CN111657229B - Method for establishing laying hen ovary oxidative stress model - Google Patents

Abstract

The invention discloses a method for establishing a laying hen ovarian oxidative stress model, which comprises the following steps: selecting Roman powder laying hens in an egg laying peak period, adopting single-factor test design, setting 6 treatments to be 1 control group and 5 tert-butyl hydroperoxide groups respectively, and feeding normal saline to the control groups; sampling 48 hours after the last feeding; the test period is 24 days, the feed is corn-soybean meal type basic feed, and the feeding management is carried out according to the conventional feeding management; the laying rate is recorded, and the concentration of estradiol and the like is measured by an ELISA method; collecting ovaries 48 hours after the last feeding of the physiological saline and the tert-butyl hydroperoxide, counting the number of original follicles after HE dyeing, and counting and measuring the number of the follicles of the previous grade and the grade; and (3) measuring the content or activity of vitamin C and the like in the ovary by using an HPLC method and a kit method. The method can increase the content of the ovary free radicals and reduce the activity of the ovary antioxidase, thereby causing the oxidative stress of the laying hens.

Description

Method for establishing laying hen ovary oxidative stress model

Technical Field

The invention belongs to the technical field of animal reproduction and toxicology, and particularly relates to a method for establishing an oxidative stress model of a laying hen ovary.

Background

The ovary of poultry is the classical model for studying ovarian biology, follicular development and ovarian cancer. The egg laying performance of poultry is mainly determined by the growth and development level of follicles in ovaries and is influenced by factors such as genetics, nutrition and environmental conditions (light time and intensity). With the technical progress in the fields of heredity and nutrition, the production performance of commercial laying hens is continuously improved, 410-430 eggs can be produced by the high-yield laying hens in 110 weeks at present, but the average laying rate of the whole period (20-110 weeks old) is only 65-70%, and a certain production potential is still remained to be excavated. In actual production, laying hens generally achieve sexual maturity and start laying at 18-20 weeks, the laying rate reaches over 95% when the laying hens are 24-25 weeks old, then the laying rate slowly decreases until the laying rate at the later laying period (45 weeks old) decreases to about 88%, then the laying rate decreases rapidly, the laying interval is increased when the laying hens are 80 weeks old to about 60%, the follicular atresia number at different development stages in ovaries is increased, and the fact that the ovarian function of the laying hens at the later laying period gradually declines, follicular development is inhibited and ovarian cancer is easily induced is shown. In addition, other stressors, such as high temperature, high feed density, heavy metals, disease, etc., can also lead to a dramatic decrease in egg production. In the production process, due to factors such as ovarian function decline or other stresses in the later period of egg laying, the egg laying number in the whole period is low, generally, the egg laying hens are eliminated before the first production period (72 weeks old) is finished, and only 280-300 eggs are provided for the lifetime, which is mainly caused by the increase of follicular atresia quantity and poor follicular quality. The study of the early stage of the school subjects discovers that the laying rate and the number of follicles of laying hens under a heavy metal cadmium and vanadium induced oxidative stress model can be improved by adding an antioxidant (tea polyphenol extract) into the laying hen feed, and the laying performance of the laying hens at the later period of laying can also be improved; previous research reports also confirmed that reproductive performance decline caused by oxidative stress may be a major cause of egg laying performance decline in laying hens at a later stage and under stress. When the poultry is hatched, 480 thousands of primary follicles exist in ovaries, but the number of ovulations in the life is only 500-1000, so that the poultry has great production potential. Oxidative stress (Oxidative stress) refers to the condition that when the body is subjected to various harmful stimuli, active molecules in the body, such as Reactive Oxygen Species (ROS) and reactive nitrogen radicals, are excessively generated, the degree of oxidation exceeds the oxide scavenging capacity, and the Oxidative system and the antioxidant system are dynamically unbalanced, thereby causing tissue damage. ROS are important regulatory molecules of cellular pathways, which regulate cell cycle and apoptosis. Numerous studies have shown that a certain amount of ROS in the follicles has important physiological effects on their development and ovulation, however, when the ROS is excessively elevated, oxidative stress will be induced, inducing follicular atresia. The research on mammals finds that oxidative stress can reduce the number of follicles in each stage of ovary, but the specific mechanism of inhibiting follicular development and inducing follicular atresia is not clear, and only the research on animals such as human and mice is mainly carried out, and the research on domestic birds is very few.

Therefore, in view of the above reasons, in order to further explore a specific mechanism of ovarian function damage caused by oxidative stress so as to reduce egg laying performance, it is necessary to establish an oxidative stress model of the ovary of the laying hen.

Disclosure of Invention

In view of the above, the invention provides a method for establishing an oxidative stress model of a laying hen ovary, which can further explore specific mechanisms of nutrition and feed factors and the reduction of egg production performance caused by aging.

In order to solve the technical problem, the invention discloses a method for establishing an oxidative stress model of a laying hen ovary, which comprises the following steps of:

selecting 28-week-old Roman powder laying hens in an egg laying peak period, adopting a single-factor test design, setting 6 treatments, each treatment being 10 times repeated, wherein 1 chicken is repeated, namely 1 control group and 5 tert-butyl hydroperoxide groups, and feeding physiological saline to the control groups respectively in 8, 15 and 22 days, 9 in the morning: 00 feeding physiological saline and tert-butyl hydroperoxide; sampling 48 hours after the last feeding; the test period is 24 days, the feed is corn-soybean meal type basic feed, and the feeding mode is quantitative feeding; feeding management is carried out according to conventional feeding management; the test is started to be pre-fed for 2 weeks, the laying rate of the laying hens is recorded by observation before the test, and the test is randomly grouped according to the laying rate;

Daily egg production was recorded and the concentrations of estradiol, progesterone, testosterone, leptin, follicle stimulating hormone, insulin-like growth factor-1 and anti-muckle hall were determined by ELISA; collecting ovaries 48 hours after the last feeding of the physiological saline and the tert-butyl hydroperoxide, counting the number of original follicles by using a correction method after HE dyeing, and counting the number of the front-grade follicles, the grade follicles and measuring the size of the grade follicles; the contents or activities of vitamin C, vitamin E, lipid peroxidation products, protein carbonyl, antioxidant enzymes (SODs, CAT, GSH, GST and TAOC) in ovary were determined by HPLC and kit method.

Alternatively, 5 t-butyl hydroperoxide groups were fed at respective concentrations of: 100. mu. mol/kg body weight, 200. mu. mol/kg body weight, 400. mu. mol/kg body weight, 800. mu. mol/kg body weight, and 1000. mu. mol/kg body weight.

Optionally, the feed comprises the following components in parts by mass: 59.064 parts of corn, 3.867 parts of wheat bran, 1.50 parts of soybean oil, 15.236 parts of soybean meal, 5.00 parts of corn protein powder, 5.00 parts of corn DDGS, 6.103 parts of granular stone powder, 2.50 parts of powdery stone powder, 0.941 part of powdery calcium hydrophosphate, 0.250 part of sodium chloride, 0.100 part of baking soda, 0.162 part of L-lysine sulfate, 0.012 part of DL-methionine, 0.100 part of choline chloride, 10.015 parts of multi-vitamin and 10.15 parts of mineral additive.

Optionally, the purity of the L-lysine sulfate is 70%; the purity of DL-methionine is 99%; the purity of choline chloride was 60%.

Optionally, during the raising management process, strict disinfection is carried out every week, the room temperature is kept at 20-22 ℃, and ventilation is taken.

Optionally, the pre-grade comprises small white, large white and small yellow follicles; the grade follicles included F1-F7 follicles.

Optionally, the antioxidant enzymes include superoxide dismutase, catalase, glutathione-S-transferase, and total antibody capacity.

Compared with the prior art, the invention can obtain the following technical effects:

according to the method, the content of ovary free radicals can be increased by feeding 800 mu mol/kg body weight of tBHP, the activity of ovary antioxidant enzyme is reduced, the apoptosis rate of ovary cells is increased, the laying rate of laying hens is reduced, and the oxidative stress of the laying hens is caused.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a comparison of the difference between the number of primordial ovarian follicles and the number of ovarian follicles at all levels of the laying hen fed with tBHP of different concentrations in the invention; wherein the 6 histograms in primordial, primary, pre-grade and atretic follicles in the abscissa are from left to right T1, T2, T3, T4, T5 and T6;

FIG. 2 is a graph of the effect of the model of the invention on the rate of ovarian apoptosis in layers.

Detailed Description

The following embodiments of the present invention will be described in detail with reference to the accompanying drawings, so that the implementation process of the present invention, which adopts technical means to solve the technical problems and achieve the technical effects, can be fully understood and implemented.

Example 1

A method for establishing a laying hen ovarian oxidative stress model comprises the following steps:

selecting Manfen laying hens at the egg-laying peak period, namely 28 weeks old, adopting single-factor test design, setting 6 treatments, each treatment being 10 times repeated, each repeated 1 chicken respectively being a control group (feeding physiological saline) and a tBHP (tert-butyl hydroperoxide) group, wherein the feeding concentration is determined as follows: 100(T2), 200(T3), 400(T4), 800(T5), 1000 μmol/kg body weight (T6), in the morning of experiment 8, 15, 22 days 9: 00 tBHP was fed. Sampling is carried out 48 hours after the last feeding. The experimental period is 24 days, the feed is corn-soybean meal type basic feed, the feed is prepared according to NRC (1994) and Chinese chicken feeding standards, specific data are shown in table 1, and the feeding mode is quantitative feeding. The test was conducted at the teaching experiment base of Sichuan university of agriculture. And (4) carrying out feeding management according to the conventional feeding management, strictly disinfecting every week, keeping the room temperature at about 20-22 ℃, and carrying out ventilation. The test was started with 2 weeks of pre-feeding, before the laying rate of the hens was recorded by observation and randomly grouped by laying rate.

The method comprises the following steps: egg production was recorded daily and the concentrations of estradiol, progesterone, testosterone, leptin, follicle stimulating hormone, insulin-like growth factor-1 and anti-muckle hall were determined by ELISA. Collecting ovaries 48h after the last tBHP feeding, counting the number of primordial follicles by a correction method after HE staining, and counting and measuring the sizes of the front-grade follicles (small white, large white and small yellow follicles) and the grade follicles (F1-F7). The contents or activities of direct antioxidant substances (vitamin C and vitamin E), lipid peroxidation products (MDA), protein peroxidation products (protein carbonyl), and antioxidases (superoxide dismutase SODs, catalase CAT, glutathione GSH, glutathione S-transferase GST, and total antibody ability TAOC) in ovary are determined by HPLC and kit.

TABLE 1 feed formulation

Note: ¹ the mineral elements and the multi-dimensional premix are configured according to the Chinese chicken feeding standard and are substituted by DSM.

The technical effects of the embodiment are as follows:

1. effect of the model of the invention on the laying Rate of a laying hen

As shown in Table 2, the laying hens are fed with 100, 200 and 400 mu mol/kg of tBHP, so that the laying rate of the laying hens is not remarkably influenced, and the laying rate of the laying hens can be remarkably reduced by feeding 800 and 1000 mu mol/kg of tBHP (P is less than 0.01).

TABLE 2 influence of feeding tBHP of different concentrations on laying rate of laying hens at later period of laying

Note: the same row is shouldered with different lower case letters to indicate significant difference (P < 0.05).

2. Effect of the model of the invention on reproductive hormones of layers

As can be seen from Table 3, after feeding 800 and 1000. mu. mol/kg of tBHP to the laying hens for 21 days, the concentrations of follicle stimulating hormone, estradiol and insulin-like growth factor-1 in the blood of the laying hens were significantly reduced (P <0.05), while feeding 100, 200 and 400. mu. mol/kg of tBHP had no significant effect on the blood hormones (P > 0.05).

TABLE 3 Effect of feeding different concentrations of tBHP on the expression of blood reproductive hormones and other metabolic hormones in egg-laying hens

Note: the same row is shouldered with different lower case letters to indicate significant difference (P < 0.05).

3. Effect of the model of the invention on ovarian morphology and follicle count at various stages in egg-laying hens

As can be seen from FIG. 1, feeding of 800 and 1000. mu. mol/kg of tBHP to layers significantly reduced the number of ovarian primordial follicles, primary follicles and pre-grade follicles of the layers, increasing the number of atretic follicles (P <0.05), while 100, 200 and 400. mu. mol/kg of tBHP had no significant effect on ovarian stage follicle counts (P > 0.05).

4. Influence of the model on the oxidative stress state and the antioxidant enzyme activity of the ovary of the laying hen

As can be seen from Table 4, the activity of total SOD, reducing GSH, antioxidant enzyme Cu-ZnSOD, MnSOD and GST in the ovary of the laying hen can be remarkably reduced by feeding 800 and 1000 mu mol/kg of tBHP (P is less than 0.05), and the tBHP with other concentrations has no remarkable influence on the activity (P is more than 0.05); as can be seen from Table 5, feeding of 800 and 1000 tBHP significantly increased the oxygen free radical content, the protein oxidation product protein carbonyl content and the lipid metabolism product MDA content in the egg hen ovary (P <0.05), and other concentrations of tBHP had no significant effect (P > 0.05).

TABLE 4 influence of feeding tBHP with different concentrations on the activity of the antioxidant enzymes of the ovaries of the laying hens

TABLE 5 Effect of feeding different concentrations of tBHP on egg hen ovarian oxidative stress products

Note: the difference was marked by shoulder-pointing different lower case letters on the same row (P < 0.05).

5. Influence of the model on the ovarian apoptosis rate of laying hens

As can be seen from FIG. 2, feeding layers with 800 and 1000. mu. mol/kg of tBHP significantly increased the rate of ovarian apoptosis (P <0.05), while 100, 200 and 400. mu. mol/kg of tBHP had no significant effect on the rate of ovarian apoptosis (P > 0.05).

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (3)

1. The method for establishing the oxidative stress model of the egg nest of the laying hen is characterized by comprising the following steps of:

Selecting a 28-week-old Manfen laying hen at an egg laying peak period, adopting a single-factor test design, setting 6 treatments, each treatment being 10 times repeated, wherein 1 chicken is repeated, the treatment is respectively 1 control group and 5 tert-butyl hydroperoxide groups, the control group is fed with normal saline, and the feeding concentration of the tert-butyl hydroperoxide group is determined as follows: 100. 200, 400, 800, 1000 μmol/kg body weight, in experiment 8, 15, 22 morning 9: 00 feeding physiological saline and tert-butyl hydroperoxide; sampling 48 hours after the last feeding; the test period is 24 days, the feed is corn-soybean meal type basic feed, and the feeding mode is quantitative feeding; feeding management is carried out according to conventional feeding management; the test is started to be pre-fed for 2 weeks, the laying rate of the laying hens is recorded by observation before the test, and the test is randomly grouped according to the laying rate;

daily egg production was recorded and the concentrations of estradiol, progesterone, testosterone, leptin, follicle stimulating hormone, insulin-like growth factor-1 and anti-muckle hall were determined by ELISA; collecting ovaries 48 hours after the last feeding of the physiological saline and the tert-butyl hydroperoxide, counting the number of original follicles by using a correction method after HE dyeing, and counting the number of the front-grade follicles, the grade follicles and measuring the size of the grade follicles; measuring the content or activity of vitamin C, vitamin E, lipid peroxidation products, protein carbonyl and antioxidant enzymes in ovary by HPLC and kit method, wherein the antioxidant enzymes comprise superoxide dismutase, catalase, glutathione S-transferase;

The concentrations of 5 tert-butyl hydroperoxide groups fed respectively are as follows: 100. mu. mol/kg body weight, 200. mu. mol/kg body weight, 400. mu. mol/kg body weight, 800. mu. mol/kg body weight, and 1000. mu. mol/kg body weight;

the feed comprises the following components in parts by mass: 59.064 parts of corn, 3.867 parts of wheat bran, 1.50 parts of soybean oil, 15.236 parts of soybean meal, 5.00 parts of corn protein powder, 5.00 parts of corn DDGS, 6.103 parts of granular stone powder, 2.50 parts of powdery stone powder, 0.941 part of powdery calcium hydrophosphate, 0.250 part of sodium chloride, 0.100 part of baking soda, 0.162 part of L-lysine sulfate, 0.012 part of DL-methionine, 0.100 part of choline chloride, 0.015 part of multi-vitamin and 0.15 part of mineral additive;

the purity of the L-lysine sulfate is 70 percent; the purity of DL-methionine is 99%; the purity of choline chloride was 60%.

2. The method for establishing the layer chicken ovarian oxidative stress model as claimed in claim 1, wherein in the process of feeding management, strict disinfection is performed every week, the room temperature is kept at 20-22 ℃, and ventilation is paid attention.

3. The method for establishing an ovarian oxidative stress model of laying hens as claimed in claim 1, wherein the pre-grade comprises small white, large white and small yellow follicles; the grade follicles include F1-F7 follicles.

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