Slov Vet Res 2015; 52(4): 185-91 UDC 636.2.082:612.664:575.117+575.22
Original Scientific Article
EFFECTS OF DGAT1 AND GH1 POLYMORPHISM ON MILK YIELD IN HOLSTEIN COWS REARED IN TURKEY
Bilal Akyuz1*, Ozgecan Korkmaz Agaoglu3, Aytac Akcay2, Ali Reha Agaoglu4
1Department of Genetics, 2Department of Biometrics, Faculty of Veterinary Medicine, University of Erciyes, Kayseri, 3Department of Animal Science, 4Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, Burdur, Turkey
Corresponding author, E-mail: bakyuz@erciyes.edu.tr
Summary: The aim of this study was to analyse the associations among the K232A polymorphism in the diacylglycerol acyltransferasel (DGAT1) gene and L127V polymorphism in the bovine growth hormone (GH1) gene and milk yield in Holstein cows. A total of 281 Holstein cows from three different dairy cattle farms in the Burdur and Kayseri provinces of Turkey were included in this study. The PCR-RFLP method was used for DGAT1 and GH1 genotyping. The frequencies of genotypes and alleles of GH1 gene were found to be 0.78 for LL, 0.18 for LV and 0.04 for VV; 0.87 for the L allele and 0.13 for the V allele. The frequencies of genotypes and alleles of the DGAT1 gene were to be 0.61 for AA, 0.30 for KA and 0.09 for KK; 0.76 for the A allele and 0.24 for the K allele. No relations were found between DGAT1-K232A genotypes and the average milk yield in the first three lactations. However, an association between GH1-L127V polymorphism and the average milk yield of Holstein cows was found in the first three lactations. Cows with LL genotype had higher milk yield compared to LV and VV cows (P<0.05).
Key words: DGAT1; GH1; Holstein; milk yield; PCR-RFLP
Introduction
Estimation of the future performance of livestock is a crucial subject in farm animal breeding. Male and female animals with superior features should be selected to accelerate genetic improvement (1). The genetic potential of breeder candidates can be directly determined using the available molecular genetic methods (2). For this purpose, quantitative genetic methods have been used. Recent developments in the field of molecular
Received: 2 August 2014
Accepted for publication: 18 August 2015
biology have been used in livestock breeding and selection methods as an additional tool (3).
Selection programmes in dairy cattle breeding are basically aimed at milk production traits, milk components and fertility. These traits are known as polygenic traits, and they are controlled by numerous genetic loci and influenced by many environmental factors. Therefore, it is thought that candidate genes with close linkage of the encoding loci can be used to estimate milk production performance (4). Several polymorphisms in different gene loci have been noted to affect production traits such as milk yield and milk composition (2). For instance, it has been reported that there are
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strong association between acylCoA-diacylglycerol acyltransferasei (DGAT1) genotypes and milk traits (5); bovine growth hormone (GH1) genotypes and milk yield (6). Many genome scans have shown that QTLs on Bos taurus autosomal (BTA) chromosomes 6, 14, 20, and 26 have significant effects on milk yield traits (7). It is thought that the selection of animals with favourable genotypes of genetic markers may be possible, thus allowing rapid genetic progress in dairy cattle breeding. Particularly in the last few decades, with regard to this topic, several potential candidate genes have been recognised in cattle.
Triglyceride synthesis is catalysed by DGAT1. This enzyme plays a major role in intestinal fat absorption, lipoprotein synthesis, the development of adipose tissue and lactation in higher eukaryotes (8). The bovine DGAT1 gene, which is located on the centromeric end of BTA14, has been reported as a candidate gene for QTLs associated with fat content and milk yield (5, 9, 10,11).
Bovine growth hormone directly or indirectly affects many physiological processes such as lactation, growth and reproduction (12). Therefore, it is thought that GH1 can be considered as a genetic marker of milk productivity in cattle (13). Previous studies have shown that there might be an association between allelic variants of the GH1 gene and milk yield traits (14). Several polymorphisms were found in the GH1 gene (1, 4). The best known of these polymorphisms is the leucine (L allele) to valine (V allele) substitution at position 127 in exon 5 of the GH1 gene. The effects of L127V polymorphism on milk production in cattle have been studied to some extent but the results obtained by various researchers have been found to be contradictory (1, 15).
The Holstein is the most commonly reared dairy cattle breed (16) and 92% of annual milk production is obtained from imported breeds such as Holstein, Simmental and Brown Swiss and their crossbreeds in Turkey. The association among GH1 and DGAT1 gene polymorphisms and milk yield has been studied in different countries (1, 11, 12). However, according to the authors' knowledge, no study has as yet been conducted on the association among GH1 and DGAT1 polymorphisms and milk yield in the Holstein breed in Turkey.
The aim of this study was to analyse the association of GH1 and DGAT1 gene polymorphisms with average (1., 2. and 3. lactations) milk yield in a Turkish Holstein-Friesian cow population.
Materials and methods
PCR-RFLP assay for DGAT1 and GH1
genotypes
A total of 281 Holstein cows, in the third lactation, were used in this study. The DNA was extracted from whole blood using phenolchloroform method (17).
For detection of DGAT1 genotypes, a 411 bp DNA fragment was amplified by PCR. PCR was carried out in a 25^L volume containing 5 pmol of each primer (forward: 5'-GCACCATCCTCTTCCTCAAG-3'; reverse: 5'-GGAAGCGCTTTCGGATG-3') (Genbank no. AJ318490.1) 1.5 mM MgC^, 200 ^M dNTP mix, 1 X PCR buffer, 1U Taq polymerase and 100 ng of genomic DNA template. PCR included the following steps: pre-denaturation of 95 oC/5 min followed by 35 cycles at 94 oC/1 min, 60 oC/1 min, 72 oC/1 min and final extension at 72 oC/10 min. Within the DGAT1 gene the non-conservative polymorphisms K232A at position 10433 and 10434 in exon 8 giving rise to a lysine by alanine amino acid substitution. The amplified PCR products were digested using the CfrI (MBI Fermentas) enzyme. The PCR mixture for the GH1 gene was prepared in the same way as for DGAT1, and a 223 bp DNA fragment was amplified. PCR products were amplified using forward 5'-GCTGCTCCTGAGGGCCCTTCG-3' and reverse 5'-GCGGCGGCACTTCATGACCCT-3' (Genbank no. JQ711182.1) primers. The cycles applied were as follows; pre-denaturation at 95 oC/4 min, followed by 35 cycles at 94oC/40sec, 60 oC/40 sec, 72oC/40 sec and the final extension at 72 oC/5 min. Amplified PCR products were digested with the Alul (MBI Fermentas). Polymorphism is the leucine (L allele) to valine (V allele) substitution at position 127 in exon 5 of the GH1 gene.
Data sets and statistical analysis
Data for daily milk production in the first three lactations were obtained from the Cattle Breeders' Association of Burdur and Kayseri. Direct counting was used to estimate the genotype and allele frequencies of the DGAT1 gene CfrI and GH1 gene AluI genetic variants. The chi-square test (x2) was used to check whether the populations were in Hardy-Weinberg equilibrium using PopGene32 software (18). Mean differences of milk yield among genotype groups were assessed by analysis
Effects of DGAT1 and GH1 polymorphism on milk yield in Holstein cows reared in Turkey
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Table 1: Allelic and genotypic frequencies of DGAT1 and GH1 genes
Loci	Frequency		Genotype		Allele Frequency		Statistical Significant (Chi-squared HWE)
		AA	KA	KK	A allele	K allele	
DGAT1	Observed Expected	172 162.2	83 102.6	26 16.2	0.76	0.24	X2 = 10.23 P<0.01 (df=1)
		LL	LV	VV	L allele	V allele	
GH1	Observed Expected	219 212.7	51 63.5	11 4.7	0.87	0.13	X2 = 10.91 P<0.001 (df=1)
HWE: Hardy-Weinberg Equilibrium; df: degree of freedom
Table 2: Means and standard error of mean (SEM), coefficient of variation of milk yields (average daily milk yield in the first three lactations) in Holstein cows with different DGAT1 and GH1 genotypes
Loci	Genotype	N	Mean ± SEM (liter)	%V	Statistical Significance (ANOVA)
	AA	172	26.55± 0.39	19.2	
DGAT1	KA	83	25.64± 0.55	19.6	F: 1.73 P>0.05
	KK	26	27.60± 0.93	17.2	
ß= 0.20	P= 0.966	R2 =	0.001		
	LL	219	26.79± 0.33a	18.4	
GH1	LV	51	24.60± 0.69b	20.0	F: 3.95 P<0.05
	VV	11	26.43± 2.03ab	25.4	
ß= -1.18	P= 0.042	R2 =	0.015		
a,b,c : Different superscripts within the same column demonstrate significant differences; %V: Coefficient of variation; p: Regression coefficient; R2: Determination coefficient
30,00"
o
ib 25,00" <T>
Figure 1: Error Bar graph with 95% Confidence interval (CI) of milk yields for GH1 genotypes in Holstein cows
Average
Lactation number
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of variance and Sidak multiple comparisons test as post hoc test. The effects on the average milk yield of GH1 and DGAT1 genotypes were estimated using regression analysis. The software SPSS for Windows Version 14.01 (License number: 986964) was used in statistical analysis data.
Results
PCR amplification yielded a 411 bp long DGAT1 gene fragment. Restriction digestion of 208 and 203 bp PCR products with the CfrI enzyme revealed three genotypes of AA (208 and 203 bp), KK (411 bp) and KA (411, 208 and 203 bp). PCR amplification yielded a 223 bp long GH1 gene fragment. Restriction digestion of 171 and 52 bp PCR products with the AluI enzyme revealed three genotypes of LL (171 and 52 bp), VV (223bp) and LV (223, 171 and 52 bp). The allelic and genotypic frequencies of the DGAT1 and GH1 genes, and the polymorphisms for the Holstein cows are given in Table 1. Significant deviation was observed from HWE in the Holstein breeds on DGAT1 (P<0.01) and GH1 (P<0.001) genes.
Our findings revealed that no-significant difference was found among cows with different DGAT1 genotypes (AA, AK, KK) in terms of average milk yield per day (P>0.05). The LL and VV genotypes of the GH1 gene were better than the LV genotype for average daily milk yield (Table 2) (P<0.05). Additionally it was found that animals who carry the LL genotype had a higher, and more homogeneous milk yield than those with the other two genotypes (Figure 1). On the other hand, it was found that VV genotype cows show a similar feature together with LL and LV genotypes in terms of daily milk yield, but VV genotype cows showed a higher variation coefficient (25%) (Table 2).
In this study, regression analysis was also performed and the regression coefficient values of genotypes were calculated. The effect of DGAT1 genotypes on average milk yield was not found significant (p=0.966). The regression coefficient (R2) value of the DGAT1 gene was found to be very low (<0.001) in the Holstein cows studied. On the other hand, the effects of GH1 genotypes on milk yield were found to be significant, and the regression coefficient (R2) value was found as 0.015.
Discussion
In this study, due to their potential roles in milk yield, the GH1 and DGAT1 genes were studied to evaluate their effects on milk yield in Holstein cows.
The frequency of the GH1-V allele was found to be lower than that of the L allele; this has been generally reported in all Holstein populations (12, 15). Similarly, in this study, the frequency of the GH1-V allele was found to be lower than that of the L allele. It was reported that the highest frequency of the L allele was found in larger sized dairy breeds such as Holstein, and this allele was also correlated with higher milk production (19). In addition, it was reported that selection for milk yield in the German Holstein cattle population has been indirectly effective in the spreading of the GH1-L allele. Hence, the frequency of the V allele was found to be very low in German Holstein sires (12). Similarly, the GH1 -L allele frequency was also found to be higher than that of the V allele in our study. The high GH1-L allele frequency in the examined Holstein cattle may have caused the deviation from HWE. On the other hand, it is aimed to increase the milk yield in Holstein breeding in Turkey for many years. Hence, it may lead to HWE in terms of GH1-L allele in the Turkish Holstein population. It is thought that this selection in GH1 gene may cause deviation from HWE in DGAT1 gene as well. In addition, Thaller et al. (11) reported that DGAT1-A allele frequency was found to be higher than that of the K allele and was fixed in German Holstein cattle population. This information may be an explanation of the deviation from HWE for GH1 and DGAT1 genes in the Holstein cows examined in our study, because an important part of the Turkish Holstein cattle population originated from Germany and the US (20). Similarly, DGAT1-A allele frequency was also found to be considerably higher than that of the K allele in our study. On the other hand, the deviation from HWE for GH1 and DGAT1 genes may be due to the number of animal population used in this study.
It was reported that there was a significant association between the GH1-L127V genotypes and milk production traits in different cattle breeds (13, 21). Nevertheless, this information is still controversial. For example, Zwierzchowski et al. (22) reported that the presence of the V allele
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may be indicative of better performance in daily milk yield and milk composition in Holstein cows. Similarly, V allele frequency was reported to be very common in best Canadian Holstein AI bulls by Sabour and Lin (23). Kovacs et al. (1) found that the V allele was preferred for increased milk production traits in a Hungarian Holstein cattle population. Khatami et al. (13) investigated the relationship between GH1 genotypes and milk production in a Russian Holstein cattle population and the Yaroslavl cattle breed, and they found that the rate of cows with high milk production (more than 6000 kg per lactation) was higher in cows with the VV genotype than in those with other genotypes. This rate was 1.75 times higher in cows with the VV genotype than in cows with LL and LV genotypes, respectively. The V allele was reported by Khatami et al. (13) to be superior commercially to the L allele, especially when present in homozygote form. On the other hand, there are studies indicating that the LV genotype is superior to other genotypes for milk yield. For instance, in Polish Holstein bulls and heifers, significant differences were reported between GH1 genotypes by Grochowska et al. (24), and the highest values for milk and protein yields were observed for cows with the LV genotype. Similarly, Kovacs et al. (1) reported that the GH1 -LV genotype compared to other genotypes was shown to have a positive effect on 305 days lactation yield. Furthermore, there have also been studies in which the L allele was associated with high milk yield. For instance, the LL genotype of the GH1 gene was significantly associated with better milk production traits, mainly with fat content in the Brown Swiss breed (6). Furthermore, cows with the LL genotype were shown to have a higher milk yield than GH1-LV cows in the first lactation. No significant differences between the genotypes and milk production traits in the second and third lactations in Polish Holstein cows were found by Dybus (15). In this study, the LL genotype of the GH1 gene was significantly better for milk yield than the other two genotypes in Turkish Holstein cows.
DGAT1 is another gene which is thought to be associated with milk yield in cattle, and this gene has two alleles. The DGAT1-K allele has been associated with high milk fat yield (25), the DGAT1-A allele has been associated with high milk yield (5, 11).
The milk fat yield has become a desired characteristic in dairy cattle breeding in recent years. This situation may have been caused by
an increase in DGAT1 -K allele frequency in some populations. As a result of selection to increase milk yield, the frequency of the DGAT1-K allele decrease from 15 to 5% between 1981 and 1990 in Israel Holstein cows (26).
The milk yield of daughters of bulls with DGAT1-AA genotypes was found on average to be 548 kg higher than that of bulls with DGAT1-KK genotypes in the Holstein breed (12). Similarly, the DGAT1-A allele was found to have economically beneficial effects in German Holstein across all lactations. However, it was reported that this allele showed better performance in Fleckvieh cattle in only the first lactation (11).
On the other hand, there have been studies in which no relationship between DGAT1 genotypes and milk yield existed. For instance, Naslund et al. (27) found no differences among the three DGAT1 genotypes and milk yield in Swedish Holsteins. Similarly, we did not find any relationship between DGAT1 alleles and milk yield in Holsteins in our study. To estimate the possible effects of DGAT1 alleles on milk yield traits more studies should be conducted in dairy cattle breeds.
Milk yield is a multifactorial trait, and milk yield traits have been shown to be primarily and considerably influenced by environmental factors such as farm environment, management and feeding. Therefore, the observed differences between the GH1 and DGAT1 genotypes and milk production characteristics could have resulted from another source of variation such as the effects of herd and sire. The effects of the GH1 and DGAT1 genotypes on the milk yield, milk fat and milk protein content should be confirmed in further studies. However, it is not yet possible to say which genotypes of GH1 and DGAT1 should be recommended for the improvement of milk production traits.
In terms of herd management in farms, high and uniform milk yield is very important. It was found in this study that LL genotype cows show uniformity in terms of daily milk yield, whereas VV genotype cows have a higher variation coefficient. The study showed that GH1 may be used in selection programmes in dairy cattle breeding. The crucial role of growth hormone in lactation initiation and maintenance is well known, thus GH1 polymorphisms and interaction with other yield traits should be the subject of further research. Furthermore, DGAT1 polymorphism was not found to have a significant effect on milk yield.
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VPLIVI POLIMORFIZMA DGAT1 IN GH1 NA MLEČNOST KRAV PASME HOLSTEIN, GOJENIH V TURČIJI
B. Akyuz, O. K. Agaoglu, A. Akcay, A. R. Agaoglu
Povzetek: Namen raziskave je bil proučiti povezave med polimorfizmom K232A v genu za diacilglicerol acyltransferaso 1 (DGAT1) in polimorfizmom L127V v genu govejega rastnega hormona (GH1) ter mlečnostjo krav holstein. Skupno je bilo v raziskavo vključenih 281 prvesnic in krav pasme holstein v drugi laktaciji s treh različnih mlečnih govedorejskih kmetij v provincah Burdur in Kayseri v Turčiji. Za genotipizacijo DGAT1 in GH1 je bila uporabljena metoda PCR-RFLP. Frekvence genotipov in alelov gena GH1 so bile 0,78 za LL, 0,18 za LV, 0,04 za VV, 0,87 za alel L in 0,13 za alel V. Frekvence genotipov in alelov gena DGAT1 so bile 0,61 za AA, 0,30 za KA, 0,09 za KK, 0,76 za alel A in 0,24 za alel K. Med genotipi DGAT1-K232A in povprečno mlečnostjo v prvih treh laktacijah niso bile ugotovljene povezave. Ugotovljena pa je bila povezava med polimorfizmom GH1-L127V in povprečno mlečnostjo krav pasme Holstein v prvih treh laktacijah. Pri kravah z genotipom LL je namreč mlečnost višja kot pri kravah z genotipoma NN in VV (p <0,05).
Ključne besede: DGAT1;GH1; holstein; proizvodnja mleka; PCR