KR102635871B1 - Method for improving Korean beef and meat quality by feeding vitamin A - Google Patents

Abstract

The present invention relates to a method of improving Korean beef meat mass and meat quality by feeding vitamin A, and more specifically, to a method of improving Korean beef meat mass and meat quality by additionally feeding vitamin A orally during the young calf period.

Description

Method for improving Korean beef meat mass and meat quality by feeding vitamin A {Method for improving Korean beef and meat quality by feeding vitamin A}

The present invention relates to a method of improving Korean beef meat mass and meat quality by feeding vitamin A, and more specifically, to a method of improving Korean beef meat mass and meat quality by additionally feeding vitamin A orally during the young calf period.

Marbling of beef is an important factor in Korea's direct fire grilling food culture, juiciness, and preference, and is therefore used as an important indicator in evaluating meat quality. However, in the process of increasing marbling in cattle, inedible fat such as subcutaneous fat and visceral fat, which acts as an economic loss, also increases. In addition, among the edible parts of intramuscular fat, marbling, which only focuses on the total amount of fat, has a negative perception among consumers in terms of health and preference. Reflecting these consumer demands, in Japan, beef with higher intramuscular fat distribution is given a higher grade even if it has the same intramuscular fat content (Kuchida, K et al., 2006) (see Figure 1). Beef with high intramuscular fat distribution is recognized as meat with high palatability and consumer preference due to the juiciness, flavor, and appetite stimulation of Korean beef, and has the effect of relatively lowering the total fat content in beef. In order to increase intramuscular fat distribution, the number of fat cells must be increased, not the size of fat cells.

The increase in fat is caused by the proliferation and growth of adipocytes. Cell proliferation and growth are divided into hyperplasia and hypertrophy. Hyperplasia refers to cell proliferation and occurs before differentiation, whereas hypertrophy refers to an increase in cell size and occurs before differentiation. It happens after it happens. Previous fat metabolism research has focused on lipid metabolism and lipid deposition, or hypertrophy, but research on hyperplasia is insufficient (Smith. S. B et al., 2009). In general, adipocytes and adipose tissue are found in the fetus even before the second trimester of pregnant cows (Berry, D.C. et al., 2012). Through many experiments in mouse models, adipocytes can be produced throughout life, but production decreases with age, so the fetal period and young calf period were predicted to be the optimal times for increasing intramuscular fat distribution (Du. M et al ., 2010). For this reason, although the fetal period and young calf period (up to 250 days after birth) are the periods in which hyperplasia of intramuscular preadipocytes occurs, and should be managed with great care, research related to this is minimal.

Among nutrients, retinoic acid (vitamin A) is reported to inhibit methylation in DNA promoters, promote genes related to preadipocytes, and have a metabolic imprinting effect related to hyperplasia in adipocytes. Results were published showing that when retinoic acid was fed to rats, it activated the extracellular-signal-reulated kinases (ERKs) pathway and induced adipogenic commitment (Bost. F et al., 2002). Adipogenic commitment is induced as a result of treatment with retinoic acid during the growth stage of stem cell lines (Dani, C et al., 1997). Retinoic acid shows different responses in preadipocytes and mature adipocytes. In preadipocytes, the ratio of CRABP-II/FABP5 is high, so there is a lot of Retinoic acid that binds to CRABP-II. This moves to RAR (Retinoic acid receptor) and promotes Pref1 and Sox9, genes related to preadipocytes, to promote differentiation. Inhibits and promotes proliferation of preadipocytes (Noy. N., 2013).

It has been shown that retinoic acid (RA) can not only induce stem cell differentiation into skeletal muscle lineage to a low extent, but also increase the muscle progenitor pool from embryonic stem cells. However, overfeeding with retinoic acid has a negative effect on myotube maturation (Yu et al. 2004; Ryan et al. 2012; Chen and Li 2016; Liu et al. 2016). As previously explained, RAR and RXR are known as receptors of RA, and they are activated by metabolites of RA following the selective ligand mechanism of RXR and RAR during myogenic differentiation (Le May et al. 2011; Jin et al. 2016). In the zebrafish model, it was revealed that RA activates muscle differentiation along the Fgf8 gene, which is known to be a muscle differentiation factor. And inhibition of RA signaling will inhibit muscle differentiation and myoD expression. (Hamade et al. 2006).

Japanese Patent Laid-open No. 2004-113216

The present invention provides a method for improving the meat mass and quality of Korean beef by feeding vitamin A. More specifically, it provides a method of improving Korean beef meat volume and meat quality by providing additional vitamin A orally during the young calf period.

The present invention provides a method for improving meat mass and quality of Korean beef, including feeding vitamin A to calves until about 2 months of age.

The administration of vitamin A may be oral administration.

The feeding amount of vitamin A may be 20,000 IU/day to 120,000 IU/day.

In the present invention, it was confirmed that feeding 45,000 IU/kg of vitamin A in DM to the milk replacer of newborn calves until 2 months of age promoted preadipocyte and muscle development in the loin tissue of calves.

It was confirmed that the growth performance (weight gain and daily weight gain) of the calves was improved when 45,000 IU/feed kg of DM was fed to newborn calves as milk substitute until 2 months of age.

Figure 1 is a photograph showing the shape according to intramuscular fat distribution within sirloin tissue (Kuchida, K et al., 2006).
Figure 2 is an experiment flow chart.
Figure 3 shows the milk substitute and feed feeding strategy until the calf is 2 months old after birth.
Figure 4 shows the results of calculating the number of muscle fibers in calves additionally fed vitamin A.
Figure 5 shows the results of gene expression levels in loin tissue of a 2-month-old calf additionally fed vitamin A.

Hereinafter, the present invention will be described in more detail through examples. The purpose, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced here are provided to enable the idea of the present invention to be sufficiently conveyed to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

The present invention provides a method for improving meat mass and quality of Korean beef, including feeding vitamin A to calves until about 2 months of age.

The administration of vitamin A may be oral administration.

The feeding amount of vitamin A may be 20,000 IU (international unit)/day to 120,000 IU/day.

Feeding calves with DM's milk substitute containing 45,000 IU/kg of feed kg of vitamin A from 5 days to 2 months of age can improve the calf's growth performance (weight gain and daily weight gain).

In the present invention, the effect on hyperplasia and muscle development of calf preadipocytes due to the metabolic imprinting effect of vitamin A during the calf period of Korean beef was confirmed. Specifically, we analyzed the effects of oral feeding of vitamin A, which is necessary for hyperplasia and muscle development of preadipocytes during the calf period, to improve meat mass and quality of Korean beef.

Preparation example 1. Preparation and management of experimental animals

The present invention was conducted on 27 Korean native (Hanwoo) cows (divided into two, initial average BW = 320 kg [SD 31.2]). All cows were randomly placed in multiple pens with stainless steel gates and automatic water troughs. Clean, dry straw was spread on the bedding floor, which was changed monthly. Experimental animals were conceived using random semen from various Korean cattle. For pregnant cows, a total of 13 kg of mixed feed was fed once a day. The amount and nutritional content of the feed were determined according to the Korean feed standards for Korean beef (Table 1). Based on a pilot study, high vitamin A supplementation (total 100,000 IU/day, retinyl acetate, Hanyou Feed, Wuxi, China) was provided during the third trimester of pregnancy (from approximately day 220 until delivery).

All newborn calves were weighed at birth and immediately separated from their mothers and placed in individual wooden cages (200 cm (L) Х 135 cm (B) Х 120 cm (H)). Newborn calves initially had similar mean body weight (BW) for the control group (n=13, 9 steers, 4 heifers) and the treatment group (n=14, 10 steers, 4 heifers). were randomly selected (initial mean BW = 31.6 kg [SD 3.98]). The experimental flow chart is shown in Figure 2. Figure 3 shows the milk substitute and feed feeding strategy until the calf is 2 months old after birth. During the first 12 hours of life, 3.6 L of commercial colostrum (625 ml of 45°C water, Headstart, NAC No. 15360020, Saskatoon Colostrum Company Ltd., Saskatoon, SsK, CA) was supplied. Water, feed and milk replacer were provided to each calf in separate buckets. Substitute milk (Nukamel Yellow, The Dawn Ltd., Seoul, Republic) is prepared by dissolving 140g of powdered milk replacer in 1L of water at 45°C and feeding the cows for 1 month from birth using an automatic calf feeder (Foerster Technik, Gerwigstraße 25, 78234 Engen, Germany). The maximum volume was set at 6.1 L (about 20% of birth weight) and fed to each calf six times a day. The supply of substitute milk was reduced by gradually increasing the supply from 1 month to 2 months (weaning period). Total feed intake (including milk replacer, concentrate and forage) was monitored daily, and dry sawdust was replaced weekly throughout the experiment. Monitor the health of the calves daily and treat those with diarrhea with biodyl, baytril, ketoprofen and oral nutritional supplements (Life-aid) mixed with Neodiaristin. Norbrook, Northamptonshire, UK) was administered. To prevent coccidiosis, Ampura-q was also administered to one-month-old calves. During the experiment, each calf in the control or vitamin A treatment group had diarrhea for approximately 1 to 3 days within the first month of life (the frequency between the control and vitamin A treatment groups was not significant and data not shown). Additional vitamin A supplement (25,000/IU per day, retinyl acetate, Hanyou Feed, Wuxi, China) was administered as a supplement to the milk replacer from 5 days of age until 2 months (weaning period) in the treatment group. Blood samples and body weight data were obtained at 5, 15, 30, 45, and 60 days after birth. Total feed intake (g/day of DM) consisting of milk replacer, feed and fiber was recorded during the experiment period, and average daily gain (ADG) and feed efficiency were calculated.

Longissimus muscle samples (12th to 13th rib, approximately 2 g) were collected via surgical biopsy from five male calf heads from each group when the calves were 2 months old. All biopsy procedures were performed by veterinarians in accordance with the “Guide for the Care and Use of Laboratory Animals” of Konkuk University. Local anesthetic was administered at 4 equidistant points around the longissimus dorsi muscle from the 12th to 13th ribs. The veterinarian excised approximately 5 kilos of skin from the target muscle area and excised approximately 2 g of the longus dorsi muscle using surgical equipment. Tissue samples were immediately placed into 5 ml DEPC-treated microtubes, snap frozen in liquid nitrogen, and placed in a box filled with dry ice until arrival at the laboratory. All muscle samples were stored in a -80 °C freezer before further analysis. The sampling procedure was performed on the day of blood draw but after blood draw at 1400. After the biopsy procedure, management was continued until 2 weeks after surgery according to the veterinarian's prescription.

Preparation Example 2. Blood sample preparation and complete blood count (CBC) measurement

All blood samples were taken from each jugular vein with an 18GX needle at 0900 hours. Blood for serum vitamin A analysis was collected in foil-wrapped coagulation activator tubes (BD Vacutainer®, Belliver Industrial Estate, Plymouth, UK). Whole blood for complete cell counts (CBC) was collected in blood collection tubes containing 7.2 mg K2 EDTA (BD Vacutainer®, Belliver Industrial Estate, Plymouth, UK). After blood collection, all tubes were transported to the laboratory in an opaque box containing an ice pack. The blood for serum vitamin A was left at room temperature (RT) for 1 hour and then centrifuged (3,500 rpm (radius: 155 mm), 15 minutes, 4°C), and the serum was transferred to a brown 1.5mL micro tube under red light. After separation, it was stored at -80°C for analysis. Upon arrival at the laboratory, CBC of whole blood samples was analyzed using a VetScanHM2 analyzer (Abaxis, Whipple Road Union City, California, USA). Whole blood for complete blood cell (CBC) analysis was collected in tubes containing 7.2 mg K2 EDTA. Complete blood count (CBC) measurements were performed according to the hematology analyzer. White blood cells (WBC), red blood cells (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin content (MCHC) , a total of 10 parameters were analyzed, including platelets, lymphocytes, and monocytes.

Preparation Example 3. Serum Vitamin A Analysis

The analysis method for vitamin A in serum was performed according to a previous study (Peng et al., 2019). Methanol, ethanol and hexane were prepared at HPLC grade. Meanwhile, retinyl acetat, retinol, and BHT (2.6-di-tert-butyl-4-methylphenol) were purchased from Sigma (Sigma-Aldrich, Seoul, Korea). The standard stock solution was prepared as 1 mg/mL retinyl acetate dissolved in 0.04% BHT-EtOH solution (0.4 g of BHT dissolved in 1 L ethanol). A stock of standard solution was prepared at 0.1 mg/0.9 mL (25 mg retinol dissolved in 225 mL of 0.04% BHT-EtOH solution). Then, 900 μL of standard stock solution was double-diluted into each tube with 0.04% BHT-EtOH solution. Finally, 100 μL diluted internal standard working solution (mixed with 20 μL stock internal standard solution and 1,980 μL 0.04% BHT-EtOH) at final concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56 for later standard curve analysis. , were added to each tube at concentrations ranging from 0.78, 0.39, 0.20, 0.10, and 0.05 ug/mL. For sample analysis, a new internal standard working solution was created by mixing 25 μL stock internal standard solution and 975 μL 0.04% BHT-EtOH solution. After thawing the target serum sample at RT in the dark under red light (to prevent degradation of vitamin A), 200 μL serum and 20 μL internal working solution were each added to a 2 mL microtube. Then, 200 μL of distilled water (DW) and 400 μL of 0.04% BHT-EtOH solution were added to the tube and vortexed vigorously. Next, 800 μL of 0.04% BHT-hexane (0.1 g of BHT dissolved in 250 mL hexane) was added and vortexed vigorously. Next, all tubes were centrifuged at 3,500 g for 10 minutes at 4°C. Afterwards, 700 μL of hexane supernatant was transferred to each new 1.5 mL brown microtube, and N2 gas was blown to completely evaporate the hexane in a dry block incubator (MG-2200, EYELA, Tokyo, Japan). All samples were dissolved in 95% methanol and then vortexed. After filtering with a syringe filter (PES, 0.22 μm, Millex, Darmstadt, Germany), each sample was transferred to brown LC vials (Infochroma AG, Goldau, Switzerland) for vitamin A analysis by HPLC (High Performance Liquid Chromatography, Agilent 1100 series). was busy. After filtering with a syringe filter (PES, 0.22 μm, Millex, Darmstadt, Germany), each sample was transferred to brown LC vials (Infochroma AG) for vitamin A analysis by HPLC (High Performance Liquid Chromatography, Agilent 1100 series, Waldbronn, Germany). , moved to Goldau, Switzerland. A control internal standard sample (20 μL of internal standard working solution mixed with 480 μL of 0.04% BHTEtOH) was also prepared for analysis. For the HPLC system for vitamin A analysis, 95% methanol was used as the mobile phase at a flow rate of 1 mL/min. A stainless steel Novapak C18 column (4-mm reversed-phase column, 3.9 mm ID · 150 mm, Waters, Dublin, Ireland) was selected for this system with an analysis temperature of 20°C and detected at 325 nm absorbance by a DAD-UV lamp. All data for retinyl acetate and retinol in the samples were obtained from the HPLC program.

Preparation Example 4. Measurement of Longissimus muscle mRNA expression

Total RNA was extracted based on the Trizol extraction method used in a previous study (Peng et al., 2019). All tubes and tips for RNA and qRT-PCR analysis were treated with DEPC-treated distilled water, autoclaved, and sterilized. All longissimus muscles were washed with DEPC-treated distilled water, and all materials were pretreated with RNase ZAPTM (Sigma-Aldrich, Seoul, Korea). Each muscle sample from the biopsy was ground to powder in liquid nitrogen. Immediately after grinding, 0.1 g of muscle sample was separated into a 2.0 mL microtube by adding Trizol reagent (Ambion®, Thermo Fisher Scientific Korea Ltd., Seoul, Republic). After homogenization using a homogenizer (IKA® T10 basic with dispersion tool S10-5G, Seoul, Republic of Korea), all samples were centrifuged at 12,000 x g for 10 minutes at 4°C. The upper aqueous phase was transferred to new 1.5 mL microtubes, and then 200 μL of chloroform was added to each tube. After vortexing, all samples were incubated for 2 min at RT and then centrifuged at 12,000 × g for 15 min at 4 °C. For the pre-step, transfer the upper aqueous phase to another 1.5 mL microtube, then 500 μL of isopropanol was added to the tube with 10 min incubation at RT and centrifugation at 12,000 x g for 10 min at 4 °C. For the RNA step, the supernatant was removed, 1 mL of 75% ethanol was added to the tube, vortexed vigorously, and centrifuged at 12,000 x g for 10 min at 4°C. Next, after completely removing the supernatant, 1 mL of 100% ethanol was added to the tube, vortexed, and all samples were centrifuged at 12,000Хg for 10 minutes at 4°C to completely remove the supernatant. The extracted RNA was dried under vacuum for 15 minutes, immediately dissolved in 30 μL of DEPC-treated distilled water, and incubated at 60°C for another 10 minutes. The concentration of total RNA was confirmed spectrophotometrically (NanoDrop 1000, Thermo Scientific, Seoul, Korea). RNA integrity (RIN) testing was performed with the RNA Nano 6000 Assay Kit on an Agilent Bioanalyzer 2100 system (Agilent Technologies, Richardson, USA) according to the manufacturer's instructions. Samples with an RNA integrity number (RIN) of 6 or higher were used for complementary DNA (cDNA) synthesis. The RIN of RNA used for real-time PCR analysis was 7.3 (SD = 0.3). cDNA synthesis using iScript ?? It was performed by cDNA synthesis kit (Bio-Rad, Seoul, Korea). 1 μg of total RNA extracted according to the protocol was added to a tube in a 20 μL volume system mixed with 5x iScript reaction mix, iScript reverse transcriptase, and nuclease-free water. The complete reaction mixture was then incubated at 25°C for 5 min, followed by 30 min at 42°C, 5 min at 85°C, and maintained at 4°C. cDNA was diluted to 200 ng/μL with DEPC-treated 3x distilled water for subsequent quantitative real-time polymerase chain reaction (qRT-PCR) experiments.

qRT-PCR was performed using the primer set shown in Table 2 below. Primer mixtures (forward and reverse), cDNA, DEPC-treated distilled water, and Cyber Green Super Mix (AccuPower® 2X GreenStarTM qPCR MasterMix, Bioneer, Seoul, Korea) were mixed in a total volume of 20 μL. The following PCR conditions were used. 3 minutes at 95°C (step 1); 95°C for 10 seconds (step 2); Applicable temperature for 30 seconds for each primer in the gene (step 3); followed by 30 seconds and plate recording at 72°C (step 4); Repeat 39 cycles from steps 2 to 4 (step 5); Stored at 95°C for 10 seconds. Finally, the melting curve was measured from 65 to 95 °C, held for 5 seconds and recorded every 0.5 °C. Relative gene expression levels were calculated based on 18s rRNA by the ΔΔCq normalized expression model using Bio-Rad CFX Manager 3.1 software (Bio-Rad, Seoul, Korea). All gene expression levels were performed by fold change compared to controls.

Example 1. Oral vitamin A supplementation of milk replacer improves growth performance of calves.

Body weight (BW) results up to 1 month showed no significant effect in the control group and the vitamin A supplement group (Table 3). However, the group fed vitamin A supplements showed a higher BW at 45 days (p=0.041) and an increasing trend at 60 days (p=0.079). However, the average daily gain (ADG) of the group that received vitamin A supplements was 5 to 30 days (p=0.395), 31 to 60 days (p=0.405), and 5 to 60 days (p= There was no difference up to 0.162). Feed intake (g/day of DM) and feed efficiency (g gain/kg of DM) did not show any differences between the two groups (p> 0.05). However, no significant sex effects were found in the growth performance of calves on days 5, 15, and 30 after birth, except for showing slightly but substantially higher body weights in steers at 45 and 60 days.

Additionally, details of milk replacer, feed, and total feed intake during the experimental period are shown in Table 4. There was no significant difference (p > 0.05) between the control and vitamin A treated groups. Muscle fiber was calculated for the group supplemented with vitamin A and the control group (Figure 4). As a result, muscle fiber increased in the group fed vitamin A compared to the control group.

Example 2. Oral vitamin A supplementation of substitute oil increases serum vitamin A levels without affecting blood parameters.

Serum vitamin A concentration was checked on days 5 (initial), 15, 30, 45, and 60 (Table 5). When compared up to 30 days, no difference was observed in serum vitamin A between the control and treatment groups. An increasing trend in serum vitamin A levels was seen in the treatment group at days 45 and 60 (p = 0.098 and p = 0.060, respectively).

However, no differences in serum vitamin A were found between steers and heifers. To determine hematological parameters, CBC analysis was performed at 5 days (early weaning) and 60 days (weaning). It was found that oral vitamin A supplementation in the first 5 and 60 days had no effect on CBC (Table 6). No gender effect was found in the above analysis (p > 0.05). Results confirmed that vitamin A intake of 45,000 IU/day had no effect on the immune response of calves after birth.

Example 3. Pre-adipocytes and muscle development were confirmed in the longissimus muscle by oral vitamin A supplementation.

Gene expression results in the longissimus muscle were confirmed, and gene expression in the treatment group showed higher levels of ERK1, ERK2, CTNNB1, Pref-1, Zfp423, MyoD, MYF6, and Myogenin than the control group. As a result, it was confirmed that vitamin A in the treatment group could improve the β-catenin/ERK pathway, pre-adipocyte development, and calf muscle development. However, no significant differences were observed in KLF2, Pax-7, Myf5, PPARγ, FABP4, C/EBPα, and SCD gene expression. It was confirmed that vitamin A supplementation in the treatment group promoted pre-adipocyte and muscle development by increasing the expression of related marker genes and pathways without affecting mature cell markers.

Claims (5)

A method for improving Korean beef meat mass and meat quality, comprising the following steps:
(a) preparing a composition for feeding calves containing vitamin A and/or retinyl acetate as active ingredients; and
(b) The composition for calf feeding is fed to the calf at a dose of 20% of the calf's body weight from immediately after birth to 1 month,
Gradually reducing the amount of the composition fed from 1 month to 2 months, and stopping feeding the composition around 2 months,
The method of claim 1, wherein the concentration of vitamin A in the composition is 45,000 IU/kg of feed.
According to paragraph 1,
In step (a), the composition for feeding calves is applied to one or more selected from the group consisting of a substitute oil composition, a feed composition, a feed additive composition, and a forage composition. A method of improving meat mass and meat quality of Korean beef.
delete According to clause 1,
A method for improving Korean beef meat mass and meat quality, characterized in that the composition for feeding calves is orally fed in step (b).
delete