Research Article

Genetic Diversity of the CSN2 Gene and its Impact on Milk Production and Quality in Indonesian Friesian Holstein Cattle

Salsabila1, Siti Kholifah3, Ronny Rachman Noor2, Jakaria2*

1Animal Production Science and Technology Study Program, Postgraduate School, IPB University, Indonesia; 2Department of Animal Production and Technology, Faculty of Animal Science, IPB University, Bogor, Indonesia; 3Livestock Breeding Center (BPTU-HPT) Baturraden, Purwokerto, Central Java, Indonesia.

Received | December 23, 2024; Accepted | March 02, 2025; Published | April 19, 2025

*Correspondence | Jakaria, Department of Animal Production and Technology, Faculty of Animal Science, IPB University, Bogor, Indonesia; Email: [email protected]

Citation | Salsabila, Kholifah S, Noor RR, Jakaria (2025). Genetic diversity of the CSN2 gene and its impact on milk production and quality in indonesian friesian holstein cattle. Adv. Anim. Vet. Sci. 13(5): 1072-1077.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.5.1072.1077

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Based on data from the Central Statistics Agency, the population of dairy cattle in Indonesia in 2022 is still tiny, namely 592,897 heads, dominated by the Friesian Holstein (FH) cattle breed. The advantages of FH cattle are that they have high milk production and good adaptability. Despite this FH cattle population, milk availability in Indonesia remains lower than in other Southeast Asian countries, such as Malaysia, which has a milk consumption rate three times higher than Indonesia. According to data from the Directorate General of PKH, Ministry of Agriculture, the amount of milk needed in Indonesia in 2022 is 4.4 million tons, while milk production is 0.95 million tons. Thus, imports from other countries meet almost 70% of domestic milk needs. On the other hand, with limited consumption, there is also a limitation on milk consumption for the Indonesian people, namely lactose intolerance due to the high lactose content in milk. Based on its composition, milk protein is classified into two groups. Namely, casein contributes around 80% of milk protein content, and the remaining 20% is whey (Marko et al., 2020). Four casein proteins are found in cow’s milk, namely αs1-casein (CSN1S1, 38% of total casein), αs2-casein (CSN1S2, 10%), β-casein (CSN2, 36%), and ĸ-casein (CSN3, 13%) (Marko et al., 2020). Thus, 36% of cow’s milk casein is β-casein (CSN2).

The β-casein gene (CSN2) is one of the primary milk protein genes with 13 variants, namely the A1 and A2. The β-casein gene (CSN2) forms up to 45% of cow’s milk casein. CSN2 is localized on chromosome 6 in cows (Miluchová et al., 2014). The forms of β-casein in dairy cattle are A1 and A2. The difference between β-Casein A1 and A2 lies in the composition of the amino acid chain at position 67, namely the amino acid histidine and the second amino acid proline (Marko et al., 2020). This difference is due to changes in single nucleotide bases (SNPs) at codon 67, namely CCT (A2, proline) or CAT (A1, histidine) so that until now, very intensive research has been carried out on the β-casein gene (CSN2) such as in dairy cows in Japan (Yamada et al., 2021). Milk with A1 protein produces β-casomorphin (BCM-7) during the digestion process, β-casomorphin (BCM-7) is also thought to show many immunological activities, such as chronic inflammatory responses, such as allergies, mucin production, lymphocyte proliferation, and skin reactions (Haq et al., 2014). Therefore, mapping the distribution of the A2A2 genotype in Indonesian FH cattle is essential to evaluate the potential benefits of A2 milk for consumers with lactose intolerance.

The preference for A2 milk in FH cattle has implications for human health and dairy production. This study focuses on the role of the A2A2 genotype in the production and quality of FH cow milk in Indonesia. Its aim is to evaluate whether FH cows with the A2A2 genotype have advantages in terms of lower lactose content or better milk quality compared to A1A1. This study, which aims to analyze the diversity of the CSN2 gene and its effect on milk production and quality in Indonesian FH cattle, is significant due to the potential impact of our findings on dairy production and the health benefits of A2 protein. Our research builds on previous reports on this topic. It provides valuable insights into the genetic diversity of the CSN2 gene and its implications for dairy production in Indonesian Friesian Holstein cattle, highlighting the importance of understanding the A2A2 genotype for the future of dairy production.

MATERIALS AND METHODS

Data Collection

This research procedure, approved by the Faculty of Medicine ethics committee, Andalas University, Padang, West Sumatra, Indonesia, with the number 558/UN.16.2/KEP-FK/2024, involved a comprehensive and meticulous data collection process. Avoid unnecessary phrases like “utmost care” unless a specific method is described to ensure accuracy. The DNA samples used for molecular analysis were 107 FH cattle which were the result of mating in Indonesia with imported cattle from New Zealand which were randomly selected in a controlled environment at the Center for Superior Livestock Breeding and HPT Baturraden, Banyumas Regency, Central Java. DNA samples were obtained from the extraction of cow blood samples using the GeneAid DNA extraction kit protocol. DNA samples were extracted at the Animal Molecular Genetics Laboratory, Faculty of Animal Husbandry, Bogor Agricultural University. FH cow milk samples used for milk content analysis were taken from as many as 107 individuals. Of the 107 DNA samples, only 21 cows were in their first lactation, so only 21 milk samples could be analyzed. This study used milk production recording data in the first lactation and milk samples collected from 21 Friesian Holsten (FH) cows representing the A2A2 and A1A1 genotypes.

CSN2 Gene Diversity Analysis and Milk Content

The analysis of CSN2 gene diversity was carried out in several stages, namely (1) DNA extraction using the GeneAid DNA extraction kit protocol, (2) amplification of the CSN2 exon seven gene with a PCR machine with primers designed using the Primer3 program (https://bioinfo.ut.ee/primer3-0.4.0/) and Primer Stats with the Genbank M55158.1 reference. The primers used were the forward primer five ′- TAA AAT CCA CCC CTT TGC CC-3’ and reverse 5′ - GAC AGT TGG AGG AAG AGG CT -3’ with a product length of 325 bp with the target SNP A>C rs43703011 (Marko et al., 2020; Yamada et al., 2021). CSN2 gene amplification was carried out using the PCR technique using the AB System PCR machine. Gene amplification using a mixture consisting of primers (forward and reverse) as much as 0.3 µL, MyTaq HS RedMix as much as 12.5 µL and nuclease-free water (NFW) 9.9 µL was put into a 1.5 uL tube. The mixture was distributed to the DNA sample extracted from 2 µL of blood, which was transferred into a 0.2 mL tube. PCR conditions included predenaturation at 95 °C for 5 minutes, denaturation at a temperature of 95 °C for 10 seconds, annealing at a temperature of 61 °C for 20 seconds, extension at a temperature of 72 °C for 30 seconds and final extension at a temperature of 72 °C for 5 minutes. The PCR product of the CSN2 gene was sequenced using the services of the 1st Base Laboratory in Selangor, Malaysia, through PT. Genetika Science Indonesia.

The first FH cows observed at BPTU-HPT Baturraden, Banyumas Regency, Central Java, were taken for milk production data and 100 mL milk samples based on known genotypes, namely genotypes A1A1 and A2A2, 11 and 10, respectively. The milk samples were analyzed for milk content, including lactose, protein, and milk fat levels, using the HPLC (High-Performance Liquid Chromatography) method through the PT. Saraswanti Indo Genetch (SIG) Taman Yasmin, Bogor Laboratory service.

Statistical Analysis

Data normality testing is carried out using the Ryan-Joiner test. If the test results show a P value > 0.05, then the data is said to be normally distributed and can be analyzed. The results of CSN2 gene sequencing were analyzed using the FinchTV program, while SNP determination was analyzed using the Molecular Evolutionary Genetic Analysis 11 (MEGA11) program. Allele frequencies, genotype frequencies, and Hardy-Weinberg equilibrium were analyzed using the Popgene 32 version 1.3 program.

 

The differences in the CSN2 gene A1A1 and A2A2 genotypes in milk production and milk quality of FH cows were analyzed using the t-test, which used the Minitab 19 program.

Table 1: Description of daily milk quality and production of FH Indonesia.

Parameter	Mean and Standard deviation (n=21)	Minimum	Maximum
Milk yield(kg/animal/day)	20.68 ± 1.37	16.65	23.49
Fat (%)	4.41 ± 1.37	2.79	6.70
Protein (%)	3.57 ± 0.61	2.78	4.94
Lactose (%)	3.64 ± 0.34	3.05	4.01
Indonesian National Standard (SNI) for Fresh Milk (3141.1:2011)	Minimum
Fat (%)	3.00
Protein (%)	2.40
Lactose (%)	-

 

RESULTS

Milk Quality and Production of FH

Table 1 shows that the daily milk production of first lactation Indonesian FH cows at BBPTU-HPT Baturraden is relatively high compared to other studies, namely 20.68 ± 1.37 kg/animal/day. The quality of fresh milk from Indonesian FH cows based on SNI, especially the fat and protein content, meets the minimum SNI requirements, while the lactose content is low compared to other studies (Table 1). This is caused by genetic and environmental factors. This study shows that the lactose content in FH Indonesian cows milk at BBPTU-HPT Baturraden is lower, so it can be beneficial for individuals with lactose intolerance.

CSN2 Gene Diversity

Amplification of the rs43703011 fragment of the CSN2 gene exon 7 in Indonesian FH cattle was carried out at an annealing temperature of 61oC and was successful (97 out of 107 cattle) with a PCR product length of 325 bp (Figure 1).

 

Table 2: Information on CSN2 gene diversity in FH.

Line- Locus	N	Genotype frequency			Allele frequency		Ho	He	χ2 test
		A1A1	A1A2	A2A2	A1	A2
FH- CSN2Gene	97	0.57	0.01	0.42	0.57	0.43	0.01	0.49	92.95*

 

Note: *= significantly different at the 5% level (χ2 test > χ2 table 0.05 = 3.84).

 

The CSN2 gene sequencing analysis results obtained three genotypes, namely genotypes A1A1, A1A2, and A2A2 (Figure 2). The diversity of CSN2 gene genotypes is presented in Table 2. The CSN2 gene has polymorphic in Indonesian FH cattle in this study. The equilibrium of the Hardy-Weinberg law in the Indonesian FH cattle population indicates an unbalanced status (Table 2). This is because selection has occurred. This is due to the selection process that has been carried out on Indonesian FH cattle at BBPTU-HPT Baturraden which originate from imported New Zealand stock. Allele frequency of the CSN2 gene has been reported in several studies in Bos taurus, Bos indicus, and its crossbreed cattle, as presented in Table 3.

Differences in CSN2 Gene Genotypes with Milk Quality and Production of FH

The results of the analysis of differences in genotypes (A1A1 and A2A2) with milk production and milk quality (fat, protein, and lactose) are presented in Table 4. Table 4 shows that the A1A1 genotype has no difference (P>0.05) in daily milk production, fat content, protein content, and lactose with the A2A2 genotype in Indonesian FH cattle. The results of this study indicate that the CSN2 gene (rs4370301) of the A2A2 genotype does not affect milk production and quality or shallow lactose content. This is caused by genetic and environmental factors. The relatively high coefficient of variation is also likely to influence this study’s results. The differences in the A1A1 and A2A2 genotypes of the CSN2 gene on milk quality are presented in Table 5.

 

Table 3: Allele frequency of CSN2 gene in numerous cattle breeds.

Species	Breeds of Cattle	N	Allele Frequency		References
			A1	A2
Bos taurus	Slovak Spotted breed	111	0.2928	0.7072	Miluchová et al. (2014)
Bos taurus	Pinzgau breed	89	0.5618	0.4382	Miluchová et al. (2014)
Bos taurus	Holstein breed	87	0.3678	0.6322	Miluchová et al. (2014)
Bos taurus	Holstein breed	189	0.4925	0.561	Soyudal et al. (2019)
Bos taurus	Pabna	24	0.04	0.96	Pabitra et al. (2022)
Bos indicus	Red Chittagong	30	0.10	0.90	Pabitra et al. (2022)
Bos indicus	Sahiwal	12	0.08	0.92	Pabitra et al. (2022)
Bos indicus	Munshigan	20	0.08	0.92	Pabitra et al. (2022)
Bos taurus x Bos indicus	Holstein-zebu crossbreed	133	0.39	0.61	Pabitra et al. (2022)
Bos taurus	Holstein breed	1478	0.2382	0.7618	Miluchová et al. (2023)
Bos taurus	Friesian Holstein	97	0.57	0.43	This study

 

DISCUSSION

Daily milk production of first lactation Indonesian FH cows in this study at BBPTU-HPT Baturraden included high milk production. The average milk production of FH cows at BPPIBTSP Bunikasih Cianjur in the first lactation was 11.36±3.70 kg (Bahri et al., 2022), and the average milk production of FH cows in Pacitan Regency was 14.5±3.15 kg (Prabowo, 2021). Domitrovska et al. (2021) stated that FH cow milk’s fat, protein, and lactose content in Southern European countries were 3.25%, 3.14%, and 4.46%, respectively. This study shows that the lactose content of Indonesian FH cows is lower than that of FH cows in Europe. Lim et al. (2020) reported that Jersey cows’ fat and protein content in Korea were 4.46% and 3.85%. The production and quality of dairy cow milk are influenced by various factors, namely genetic, management, and health factors (Kolo et al., 2023).

 

Table 4: Comparison of milk quality and production between CSN2 gene genotypes in Indonesian FH cattle.

Parameter	A1A1 Genotype (X±SD) (n=11)	Coefficient of Variation (%)	A2A2 Genotype (X±SD) (n=10)	Coefficient of Variation
Lactose(%)	3.47 ± 0.70	16	3.52 ± 0.64	8
Protein(%)	3.48 ± 0.48	38	3.80 ± 1.15	47
Fat (%)	4.03 ± 1.54	14	4.90 ± 2.31	30
Daily Milk Production (kg)	20.13 ± 1.51	20	21.31 ± 1.03	18

 

Note: n: number of individuals; X: mean; SD: standard deviation.

 

Table 5: References for differences in CSN2 gene genotypes on milk production and milk quality in numerous cattle breeds.

Breeds of Cattle	Genotypes	Fat (%)	Protein (%)	Lactose %	References
Holstein Slovakia	A2A2	3.78	3.32	-	Miluchová et al. (2023)
	A1A1	3.75	3.27	-
Montbèliarde	A2A2	4.08	3.41	4.53	Bugeac et al.(2019)

 

A locus is polymorphic if it has an allele frequency of ≤ 0.99 for large populations and ≤ 0.95 for smaller populations (Allendorf et al., 2010). Anggraeni et al. (2021) stated that heterozygosity is used to estimate the level of genetic polymorphism in a population so that if the observed heterozygosity value (Ho) is close to 0, it means that the level of heterozygosity is low. The equilibrium of the Hardy-Weinberg law in the Indonesian FH population indicates an unbalanced status (Table 2). Factors that influence genetic balance in a population (Hardy-Weinberg Law) are when a large population has a constant frequency of dominant and recessive genotypes from generation to generation, and there is no selection, mutation, migration, or genetic drift (Noor, 2010). Selection is one factor that can quickly change the balance in a population (Saputra et al, 2020).

Bos taurus species in several studies, including this study, have a higher frequency of A1 alleles than A2. However, several other studies show a higher frequency of A2 alleles than A1. Bos indicus species in all studies have a higher frequency of A2 alleles than A1. Cows resulting from crossbreeding Bos taurus and Bos indicus also show a more dominant frequency of A2 alleles than A1. Differences in CSN2 Gene Genotypes with Milk Production and Milk Quality of FH Cows. This is due to differences in species of dairy cattle. Haq (2020) stated that Bos indicus species cows show a higher frequency of A2 alleles while Bos taurus and Bos indicus crossbreed cows show the presence of A1 and A2 alleles, but with a higher frequency of A2 alleles.

The results of this study differ from several other studies. The A2A2 genotype of the CSN2 gene of Holstein Slovak cattle (Miluchová et al., 2023) and FH Chinese cattle (Zhou et al., 2019) have associations with milk protein. Marko et al. (2020) also reported that the CSN2 gene has associations with milk production, but not with milk protein and fat for the A2A2 and A1A1 genotypes of the CSN2 gene. The results of this study are also likely influenced by the high coefficient of diversity and the number of samples being too small and not yet representative of the genotype. To improve the A2A2 genotype in Indonesian FH cattle, a more diverse sample analysis representing the Indonesian FH cattle population and genotyping analysis on Indonesian FH bulls are necessary. In addition, further research is needed on the effect of the A1A1 and A2A2 genotypes on Indonesian FH cattle calves.

Diversity is said to be low if the coefficient of diversity is below 5%; if the coefficient of variation is between 6-14%, it is said to be moderate, and high if the coefficient of variation is above 15% (Kurnianto, 2009). The higher the coefficient of variation of a variable indicates that the variable tends to vary and is less stable because it is greatly influenced by genetics and the environment (Hartati et al., 2010).

CONCLUSIONS AND RECOMMENDATIONS

The CSN2 exon-7 gene (rs43703011) in Indonesian FH is polymorphic, and the A2A2 genotype is found. The difference in the A1A1 and A2A2 genotypes of the CSN2 gene does not affect milk production, fat, protein, and lactose levels in Indonesian FH cattle. The role of the A2A2 genotype of the CSN2 gene needs further research as a candidate marker of milk production and quality, especially the low-lactose component in Indonesian FH cattle by increasing the population sample size.

ACKNOWLEDGMENTS

The author would like to thank the Ministry of Education, Culture, Research and Technology for funding the research through the Master’s Thesis Research (PTM) scheme with contact number 22256/IT3.D10/PT.01.03/P/B/2024.

NOVELTY STATEMENTS

Indonesian FH cattle were found to exhibit genotype diversity of A1A1 and A2A2 in the CSN2 gene.

AUTHOR’S CONTRIBUTIONS

All authors contributed equally to the manuscript.

Conflict of Interest

The author declares that we have no conflict of interest.

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