Application of alpha KG in improving growth performance of ruminant offspring

Abstract

The invention discloses application of alpha KG in improving growth performance of ruminant offspring, and belongs to the technical field of feed additives. The alpha KG is used for improving the trans-generation daily gain and the heat production metabolism of the body of the ruminant for the first time, a remarkable and long-acting daily gain trans-generation programming effect is generated on the offspring calves through maternal feeding, the postpartum daily gain of the offspring calves is increased, the BAT development and heat production glycolipid metabolism functions are activated, and the glucose utilization efficiency of the body is optimized in a long-term mode. Experimental results show that after the alpha KG is added into the dry dairy cow TMR, the postpartum daily gain of the offspring calves can be directly increased, the BAT development and heat production glycolipid metabolism functions are activated, and the glucose utilization efficiency of the body is optimized in a long-term mode.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of feed additives, and particularly relates to application of alphaKG in improving growth performance of offspring of ruminants. BACKGROUND

[0002] In the cattle industry, postpartum calf average daily gain (ADG) is a key indicator for measuring the growth and health of the calf and the future production potential. However, in large-scale ranches, about 15%-25% of newborn calves face a difficult problem: although their birth weight is normal, and postpartum feeding management and nutrition supply are at the best level, they still show severe growth retardation and abnormal glucose and lipid metabolism before weaning, and their ADG (usually only 0.2-0.4 kg / d) is significantly lower than the normal level (about 0.6 kg / d). Studies have found that this growth retardation is closely related to the adaptability of the calf to the low-temperature environment outside the uterus after birth, and the core is the development and function of the brown adipose tissue (BAT), a key thermogenic organ. The development of BAT is not determined after birth, but is largely programmed by the nutritional metabolism status of the pregnant cow during the gestation period, especially during the fetal period.

[0003] However, current interventions for postpartum growth retardation of calves are mostly focused on the postpartum stage, such as optimizing starter feed, improving feeding environment, etc. These conventional methods have a fundamental limitation: they cannot reverse or correct the BAT development and metabolic programming defects caused by abnormal maternal nutrition metabolism during the fetal period. In other words, the field has long lacked an effective nutritional intervention strategy that targets improving fetal BAT development from the maternal gestation nutrition, and then preventing and solving the problem of postpartum growth retardation of offspring calves from the source. Therefore, there is an urgent need in the field for a new method that can break through the limitations of the prior art and achieve long-term, targeted regulation of offspring ADG through maternal nutrition. SUMMARY

[0004] To solve the above technical problems, the application provides application of alphaKG in improving growth performance of offspring of ruminants. Through maternal gestation nutrition intervention, the application targets to improve the innate growth potential and fat thermogenesis metabolism health of offspring calves.

[0005] To achieve the above purpose, the application provides the following technical solutions:

[0006] The application provides application of alphaKG (alpha-ketoglutaric acid) or a pharmaceutically acceptable salt thereof in improving growth performance of offspring of ruminants, and the application comprises:

[0007] 1) increasing postpartum average daily gain of offspring of ruminants;

[0008] 2) increasing weight of brown adipose tissue of offspring of ruminants;

[0009] 3) enhancing the body heat production function of the offspring of ruminants;

[0010] 4) improving the efficiency of sugar and lipid metabolism of the offspring of ruminants.

[0011] Further, the application comprises feeding the pregnant ruminant mother with a diet containing alpha KG during the gestation period.

[0012] Further, the ruminant mother is fed with a diet containing alpha KG 20 days to 80 days before delivery.

[0013] Further, the ruminant mother is fed with a diet containing alpha KG 60 days before delivery to delivery.

[0014] Further, the amount of alpha KG added is 6.5-19.5 g / head / day.

[0015] Further, the amount of alpha KG added is 13 g / head / day.

[0016] Further, the ruminant is a cow.

[0017] Further, the ruminant is a dairy cow.

[0018] Further, the alpha KG or its pharmaceutically acceptable salt comprises alpha-ketoglutarate sodium.

[0019] The present application provides a feed additive for improving the growth performance of the offspring of ruminants, which is alpha KG or a pharmaceutically acceptable salt thereof.

[0020] The present application also provides a method for improving the growth performance of the offspring of ruminants, which comprises feeding the pregnant ruminant mother with a diet containing the feed additive as described in the above technical solution during the gestation period.

[0021] Compared with the prior art, the present application has the following advantages and technical effects:

[0022] The present application first uses alpha KG to improve the cross-generation daily weight gain and body heat production metabolism of ruminants, and through maternal feeding, a significant and long-acting cross-generation programming effect on daily weight gain of offspring calves is produced, the postpartum daily weight gain of offspring calves is increased, and the development of BAT and the function of heat production and sugar and lipid metabolism are activated, and the efficiency of glucose utilization in the body is long-acting optimized.

[0023] The mechanism of the application for improving the growth performance of offspring of ruminants by using alpha-ketoglutarate (alpha-KG) is as follows: taking brown adipose tissue (BAT) as a target tissue, by inhibiting the cGAS / STING inflammation pathway in BAT, the chronic inflammation caused by mitochondrial DNA leakage is reduced, so that the normal development and thermogenic metabolism function of BAT are restored, and the BAT thermogenic capacity is directly related to the offspring body temperature regulation and glucose metabolic rate, which ultimately drives the ADG growth, and the growth rate and body health level of the calf are fundamentally improved.

[0024] The application discloses for the first time that alpha-KG can improve the mitochondrial function and thermogenic metabolism by regulating the cGAS / STING signal pathway of BAT of offspring calves, which provides a new patent technology path for fundamentally solving the postpartum growth retardation problem of calves, and has a wide industrial application prospect in the field of ruminant breeding.

[0025] The experimental results of the application show that the addition of alpha-KG in dry cow TMR (total mixed ration) can directly increase the postpartum daily weight gain of offspring calves, and activate the development and thermogenic glycolipid metabolism function of BAT, and long-acting optimize the glucose utilization efficiency of the body. The application not only opens up a new application field for alpha-KG, but also provides a safe, efficient and easy-to-promote method for improving the growth performance of young ruminants and improving the economic benefits of animal husbandry. BRIEF DESCRIPTION OF DRAWINGS

[0026] The accompanying drawings, which form a part of this application, are used to provide a further understanding of the application, and the illustrative embodiments of the application and their description serve to explain the application without imposing undue limitation on the application. In the drawings:

[0027] Figure 1 For the growth and development, glucose metabolism characteristics and maternal and offspring alpha-KG level analysis of the slow-growing calves in Example 1, A is daily weight gain, B is glucose level, C is glucose tolerance accumulation, D is insulin resistance index, E is serum, rumen fluid and umbilical cord metabolite Venn diagram, F is serum, rumen fluid and umbilical cord alpha-KG level, G is significant umbilical cord metabolite of slow-growing calves, H is correlation between serum metabolites and growth indicators, and I is correlation between rumen metabolites and growth indicators.

[0028] Figure 2 For the daily weight gain of the control group and the three alpha-KG addition groups in Example 2.

[0029] Figure 3 For the influence of maternal alpha-KG supplementation during pregnancy on the growth rate and glucose metabolism efficiency of calves in Example 3, A is 1-month-old weight, B is weight growth curve, C is glucose concentration, D is glucose tolerance accumulation, and E is insulin resistance index.

[0030] Figure 4For the evaluation of brown adipose tissue development and metabolic function of 1-month-old calves in the control group and the slow growth group in Example 1, A is the weight of perirenal brown adipose tissue, B is the number and structure of adipocytes, C is the expression of brown adipose tissue metabolism-related genes, and D is the expression of brown adipose tissue metabolism-related proteins;

[0031] Figure 5 For the evaluation of brown adipose tissue development and metabolic function of 1-month-old calves in the control group and the slow growth group in Example 1, A is the weight of perirenal brown adipose tissue, B is the number and structure of adipocytes, C is the expression of brown adipose tissue metabolism-related genes, and D is the expression of brown adipose tissue metabolism-related proteins;

[0032] Figure 6 For the evaluation of brown adipose tissue development and metabolic function of 1-month-old calves in the control group and the slow growth group in Example 1, A is the weight of perirenal brown adipose tissue, B is the number and structure of adipocytes, C is the expression of brown adipose tissue metabolism-related genes, and D is the expression of brown adipose tissue metabolism-related proteins;

[0033] Figure 7 For the evaluation of brown adipose tissue development and metabolic function of 1-month-old calves in the control group and the slow growth group in Example 1, A is the weight of perirenal brown adipose tissue, B is the number and structure of adipocytes, C is the expression of brown adipose tissue metabolism-related genes, and D is the expression of brown adipose tissue metabolism-related proteins; DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be described clearly and completely in the embodiments of the present application combined with the drawings. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0035] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be further described in detail below combined with the drawings and specific embodiments.

[0036] In the following examples, hematoxylin was purchased from Shanghai Micron Biotech Co., Ltd., eosin was purchased from Beijing Solabio Technology Co., Ltd., a body weight meter was purchased from Xingzhi (Shanghai) Technology Co., Ltd., and alpha-ketoglutarate sodium was purchased from Beijing Yueyang Biological Technology Co., Ltd.;

[0037] The molecular formula of alpha-ketoglutarate sodium (αKG) is C5H4O5Na2, and the structural formula is: .

[0038] Example 1 Growth Metabolic Characteristics and αKG Content Analysis of Slow Growth Calves

[0039] Select Holstein newborn calves with similar birth date and birth weight, and use the same feeding and management methods from birth to 30 days. The feeding and management refers to the Chinese reserve cow feeding and management regulations. The specific management measures are as follows: the calves are individually fed on the calf island, can drink water freely, and are fed milk twice a day in the morning and afternoon respectively; the calves are fed 4 liters of colostrum within 1 hour of birth, 2 liters of colostrum within 8 hours of 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 at 7 weeks of age, and 4 liters of milk at 8 weeks of age; start to feed starter after 4 days of age;

[0040] The corresponding cows of all the above calves are fed with dry cow TMR diet 60 days before production. The diet is strictly designed in accordance with the international NRC (2001) dry cow nutritional needs. The formula raw materials are selected from common cow feed raw materials such as corn, soybean meal, whole plant corn silage feed and alfalfa hay, etc., to ensure that the nutritional ingredients fully meet the nutritional needs of dry cows. After the diet is prepared according to the corresponding proportion, a double-shaft mixer is used to stir and mix for 30 minutes at a speed of 30 rpm. The cows are fed in a scattered stall feeding mode, and can drink water freely;

[0041] According to the daily weight gain within 30 days after birth, all the above calves are divided into: a control group (n=8) and a slow growth group (n=8);

[0042] Blood and rumen fluid are collected from cows before production, and umbilical cord blood is collected during production. The body weight and blood of calves are sampled and detected once a week, and the results are shown in Figure 1 .

[0043] Figure 1 For the growth and development, glucose metabolism characteristics and maternal and child source αKG level analysis of slow growth calves in Example 1, A is daily weight gain, B is glucose level, C is glucose tolerance, D is insulin resistance index, E is serum, rumen fluid and umbilical cord metabolites Venn diagram, F is serum, rumen fluid and umbilical cord αKG level, G is slow growth significantly reduced characteristic umbilical cord metabolite, H is serum metabolite correlation with growth index, and I is rumen metabolite correlation with growth index. Figure 1 The results show that the αKG levels in the rumen fluid, blood and umbilical cord blood of the cows of the postpartum slow growth calves are significantly lower than those of the normal population (as shown in parts E, F and G of Figure 1 Correspondingly, these offspring calves show severe growth and metabolic defects at one month of age: the daily weight gain (ADG) is extremely significantly reduced (P<0.0001, see part A of Figure 1 The glucose metabolism rate is significantly decreased (P<0.05, see Figure 1B, C, D in FIG. 1), indicating that its glucose metabolism function was impaired. Correlation analysis further revealed that the content of aKG in maternal blood during pregnancy and rumen fluid was highly positively correlated with the ADG of offspring calves and the weight of brown adipose tissue (BAT) (P<0.01, see Figure 1 H, I in FIG. 1). This series of evidence indicates that the lack of maternal aKG during pregnancy is a key maternal factor leading to the growth retardation and metabolic disorder of offspring.

[0044] Example 2 Determination of suitable addition concentration of aKG during dry period

[0045] Holstein dry cows with similar body condition, pregnancy days and parity were selected and randomly divided into four groups at 60 days before delivery: a control group (n=8) and three aKG addition groups (low, medium and high dose groups, n=8 for each group), which were respectively added with 0.0 g / head / day (control group), 6.5 g / head / day (low dose group), 13.0 g / head / day (medium dose group) and 19.5 g / head / day (high dose group) of aKG.

[0046] All test cows were fed in a scattered stall, and free access to feed and water was provided. All cows were fed with a dry cow TMR diet, which was designed to follow the international NRC (2001) dry cow nutritional needs, and the formula was mainly based on corn, soybean meal, whole-plant corn silage feed and alfalfa hay, etc. common dairy feed raw materials to ensure that the nutritional ingredients meet the nutritional needs of dry cows. The aKG addition groups were added with aKG in the dry cow total mixed ration according to the proportion, and a double-shaft mixer was used to mix for 30 min at a speed of 30 rpm. The body weight changes of offspring calves produced by each group of cows were recorded every week to evaluate the effect of maternal aKG supplementation on the growth performance of calves.

[0047] Figure 2 The daily gain of the control group and the three aKG addition groups in Example 2. Figure 2 The results showed that compared with the control group, the low-dose aKG group (6.5 g / head / day) had no significant promoting effect on the growth rate of calves (P>0.05); while the medium and high-dose aKG groups (13 g / head / day and 19.5 g / head / day) could significantly improve the growth rate of calves (P<0.05), and there was no significant difference between the two dose groups (P=0.15). Considering the effect and the cost of addition, the suitable addition amount of aKG during the dry period was determined to be 13 g / head / day.

[0048] Example 3 aKG feeding during dry period significantly improves the growth rate and glucose metabolism efficiency of calves

[0049] Holstein dry cows with similar body condition, pregnancy days and parity were selected, and dry cows with similar body condition were randomly divided into a control group (n=16) and an aKG feeding group during dry period (aKG group, n=16) at 60 days before delivery.

[0050] All cows were fed dry cow TMR diet, which was designed to follow the international NRC (2001) dry cow nutritional needs, and the formula was mainly composed of corn, soybean meal, whole-plant corn silage feed and alfalfa hay, etc. Common dairy feed raw materials were used to ensure that the nutritional ingredients met the nutritional needs of dry cows. The aKG group added aKG to the dry cow total mixed ration at a rate of 13 g per head per day, and used a double-shaft mixer to mix for 30 min at a speed of 30 rpm. All cows were raised in a scattered way, and free to eat and drink water.

[0051] The body weight and blood of the offspring calves corresponding to the treatment cows were detected every week, and the results are shown in Figure 2 .

[0052] Figure 3 For the influence of maternal aKG supplementation during pregnancy on the growth rate and glucose metabolism efficiency of calves in Example 3, A is the one-month-old body weight, B is the body weight growth curve, C is the glucose concentration, D is the glucose tolerance, and E is the insulin resistance index. As can be seen from Figure 3 , the average daily gain (ADG) of the offspring calves fed with aKG during the dry period before one month of age increased by 19.0% (P<0.001, see A, B part of Figure 3 ), which is much higher than the expected effect of conventional nutritional regulation. At the same time, the glucose metabolism rate of the calves in the aKG group was significantly improved (see C, D, E part of Figure 3 ), indicating that the overall energy utilization efficiency was fundamentally improved. In summary, aKG supplementation has a significant and long-term cross-generation programming effect on the daily weight gain of offspring calves.

[0053] Example 4: aKG feeding during pregnancy improves the development and thermogenic metabolic function of brown adipose tissue of offspring calves

[0054] The offspring newborn calves BAT (n=8) of the control group and the slow growth group of Example 1 and the control group and the aKG group of Example 3 were collected respectively, the body weight and BAT weight of the calves were weighed, and then the collected BAT tissue samples were immediately fixed in paraformaldehyde for H&E staining immunohistochemical analysis; the specific processing steps are as follows: the BAT tissue samples fixed with paraformaldehyde are washed with running water for 24 h, and then dehydrated with alcohol, including 70% alcohol, 80% alcohol for 2 h each, 95% alcohol for 2 h (twice), anhydrous alcohol for 1 h (twice), alcohol and xylene mixture (volume ratio 1:1) for 30 min, xylene for 15 min (twice), wax immersion for 1.5 h (twice, one overnight), embedding, slicing (thickness 5 μm), staining with hematoxylin and eosin, and after staining, the slices are dried, neutral gum is added on the specimen and covered with a cover glass for long-term storage.

[0055] Figure 4 For the evaluation of brown adipose tissue development and metabolic function of 1-month-old calves in the control group and the slow growth group in Example 1, A is the weight of perirenal brown adipose tissue, B is the number and structure of adipocytes, C is the expression of brown adipose tissue metabolism-related genes, and D is the expression of brown adipose tissue metabolism-related proteins. From Figure 4 It can be seen that the weight of BAT in the body of slow growth calves is significantly reduced, the morphology of brown adipocytes is damaged, and the expression of corresponding thermogenesis and glycolipid metabolism-related genes is significantly reduced.

[0056] Figure 5 For the evaluation of brown adipose tissue development and thermogenic metabolism function of calves in the control group and the aKG group in Example 3, A is the weight of brown adipose tissue in newborn calves, B is the rectal temperature of newborn calves, C is the number and structure of adipocytes, D is the expression of thermogenic-related genes, E is the expression of thermogenic-related proteins, F is the weight of perirenal brown adipose tissue in 1-month-old calves, G is the expression of thermogenic-related genes in 1-month-old calves, and H is the expression of thermogenic-related proteins in 1-month-old calves. Figure 5 The results show that compared with the control group, the BAT weight of the newborn calf in the aKG group added during pregnancy increases by 34.6%, and the adipocyte morphology is more compact, the expression of thermogenic key genes (UCP-1, PPARGC1a) is significantly up-regulated, and the body temperature stability is enhanced (see Figure 5 Part A-E), which indicates that feeding aKG during pregnancy significantly increases the weight of brown adipose tissue in the offspring newborn calf, improves its body temperature regulation ability, and improves the development of brown adipocyte and the expression of thermogenic genes. Moreover, the above-mentioned effects have long-term effects, and by the time the offspring calf is one month old, the BAT weight of the offspring calf is further increased by 41.4%, and the thermogenic genes are continuously highly expressed (see Figure 5 Part F-H). This proves that the maternal nutritional intervention has a lasting "imprinting" effect on the offspring thermogenic metabolism system, promotes the expression of thermogenic genes and proteins, reduces the adaptability to environmental temperature, and improves the body thermogenesis ability of the offspring calf after birth, thereby improving the daily gain.

[0057] Example 6 Mechanism of the effect of aKG feeding during pregnancy on the development and metabolic function of brown adipose tissue in offspring calves

[0058] According to the experimental results of Example 4, feeding aKG during pregnancy significantly improves the growth and thermogenic metabolism function of brown adipose tissue in newborn (n=8) and weaned calves (n=8), and improves the postnatal body weight gain and glucose sensitivity. In order to further study the mechanism of aKG feeding during pregnancy on brown adipose tissue, the mitochondrial inflammation pathway of BAT in slow growth calves and aKG-fed calves was studied, and the results are shown in Figure 6 and Figure 7 .

[0059] Figure 6Gene expression and relative amount of cytoplasmic mitochondrial DNA of the control group and the slow growth group in Example 1, wherein A is the expression of cGAS related protein, B is the expression of cGAS related gene, C is the expression of inflammation related gene, and D is the relative amount of cytoplasmic mitochondrial DNA. Figure 6 The results show that there is obvious mitochondrial dysfunction in the BAT of the slow growth calf, which is manifested by the significant activation of the cGAS / STING inflammation pathway, and the activation of the pathway leads to the release of downstream inflammatory factors (IL6, CCL2, TGFbeta, IL10, IL4, IL1beta), which directly inhibits the development of BAT, mitochondrial thermogenic capacity and glycolipid metabolism function.

[0060] Figure 7 Gene expression and relative amount of cytoplasmic mitochondrial DNA of the control group and the alpha KG group in Example 3, wherein A is the expression of cGAS related protein, B is the expression of cGAS related gene, C is the expression of inflammation related gene, and D is the relative amount of cytoplasmic mitochondrial DNA. Figure 7 The results show that the alpha KG feeding intervention during pregnancy significantly alleviates the overactivation of the cGAS / STING pathway in the BAT of the offspring calf and reduces the mitochondrial inflammation level. This indicates that one of the core mechanisms of alpha KG is its novel anti-mitochondrial inflammation activity. Through this mechanism, the present application successfully restores the normal development and thermogenic metabolism function of BAT, ultimately improving the growth rate and body health level of the calf. The alpha KG feeding alleviates the expression of the mitochondrial cGAS-STING pathway and inflammatory factors in the brown adipose cells of the one-month-old offspring calf and improves the mitochondrial function. Therefore, it can be concluded that the alpha KG feeding during pregnancy improves the brown adipose development and thermogenic metabolism function of the offspring calf through the regulation of the cGAS-STING pathway, thereby affecting the growth rate and glycolipid metabolism health of the whole body.

[0061] In summary, as a natural active substance, alpha KG has significant application value in promoting the growth rate of the offspring calf and improving the development and thermogenic metabolism function of the brown adipose tissue of the body when fed during pregnancy, and can provide strong support for improving the growth potential of the calf.

[0062] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, which should be covered within the protection scope of the present application.

Claims

1. Use of alpha KG or a pharmaceutically acceptable salt thereof in improving the growth performance of offspring of a ruminant, characterized in that, The application comprises: 1) increasing the postpartum daily weight gain of offspring of ruminants; 2) increasing the weight of brown adipose tissue of offspring of ruminants; 3) enhancing the body heat production function of offspring of ruminants; 4) improving the glycolipid metabolism efficiency of offspring of ruminants.

2. Use according to claim 1, characterized in that, Comprise: feeding the ruminant mother during the gestation period with a diet containing αKG.

3. Use according to claim 2, characterized in that, feeding the ruminant mother with a diet containing αKG 20 days to 80 days before parturition.

4. Use according to claim 3, characterized in that, feeding the ruminant mother with a diet containing αKG 60 days before parturition to delivery.

5. Use according to claim 2, characterized in that, The added amount of αKG is 6.5-19.5 g / head / day.

6. Use according to claim 5, characterized in that, The added amount of αKG is 13 g / head / day.

7. The use according to claim 1, characterized in that, The ruminant is a cow.

8. The use according to claim 1, characterized in that, The αKG or pharmaceutically acceptable salt thereof comprises α-ketoglutarate sodium.

9. A feed additive for improving the growth performance of offspring of ruminants, characterized in that, The feed additive is αKG or a pharmaceutically acceptable salt thereof.

10. A method of improving the growth performance of offspring of a ruminant animal, characterized in that, feeding the ruminant mother during the gestation period with a diet containing the feed additive of claim 9.

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