extract on the boar semen quality.

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0022] 1. Preparation of the mixture additive of tomato extract and eugenol:

[0023] a) Remove tomato residue leaves, etc., wash, remove surface dirt such as dust and soil, then crush and homogenize, add 2 times the weight of 0.25 M concentration acetic acid for dilution to obtain a homogenate;

[0024] b) Add 200 ppm of β-glucanase, 500 ppm of cellulase, 300 ppm of pectinase and 350 ppm of xylanase to the homogenate, mix well, and carry out enzymatic hydrolysis treatment in a 55 °C constant temperature water bath for 2 h;

[0025] c) After enzymatic hydrolysis is completed, use ultrasonic-assisted extraction of the enzymatic hydrolysate under the conditions of ultrasonic power of 600 W, temperature of 50 °C, and time of 1 h. After ultrasonic treatment, centrifuge the product at 4 °C and 3000 g for 20 min, and take the supernatant for freeze-drying to obtain dry tomato extract powder.

[0026] d) Mix the dry tomato extract powder and eugenol in an appropriate ratio to obtain the required mixture additive of tomato extract and eugenol in combination.

[0027] 2. Boar feeding experiment.

[0028] a) Use the mixture additive of the present invention to conduct an 8-week experiment on boars. Among them, a 2-week pre-feeding transition period is carried out before formal feeding.

[0029] b) Select 20 healthy boars with normal reproductive performance, similar body weights, about 18 months old, and similar genetic backgrounds, and divide them into four groups on average. Among them, the control group is fed a basal diet, and experimental group 1 is prepared by mixing tomato extract and eugenol in a mass ratio of 1:1 to form an additive, and 1.5‰ of the mixture additive is added to the basal diet for feeding. Similarly, the mixture additives used in experimental group 2 and experimental group 3 are prepared by mixing tomato extract and eugenol in mass ratios of 2:1 and 3:1, respectively. All groups of boars are managed according to normal production procedures. By detecting the difference in semen quality between the two groups, the reproductive ability of boars is comprehensively evaluated.

[0030] Table 1 Composition of boar basal diet

[0031] Raw material Content % Corn 63 Soybean meal 16 Wheat bran 9 Fat powder 4 Fish meal 4 Premix 4 Total 100

[0032] Note: The premix provides per kilogram of diet: Vitamin A 20,000 IU, Vitamin B1 10 mg, Vitamin B2 80 mg, Vitamin B6 30 mg, Vitamin B12 25 μg, Vitamin K3 15 mg, Vitamin D3 250,000 IU, Vitamin E 100 mg, Vitamin K 31.96 mg, Pantothenic acid 20.00 mg, Niacin 1,100 mg, Biotin 1.3 mg, Choline 9.0 g, Folic acid 19 mg, Manganese 20 mg, Zinc 100 mg, Iron 80 mg, Copper 50 mg, Iodine 0.15 mg, Selenium 0.15 mg, Cobalt 30 mg.

[0033] c) Semen collection and related determination methods

[0034] ① Boar semen is collected by the hand-grip method, once a week. Record the semen volume each time, and determine sperm motility, sperm density, and sperm malformation rate, and calculate the average results.

[0035] ② Sperm motility is determined using the CASA system.

[0036] [[ID=!2]]③ Sperm density is counted using a hemocytometer.

[0037] ④ The semen volume is weighed using an electronic balance and recorded as the semen volume.

[0038] ⑤ The sperm malformation rate is determined using the Giemsa staining method, as follows:

[0039] Semen smear preparation: Drop a drop of semen at one end of a glass slide. Use the tip of another glass slide to touch the semen drop at a 35° angle and gently pull it to the other end to evenly smear the semen on the glass slide. Let it air-dry naturally for 5 minutes; Fixation and staining: Stain with Giemsa stain for 5 minutes, then wash with water and air-dry. Place the air-dried slide under a microscope for microscopic examination. According to the sperm malformation determination standard, calculate the number of malformed sperm. Sperm malformation rate = number of malformed sperm / total number of sperm × 100%. In the malformation rate statistics, the same sample is counted twice. If the error between the two counts is greater than 10%, a third check is made. Finally, take the average of the two counts with an error not exceeding 10% in the three checks, which is the determined sperm malformation rate.

[0040] ⑥ Analysis of sperm malondialdehyde (MDA) content:

[0041] The MDA analysis uses the malondialdehyde assay kit from Nanjing Jiancheng Bioengineering Institute. The method is as follows: Take 100 μL of semen, centrifuge at 800 g for 10 min, wash twice with PBS, add 100 μL of pre-cooled RIPA lysis buffer, incubate on ice for 10 - 15 min, sonicate, centrifuge at 12,000 g for 3 - 5 min, and take the supernatant for protein concentration determination and MDA analysis. Set different assay groups for the supernatant samples as shown in the following table, and add each reagent in the corresponding order.

[0042]

[0043] After adding the reagent, cover the centrifuge tube with a lid and prick a small hole in the lid. Vortex to mix well, place in a boiling water bath at 95 °C for 40 min, take out and cool with running water, then centrifuge at 4000 rpm for 10 min. Take the supernatant, set the zero point with distilled water according to the parameters of 532 nm and 1 cm optical path, and measure the absorbance value of each tube. The calculation formula for the MDA content in the liquid sample is as follows:

[0044]

[0045] ⑦ Analysis of total antioxidant capacity (T-AOC) of sperm

[0046] For T-AOC analysis, use the T-AOC assay kit from Nanjing Jiancheng Bioengineering Institute, and process the semen samples as above. Prepare the reagents according to the kit instructions. Set up different assay groups as shown in the following table and add each reagent in the corresponding order.

[0047]

[0048] After adding the reagent, vortex to mix well, let stand for 10 min, set the zero point with distilled water, measure the absorbance of each tube according to the parameters of 1 cm optical path and 520 nm.

[0049]

[0050] d) Feeding experiments were conducted on Duroc boars with this product, and the results are as follows:

[0051] Table 2 Detection results of semen quality of Duroc boars in different treatment groups.

[0052] Item Control group Experimental group 1 Experimental group 2 Experimental group 3 Semen volume (mL) 229.6 256.7* 282.8* 271.8* Sperm motility (%) 70.7 78.6* 82.3* 77.3* <![CDATA[Sperm density (10 8 spz / mL)]]> 3.31 4.54* 4.76* 3.63 Sperm malformation rate (%) 15.1 10.1 6.3** 13.1

[0053] Note: Each experimental group was statistically analyzed with the control group respectively. * indicates significant difference, and ** indicates extremely significant difference.

[0054] The antioxidant effect of the mixture additive on pig semen is as Figure 1 shown.

[0055] From the above data, it can be seen that compared with the boars in the control group, the average semen volume, average sperm motility and density in experimental group 1 were significantly higher than those in the control group, and the average malformation rate decreased, but the difference was not significant; the average semen volume, average sperm motility and density in experimental group 2 were significantly higher than those in the control group, and the average malformation rate decreased extremely significantly; the average semen volume and average sperm motility in experimental group 3 were significantly increased, but there was no significant difference in density and average malformation rate compared with the control group; this indicates that the mixture additive of tomato extract and eugenol can improve the semen quality of boars, and the feeding effect is the best when tomato extract and eugenol are mixed at a mass ratio of 2:1.

[0056] The antioxidant capacity of pig semen within 3 days after semen collection was measured. The results of MDA content measurement showed that in experimental group 2, it was significantly lower than that in the control group on the first day, and extremely significantly lower than that in the control group on the second and third days. In experimental group 1, there was no significant difference from the control group on the first day, it was significantly lower than the control group on the second day, and there was no significant difference from the control group on the third day. In experimental group 3, there was no significant difference from the control group on the first day, and it was significantly lower than the control group on the second and third days. The effect of experimental group 2 was the best. Similarly, for the results of T-AOC capacity measurement, T-AOC in experimental group 2 was extremely significantly higher than that in the control group within 3 days, while there was no significant difference in T-AOC in experimental group 1 from the control group within 3 days. T-AOC in experimental group 3 was extremely significantly higher than that in the control group on the first day, and significantly higher than that in the control group on the second and third days. This result indicates that feeding with a mixture of tomato extract and eugenol at a mass ratio of 2:1 can significantly improve the antioxidant capacity of pig sperm. In summary, the tomato extract and eugenol prepared in the present invention in a mass ratio of 2:1 can effectively promote spermatogenesis in boars, enhance sperm motility and other effects, improve reproductive performance, reduce the consumption of manpower, material resources and financial resources in production, reduce the breeding cost, and thus improve the breeding efficiency.

[0057] It should be noted that the above-described embodiments should be understood as illustrative and not limiting the protection scope of the present invention. The protection scope of the present invention is subject to the claims. For those skilled in the art, without departing from the essence and scope of the present invention, some non-essential improvements and adjustments made to the present invention still fall within the protection scope of the present invention.

Claims

1. Application of the combination of tomato extract and eugenol in improving the reproductive performance of boars, wherein the tomato extract is prepared by the following method: Step 1: Remove tomato residual leaves, wash, remove surface dirt, then break and homogenize, add 2 times the weight of 0.25M acetic acid for dilution to obtain a homogenate; Step 2: Add 200 ppm of β-glucanase, 500 ppm of cellulase, 300 ppm of pectinase and 350 ppm of xylanase to the homogenate, mix well, and carry out enzymatic hydrolysis treatment in a constant temperature water bath at 55°C for 2 h; Step 3: After enzymatic hydrolysis is completed, perform ultrasonic-assisted extraction on the enzymatic hydrolysate under the conditions of ultrasonic power of 600 W, temperature of 50°C, and time of 1 h; after ultrasonic treatment, centrifuge the product at 4°C and 3000 g for 20 min, take the supernatant and perform freeze-drying to obtain tomato extract dry powder.

2. Application of the combination of tomato extract and eugenol in preparing a feed mixture additive for improving the semen quality of boars, wherein the tomato extract is prepared by the following method: Step 1: Remove tomato residual leaves, wash, remove surface dirt, then break and homogenize, add 2 times the weight of 0.25M acetic acid for dilution to obtain a homogenate; Step 2: Add 200 ppm of β-glucanase, 500 ppm of cellulase, 300 ppm of pectinase and 350 ppm of xylanase to the homogenate, mix well, and carry out enzymatic hydrolysis treatment in a constant temperature water bath at 55°C for 2 h; Step 3: After enzymatic hydrolysis is completed, perform ultrasonic-assisted extraction on the enzymatic hydrolysate under the conditions of ultrasonic power of 600 W, temperature of 50°C, and time of 1 h; after ultrasonic treatment, centrifuge the product at 4°C and 3000 g for 20 min, take the supernatant and perform freeze-drying to obtain tomato extract dry powder.

3. A feed mixture additive for improving the semen quality of boars, characterized in that: The feed mixture additive contains tomato extract and eugenol, and the tomato extract is prepared by the following method: Step 1: Remove tomato residual leaves, wash, remove surface dirt, then break and homogenize, add 2 times the weight of 0.25M acetic acid for dilution to obtain a homogenate; Step 2: Add 200 ppm of β-glucanase, 500 ppm of cellulase, 300 ppm of pectinase and 350 ppm of xylanase to the homogenate, mix well, and carry out enzymatic hydrolysis treatment in a constant temperature water bath at 55°C for 2 h; Step 3: After enzymatic hydrolysis is completed, perform ultrasonic-assisted extraction on the enzymatic hydrolysate under the conditions of ultrasonic power of 600 W, temperature of 50°C, and time of 1 h; after ultrasonic treatment, centrifuge the product at 4°C and 3000 g for 20 min, take the supernatant and perform freeze-drying to obtain tomato extract dry powder; perform ultrasonic-assisted extraction; after ultrasonic treatment, centrifuge the product at 4°C and 3000 g for 20 min, take the supernatant and perform freeze-drying to obtain tomato extract dry powder.

4. The mixture additive according to claim 3, characterized in that: The mass ratio of the tomato extract to eugenol is 1:1 to 3:

1.

5. The mixture additive according to claim 4, characterized in that: The mass ratio of the tomato extract to eugenol is 2:

1.

6. A feeding method for improving the semen quality of boars, characterized in that: The mixture additive described in any one of claims 3-5 is added to the basal diet at a ratio of 1.5‰ for feeding.

7. The feeding method according to claim 6, wherein: On a mass percentage basis, the basal diet comprises: 63% corn, 16% soybean meal, 9% wheat bran, 4% fat powder, 4% fish meal, and 4% premix; the premix provides per kilogram of diet: vitamin A 20,000 IU, vitamin B1 10 mg, vitamin B2 80 mg, vitamin B6 30 mg, vitamin B12 25 μg, vitamin K3 15 mg, vitamin D3 250,000 IU, vitamin E 100 mg, vitamin K 31.96 mg, pantothenic acid 20.00 mg, niacin 1,100 mg, biotin 1.3 mg, choline 9.0 g, folic acid 19 mg, manganese 20 mg, zinc 100 mg, iron 80 mg, copper 50 mg, iodine 0.15 mg, selenium 0.15 mg, cobalt 30 mg.

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