KR102720405B1 - Composition for improving anti-stress of ruminants and meat productivity comprising l-glutamine as an active ingredient - Google Patents

Abstract

The present invention relates to a method for enhancing immunity and reducing stress in ruminants using L-glutamine. An anti-stress composition manufactured according to the present invention contains a specific amount of L-glutamine, and when fed to ruminants, it can enhance immune responses such as a decrease in the expression level of HSP 90 and an increase in white blood cells, lymphocytes, and granulocytes in the blood, and reduce high temperature stress.

Description

{COMPOSITION FOR IMPROVING ANTI-STRESS OF RUMINANTS AND MEAT PRODUCTIVITY COMPRISING L-GLUTAMINE AS AN ACTIVE INGREDIENT}

The present invention relates to a method for enhancing immunity and reducing stress in ruminants using L-glutamine, and a method for increasing meat productivity. More specifically, the present invention was completed by confirming that intake of L-glutamine reduces immunity and high-temperature stress in ruminants, and provides an anti-stress feed containing L-glutamine and a method for reducing stress in ruminants.

The average summer temperature on the Korean Peninsula, which is surrounded by the sea on three sides, from 2009 to 2019 was 24.2℃, the average low temperature was 20.5℃, the average high temperature was 28.8℃, and the average relative humidity was 77.4%, indicating that summers in South Korea are hot and humid (Statistics Korea, 2020).

Cows, which are ruminants, also show a "negative energy balance" in which the amount of energy consumed is greater than the amount of energy consumed due to an increase in energy consumption, a lack of nutrients necessary for growth, production, and maintenance, an increase in water intake, and an increase in panting and sweating to dissipate heat, which leads to an increase in maintenance energy (NRC, 2001).

In a state of high temperature stress, the secretion of insulin, a hormone that increases intracellular sugar absorption and has an anti-lipolysis effect that inhibits the decomposition of fat, increases, thereby inhibiting the action of beta-oxidation, which breaks down fat tissue accumulated in the body to create energy, and thus increases the availability of monosaccharides such as glucose and amino acids (Wheelock et al., 2009, Cowley et al., 2015). When exposed to a state of high temperature stress, protein metabolism in particular is inhibited. First, in order to dissipate heat, blood flows to peripheral tissues more turbidly, which reduces blood flow to internal organs and decreases the absorption of amino acids (McGuire et al., 1989). Second, ruminants specifically decompose and absorb the body proteins of ruminant microorganisms living in the rumen, and although the amount varies between diets, species, and individuals, they supply approximately 59% of the protein. High temperature stress reduces the ammonia utilization of microorganisms and body protein synthesis, which reduces the supply of microbial body proteins (Clark et al., 1992, Bernabucci and Calamari, 1998). Third, according to a previous study, the production of heat shock proteins, which are proteins produced in the body to prevent shock in preparation for high temperature stress, increased 200-fold in lymphocytes than heat shock protein 70. This confirms that heat shock proteins are produced in large quantities to prevent the denaturation of other proteins. Finally, in a heat stress situation, an insufficient energy balance is observed, so amino acids are used for energy production, and the amount of amino acids in the blood decreases by approximately 17.1% (Gao S.T. et al., 2017).

In this way, the absorption of protein decreases in high temperature stress situations and the availability of amino acids to reduce high temperature stress increases, which causes a decrease in the productivity of ruminants. Amino acids in the body are stored in organs such as blood, muscles, and liver, and L-glutamine is the second largest amount of free amino acids in the sirloin of Korean beef, after L-alanine, and accounts for an average of about 15% (Jeong et al., 2012; Cho et al., 2013).

L-glutamine has various functions in the body. It is the basic skeleton of amino acids that can be synthesized in the body, and is an amino acid involved in the proliferation and maintenance of immune cells, and in the promotion and suppression of cytokine secretion. It is also used as an energy source for intestinal epithelial cells and muscle cells, and is also involved in the size increase of muscle cells (Curi et al., 2005).

The present inventors comprehensively confirmed the phenomena occurring in fattening cattle under high temperature stress by confirming the changes in the composition of blood amino acids, changes in blood cells and metabolites, growth performance, expression levels of heat shock proteins in peripheral mononuclear cells and hair roots, and gene expression levels in loin tissue when L-glutamine is fed to fattening Hanwoo cattle under high temperature stress conditions with reduced protein and energy, and confirmed that the intake of an appropriate amount of L-glutamine by fattening cattle can reduce temperature stress, thereby completing the present invention.

The present invention aims to confirm the effects of adding L-glutamine to basal feed under high temperature stress on growth performance, blood indices, changes in heat shock protein (HSP) expression in hair roots and blood, and gene expression in loin tissue.

Another purpose of the present invention is to confirm that the addition of L-glutamine to the basal feed has a resistance effect against high temperature stress through the enhancement of immune responses such as a decrease in the expression of HSP 90 in the hair roots of Korean cattle steers and an increase in white blood cells, lymphocytes, and granulocytes in the blood.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

To achieve the above purpose, the present invention provides a composition for anti-stress in ruminants containing L-Glutamine as an effective ingredient.

In the present specification, the composition may be for reducing temperature stress. The feed additive composition may be a feed additive composition for fattening cattle, and in particular may be for reducing high temperature stress.

The term "temperature stress" in this specification means a decrease in the productivity of cattle due to a change in temperature, and specifically, it can mean a decrease in the productivity of cattle due to a temperature outside the optimal temperature range or a sudden change in temperature, and for example, it can be low temperature stress or high temperature stress, but is not limited thereto. Specifically, the low temperature stress can be a stress at a temperature below the optimal temperature range, below the low temperature threshold, or a suddenly decreased temperature, and the high temperature stress can be a stress at a temperature above the optimal temperature range, above the high temperature threshold, or a suddenly increased temperature, but is not limited thereto. The low temperature can be +10°C or below, -50°C to +10°C, -40°C to +10°C, -30°C to +10°C, -20°C to +10°C, -10°C to +10°C, or -10°C to 0°C, but is not limited thereto. The above high temperature may be, but is not limited to, 10℃ or higher, 15℃ or higher, 20℃ or higher, 10℃ to 50℃, 15℃ to 50℃, 20℃ to 50℃, 30℃ to 50℃, 10℃ to 40℃, 15℃ to 40℃, 20℃ to 40℃, 30℃ to 40℃, 15℃ to 35℃, or 20℃ to 35℃. Reduction of temperature stress may be determined by, but is not limited to, whether the daily weight gain and feed intake of cattle increase.

In the present invention, L-glutamine refers to a compound having a structure represented by the following chemical formula 1. For the purpose of the present invention, the L-glutamine can be used without limitation on its source, and the production of the L-glutamine can be performed using various methods known in the art.

[Chemical Formula 1]

According to one specific example of the present invention, the content of L-Glutamine of the present application can be determined by a person skilled in the art in consideration of the feeding target, the species of the feeding target, body weight, feeding period, type of feeding feed, feeding purpose, etc. Specifically, the L-Glutamine can be included in a proportion of 0.1 to 1.0 wt%, 0.2 to 0.7 wt%, or 0.3 to 0.6 wt% of the total composition weight. When the composition of the present invention is added to compound feed, if the amount added is too small, the effect of reducing high temperature stress (heat stress) cannot be expected, and if the amount is more than the appropriate amount, feed intake may decrease and economic feasibility is poor. The composition of the present invention can be directly added to feed and mixed, and can be variously manufactured and mixed in the form of other raw materials (single feed) or premix, and thus can be applied to all livestock feed.

In addition to L-Glutamine, the substance included in the anti-stress composition of the present invention is not particularly limited as long as it is a substance used in a typical livestock feed composition.

The anti-stress composition of the present invention can be used for livestock that are subjected to high temperature stress during the high temperature season in summer, which lowers their immunity and affects their productivity. Suitable animals include livestock such as poultry (laying hens, ducks, turkeys, geese, etc.), ruminants (cattle, horses, goats, sheep, etc.), pigs (pigs), rodents (rabbits), and fish, and can be used very widely and usefully, and most preferably, ruminants can be used. The ruminants of the present invention may include cattle, goats, sheep, giraffes, American bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeests, antelopes, pronghorns, and nilgai, but are not limited thereto, and most preferably, cattle, and among them, Korean cattle.

In this specification, the term "beef cattle" refers to cattle raised as livestock for the purpose of meat production. The beef cattle in the present invention correspond to cattle capable of meat production, and are all included in the scope of this application regardless of breed, gender, etc. More specifically, it may be Korean native cattle, but is not limited thereto.

The composition of the present invention can be used as a feed additive, and the term "feed additive" in this specification means a substance added to feed. The feed additive may be for improving the productivity of the target organism, improving a specific purpose, or promoting health, but is not limited thereto. In addition, the feed additive may correspond to supplementary feed under the Feed Management Act.

When the composition of the present invention is used as a feed additive, the feed intake and body weight of ruminants increase, so that the productivity of meat can increase compared to the non-intake group. In the present invention, there is no statistical difference in the productivity (weight gain, feed intake) of Hanwoo steers, but the average weight gain was 3.4 kg higher in the experimental group after a total of 10 weeks of feeding (control group 12.9 kg vs. experimental group 16.3 kg).

In this specification, “productivity” may mean the number of ruminants, their weight class, or the quantity or quality of meat slaughtered.

The feed additive composition of the present invention may additionally contain, in addition to L-Glutamine, nutrients such as nucleotides, amino acids, calcium, phosphoric acid, and organic acids for improving the productivity or health of the target organism, but is not limited thereto.

The present invention provides a method for reducing high temperature stress in ruminants, which comprises the step of feeding the anti-stress composition according to the present invention to the ruminant. The method may be fed according to a general feeding management method, and specifically, may be fed at a certain time every day, but is not limited thereto. In addition, the feeding amount is not particularly limited.

The present invention provides the above-mentioned anti-stress composition, which has the effect of reducing high temperature stress through enhancing immune response such as decreasing the expression level of HSP 90 and increasing white blood cells, lymphocytes, and granulocytes in the blood when fed to ruminants, etc.

The present invention also provides a content of L-glutamine that can exhibit a heat stress reduction effect when consumed by ruminants.

Figure 1 is a graph showing the changes in the minimum, average, and maximum temperature-humidity index during the 1-week adaptation period and the 10-week experimental period of Hanwoo fattening cattle according to one embodiment of the present invention.
Figure 2 shows the results of comparing the expression levels of heat shock proteins (HSP) 70 and 90 in hair roots according to the addition of L-glutamine to Hanwoo fattening cattle under high temperature stress conditions.
Figure 3 shows the results of comparing the expression levels of heat shock proteins (HSP) 70 and 90 in peripheral blood mononuclear cells according to the addition of L-glutamine to Hanwoo beef cattle under high temperature stress conditions.
Figure 4 shows the results of comparing the expression of genes related to muscle and fat development in the loin muscle according to the addition of L-glutamine to Hanwoo beef cattle under high temperature stress conditions.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example 1.

1-1. Experimental animals and management

All experiments were conducted in accordance with the guidelines specified by the "Institutional Animal Experiment Ethics Committee of Konkuk University (KU 20001)". Eight Hanwoo mid-fattening (starting weight = 570.7 kg (SD = 43.6), 22.3 months of age (SD = 0.88)) were used and divided into control and treatment groups of four each. The control group was a group to which L-glutamine was not added, and the treatment group was a group to which 0.5% of the concentrate feed based on the raw material was additionally added as L-glutamine. The feed consisted of concentrate (Cargill Purina, Hanwoo Love Finishing) and forage (rice straw). The concentrate feed was individually fed at 1.5% of the body weight per day based on the raw material according to the feeding guide of Cargill Purina, and 1.5 kg of rice straw was fed based on the raw material. Feeding was divided into two times every day, at 0800 h and 1600 h, and the remaining amount was measured every day at 0700 h.

Table 1 below shows the results of analyzing the chemical composition of the concentrated feed and rice straw used in this experiment.

Results of general chemical composition analysis of concentrated feed and rice straw designation Concentrated feed ¹ Straw Chemical composition (DM), %) Moisture 4.70 12.29 Crude protein 15.35 4.45 Ether extraction 3.37 1.74 Neutral detergent fiber 25.43 66.70 Acid detergent fiber 7.66 45.13 Non-fiber carbohydrate ² 47.58 12.43 Crude ash 8.27 14.68 Ca 0.78 0.33 P 0.44 0.13 Total digestible nutrition ³ 75.0 43.66 Digestible energy ⁴ 3.31 1.93

¹ Commercial feed (Cargill agripurina, Seoul, Korea)

² Calculated value using NRC, 2001 equation is 100 - (CP + EE + NDF - NDIP + ASH)

³ Calculated value using NRC, 2001 equation is truly digestible (td)NFC + tdCp + (tdFA Х 2.25) + tdNDF -7

⁴ Calculated value using NRC, 1989 equation is 0.04409 Х TDN

1-2. Calculating growth performance

In Example 1-1, the body weights of the control and treatment groups were measured four times in total: at weeks 0, 3, 6, and 10 from the start of the experiment. The average daily gain and gain:feed were calculated using formulas.

1-3. Blood collection

Before measuring the body weight of the control and treatment groups, blood was collected four times in total: at weeks 0, 3, 6, and 10, before meals. Serum and plasma (heparin tubes) were separated from the collected blood, and blood for general blood tests was collected using tubes treated with K2 EDTA.

1-4. Complete blood cell (CBC)

A general blood test was performed on the blood treated with EDTA according to the above Example 1-3 within 2 hours. The evaluation items in this test are as follows.

1) Hemoglobin (HGB; g/dL), hematocrit (HCT; %), red blood cell count (RBC; 106/uL)

2) Red blood cell indices: ① mean corpuscular volume (MCV; fL), ② mean corpuscular hemoglobin (MCH; pg), ③ mean corpuscular hemoglobin concentration (MCHC; g/dL), ④ red blood cell distribution width (RDW; %)

3) White blood cell count and differential count (WBC differential count): ① Neutrophil (103/ul and %/WBC), ② Lymphocyte (103/ul and %/WBC), ③ Monocyte (103/ul and %/WBC)

4) Platelet count (platelet, 103/uL)

5) Platelet indices: ① mean platelet volume (MPV; fL), ② platelet volume (plateletcrit; PCT; %), ③ platelet distribution width (PDW; %)

1-5. Measurement of metabolites in plasma

After blood collection according to the above examples 1-3, the separated plasma was used to confirm metabolites (Glucose, Total protein, Albumin, Globulin, Blood urea nitrogen, creatine phosphokinase) using a Dri chem 7000i biochemistry analyzer.

1-6. Measurement of free amino acids in plasma

After removing proteins by treating plasma with 10% sulfosalicylic acid, the proteins were analyzed using an amino acid analyzer (Sykam S433, Sykam GmbH, Germany).

1-7. Total RNA extraction and cDNA synthesis in the hamstring muscle, pubic hair, and peripheral blood mononuclear cells, and genetic analysis through real-time PCR

According to Example 1-1, the loin muscles (2 g) of the control and treatment groups raised were collected by biopsy on the last day of the experiment in accordance with the guidelines specified by the "Animal Experiment Ethics Committee of Konkuk University (KU 20001)", and the hair roots and peripheral blood mononuclear cells were collected four times at 0, 3, 6, and 10 weeks. The hair roots were stored in an RNA later product until RNA extraction using TRIzol, and the peripheral blood mononuclear cells were isolated using Histophaque.

Total RNA was extracted using TRIzol reagent, the amount of RNA was confirmed using NanoDrop, and the quality of RNA was checked by RNA integrity number using RNA 6000 Nano lab chip kit (Agilent, Palo Alto, CA, USA). Only RNA with a number higher than 6.5 was synthesized into cDNA.

cDNA was synthesized using a cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) and diluted to obtain the same amount of cDNA (20 ng/ul). - The gene expression level was confirmed by checking the light amplification of SYBR-Green® using real-time PCR, and the primer information is as shown in Table 2 below.

As housekeeping genes, 18S, GAPDH, and RPLP0 were used in loin tissue, GAPDH in hair roots, and GAPDH, B2M, and RPS15A in peripheral blood mononuclear cells.

Primer name, annealing temperature, and sequence genetic symbol designation Annealing temperature order FABP4 Fatty acid binding protein 4 60.8℃ Forward primer (5'→3') TGTCACTGCCACCAGAGTTT Reverse primer (5'→3') TGGACAACGTATCCAGCAGA A GDP glycerophosphate dehydrogenase 65.0℃ Forward primer (5'→3') CACGAAGTCCATCTCCCGAA Reverse primer (5'→3') GTTGTCCACTTTCCACCTGCT LPL Peroxisome proliferator-activated receptor gamma 60.0℃ Forward primer (5'→3') TACCCTGCCTGAAGTTTCCAC Reverse primer (5'→3') CCCAGTTTCAGCCAGACTTTC PPARγ Peroxisome proliferator-activated receptor gamma 61.0℃ Forward primer (5'→3') ACTTTGGGATCAGCTCCGTG Reverse primer (5'→3') TCCTCATAGTGCGGAGTGGA SCD Stearoyl-CoA desaturase 60.0℃ Forward primer (5'→3') TCCGACCTAAGAGCCGAGAA Reverse primer (5'→3') GCAGGATGAAGCACAACAAC AG HSPB1 Heat shock protein beta 1 60.0℃ Forward primer (5'→3') CCTGGACGTCAACCACTTC Reverse primer (5'→3') GCTTGCCAGTGATCTCCAC MYOD Myoblast determination protein 59.6℃ Forward primer (5'→3') AGAGTTGCTTTGCCAGAG Reverse primer (5'→3') CTGCCTGCCGTATAAACA MYF5 Myogenic factor 5 62.5℃ Forward primer (5'→3') TCCTGATGTACCAAATGTATAT GCC Reverse primer (5'→3') ATCCAGGTTGCTCTGAGTTGG MYF6 Myogenic factor 6 60.7℃ Forward primer (5'→3') GAAGGAGGGACAAGCATTGA Reverse primer (5'→3') GAGGAAATGCTGTCCACGAT MYOG Myogenin 65.0℃ Forward primer (5'→3') TACAGACGCCCACAATCTGC Reverse primer (5'→3') GGTTTCATCTGGGAAGGCCG HSP70 Heat shock protein 70 60.0℃ Forward primer (5'→3') TACGTGGCCTTCACCGATAC Reverse primer (5'→3') GTCGTTGATGACGCGGAAAG HSP90 Heat shock protein 90 60.0℃ Forward primer (5'→3') GGAGGATCACTTGGCTGTCA Reverse primer (5'→3') GGGATTAGCTCCTCGCAGTT 18S 18S ribosomal RNA 51.0℃ Forward primer (5'→3') ACCCATTCGAACGTCTGCCCTATT Reverse primer (5'→3') TCCTTGGATTGTGGTAGCCGTTTCT GAPDH Glyceraldehyde-3-phosphate dehydrogenase 60.0℃ Forward primer (5'→3') CGTGGAGGGACTTATGACCAC Reverse primer (5'→3') CGCCAGTAGAAGCAGGGATG RPLP0 Ribosomal protein lateral stalk subunit P0 62.5℃ Forward primer (5'→3') CAACCCGGCTCTGGAGAAACTG Reverse primer (5'→3') ACTTCACACGGCGCTATGG B2M beta-2-microglobulin 60.0℃ Forward primer (5'→3') GACACCCACCAGAAGATGGA Reverse primer (5'→3') CAGGTCTGACTGCTCCGATT RPS15A ribosomal protein S15a 60.0℃ Forward primer (5'→3') CCGTGCTCCAAAGTCATCGT Reverse primer (5'→3') GGGAGCAGGTTATTCTGCCA

1-8. Statistical Analysis

In this experiment, significant differences were analyzed using the Proc MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The fixed factors were groups according to L-glutamine treatment, and the random variables were individuals, weekly measurement values, and initial values. - A significant difference was set at p<0.05, and a trend was set at 0.05<P≤0.1.

Example 2.

2-1. Comparative analysis of blood amino acids according to the addition of L-glutamine to Korean beef cattle under high temperature stress conditions

As a result of analyzing the content of blood amino acids according to the addition of 0.5% L-glutamine to the concentrate feed based on the raw material, it was confirmed that L-glutamic acid increased by 2.27 times (p = 0.083) and L-citrulline increased by 1.30 times (0.061) in the L-glutamine-added group (see Table 3).

When immune cells utilize L-glutamine, L-glutamic acid, L-aspartate, lactic acid, and carbon dioxide are produced (Curi et al., 1999), and when intestinal epithelial cells utilize it, L-citrulline, L-proline, L-alanine, organic acids, and ammonia are decomposed or synthesized (Herbert et al., 1974).

Based on this, it can be confirmed that the addition of L-glutamine has an effect on immune cells and intestinal epithelial cells.

Comparative analysis of blood amino acids according to the addition of L-glutamine to Korean beef cattle under high temperature stress conditions substance, μmol/L Control (CON) Treatment area (TRT) SEM P-value Ammonium chloride 50.9 46.8 3.44 0.278 Glycine 62.1 65.5 3.88 0.410 L-Alanine 48.8 52.6 4.05 0.378 L-Arginine 22.7 26.0 1.84 0.123 L-Citrulline 23.3 30.2 3.01 0.061 L-Glutamic acid 12.4 28.2 7.58 58 0.083 L-Histidine 11.7 11.5 1.21 0.896 L-Isoleucine 24.0 26.4 2.86 0.421 L-Leucine 36.6 43.1 5.00 0.244 L-Lysine 37.1 37.7 8.72 0.944 L-Methionine 6.3 5.5 0.66 0.302 L-Ornithine-monohydrochloride 21.4 24.6 3.45 0.391 L-Phenylalanine 23.8 21.1 1.44 0.104 L-Tryptophan 6.3 7.7 0.76 0.132 L-Tyrosine 19.6 19.2 2.07 0.840 L-Valine 56.8 65.5 8.30 0.338 Taurine 12.0 14.4 1.93 0.256

2-2. General blood test after adding L-glutamine to Korean beef cattle under high temperature stress

The addition of 0.5% L-glutamine to the raw feed increased white blood cells (WBC) in the blood of Hanwoo by 1.26 times (p = 0.058), lymphocytes (LYM) by 1.30 times (p = 0.068), and granulocytes (GRA) by 1.28 times (p = 0.031), but did not affect the number of monocytes (MON), confirming a decrease in the ratio (MON, %WBC) (p = 0.058) (see Table 4).

It is known that L-glutamine is utilized rapidly in intestinal epithelial cells and immune cells, and among immune cells, L-glutamine utilization is known to be particularly rapid in lymphocytes (LYM) and monocytes (MON) (McCauley et al., 1998; Ardawi et al., 1982).

Results of general blood tests after adding L-glutamine to Korean beef cattle under high temperature stress conditions substance Control (CON) Treatment area (TRT) SEM P-value Leukocyte indices WBC, 10 ³ /μL 10.20 12.88 1.15 0.058 LYM, 10 ³ /μL 5.78 7.51 0.78 0.068 GRA, 10 ³ /μL 3.96 5.07 0.40 0.031 MON, 10 ³ /μL 0.45 0.31 0.21 0.520 LYM, %/WBC 57.19 58.06 2.50 0.740 GRA, %/WBC 38.59 39.71 2.31 0.644 MON, %/WBC 4.53 2.33 0.94 0.058 Erythrocyte indices RBC, 10 ⁶ /μL 9.93 8.84 0.73 0.185 HGB, g/dL 14.24 13.56 0.64 0.328 HCT, % 40.01 38.06 1.86 0.334 MCH, pg 14.41 15.42 0.68 0.188 MCHC, g/dL 35.64 35.63 0.56 0.988 MCV, fL 40.26 43.15 2.01 0.200 RDW, % 21.75 20.48 0.63 0.090 Platelet indices PLT, 10 ³ /μL 352.35 310.92 60.84 0.521 PCT, % 0.25 0.22 0.05 0.600 MPV, fL 6.85 7.15 0.24 0.259 PDW, % 33.25 33.07 1.19 0.888

WBC: white blood cell LYM: lymphocyte; GRA: granulocyte; MON: monocyte; RBC: red blood cell; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin concentration; MCH: mean corpuscular hemoglobin; RDW: red blood cell width; PLT: platelet; MPV: mean platelet volume; PCT: plateletcrit; PDW: platelet distribution width

2-3. Results of plasma metabolite analysis according to addition of L-glutamine to Korean beef cattle under high temperature stress

L-glutamine intake can be used as an energy source in immune cells, muscles, liver, and digestive organs (David et al., 1996). According to previous studies, in livestock, a negative energy balance is shown due to an increase in maintenance energy caused by a decrease in intake and an increase in heat dissipation such as panting and sweating in a high temperature stress situation (Wheelock et al., 2009).

However, in Table 6 of the results of this experiment, L-glutamine supplementation did not show a difference in blood sugar (glucose) that can be used as an energy source in the body (p = 0.104), and blood urea nitrogen discharged during the breakdown of L-glutamine also did not show a difference (p = 0.837). Creatine phosphokinase, an enzyme discharged during the breakdown of muscle tissue, also did not show a difference (p = 0.684). Additional L-glutamine supplementation tended to decrease total protein (p = 0.097) and albumin (p = 0.059). It is judged that total protein decreased due to the decrease in albumin. Albumin is a multifunctional blood protein that has the functions of maintaining plasma colloid osmotic pressure, transport, and nutrient supply, and is known to increase when L-glutamine is supplemented additionally, but the opposite result was shown in this experiment.

Results of plasma metabolite analysis after adding L-glutamine to Korean beef cattle under high temperature stress conditions substance Control (CON) Treatment area (TRT) SEM P-value Blood urea nitrogen, mg/dL 18.21 17.84 1.68 0.837 Glucose, mg/dL 71.88 66.68 2.71 0.104 Total protein, g/dL 7.50 7.12 0.19 0.097 Albumin, g/dL 3.43 3.25 0.08 0.059 Globulin, g/dL 4.06 3.84 0.14 0.146 Creatine phosphokinase, u/L 111.50 121.50 23.41 0.684

2-4. Comparative analysis of intake and growth performance according to addition of L-glutamine to Korean beef cattle under high temperature stress

The inventors of the present invention have confirmed in an experiment to determine the optimal amount of L-glutamine for Korean cattle that an additional 0.5% of the original intake of concentrated feed is the most appropriate amount. In this experiment, based on a previous study, the results of a comparative analysis of intake and growth performance according to the addition of L-glutamine to Korean cattle fattening under high temperature stress are as follows (see Table 6).

After prior group separation to ensure that there was no difference in initial body weight (0 weeks; p = 0.950), a 10-week feeding experiment was conducted, but there was no significant difference in average body weight, intake, weight gain, daily weight gain, feed utilization, etc., intake and growth performance depending on whether L-glutamine was added (p > 0.05).

Comparative analysis of intake and growth performance according to addition of L-glutamine to Korean beef cattle under high temperature stress conditions substance Control (CON) Treatment area (TRT) SEM P-value Week 0 Average BW 571.8 569.6 23.5 0.950 Week 3 Average BW ¹ 578.0 579.5 22.8 0.964 Increment ¹ 6.25 9.94 3.82 0.537 Average daily gain 0.30 0.47 0.18 0.533 Feed intake 9.21 9.88 0.13 0.015 Gain:Feed 0.03 0.05 0.02 0.529 Week 6 Average BW 584.5 591.1 25.2 0.859 Increment 6.50 11.6 3.90 0.388 Average daily gain 0.31 0.55 0.19 0.388 Feed intake 9.21 9.48 0.16 0.182 Gain:Feed 0.03 0.06 0.02 0.360 Week 10 Average BW 610.5 618.4 22.1 0.809 Increment 26.0 27.3 4.92 0.863 Average daily gain 0.90 0.94 0.17 0.863 Feed intake 9.40 9.47 0.19 0.738 Gain:Feed 0.10 0.10 0.02 1.000 Total Increment BW 12.9 16.3 3.71 0.395 Average daily gain 0.50 0.66 0.17 0.383 Feed intake 9.28 9.60 0.63 0.625 Gain:Feed 0.06 0.07 0.02 0.449

2-5. Comparative analysis of expression levels of heat shock proteins (HSP) 70 and 90 in hair roots and peripheral blood mononuclear cells according to addition of L-glutamine to Hanwoo beef cattle under high temperature stress conditions

During the experimental period (1 week of adaptation period, 10 weeks of experimental period), temperature and relative humidity were measured every minute using an automatic measurement sensor (MHB-382SD, ZL 2008 2, Lutron, China), and the temperature humidity index (THI) was calculated by applying the calculation formula (NRC, 2001).

THI = (1.8 x Tdb + 32) - [(0.55 - 0.0055 x RH) x (1.8 x Tdb - 26.8)]

Tdb: dry bulb temperature (°C); RH: relative humidity (%)

During the adaptation and experimental period (10 weeks), the average temperature and humidity index was below 72 for approximately 17 days, and the temperature and humidity index, which is recognized as heat stress for livestock, was above 72 for the remaining 60 days (see Figure 1).

In particular, it was confirmed that the temperature and humidity index was the highest before and after the 6-week experimental period, and the change in this temperature and humidity index showed a similar trend to the gene expression change of HSP 70 and HSP 90 in the hair roots and peripheral blood mononuclear cells of livestock (see Figs. 2 and 3). In addition, it was confirmed that it well reflects high temperature stress as in a previous study (Kim et. 2020), showing a significant difference in the hair roots than in the peripheral blood mononuclear cells (see Figs. 2 and 3).

When comparing HSP 90 in the root at 10 weeks, a decrease in expression was confirmed in the L-glutamine-fed treatment group, suggesting that L-glutamine intake is effective in reducing heat stress (p < 0.05) (see Figure 2).

2-6. Comparison of gene expression levels related to muscle and fat development in loin tissues according to addition of L-glutamine to Hanwoo beef cattle under high temperature stress conditions

Feeding 0.5% L-glutamine to the raw feed of Hanwoo under high temperature stress did not show any difference in the expression of genes (FABP4, GDP, LPL, PPARγ, SCD) related to the development of adipose tissue in the loin tissue, and no significant difference was shown in the muscle-related genes HSPB1, MYF5, MYF6, MyoD, and MyoG (p > 0.05).

That is, it can be seen that the addition of 0.5% L-glutamine to the basal feed has the effect of increasing the number of white blood cells, lymphocytes (LYM) and granulocytes (GRA) in the blood and decreasing the expression of heat shock protein (HSP) 90 in the hair root in the high temperature stress situation of Hanwoo steers compared to the control group, indicating that it has the effect of resisting high temperature stress through enhancing immunity.

Claims (7)

Contains L-Glutamine as an active ingredient, added at 0.5 wt% of the total composition weight,
A composition for reducing high temperature stress in ruminants, which exhibits a high temperature stress resistance effect through enhancing immunity, when the temperature humidity index (THI) of the following formula 1 is 72 or higher at 10 weeks after ingestion of the above composition:
[Formula 1]
THI = (1.8 x Tdb + 32) - [(0.55 - 0.0055 x RH) x (1.8 x Tdb - 26.8)].
(where, Tdb = dry bulb temperature (°C); RH = relative humidity (%))

delete delete In the first paragraph,
A composition for reducing heat stress in ruminants, wherein the ruminant is any one animal selected from the group consisting of cattle, goats, sheep, giraffes, American bison, European bison, yak, water buffalo, deer, camel, alpaca, llama, wildebeest, antelope, pronghorn and nilgai. A feed additive for improving the productivity of ruminants, comprising a composition according to any one of claims 1 and 4. A method for reducing heat stress in ruminants, comprising the step of feeding a composition of any one of claims 1 and 4 to the ruminant. In Article 6,
A method for reducing heat stress in ruminants, wherein the ruminant is any one animal selected from the group consisting of cattle, goats, sheep, giraffes, American bison, European bison, yaks, water buffalos, deer, camels, alpacas, llamas, wildebeests, antelopes, pronghorns and nilgai.