A method for improving semen quality and testicular antioxidant capacity of heat-stressed boars

Abstract

The present invention discloses a method for improving the semen quality and testicular antioxidant capacity of heat-stressed boars, comprising the following steps: S1: preparing a retinoic acid composition; S2: preparing pig feed; S3: selecting several boars with similar production performance and dividing them into a control group and a retinoic acid group; S4: feeding the control group with a basic diet, a retinoic acid group and a composition; S5: collecting the semen of the boars after feeding the boars for one week, and evaluating the effect of feeding retinoic acid on the semen quality of the boars; S6: establishing a tissue culture model for improving the antioxidant function of the boar testicles. The method for improving the semen quality and testicular antioxidant capacity of heat-stressed boars described in the present invention can improve the semen quality of the boars by feeding the boars with a retinoic acid composition for one week during the high temperature period in summer, including reducing the boar sperm deformity rate, improving the semen antioxidant capacity, and regulating the gene expression of testicular tissue.

Description

Technical Field

[0001] The invention relates to the field of animal husbandry, and in particular to a method for improving semen quality and testicular antioxidant capacity of heat-stressed boars. Background Art

[0002] During the high temperatures in summer, boars suffer from heat stress and the quality of their semen declines, seriously affecting their breeding value. Boars' sweat glands are underdeveloped, and their subcutaneous fat tissue is thick, with poor heat dissipation performance and they are very sensitive to rising temperatures. Under high temperature conditions, the poor quality of boar semen is mainly manifested in a significant decrease in sperm count, sperm motility and semen volume.

[0003] Retinoic acid is the active metabolite of vitamin A in the body. Retinoic acid deficiency can cause disorders in spermatogenesis and reproductive abnormalities. Retinoic acid can maintain the redox balance of sperm. Sperm treated with retinoic acid has higher antioxidant enzyme activity and better tolerance to oxidative stress. This suggests that retinoic acid may be a potential substance to alleviate the decline in boar semen quality caused by high temperatures in summer.

[0004] In order to more comprehensively develop methods and applications of retinoic acid in improving boar testicular spermatogenesis disorders, this patent combines in vivo and in vitro testicular tissue culture methods to determine the effect of feeding retinoic acid on the semen quality of boars during the high temperature period in summer, collects testicular tissue blocks of boars treated with retinoic acid, detects the expression of genes related to spermatogenesis disorders, and proposes the effectiveness of using retinoic acid to improve the semen quality of boars during the high temperature period in summer. This provides a nutritional intervention strategy to eliminate the damage caused by high temperature to the testicular spermatogenesis function of boars and effectively improve the poor semen quality in the high temperature season. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for improving the semen quality and testicular antioxidant capacity of heat-stressed boars, which can effectively solve the problems in the background technology.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for improving semen quality and testicular antioxidant capacity of heat-stressed boars, comprising the following steps:

[0008] S1: preparing a retinoic acid composition, comprising: retinoic acid, dimethyl sulfoxide and corn oil, wherein by weight, the retinoic acid accounts for 4-8%, the dimethyl sulfoxide accounts for 40-50%, and the corn oil accounts for 50-60%;

[0009] S2: preparing pig feed: by weight, comprising: the retinoic acid composition prepared in S1 and a basic diet, wherein by weight, the retinoic acid composition: the basic diet = 1: (100-200), the basic diet comprising corn, wheat, hulled barley, soybean meal, sunflower meal, and fish meal, wherein by weight, the proportion of corn is 30-35%, the proportion of wheat is 20%, the proportion of hulled barley is 30%, the proportion of soybean meal is 6-7%, the proportion of sunflower meal is 5-6%, and the proportion of fish meal is 3%;

[0010] S3: Select several boars with similar production performance and divide them into a control group and a retinoic acid group;

[0011] S4: The control group was fed with a basic diet, and the retinoic acid group was fed with a basic diet every day. At the same time, the retinoic acid composition was mixed into the basic diet and fed to boars together for 1 week. The feeding amount of the retinoic acid composition was 5-10 mg / kg body weight.

[0012] S5: After feeding the boars for 1 week, the semen of the boars was collected to evaluate the effect of feeding the retinoic acid composition on the semen quality of the boars;

[0013] S6: Establishment of a tissue culture model for improving the antioxidant function of boar testis, including the following steps:

[0014] A: Testicles of 21-day-old boars were collected, cut into small pieces, placed in culture plates, and divided into control group, heat treatment group, and heat treatment + retinoic acid group, and cultured in a carbon dioxide incubator;

[0015] B: Retinoic acid was added to the culture medium of the heat treatment + retinoic acid group to a concentration of 10 μmol / L, and the culture medium was placed in a carbon dioxide incubator. After 4 hours of incubation, the testicular tissue blocks were collected, the morphology of the testicular tissue blocks was observed, the tissue antioxidant parameters were determined, and the effect of retinoic acid on improving the antioxidant capacity of the testicular tissue blocks was evaluated.

[0016] Preferably, the retinoic acid group includes a low-dose retinoic acid group and a high-dose retinoic acid group, and the supplementary feeding amounts are 5 mg / Kg body weight and 10 mg / Kg body weight, respectively.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention can improve the semen quality of boars by continuously feeding the boars with a composition containing retinoic acid for one week during the high temperature period in summer. It is appropriate to feed the boars 1-2g of retinoic acid every day. Further, an in vitro culture model of boar testicular tissue is established to explore the effect of retinoic acid in improving the antioxidant capacity of testicular tissue and resisting the adverse factors of heat treatment, so as to provide a research basis and nutritional intervention strategy for the problem of poor semen quality of boars during the high temperature period in summer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a result diagram of boar sperm deformity rate in the present invention;

[0020] Figure 2 It is a flow chart of the tissue culture model of the antioxidant function of boar testis in the present invention;

[0021] Figure 3 This is a morphological result diagram of HE staining of boar testicular tissue in the present invention;

[0022] Figure 4 This is a diagram showing the expression results of the antioxidant gene in boar testicular tissue in the present invention. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The present invention relates to a method for improving the semen quality and testicular antioxidant capacity of heat-stressed boars, comprising the following steps:

[0025] S1: preparing a retinoic acid composition, comprising: retinoic acid, dimethyl sulfoxide and corn oil, wherein by weight, the proportion of retinoic acid is 4-8%, the proportion of dimethyl sulfoxide is 40-50%, and the proportion of corn oil is 50-60%;

[0026] S2: preparing pig feed: by weight, comprising: the retinoic acid composition prepared in S1 and a basic diet, wherein by weight, the retinoic acid composition: the basic diet = 1: (100-200), the basic diet comprises corn, wheat, hulled barley, soybean meal, sunflower meal, and fish meal, wherein by weight, the proportion of corn is 30-35%, the proportion of wheat is 20%, the proportion of hulled barley is 30%, the proportion of soybean meal is 6-7%, the proportion of sunflower meal is 5-6%, and the proportion of fish meal is 3%;

[0027] S3: Select several boars with similar production performance and divide them into a control group and a retinoic acid group. The retinoic acid group includes a low-dose retinoic acid group and a high-dose retinoic acid group, and the supplementary feeding amount is 5 mg / Kg body weight and 10 mg / Kg body weight respectively;

[0028] S4: The control group was fed with a basic diet, and the retinoic acid group was fed with a basic diet every day. The retinoic acid composition was mixed into the basic diet and fed to boars together for 1 week. The feeding amount of the retinoic acid composition was 5-10 mg / kg body weight.

[0029] S5: After feeding the boars for 1 week, the semen of the boars was collected to evaluate the effect of feeding the retinoic acid composition on the semen quality of the boars;

[0030] S6: Establishment of a tissue culture model for improving the antioxidant function of boar testis, including the following steps:

[0031] A: Testicles of 21-day-old boars were collected, cut into small pieces, placed in culture plates, and divided into control group, heat treatment group, and heat treatment + retinoic acid group, and cultured in a carbon dioxide incubator;

[0032] B: Retinoic acid was added to the culture medium of the heat treatment + retinoic acid group to a concentration of 10 μmol / L, and the culture medium was placed in a carbon dioxide incubator. After 4 hours of incubation, the testicular tissue blocks were collected, the morphology of the testicular tissue blocks was observed, the tissue antioxidant parameters were determined, and the effect of retinoic acid on improving the antioxidant capacity of the testicular tissue blocks was evaluated. Specific embodiment one:

[0034] This example provides a composition for improving boar semen quality, comprising, by weight, 4-8% retinoic acid, 40-50% dimethyl sulfoxide and 50-60% corn oil, wherein the index for improving boar semen quality is boar sperm deformity rate.

[0035] Preferably, the composition comprises retinoic acid 4%, dimethyl sulfoxide 40% and corn oil 56% by weight. Specific embodiment 2:

[0037] This embodiment provides a use of the composition described in Example 1 in the preparation of a drug for improving the semen quality of heat-stressed boars during the high temperature period in summer. Specific embodiment three:

[0039] The present embodiment provides a pig feed, which comprises, by weight, the composition described in Example 1 and a basic diet, and by weight, the composition: the basic diet = 1: (100-200), preferably 1: 180, and in the present embodiment, by weight, the basic diet comprises 30-35%, preferably 32%, corn, 20%, wheat, 30% hulled barley, 6-7%, preferably 6.5%, soybean meal, 5-6%, preferably 5.8%, sunflower meal, and 3%, preferably 2.5%, fish meal. Specific embodiment four:

[0041] This embodiment provides a method for improving the semen quality of heat-stressed boars during the high temperature period in summer, comprising the following steps:

[0042] S1: During the high temperature period in summer, several boars with similar production performance were selected and divided into a control group of 5 boars, a low-dose retinoic acid group of 5 boars and a high-dose retinoic acid group of 5 boars.

[0043] S2: The control group was fed a basal diet, and the retinoic acid group was fed boars every day. At the same time, any one of the compositions described in 1-3 (composition:basic diet=1:(100-200)) was mixed into the feed and fed to the boars together for 1 week. The specific feeding plan is shown in Table 1. The feeding amounts were 5 mg / kg boar body weight for the low-dose retinoic acid group and 10 mg / kg boar body weight for the high-dose retinoic acid group.

[0044] Table 1 Experimental design of feeding composition to boars:

[0045]

[0046] S3: After feeding the boars for 1 week, the boar semen was collected to evaluate the effect of feeding the retinoic acid composition on the quality of the boar semen. Specifically, Figure 1 The effect of retinoic acid on sperm deformity rate in boars is given.

[0047] from Figure 1 It can be seen that after feeding retinoic acid, the semen of boars was collected continuously for 3 weeks and analyzed, and it was found that retinoic acid had a significant effect on reducing the sperm deformity rate of boars. The sperm deformity rate of boars in the retinoic acid feeding group was lower than that in the control group.

[0048] As can be seen from Table 2, after feeding retinoic acid, the comparison of the first semen samples collected found that retinoic acid had a significant effect on increasing glutathione peroxidase (GSH-PX) in boar semen, which is beneficial to improving the antioxidant capacity of semen. Specific embodiment five:

[0050] This embodiment provides a method for establishing a tissue culture model for improving the antioxidant function of boar testicles, comprising the following steps:

[0051] S1: Testicles of 21-day-old boars were collected, cut into small pieces, and placed in culture plates. They were divided into a control group (CON group), a heat treatment group (HT group), and a heat treatment + retinoic acid group (HT+RA group), and cultured in a carbon dioxide incubator.

[0052] S2: Figure 2 As shown in the technical route, retinoic acid was added to the culture medium of the heat treatment + retinoic acid group to a concentration of 10 μmol / L, and the culture medium was placed in a carbon dioxide incubator. After 4 hours of incubation, the testicular tissue blocks were collected, the morphology of the testicular tissue blocks was observed, the tissue antioxidant parameters were determined, and the effect of retinoic acid on improving the antioxidant capacity of the testicular tissue blocks was evaluated.

[0053] from Figure 3It can be seen that after 4 hours of heat treatment at 39°C, the testicular spermatogonia in the HT group fell off and were scattered in the lumen. The cell nuclei were significantly reduced in size. The morphology of the testicular tissue blocks in the HT+RA group was close to that in the CON group. Further analysis of the cell nuclei revealed that compared with the CON group, the diameter and area of ​​the cell nuclei in the HT group were significantly reduced, and compared with the HT group, the diameter and area of ​​the cell nuclei in the HT+RA group were significantly increased.

[0054] As can be seen from Table 3, compared with the CON group, the activities of T-SOD and GSH-PX in the HT group decreased to a certain extent. Compared with the HT group, the activity of GSH-PX in the HT+RA group increased from 78.54 to 90.97, and the tissue antioxidant capacity was improved.

[0055]

[0056]

[0057] from Figure 4 It can be seen that compared with the CON group, 4h heat treatment significantly increased the expression of Nrf2 and GCLC genes in testicular tissue. Compared with the HT group, RA treatment significantly reduced the relative expression of Nrf2 gene in testicular tissue, and there was no significant difference in the relative expression of Nrf2 and GCLC genes between the RA treatment group and the CON group. The coding gene of the antioxidant enzyme GSH-PX is GCLC, and the expression of GCLC gene is regulated by the nuclear transcription factor Nrf2. When GSH-PX decreases, Nrf2 expression increases, promoting GCLC gene transcription and increasing GSH-PX synthesis. On the contrary, when GSH-PX is high, negative feedback reduces Nrf2 expression, reduces GCLC gene transcription and reduces GSH-PX synthesis. Figure 4 The results showed that retinoic acid effectively maintained the level of GSH-PX and antioxidant capacity in heat-treated testis.

[0058] In summary, the present invention feeds boars with a retinoic acid combination for one week during the high temperature period in summer, thereby improving the quality of boar semen, including reducing the boar sperm deformity rate, improving semen antioxidant capacity, and regulating testicular tissue gene expression, among which the best effect is obtained after feeding the boars with the composition at a rate of 10 mg / kg body weight. A model is further established with pigs as the subject to explore the effect of retinoic acid on improving the antioxidant capacity of boar testicular tissue, providing a solid research foundation for the research and treatment of decreased semen quality in people working in high temperatures.

[0059] The present invention can improve the semen quality of boars by continuously feeding the boars with a composition containing retinoic acid for one week during the high temperature period in summer. It is appropriate to feed the boars with 1-2g of retinoic acid every day. An in vitro culture model of boar testicular tissue is further established to explore the effect of retinoic acid in improving the antioxidant capacity of testicular tissue and resisting the adverse factors of heat treatment, thereby providing a research basis and nutritional intervention strategy for the problem of poor semen quality of boars during the high temperature period in summer.

[0060] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving semen quality and testicular antioxidant capacity of heat-stressed boars, Features: The steps include: S1: preparing a retinoic acid composition, comprising: retinoic acid, dimethyl sulfoxide and corn oil, wherein by weight, the retinoic acid accounts for 4-8%, the dimethyl sulfoxide accounts for 40-50%, and the corn oil accounts for 50-60%; S2: preparing pig feed: comprising: the retinoic acid composition prepared in S1 and a basic diet, wherein by weight, the retinoic acid composition: the basic diet = 1: (100-200), the basic diet comprises corn, wheat, hulled barley, soybean meal, sunflower meal, and fish meal, and by weight, the proportion of the corn is 30-35%, the proportion of the wheat is 20%, the proportion of the hulled barley is 30%, the proportion of the soybean meal is 6-7%, the proportion of the sunflower meal is 5-6%, and the proportion of the fish meal is 3%; S3: Select several boars with similar production performance and divide them into a control group and a retinoic acid group; S4: The control group was fed with a basic diet, and the retinoic acid group was fed with a basic diet every day. At the same time, the retinoic acid composition was mixed into the basic diet and fed to boars together for 1 week. The feeding amount of the retinoic acid composition was 5-10 mg / kg body weight. S5: After feeding the boars for 1 week, the semen of the boars was collected to evaluate the effect of feeding the retinoic acid composition on the semen quality of the boars; S6: Establishment of a tissue culture model for improving the antioxidant function of boar testis, including the following steps: A: Testicles of 21-day-old boars were collected, cut into small pieces, placed in culture plates, and divided into control group, heat treatment group, and heat treatment + retinoic acid group, and cultured in a carbon dioxide incubator; B: Retinoic acid was added to the culture medium of the heat treatment + retinoic acid group to a concentration of 10 μmol / L, and the culture medium was placed in a carbon dioxide incubator. After 4 hours of incubation, the testicular tissue blocks were collected, the morphology of the testicular tissue blocks was observed, the tissue antioxidant parameters were determined, and the effect of retinoic acid on improving the antioxidant capacity of the testicular tissue blocks was evaluated.

2. A method for improving semen quality and testicular antioxidant capacity of heat-stressed boars according to claim 1, Features: The retinoic acid groups include a low-dose retinoic acid group and a high-dose retinoic acid group, and the supplementary feeding amounts are 5 mg / Kg body weight and 10 mg / Kg body weight respectively.

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