To solve the climate crisis and problems in livestock farming, a new ‘cultured meat’ industries has emerged. Cultured meat is a laboratory-grown meat that consist of satellite cells, adipose cells and scaffolds, which is a future food technology that can address the problems of conventional livestock farming. The positive effects of cultured meat do not require deforestation and excessive water use like conventional livestock farming, resulting in reduced environment pollutions (Post, 2012).

The satellite cells are the major material for producing a slice of cultured meat, isolated from the muscle mass of the livestock with various enzymes. Producing a cultured meat requires a considerable population of satellite cells. Also, the efficiency of differentiation is required to produce cultured meat. However, satellite cells cultured in vitro have limitations in maintaining its population when it cultured for long-term. Previous studies have shown that the expression levels of Pax7 gene, which is core factor for maintaining satellite cells, continuously decreased as it cultured for long-term (Ding et al., 2017; Ding et al., 2018). Much researchers have attempted to maintain the satellite cells population in vitro culture by regulating signal pathways and adding cytokines and inhibitors (Adams and Haddad, 1996; Pawlikowski et al., 2017).

There are two methods to obtain the major ingredient, satellite cells, for cultured meat production. The first method involves using induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) to differentiate into muscle cells. The second major ingredient for the producing cultured meat are primary culture, where satellite cells were isolated from muscle tissues in livestock and cultured in vitro. Since these cells originate from livestock, they are safe for human consumption, and due to their muscle origin, they can be easily differentiated in vitro. However, the satellite cells have limitation in long-term proliferation and maintenance in vitro. Until now, many research and cultured meat production have relied on the latter method, isolating muscle stem cells from muscle tissues and culturing the satellite cells in vitro. Therefore, the establishment of livestock satellite cells with high proliferation, maintenance and differentiation capabilities is crucial technology for advancing the cultured meat industry (Reiss et al., 2021).

Single nucleotide polymorphism (SNP) is a genetics change or variation of single base nucleotide in DNA base sequence. Also, SNP accounts for the most frequent alleles in a population, and this portion should not exceed 99%. In other words, SNP refers to the position of a nucleotide sequence in which a variation occurs with a frequency of 1% or more in a population. Millions of combinations of SNPs result in the differences between every individual. SNP research is a field that can identify individual disease prevention and diagnosis, and susceptibility to drug treatments.

In the field of livestock farming, technology has been developed to predict the expected four economic traits of cattle for high-grade meat production. ‘Hanwoo 50K SNP bead chip’ is a chip that has 50,000 SNPs that are related to traits of Hanwoo (Korean cattle), and provides SNP information regarding the traits of the individual. By using Hanwoo 50K SNP bead chips, the information of phenotype and genomic (SNP) information from thousands of individuals is converted into data to create a reference group. Next, the genomic estimated breeding value (GEBV) for four economic traits of the new individuals is measured along with the existing data using the genomic best linear unbiased prediction (GBLUP) method. Next, after the standardizing each individual, the GEBV score is measured by applying the income-optimal selection formula. Cattle with a high GEBV score are expected to have excellent four economic traits, increasing the probability of receiving high-grade meat.

To determine whether genotype with high scores for the bovine traits were applicable to in vitro culture, we selected Hs-GEBV and Ls-GEBV bovine by Hanwoo 50K SNP bead chip and GBLUP methods, we analyzed cell proliferation, Immunocytochemistry (ICC) and expression levels of Pax7, MyoD1 and Pax3 genes in Hs-GEBV and Ls-GEBV bovine satellite cells at short- and long-term culture. We also analyzed the differentiation capability of Hs-GEBV and Ls-GEBV bovine satellite cells at short-term culture by using the ICC and examining the expression levels of MyoG and MyHC genes, which are known as myogenic regulator factors.

Our results showed that the proliferation of Hs-GEBV bovine satellite cells was significantly higher than that of Ls-GEBV bovine satellite cells at short- and long-term culture. However, there were no significant differences in the expression levels of Pax7 between Hs-GEBV and Ls-GEBV bovine satellite cells at short-and long-term culture. Additionally, the expression levels of MyoD1 were significantly higher in Ls-GEBV bovine satellite cells at short-term culture. However, the expression levels of MyoD1 were higher in HS-GEBV bovine satellite cells at long-term culture. Additionally, the expression levels of Pax3 were significantly higher in Hs-GEBV bovine satellite cells cultured at long-term. In terms of differentiation capability, the Ls-GEBV bovine satellite cells had a higher expression levels of MyoG than Hs-GEBV bovine satellite cells. Also, the expression levels of MyHC were significantly higher in Ls-GEBV bovine satellite cells compared to Hs-GEBV bovine satellite cells. Our results indicated that Hs-GEBV and the high expression levels of Pax3 could be correlated in the in vitro culture of bovine satellite cells. Also, Hanwoo 50K SNP bead chip could be effective selection methods for massive producing of bovine satellite cells.

DNA isolation

The DNA was extracted from the fifteen bovine tail hairs by DNA ethanol precipitation methods. The bovine hair root was incubated in 55℃ with 500 μL of DNA lysis buffer and 20 μL of Proteinase K (Qiagen, Hilden, Germany, #19131) for 3 hours. Next, 500 μL of Phenol: Chloroform: Isoamylalcohol (PCI, 25:24:1) (Bionsesang, Gyeonggi-do, Republic of Korea, #PC2026-050-80) was added and inverted 3-4 times. The samples were centrifuged at 13,000 RPM for 20 minutes at 4℃. The supernatant was moved to a new tube and added 1 mL of 100% ETOH (Merck, Darmstadt, Germany, #100983). Next, the samples were centrifuged at 13,000 RPM for 20 minutes at 4℃. The supernatants were removed, and the pellet was washed with 70% ETOH and centrifuged at 13,000 RPM for 5 minutes at 4℃. The pellet was dried for 5-10 minutes and added 50 μL of deionized distilled water. The samples were analyzed with Hanwoo 50K SNP bead chip.

Material for disclosure and statistical analysis of GBLUP

In this study, the data from the evaluation group were collected by sampling fifteen tail hairs and conducting SNP chip analysis for fifteen individuals. A total of 20,380 Hanwoo cattle genomic data from animals shipped nationwide were used to construct the reference population for GBLUP analysis (Rural Development Administration, Next BioGreen 21). Phenotypic information for the reference population of 20,380 animals, including carcass weight, eye muscle area, back fat thickness, marbling score, gender, birthdate, slaughter date, and slaughterhouse name, was collected through inquiries with the Korea Institute for Animal Products Quality Evaluation and the Nonghyup’s Hanwoo Improvement Project Office. Pedigree information for both the evaluation group and reference population was obtained through the Korean Animal Improvement Association, including individual identification numbers for sires and Korean proven bulls number (KPN).

To estimate the breeding values for genetic analysis, the estimation of genetic parameters was performed using the REMLF90 program (Misztal, http://nce.ads.uga.edu/~ignacy/newprograms.html), and the estimation of breeding values was carried out using the BLUPF90 program (Misztal, http://nce.ads.uga.edu/wiki/doku.php?id=documentation) For the estimation of genetic parameters for four traits in Hanwoo cattle, the pedigree relationship matrix (A-matrix) and the genomic relationship matrix (G-matrix) were used, and single-trait analyses were conducted for each trait. After estimating the genetic and environmental variances for each trait, the estimation of breeding values for the four traits was conducted. To construct the mixed linear model for estimating the genetic parameters of the additive genetic effects for each trait, the following equation was used:

Y represents the observed values for the body traits, X is the coefficient matrix for fixed effects, Z is the random effect vector for individuals, β is the vector of estimated values for fixed effects, u represents the additive genetic effects (breeding values), e is the vector of random errors, and G denotes the genomic relationship matrix between individuals. I denotes the identity matrix, σ_a² and σ_e² represent the additive genetic variance and residual environmental variance, respectively, for each trait.

Using the above model, the genetic parameters and breeding values for each trait were estimated. The accuracy of the estimated breeding values was calculated using the solution values provided by the BLUPF90 program analysis, including the prediction error variance (PEV) of individual breeding values and the estimated additive genetic variance for each trait obtained from the REMLF90 program analysis. The accuracy (Acc) of the estimated breeding values can be calculated as follows: Acc represents the accuracy of the estimated breeding values, PEV denotes the prediction error variance for each breeding value, and σ_A² represents the additive genetic variance for the respective trait.

Bovine satellite cell isolation

The chuck muscle tissues were isolated from adult Korean native cattle. Chuck muscle tissues were washed with 10% Anti-Anti (A.A) (Gibco, Carlsbad, CA, USA, #15240112) with PBS after 75% ETOH wash. The tissues were weighed to 5 g. 5 g of chuck muscle tissues were minced for 5 minutes with surgical scissors. Then the tissue was dissociated with Digest Solution (1 g/mL) containing DMEM/F12 (Gibco, Carlsbad, CA, USA, #11320-033), 0.25% Trypsin-EDTA (TE) (Gibco, #25200-072), Collagenase 2 (Worthington, Lakewood, NJ, #CLS-2, 5 g), DispaseII (Roche, Indianapolis, IN, USA #4942078001, 1 U/mL) and supplemented with 10% A.A for 2 hours in 37℃ and with gently inverted every 10 minutes. After digestion, muscle fragment was neutralized with Neutralized media containing DMEM (Gibco, #11885092), 15% Fetal Bovine Serum (FBS) (Gibco, 16000-044, 26140079) supplemented with 1% A.A. The mixture was centrifuged 80 g for 3 min at 4℃. Then supernatant was collected as mononuclear cell suspension. The muscle fragment was triturated with neutralized media and centrifuged 80 g for 3 min at 4℃ again. The supernatant was collected again and add up to the former supernatant. The mononuclear cells were centrifuged 1,000 g for 5 minutes and washed with PBS twice. After that, cells were filled with neutralized media and filtered through 100 μm strainer followed by 40 μm cell strainer. Cells then centrifuged 1,000 g for 5 minutes. Red Blood Cell lysing buffer (RBC) (Sigma-Aldrich, St. Louis, MO, USA, #R7757-100 mL) was used to lysate red blood cell and washed with PBS for twice. Cells were then reconstituted with Primary Culture media containing Ham’s F-10 (Gibco, #15240-062), 20% FBS, 1% A.A, basic fibroblast growth factor (bFGF) (R&D System, Minneapolis, MN, USA #223-FB-500/CF, 5 ng/mL), Primocin (Invivogene, Pak Shek Kok, New Territories, Hong Kong, ant-pm-2, 100 μg/mL). Cells were seeded in 100 mm dish coated with 0.1% gelatin (Sigma-Aldrich, St. Louis, G1319) and cultured for density of 70-80%. For the experiment primary culture media wash changed to culture media which Primocin was discarded and A.A was changed to Penicillin-Streptomycin (PS) (Gibco #15140-122). Cells were frozen by culture media, FBS and DMSO (Gibco, #D2650) with ratio of 7:2:1.

The frozen cells were thawed and washed with PBS twice. Cells were reconstituted in FACS buffer containing 1% BSA (Sigma, #A8412) with PBS and stained with APC anti-human CD29 Antibody (1:20, BioLegend, #303008), PE-CyTM7 anti-human CD56 antibody (1:20, BD Bioscience, Franklin Lakes, NJ, USA, #335826), Hoechst 34580 (1 μg/mL, Invitrogen, Carlsbad, CA, USA, H21486) for 45 minutes on ice. After antibody staining cells were washed with PBS twice. Then cells were reconstituted with Ham’s F-10 supplemented with 20% FBS. CD29⁺ CD56⁺ population cells were sorted by FACS AriaⅢ (BD Biosciences) installed at the Center for University Research Facility (CURF) at Jeonbuk National University.

Bovine satellite cell culture and differentiation

Bovine satellite cells were cultured on 0.1% gelatin coated dishes and seeded in density of 2,000/cm². The culture media were changed every day and passaging was done every 3 days. The short-term culture analysis was performed at Passage 3 (P3) and Long-term culture analysis were performed at Passage 7 (P7). For bovine satellite cells differentiation, the cells were seeded at a density of 10,000/cm². Differentiation was induced when the bovine satellite cell population reached 90% confluence at P3. Bovine satellite cells were then changed to differentiate media containing DMEM with 5% Horse Serum (Gibco, #16050-122), 1% PS and cultured for 3 days.

Cell growth analysis

Bovine satellite cell was seeded in 96 well with density of 2,000/cm². Cells were cultured in culture media for 3 days. Growth analysis was detected by Cell counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan #CK04-11). Cells were treated with CCK-8 solution following the manufacturer’s instruction, and incubated at 37℃ for 3 h. Growth were measured using a microplate reader of wavelength of 450 nm.

Immunofluorescence staining bovine satellite cell

Bovine satellite cells were seeded in 4 well plate with density of 2,000/cm² and cultured for three days. After three days, cells were washed with PBS twice. Then cells were fixed by 4% paraformaldehyde for 20 minutes at 4℃. After fixation cells were washed with PBS three times. Then cells were incubated in blocking solution which is washing solution + 3% Bovine serum Albumin (Bovogen, Keilor East, Australia, Bovostar) and washing solution, 0.3% Triton X-100 (Gibco, 10010-023) with PBS for 2 hours. After blocking, cells were washed with washing solution. Then cells were stained overnight with primary antibodies against anti-paired box 7 (Pax7) (Pax7 monoclonal, 1:50, DHSB, Iowa, IA, USA) at 4℃. For differentiated cell, cells were stained with myosin heavy chain (MHC) (1:10, DHSB, #MF20-s). Then Cells were incubated with Alexa488 labeled anti-mouse (Molecular Probes, Eugene, OR, USA) antibodies, Alexa586 labeled anti-rabbit (molecular probe) antibodies in room temperature for 2 hours. 1 μg of 4’6-diamidino-2-phenylindole (DAPI) was stained for 10 min. After DAPI staining, cells were washed with washing solution for 10 min. The analysis was performed with (Leica 9900) and captured triplicate.

Gene expression analysis by qRT-PCR

RNA was extracted form cell using AccuPrep® Universal RNA Extraction kit (Bioneer, Seoul, Korea) following the manufacturer’s protocol. 1 μg of total RNA was reverse-transcribed with Accupower® CycleScript RT Premix (Bioneer, Seoul, Korea) according to manufacturers’ instruction. Relative gene expression was performed in triplicated using Powerup SYBR Green master mix (Applied Biosystem, CA, USA). The primer used for qRT-PCR were as follows: GapDH sense 5’-CACCCTCAAGATTGTCAGC-3’, GapDH antisense 5’-TAAGTCCCTCCACGATGC-3’, Pax7 sense 5’-CTCC CTCT GAAG CGTA AGCA-3’, Pax7 antisense 5’-GGGT AGTG GGTC CTCT CGAA-3’, MyoG sense 5’-GCGCAGACTCAAGAAGGTGA-3’, MyoG antisense 5’-TGCAGGCGCTCTATGTACTG-3’, MyHC sense 5’-AGAGCAGCAAGTGGATGACCTTGA-3’, MyHC antisense 5’-TGGACTCTTGGGCCAACTTGAGAT-3’, MyoD1 sense 5’-TTTGCCAGAGCAGGAGCCCCTC-3’, MyoD1 antisense 5’-TTCGAACACCTGAGCGAGCGC-3’ Pax3 sense 5’-CAAAGCTTACAGAGGCCCGA -3’, Pax3 antisense 5’-GGTCTCTGACAGCTGGTACG-3’.

Statistical analysis

All of the experiments were performed more than three times, and the data of all repetitions of each experiment were collated and expressed as means ± standard error (SE) of the mean. Statistical tests were conducted using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and statistical differences were performed with Student’s t-test or analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test for post-hoc comparisons. A p-value < 0.05 was regarded as significant.

Selection of Hs-GEBV and Ls-GEBV Hanwoo by Hanwoo 50K SNP analysis bead chip and GBLUP methods

To determine the expected GEBV of bovine, we isolated DNAs from the fifteen bovines, and analyzed the SNPs of the bovine DNA with Hanwoo 50K SNP bead chip. We obtained 50,000 SNPs of genotype of each bovine individuals from Hanwoo 50K SNP bead chip. Using the genotype (SNP) information of bovine individuals, we estimated SNP effect values for 4 economics traits, Carcass weight (CWT), Eye muscle area (EMA), Back fat thickness (BFT), Marbling score (MSC) through the GBLUP method. The GEBV was calculated by using the GBLUP method. The score and accuracy was shown in Table 1. We then standardized the GEBV score as the units of the trait values varied across individuals. We then applied the income-optimal selection formula which weight values to each of 4 economic traits. The formula of income-optimal are CWT*7, EMA*3, BFT*-3 and MSC*4. We then selected high- and low- scoring GEBV bovine with same sex and similar age. The GEBV of No. 1 Hanwoo was CWT 16.46, EMA 5.21, BFT -3.77, MSC 4.61 with in total 25.91 (Table 2). The GEBV of No. 11 Hanwoo were CWT -3.45, EMA -1.78, BFT -7.78, MSC 2.41 with in total -7.57 (Table 1). In the 20,380 references group, the rank of No. 1 Hanwoo was top 2.3% and it was named as ‘high scored GEBV (Hs-GEBV)’ bovine. While the rank of No. 11 Hanwoo was top 76.9% and it was named as ‘low scored GEBV (Ls-GEBV)’ bovine (Table 2).

Table 1. Analysis of GEBV and accuracy in fifteen Hanwoo.

Sex	Age (month)	CWT	CWT SE	CWT Acc	EMA	EMA SE	EMA Acc	BFT	BFT SE	BFT Acc	MSC	MSC SE	MSC Acc
F	43	-30.36	18.99	0.75	-3.3	4.45	0.74	1.69	1.91	0.74	-1.04	0.72	0.77
M	35	6.24	20.57	0.7	-2.22	4.81	0.69	-1.91	2.07	0.69	-0.84	0.79	0.72
M	25	54.57	21.22	0.68	9.58	4.95	0.66	5.71	2.13	0.66	1.37	0.82	0.7
F	42	-14.45	20.8	0.69	-1.71	4.86	0.68	1.19	2.09	0.68	-1.34	0.8	0.72
M	31	-11.71	21.25	0.67	-3.05	4.96	0.66	3.07	2.13	0.66	0.51	0.82	0.7
F	30	0.66	19.06	0.75	-7.13	4.46	0.74	-0.17	1.92	0.74	-0.89	0.73	0.77
F	38	13.66	20.37	0.71	2.62	4.77	0.69	-4.58	2.05	0.7	-1.44	0.78	0.73
F	51	45.59	18.93	0.75	6.59	4.43	0.74	-1.11	1.91	0.74	0.58	0.72	0.77
F	63	14.95	19.22	0.74	-1.72	4.5	0.73	7.12	1.93	0.73	0.34	0.73	0.77
F	35	48.49	21.13	0.68	3.53	4.93	0.67	0.12	2.12	0.67	-0.76	0.81	0.7
F	37	-0.17	18.86	0.76	-3	4.41	0.75	-1.68	1.9	0.75	0.69	0.72	0.78
M	27	55.63	18.1	0.78	9.05	4.24	0.77	0.24	1.82	0.77	0.98	0.69	0.8
M	20	31.98	20.24	0.71	6.75	4.73	0.7	1.39	2.04	0.7	0.87	0.77	0.73
M	1	50.8	21.38	0.67	7.9	4.99	0.66	-0.46	2.15	0.66	0.7	0.82	0.7
M	12	-26.61	22.94	0.6	-7.12	5.34	0.59	-0.68	2.3	0.59	-0.95	0.89	0.63

GEBV was analyzed in fifteen Hanwoo by using Hanwoo 50K bead chips and GBLUP methods. The score of GEBV, standard error (SE), and accuracy (Acc) of 4 traits in each individual are shown. The CWT refers to carcass weight, EMA refers to eye muscle area, BFT refers to back fat thickness, and MSC refers to marbling score..

Table 2. Selection of Hs-GEBV and Ls-GEBV Hanwoo through GEBV standardization and profit-optimal selection.

No	Sex	Age (month)	CWT	EMA	BFT	MSC	Total	Rank (%)
1	Male	27	16.46342	5.214229	-0.37728	4.612486	25.91286	2.3
2	Male	1	15.03656	4.555933	0.70918	3.300959	23.60263	3.2
3	Female	51	13.49478	3.801252	1.727493	2.74688	21.77041	4
4	Male	25	16.15143	5.520298	-8.88696	6.415442	19.2002	5.5
5	Male	20	9.46912	3.893001	-2.1631	4.107491	15.30651	8.6
6	Female	35	14.3522	2.040973	-0.187	-3.5398	12.66637	11.8
7	Female	38	4.049561	1.513724	7.134658	-6.69591	6.002035	25.9
8	Female	37	-0.04118	-1.7154	2.615419	3.263941	4.122781	32.3
9	Female	35	1.854654	-1.26681	2.980968	-3.88619	-0.31738	49.8
10	Female	63	4.432083	-0.98027	-11.0863	1.615496	-6.01897	72
11	Male	31	-3.45371	-1.74819	-4.78104	2.412942	-7.57	76.9
12	Female	30	0.203172	-4.09334	0.261295	-4.12679	-7.75566	77.4
13	Female	42	-4.26606	-0.9719	-1.85186	-6.242	-13.3318	89.2
14	Male	12	-7.86186	-4.08753	1.064034	-4.41596	-15.3013	92
15	Female	43	-8.97038	-1.88868	-2.63344	-4.85511	-18.3476	95

The Hs-GEBV and Ls-GEBV Hanwoo were selected in fifteen Hanwoo from 20,380 reference groups through standardization and income-optimal selection formula for each individual after obtaining GEBV using the 50K bead chip and GBLUP method. GEBV score was measured by applying the income-optimal selection formula..

Analysis of characteristics of short-term culture of Hs-GEBV and Ls-GEBV bovine satellite cells in vitro

The Hs-GEBV and Ls-GEBV bovine satellite cells were purely sorted using bovine satellite cells specific marker CD29 and CD56 (Fig. 1A). First, we stained PAX7 in Hs-GEBV and Ls-GEBV bovine satellite cells cultured at short-term (Fig. 1B). To assess the growth of Hs-GEBV and Ls-GEBV bovine satellite cells at short-term culture, we analyzed the proliferation with CCK-8. Our results showed that Hs-GEBV bovine satellite cells had a significantly higher proliferation compared to Ls-GEBV bovine satellite cells (p < 0.05) (Fig. 1C). For comparing the maintenance of Hs-GEBV and Ls-GEBV bovine satellite cells at short-term culture, we analyzed the expression levels of Pax7 genes by qRT-PCR. Our results showed that there were no significant differences between Hs-GEBV and Ls-GEBV bovine satellite cells at short-term culture (Fig. 1D). To analyze the myogenic commitment of bovine satellite cells, we analyzed the expression levels of MyoD1 in Hs- and Ls-GEBV bovine satellite cells at short-term culture. The expression levels of MyoD1 were significantly higher at Ls-GEBV bovine satellite cells compared to Hs-GEBV bovine satellite cells at short-term culture (p < 0.001) (Fig. 1D). These results indicated that Hs-GEBV bovine satellite cells could be effective in proliferation during in vitro culture, however, the expression levels of MyoD1 were significantly higher in Ls-GEBV satellite cells, suggesting that the population of myoblasts was higher in Ls-GEBV bovine satellite cells at short-term culture.

Figure 1.Isolation of Hs-GEBV and Ls-GEBV bovine satellite cells and comparative analysis of characteristics of Hs-GEBV and Ls-GEBV bovine satellite cells in vitro at short-term culture. (A) Isolation of Hs-GEBV and Ls-GEBV bovine satellite cells by using bovine satellite cells specific marker CD29 and CD56. The CD29 and CD56 positive cells were sorted by FACS sorting. (B) Morphology and immunocytochemistry of Hs-GEBV and Ls-GEBV satellite cells at short-term culture were stained with Pax7 (green) antibody and DAPI (blue). Scale bar 100 μm. (C) The proliferation of Hs-GEBV and Ls-GEBV bovine satellite cells at short-term culture. The analysis was performed after treating with CCK-8 for 3 hours. n = 12, The asterisks represent significant differences in expression levels (Student’s t-test): *p < 0.05; **p < 0.01; ***p < 0.001. (D) Gene expression of Pax7 and MyoD1 in Hs-GEBV and Ls-GEBV bovine satellite cells at short-term culture. n = 3, The asterisks represent significant differences in expression levels (Student’s t-test): ***p < 0.001.

Analysis of characteristics of long-term culture of Hs-GEBV and Ls-GEBV bovine satellite cells in vitro

It is known that the bovine satellite cells cultured for long-term in vitro experience a rapid decrease in Pax7 expression levels. We next analyzed the characteristics of Hs-GEBV and Ls-GEBV bovine satellite cells at long-term culture. First, we stained PAX7 in Hs-GEBV and Ls-GEBV bovine satellite cells at long-term culture (Fig. 2A). Next, we analyzed the proliferation of Hs-GEBV and Ls-GEBV bovine satellite cells with CCK-8. Our results revealed that the Hs-GEBV had significantly higher proliferation compared to Ls-GEBV bovine satellite cells (p < 0.05) (Fig. 2B). To analyze the maintenance of Hs-GEBV and Ls-GEBV bovine satellite cells at long-term culture, we analyzed the expression levels of Pax7 gene by qRT-PCR. There were no significant differences in expression levels of Pax7 between Hs-GEBV and Ls-GEBV bovine satellite cells. The expression level of MyoD1 was 3.8-fold higher in Hs-GEBV bovine satellite cell compared to Ls-GEBV bovine satellite cells at long-term culture (Fig. 2C). We then analyzed the expression level of Pax3, which regulates the myogenic commitment along with Pax7 at short- and long-term culture. The expression levels of Pax3 were significantly high at Hs-GEBV at long-term culture (P7) (p < 0.001) (Fig. 2D). Our result indicated that Hs-GEBV bovine satellite cells could proliferate effectively at long-term culture compared to Ls-GEBV bovine satellite cells and could have increased population of myoblast by expressing high levels of MyoD1 compared to Ls-GEBV bovine satellite cells in vitro.

Figure 2.Comparative analysis of characteristics of Hs-GEBV and Ls-GEBV bovine satellite cells in vitro at long-term culture. (A) Morphology and immunocytochemistry of Hs-GEBV and Ls-GEBV satellite cells at long-term culture with Pax7 (green) antibody and DAPI (blue). Scale bar 100 μm. (B) The proliferation of Hs-GEBV and Ls-GEBV bovine satellite cells at long-term culture. The analysis was performed after treating with CCK-8 for 3 hours. n = 12, The asterisks represent significant differences in expression levels (Student’s t-test): *p < 0.05. (C) Gene expression of Pax7 and MyoD1 in Hs-GEBV and Ls-GEBV bovine satellite cells at long-term culture. (D) Gene expression of Pax3 in Hs-GEBV and Ls-GEBV bovine satellite cells at short- and long-term culture. n = 3, Each letter (a, b, c, d) represents significant differences (p < 0.0001).

Capability of differentiation of Hs-GEBV and Ls-GEBV bovine satellite cells

To find the differentiation capability of Hs-GEBV and Ls-GEBV bovine satellite cells in vitro, we differentiated Hs-GEBV and Ls-GEBV bovine satellite cells at short-term culture. The formation of myotubes started at day 2 of differentiation and the myotubes matured and enlarged by day 3 (Fig. 3A and 3B). Next, we stained MYHC in differentiated Hs-GEBV and Ls-GEBV bovine satellite cells at day 3. We then analyzed the expression levels of MyoG and MyHC at differentiated Hs-GEBV and Ls-GEBV bovine satellite cells. Ls-GEBV bovine satellite cells had a 2-fold higher expression levels of MyoG compared to Hs-GEBV bovine satellite cells. Also, the expression levels of MyHC were significantly higher in Ls-GEBV bovine satellite cells compared to Hs-GEBV bovine satellite cells (p < 0.05) (Fig. 3C). This result indicated that Ls-GEBV bovine satellite cells had significant differentiation capability compared to Hs-GEBV bovine satellite cells.

Figure 3.Comparative analysis of differentiation capability of Hs-GEBV and Ls-GEBV bovine satellite cells in vitro at short-term culture. (A) Morphology of differentiated Hs-GEBV and Ls-GEBV satellite cells at day 0 to day 3. Scale bar 100 μm. (B) The Hs-GEBV and Ls-GEBV satellite cells were differentiated for 3 days and stained with MYHC (green) antibody and DAPI (blue). Scale bar 100 μm. (C) Gene expression of MyoG and MyHC in Hs-GEBV and Ls-GEBV bovine satellite after 3 days of differentiation. n = 3, The asterisks represent significant differences in expression levels (Student’s t-test): *p < 0.05.

Continuous breeding efforts are made by improving the four economic traits and maintaining the high-quality to get a high grade of Hanwoo. Strict selection of Korean proven bull for artificial insemination has led to the nationwide distribution of semen with high quality of traits. Additionally, by evaluating the GEBV of calf individuals, it is possible to predict whether a particular calf has a high probability of receiving a good grade. As mentioned earlier, one of the key technologies in cultured meat production is the establishment of cell lines with high potential of growth, maintenance and differentiation. Research on establishing bovine satellite cell lines with high growth potential in vitro using SNP technology is required.

In this study, we selected high scored GEBV and low scored GEBV using Hanwoo 50K SNP bead chips and GBLUP methods in fifteen bovines. We sorted pure Hs- and Ls- GEBV bovine satellite cells using bovine specific surface markers CD29 and CD56. Next, we analyzed the characteristics of Hs-GEBV and Ls-GEBV bovine satellite cells cultured in vitro at short- and long-term culture. Our results showed that the proliferation was significantly higher in Hs-GEBV bovine satellite cells at short- and long-term culture. However, there were no significant differences in the expression levels of Pax7 between Hs-GEBV and Ls-GEBV bovine satellite cells cultured at short- and long-term culture in vitro. The expression levels of MyoD1 were significantly higher in Ls-GEBV bovine satellite cells at short-term culture, however, the expression levels of MyoD1 at long-term culture were higher at Hs-GEBV bovine satellite cells. The expression levels of Pax3 were significantly higher at long-term culture of Hs-GEBV bovine satellite cells. The Ls-GEBV cells had high expression levels of MyoG at differentiated bovine satellite cells. Also, the expression levels of MyHC was significantly high at Ls-GEBV cells compared to Hs-GEBV cells at differentiation.

The proliferation of Hs-GEBV bovine satellite cells were significantly higher compared to Ls-GEBV bovine satellite cells at short- and long-term culture. The proliferation of bovine satellite cells could be related with the score of CWT and EMA. The high score of CWT and EMA indicates that the cattle has a high chance of yielding many meats. The score of muscle related traits CWT and EMA were high in Hs-GEBV compared to Ls-GEBV (Table 1). Previous studies showed that the proliferation of myoblast in Wagyu X, Angus and Hereford bovine were correlate with the live weight and carcass weight, indicating that bovines with high live weight and carcass weight caused significant increase in satellite cells and myoblast in vitro culture (Coles et al., 2015). Similarly, callipyge ovine, which has extensive hypertrophy muscle in the hind limb and back muscles. Callipyge mutation was identified as differences in nucleotide sequences in specifying regions between DLK1 and MEG3 genes. The callipyge ovine had high proliferation in semitendinosus derived myoblast in vitro compared to normal ovine. However, there were no significant differences in proliferation of longissimus dorsi derived myoblast and the differentiation capability between callipyge ovine and normal ovine. The authors described that the hypertrophy of callipyge semitendinosus muscles could be related to the increased proliferation of semitendinosus myoblast in vitro (Lavulo et al., 2008). Consistent with the result of callipyge sheep, the Hs-GEBV bovine satellite cells had no significant differences in maintenance, and indeed, our results of differentiation capability indicated that the muscle regenerations were not related with the GEBV in vitro.

Pax3/Pax7 genes regulate the entry of satellite cells into myogenic program by activation myogenic regulator factor Myf5 (Lagha et al., 2008). Recent studies in bovine satellite cells showed that the expression levels of Pax3 were higher at long-term culture compared to short term-culture (Stout et al., 2022). Consistent with the previous results, our results showed significantly high expression levels of Pax3 at long-term culture. Recent studies in mice found out that when the satellite cells are exposed to environmental stress, the Pax7 expression with Pax3 negative cells specifically decreased the subpopulations and impaired survival compared to co-expression of Pax3 and Pax7 positive muscle stem cells (Der Vartanian et al., 2019). Similarly, enriched expression of Pax3 muscle stem cells contributed to muscle repair upon irradiations compare to Pax3 negative muscle stem cells (Scaramozza et al., 2019). Our result could be explained that as the long-term culture cause the stress of bovine satellite cells, only the population of bovine satellite cells with expressing Pax3 that are resistant to stress is expected to be advantageous for survival. The Hs-GEBV bovine satellite cells could have competence of anti-stress and could effectively maintain the satellite cells populations compared to Ls-GEBV bovine satellite cells. However, additional in-depth studies are required for the correlation of bovine satellite cells with high expression levels of Pax3 and the Hs-GEBV.

To produce cultured meat, a significant amount of satellite cells is required. The diverse efforts of continuous satellite cell supply have been developing including, iPSCs, enhancing cell growth using growth factors and ECM (extracellular matrix), and promoting better cell proliferation through scaffold materials. While these efforts are important in cultured meat production, fundamentally, selecting cells with high growth potential is crucial. Previous research has shown that satellite cells had better proliferation abilities in calves compared to adult cattle (Kim et al., 2023). In cultured meat production, it could be effectively achieved to predict the high growth potential of bovine satellite cells in vitro through SNP bead chips technology.

In summary, we selected Hs- and Ls-GEBV of bovine by Hanwoo 50K SNP bead chips and isolated the bovine satellite cells. We then analyzed the characteristics of Hs- and Ls-GEBV of bovine satellite cells cultured in vitro at short- and long- term culture. The proliferation of bovine satellite cells was significantly high at Hs-GEBV bovine satellite cells, indicating the proliferation could be correlated with the traits of CWT and EMA. However, there were no significant differences in the expression levels of Pax7 between Hs- and Ls-GEBV satellite cells. Also, the differentiation related genes, MyoG and MyHC were significantly higher in Ls-GEBV satellite. The expression levels of Pax3 were significantly high in Hs-GEBV bovine satellite cell at long-term culture. Our results indicated that selection of bovine satellite cells by using Hanwoo 50K SNP bead chips could be effective in proliferation in in vitro culture.

This work was supported by IPET through ‘High Value-added Food Technology Development Program’, and was funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (322006-05-1-CG000).

Data curation, J.H.H., J.S.Y., D.H.K.; Formal analysis, J.H.H., J.S.Y., D.H.K.; Methodology, J.H.H., J.S.Y., D.H.K.; Project administration, H.W.C.; Supervision, H.W.C.; Writing-original draft, J.H.H; Writing-review & editing, J.H.H., H.W.C.

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All animal procedure was conducted by the Animal Ethics Committee of Jeonbuk National University (JBNU, NON2022-018-002), Republic of Korea. All of the experiment and procedures were performed in accordance with ethical standards guidelines and regulation of Jeonbuk National University.

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No potential conflict of interest relevant to this article was reported.