Application of alpha-ketoglutaric acid in feed additive for relieving pregnancy heat stress induced skeletal muscle development injury of offspring calves

Abstract

The invention discloses an application of alpha-ketoglutaric acid (alpha-KG) in preparation of a feed additive for relieving skeletal muscle development injury of pregnancy heat stress induced offspring calves. According to the additive, sodium alpha-ketoglutarate (C5H4O5Na2) accounting for 0.1% of dry matter is uniformly mixed into dry dairy cow total mixed ration (TMR), and the dairy cow is fed with heat stress (THIgt; 72) pregnant Holstein cows, the weight of fetal cattle is increased by 23.2%, and the weight of skeletal muscles is increased by 31.3%. Meanwhile, during pregnancy, alpha-KG is fed, so that the weight of the offspring weaned calves is increased by 10.5%, the weight of skeletal muscles is increased by 28.6%, and the fiber density of the skeletal muscles is improved. According to the method, the skeletal muscle growth and fiber density of the offspring calves are subjected to targeted cross-generation regulation and control through maternal nutrition intervention for the first time, the regulation and control bottleneck of heat stress cross-generation injury in the prior art is broken through, and a solution is provided for improving animal health and growth potential for ruminant heat stress management.

Description

Technical Field

[0001] The present invention relates to ruminant nutrition and functional feed additives, and in particular to the use of α-ketoglutarate (α-KG) as a core active ingredient in the preparation of a feed additive for alleviating the damage to skeletal muscle development of fetal cattle and offspring weaned calves caused by heat stress during pregnancy, including a preparation method, an action mechanism and quantitative application parameters thereof. Background Art

[0002] In summer and autumn, when the greenhouse index is ≥72, more than 80% of dairy cows will enter a state of heat stress. Heat stress not only causes a 15-30% decrease in milk production and a 20% decrease in dry matter intake in dairy cows, but also significantly inhibits the development of fetal skeletal muscle during pregnancy, causing the weaning weight of offspring calves to decrease by 12-18% and the skeletal muscle fiber density to decrease by 20-25%, which is extremely harmful to the growth, health and production potential of offspring calves. Conventional heat stress intervention measures (such as spray cooling and electrolyte supplementation) can only alleviate short-term maternal symptoms, but cannot solve the following core problems: heat stress causes damage to the growth and development of fetal skeletal muscle, and conventional nutritional regulation lacks specificity; at the same time, existing additives (such as vitamin C and betaine) have no significant improvement on offspring growth and development and skeletal muscle growth. Therefore, there is currently a lack of effective nutritional intervention measures for heat stress-induced damage to the growth and development of skeletal muscle of fetal cows and offspring calves. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a targeted nutritional intervention program based on α-KG to solve the problems of fetal bovine skeletal muscle development disorders and poor offspring growth performance caused by heat stress, and to break through the bottleneck of the lack of maternal-offspring co-effect regulation in the prior art. In view of the defects of the prior art, this study shows that α-KG can be fed to pregnant cows in the form of feed additives under heat stress conditions during pregnancy. The α-KG ingested by the mother can improve the early development of skeletal muscle fibers by regulating the energy metabolism and redox balance of fetal bovine skeletal muscle, thereby promoting offspring muscle growth and increasing weaning weight.

[0004] This study aims to explore the application of α-KG feeding during pregnancy in improving the growth and development of skeletal muscle in fetal cows and postpartum calves under heat stress. We will select an appropriate amount of α-KG as an additive to feed pregnant cows and observe its effects on the weight and skeletal muscle growth and development of fetal cows and weaned calves. Through this study, it aims to provide a theory and regulation strategy for nutrient intervention during pregnancy to alleviate heat stress-induced damage to skeletal muscle development in intrauterine fetal cows and improve muscle growth and weaning weight of offspring calves.

[0005] Technical solution: Use of α-ketoglutarate (α-KG) or a pharmaceutically acceptable salt thereof in the preparation of a feed additive for alleviating skeletal muscle development damage in offspring ruminants induced by heat stress during pregnancy.

[0006] The ruminant is selected from cattle.

[0007] Application of alpha-ketoglutarate (alpha-KG) or a pharmaceutically acceptable salt thereof in the preparation of a feed additive for alleviating skeletal muscle development damage induced by heat stress during pregnancy in fetal cattle or offspring calves.

[0008] Alpha-ketoglutarate is added to dairy cow feed from 60 days before the expected date of calving until farrowing.

[0009] α-Ketoglutarate is added to dairy cow feed at 0.01-0.2% of dry matter intake.

[0010] The α-ketoglutaric acid or a pharmaceutically acceptable salt thereof includes sodium α-ketoglutarate.

[0011] The feed additive increases the weight of clinical fetuses and the weight of skeletal muscle tissue.

[0012] The feed additive increases the body weight of offspring weaned calves and increases the weight of skeletal muscle tissue.

[0013] The ruminant is selected from dairy cows.

[0014] Application of alpha-ketoglutarate (alpha-KG) or a pharmaceutically acceptable salt thereof in the preparation of a medicine for alleviating skeletal muscle development damage induced by heat stress during pregnancy in fetal bovines or offspring calves.

[0015] Further preferred is the use of α-KG in promoting the growth and development of skeletal muscle of offspring ruminants during pregnancy.

[0016] The α-KG of the present invention is used in the preparation of a feed additive for promoting the growth and development of skeletal muscle of offspring subjected to heat stress during pregnancy.

[0017] Wherein, the animal is a ruminant suffering from heat stress during pregnancy.

[0018] Furthermore, the α-KG is used as a natural feed additive based on animal feed during pregnancy to promote the growth and development of skeletal muscle in offspring calves.

[0019] Preferably, the α-KG feeding content is 0.1% of the dry matter content and is evenly mixed into a dry total mixed ration (TMR) for dairy cows, and is stirred in the TMR (speed 30 rpm, time 30 min) for feeding.

[0020] The animal feeding period is from 60 days before birth to delivery (covering the rapid growth period of fetal bovine skeletal muscle).

[0021] Among them, the feeding targets are pregnant Holstein cows in a heat stress environment (THI>72).

[0022] In order to deal with the above problems faced by ruminant feeding under heat stress conditions during pregnancy, the present invention observed in the early stage that when dry cows were fed with less than 0.1% of α-KG (0.01%, 0.03%, 0.06%), the effect of increasing the dose of α-KG in the blood of cows was not obvious (such as Figure 1 ); the addition level of α-KG ≥ 0.1% dry matter can effectively increase the content of a-KG in the blood of dairy cows, indicating that 0.1% is the appropriate addition amount of α-KG (such as Figure 2 Heat stress leads to lower α-KG levels in pregnant cows and umbilical cord blood (e.g. Figure 2 A, 2B), feeding 0.1% α-KG to pregnant cows significantly increased the α-KG content in cows and umbilical cord blood (such as Figure 2 A, 2B). At the same time, feeding α-KG during pregnancy reduced the loss of body weight and back fat in dairy cows due to heat stress (e.g. Figure 3 A, 3B). For fetuses and offspring calves, heat stress during pregnancy impairs fetal skeletal muscle development, including reduced fetal body weight before delivery (e.g. Figure 4 A) posterior skeletal muscle (such as Figure 4 B) and posterior rectus skeletal muscle (eg Figure 4 C) weight; Adding α-KG during pregnancy can alleviate the effect of heat stress on the weight of fetuses (such as Figure 4 A) posterior skeletal muscle (such as Figure 4 B) and posterior rectus skeletal muscle (such as Figure 4 C) and improved skeletal muscle fiber density (e.g. Figure 5 Regarding the effect on skeletal muscle growth of offspring calves, we observed that the addition of α-KG to dry cows during pregnancy can significantly increase the body weight of weaned calves (e.g. Figure 6 A) posterior skeletal muscle (such as Figure 6 B) and posterior rectus skeletal muscle (eg Figure 6 C) weight, the effect of heat stress during pregnancy on the weight of postpartum lactating calves (such as Figure 6 A) The second half (such as Figure 6 B) and the damaging effects of posterior rectus skeletal muscle growth (e.g. Figure 6 C); H&E staining of skeletal muscle fibers of weaned calves revealed that feeding α-KG during pregnancy also effectively improved the side effects of heat stress on the reduction of skeletal muscle fiber density in weaned calves (such as Figure 7 ).

[0023] In summary, the experiment found that feeding α-KG to dry cows under heat stress during pregnancy can significantly alleviate the effects of intrauterine heat stress on fetal skeletal muscle growth and weight loss, and also has a significant effect on increasing the weight and skeletal muscle growth of postpartum weaned calves.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the α-KG used in the present invention targets the critical period of fetal bovine skeletal muscle development (60 days before delivery), and proposes a new function of α-KG, which can be added to the TMR of heat-stressed dry cows during pregnancy to alleviate the damaging effect of intrauterine heat stress on the growth and development of skeletal muscle of fetal bovines and offspring calves, and improve the weight gain of calves; in the experiment, α-KG significantly increased the skeletal muscle weight of heat-stressed fetal bovines in labor by 31.3% and body weight by 23.2%, and increased the skeletal muscle weight of offspring weaned calves by 28.6% and body weight by 10.5%. It shows that α-KG can be used as a feed additive for pregnant dry cows under heat stress conditions to improve the growth and weight gain of skeletal muscle fibers in intrauterine fetal cows and postpartum calves. Therefore, α-KG can be used as an effective feed additive to promote the growth and development of skeletal muscle of intrauterine fetuses and offspring under heat stress conditions during pregnancy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of α-KG feeding concentration on the α-KG content in the blood of dairy cows;

[0026] Figure 2 α-KG feeding significantly increased the α-KG content in the blood and umbilical cord blood of heat-stressed dairy cows;

[0027] Figure 3 α-KG feeding significantly alleviates the effect of heat stress on the reduction of body weight and subcutaneous fat in pregnant dairy cows;

[0028] Figure 4 α-KG feeding significantly improves the body weight and skeletal muscle growth of intrauterine fetal cattle under heat stress;

[0029] Figure 5 α-KG feeding significantly increased the skeletal muscle fiber density of fetal bovines under heat stress during pregnancy;

[0030] Figure 6 Feeding α-KG to treat heat stress during pregnancy significantly increased the body weight and skeletal muscle tissue growth of offspring weaned calves;

[0031] Figure 7 Feeding α-KG to treat heat stress during pregnancy significantly increased skeletal muscle fiber density in offspring weaned calves. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0033] The raw materials and instruments used in the examples are commercially available.

[0034] Hematoxylin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0035] Eosin was purchased from Background Solebao Technology Co., Ltd.

[0036] The body weight scale was purchased from Xingzhi (Shanghai) Technology Co., Ltd.

[0037] Sodium α-ketoglutarate was purchased from Beijing Yueyang Biotechnology Co., Ltd., China.

[0038] The molecular formula of α-ketoglutarate sodium is: 5 H 4 O 5 Na 2 , the structural formula is as follows:

[0039]

[0040] Example 1

[0041] Holstein dry cows with similar body condition, lactation days and parity were selected and randomly divided into six groups: control group (n = 8), α-KG 0.01% DM group (n = 8), α-KG 0.03% DM group (n = 8), α-KG 0.06% DM group (n = 8), α-KG 0.1% DM group (n = 8) and α-KG 0.2% DM group (n = 8). All cows were fed with dry cow TMR diets. The diet design was based on the nutritional requirements of dry cows in the international NRC (2001). The formula ingredients included corn, soybean meal, whole corn silage and alfalfa hay, which were commonly used feeds for dairy cows, to ensure that the nutritional components met the nutritional requirements of dry cows. After the diets were prepared in the corresponding proportions, they were mixed with a double-shaft mixer at a speed of 30 rpm for 30 minutes. The cows were raised in free stalls and had free access to food and water.

[0042] After 3 days of feeding, blood samples were collected from the cows’ tail venous roots. Figure 1 It shows that adding 0.1% DM to α-KG can significantly increase the content of α-KG in the blood, while the addition of more than 0.1% DM has no significant effect on the content of α-KG in the blood. Therefore, 0.1% DM is the optimal addition amount.

[0043] Example 2

[0044] Holstein dry cows with similar body condition, lactation days and parity were selected. Dry cows with similar body condition were randomly divided into a non-heat stress (THI < 72) control group (n = 8, pasture cooling measures such as spraying and fans were implemented), a heat stress group (THI > 72) during the dry period (heat stress group, n = 8, pasture cooling measures such as spraying and fans were not implemented), and a heat stress feeding α-KG (addition amount 0.1% DM) group during the dry period (α-KG group, n = 8, THI > 72, pasture cooling measures such as spraying and fans were not implemented). All cows were fed a dry cow TMR diet. The diet design was based on the nutritional requirements of dry cows set by the International NRC (2001). The formula ingredients were mainly corn, soybean meal, whole corn silage, alfalfa hay and other common feed ingredients for dairy cows. The nutrients met the nutritional needs of dry cows. The dry cow TMR diet was added at a ratio of 0.1% and mixed using a twin-shaft mixer (30rpm×30mmin). The cows were raised in free stalls and had free access to food and water.

[0045] One week before delivery, the tail venous blood and umbilical cord blood of dairy cows were collected. Figure 2 The results showed that heat stress significantly reduced the content of α-KG in the serum and umbilical cord blood of pregnant cows, while feeding α-KG during pregnancy increased the content of α-KG in the blood of cows and umbilical cords; before delivery, the cows were weighed and the thickness of subcutaneous fat was measured. Figure 3 The results showed that heat stress in dry cows significantly reduced the body weight and subcutaneous fat thickness of cows, while the addition of α-KG could alleviate the negative effects of heat stress on the body weight and subcutaneous fat thickness of pregnant cows.

[0046] Example 3

[0047] The posterior half and posterior rectus skeletal muscles of the fetuses of the above-mentioned experimental pregnant dry cows were collected, the body weight and skeletal muscle weight were weighed, and the posterior half skeletal muscle tissue samples were immediately fixed in paraformaldehyde for H&E staining and histochemical analysis, such as Figure 4 It showed that heat stress during pregnancy significantly reduced the body weight and skeletal muscle tissue weight of fetal cows.

[0048] Example 4

[0049] The paraformaldehyde-fixed posterior half of the skeletal muscle tissue samples were rinsed with running water for 24 hours, dehydrated with alcohol using 70% alcohol, 80% alcohol for 2 hours, 95% alcohol for 2 hours twice, anhydrous alcohol for 1 hour twice, alcohol and xylene mixture (1:1) for 30 minutes, xylene for 15 minutes twice, wax dipped for 1.5 hours twice (overnight), embedded, sliced ​​(5um), and stained. After staining, dry, add neutral gum to the specimen and cover with a coverslip for long-term storage. Figure 5 The results showed that heat stress during pregnancy significantly reduced the skeletal muscle fiber density of fetal bovines, while feeding α-KG during pregnancy under heat stress significantly increased the skeletal muscle fiber density of fetal bovines.

[0050] Example 5

[0051] The postpartum calves corresponding to the dry cows in the above experimental group were fed the same feeding and management from birth to 56 days before weaning, and the feeding and management referred to the Chinese reserve dairy cow feeding and management regulations. The main procedures included: calves were fed individually in the calf island, had free access to water, and were fed milk once in the morning and afternoon each day; calves were fed 4 liters of colostrum within 1 hour after birth, 2 liters of colostrum within 8 hours after birth, 6 liters of pasteurized milk per day within 1 week of age, 8 liters of milk per day from 2 to 6 weeks of age, 6 liters of milk per day at 7 weeks of age, and 4 liters of milk at 8 weeks of age; and starter feed was consumed after 4 days of age.

[0052] Before weaning, the body weight was measured and the posterior hemi- and posterior rectus skeletal muscles were collected and weighed. Figure 6 The results showed that heat stress during pregnancy significantly reduced the body weight, hind half and posterior rectus skeletal muscle weight of weaned calves; and the addition of α-KG could significantly alleviate the negative effects of heat stress during pregnancy on the body weight and skeletal muscle growth of weaned calves.

[0053] Example 6

[0054] The hind half skeletal muscle of the weaned calf collected above was immediately fixed in paraformaldehyde for 48 hours, rinsed with running water for 24 hours after fixation, and dehydrated with alcohol through 70% alcohol, 80% alcohol for 2 hours, 95% alcohol for 2 hours twice, anhydrous alcohol for 1 hour twice, alcohol and xylene mixture (1:1) for 30 minutes, xylene for 15 minutes twice, wax immersion for 1.5 hours twice (overnight), embedded, sliced ​​(5um), and stained. After staining, dry, add neutral gum to the specimen and cover with a cover glass for long-term storage. Figure 7 The results showed that heat stress during pregnancy significantly reduced the skeletal muscle fiber density of weaned calves, while feeding α-KG to cows subjected to heat stress during pregnancy could significantly increase the skeletal muscle fiber density of calves.

[0055] in conclusion:

[0056] In summary, as a natural active substance, α-KG has significant application value in improving the growth and development of skeletal muscle of fetal cows and postpartum calves under heat stress, increasing fetal weight and weaning weight. Through reasonable technical solutions and designs, good application effects can be achieved, providing support for the development of animal husbandry.

Claims

1. Use of α-ketoglutaric acid or a pharmaceutically acceptable salt thereof in the preparation of a feed additive for alleviating skeletal muscle development damage in offspring ruminants induced by heat stress during pregnancy.

2. The use according to claim 1, characterized in that: The ruminant is selected from cattle.

3. Use of α-ketoglutaric acid or a pharmaceutically acceptable salt thereof in the preparation of a feed additive for alleviating skeletal muscle development damage induced by heat stress during pregnancy in fetal cattle or offspring calves.

4. The use according to claim 1 or 3, characterized in that: Alpha-ketoglutarate is added to dairy cow feed from 60 days before the expected date of calving until farrowing.

5. The use according to claim 1 or 3, characterized in that: α-Ketoglutarate is added to dairy cow feed at 0.01-0.2% of dry matter intake.

6. The use according to claim 1 or 3, characterized in that: The α-ketoglutaric acid or a pharmaceutically acceptable salt thereof includes sodium α-ketoglutarate.

7. The use according to claim 1 or 3, characterized in that: The feed additive increases the weight of clinical fetuses and the weight of skeletal muscle tissue.

8. The use according to claim 1 or 3, characterized in that: The feed additive increases the body weight of offspring weaned calves and increases the weight of skeletal muscle tissue.

9. The use according to claim 1 or 3, characterized in that: The ruminant is selected from dairy cows.

10. Use of α-ketoglutaric acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for alleviating skeletal muscle development damage induced by heat stress during pregnancy in fetal bovines or offspring calves.

Images