Effect of DGAT 1 , leptin and TG gene polymorphisms on some milk production traits in different dairy cattle breeds in Hungary

The objective of this study was to estimate the effect of acylCoA-diacylglycerol-acyltransferase 1 (DGAT1), leptin and thyroglobulin (TG) loci on the milk fat, milk protein and milk yield in Holstein Friesian, Jersey and Hungarian Simmental cows. Leptin and DGAT1 genotypes were determined by qPCR assay, while TG genotypes were identified using the polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP). DGAT1 GC/GC cows had the highest 305-day milk yield values. Difference between AA/AA and GC/GC genotypes was significant (P<0.05). Leptin CC animals produced significantly higher 305-day milk protein percent values (P<0.05) than other genotypes in Hungarian Simmental breed. At TG locus TT cows showed the highest 305-day milk fat percent values, although differences between genotypes proved to be significant (P<0.05) only in Jersey breed.


Introduction
DGAT1 is a microsomal enzyme that catalyses the final step of triglyceride synthesis.A lysine/alanine (K232A) polymorphism in DGAT1 has been shown to affect milk fat content (Grisart et al. 2002).The lysine allele of DGAT1 gene has a positive effect on milk fat content in different cattle breeds (Winter et al. 2002, Strzałkowska et al. 2005).Spelman et al. (2002) investigated the effect of the DGAT1 polymorphism in different dairy breeds and significant differences were demonstrated for milk yield, as well as milk fat and protein content.Highly significant differences were observed for milk fat, milk protein and milk yield in Holstein-Friesian cows (Thaller et al. 2003, Bennewitz et al. 2004).Variable numbers of tandem repeat (VNTR) polymorphisms in the promoter region of the DGAT1 gene also seem to affect milk production traits (Kühn et al. 2004).Gautier et al. (2007) concluded that VNTR polymorphism in Normande and French Holstein breeds was responsible only for a small fraction of the variance of the QTL, while the K232A polymorphism had a large effect on milk production and milk composition.Sanders et al. (2006) reported about a specific allele of the DGAT1 promoter VNTR, which showed significant effects on the lactose content and milk energy content, compared with other alleles.
Leptin is the hormone product of the obese gene synthesised and secreted predominantly by adipocytes and its expression is regulated by body fatness and energy balance.This protein is supposed to be involved in the regulation of body weight by transmission of a lipostatic signal from adipocytes to the leptin receptor in the hypothalamus resulting in appetite suppression and increased thermogenesis (Zhang et al. 1994, Ji et al. 1998).The leptin gene has been mapped to bovine chromosome 4 (Stone et al. 1996).Polymorphisms in the leptin gene have been associated with milk yield in dairy cattle (Liefers et al. 2002).Animals with TT genotype of the R25C single nucleotide polymorphism in the bovine leptin gene showed increased milk production and milk protein yield compared with CC and CT genotypes, respectively (Buchanan et al. 2003).TT animals of the UASMS2 polymorphism in the bovine leptin promoter region could be associated with the highest marbling score, serum leptin concentration and backfat thickness (Nkrumah et al. 2005).
TG is a glycoprotein precursor of hormones that influence lipid metabolism and is synthesised in the thyroid gland.The effect of a C/T single nucleotide polymorphism (SNP) in the 5'-untranslated region of TG gene has been deduced to affect intramuscular fat content in cattle (Barendse 1999).TG and DGAT1 genes physically mapped to the centromeric region of the bovine chromosome 14 (Coppieters et al. 1998, Winter et al. 2002) and are separated by about 25cM (Moore et al. 2003, Thaller et al. 2003).Many studies mentioned in this chromosomal region a QTL effect on milk fat yield and milk fat percentage in dairy cattle (Khatkar et al. 2004).Based on previous QTL studies in different cattle breeds and on the impact of this gene on fat metabolism, Khatib et al. (2007) investigated the effect of the TG gene on milk production traits.Analysis of 29 sire families showed no significant association between TG variants and milk production traits.
The objective of this study was to estimate the effect of DGAT1, leptin and TG loci on some milk production traits in different dairy cattle breeds in Hungary.Considering that previous results for the possible use of the mentioned polymorphisms in selection to improve milk production traits are rather contradictory, it has been decided to carry out studies in different Hungarian dairy cattle populations with the aim to provide additional data to this particular subject.

Material and methods
1 236 blood samples were collected from Holstein Friesian (n=415), Jersey (n=340) and Hungarian Simmental (n=481) cows.Genomic DNA was isolated from whole blood (Zsolnai et al. 2003).Blood samples were stored at −20 °C until DNA extraction.Genotyping of the DGAT1 and leptin polymorphisms was performed using a TaqMan allelic discrimination method in a Rotor-Gene RG 3000 Real-Time PCR system (Corbett Research UK Ltd, Cambridge, UK).Primers and labeled oligonucleotide probes were designed based on the leptin sequence (UASMS2, C/T substitution at position 528 according to GenBank acc.no.AB070368): forward, 5'-AGG TGC CCA GGG ACT CA-3'; reverse, 5'-CAA CAA AGG CCG TGT GAC A-3'; FAM probe, 5'-CAA GCT CTA GAG CCT GTG T-3'; HEX probe, 5'-AAG CTC TAG AGC CTA TGT-3'.PCR cycling conditions were: 95 °C for 10 min, followed by 40 cycles of 95 °C for 7 s, 55 °C for 7 s and 72 °C for 15 s.For the DGAT1 sequence (SNPs are located in position 10 433 and 10 434 of the sequence under GenBank acc.no.AJ318490) primers and labeled oligonucleotide probes were designed as follows: forward, 5'-CGC TTG CTC GTA GCT TTG G-3'; reverse, 5'-CGC GGT AGG TCA GGT TGT C-3'; FAM probe, 5'-TTG GCC GCC TTA C-3'; HEX probe, 5'-CGT TGG CCT TCT TAC-3'.In this case PCR conditions were: 95 °C for 10 min, followed by 15 cycles of 94 °C for 20 s, 62 °C for 30 s and 72 °C for 30 s and 35 cycles of 94 °C for 20 s, 38 °C for 20 s and 72 °C for 20 s.TG polymorphism was detected using the PCR-RFLP method described by Barendse (1999).Digested PCR products were separated in 4 % Meta-Phor agarose gel (Rockland, ME, USA) in 1× TBE buffer and stained with ethidium bromide.As for each polymorphism about 5 % of samples were genotyped in duplicate and repeatability was in all cases perfect.Milk production data of the genotyped cows were collected throughout three consecutive lactations and statistical analyses have been carried out to find association between genotypes and milk production traits.

Statistical analysis
Dataset was analysed with SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA).Multivariate analysis of variance (general linear model, GLM) was applied to determine differences in milk production traits in case of all polymorphisms in all studied breeds.DGAT1, TG and leptin genotypes, birth year, number of lactations and calving season were included as fixed effects in mathematical models and 305-day-milk yield, 305-day-milk fat percentage and 305-daymilk protein percentage were considered as dependent variables.
So the formula of general linear model (GLM) was as follows in Hungarian Simmental breed: (1) Lepi × DGATk + Lep i ×TG j + e ijklmn can be written in Holstein-Friesian breed as follows: Finally GLM equation can be described in Jersey breed as follows: where y is the phenotypic record of the studied traits (e.g.milk %), μ is the general mean, Lep is the leptin hormone genotype (CC, TC, TT), TG refers to the TG polymorphism (CC, TC, TT), DGAT represents the effect of DGAT1 genotypes (AA/AA, AA/GC, GC/GC), birth year means the year of birth, calving no stands for the number of completed lactations, calving season refers to the season in which the given cow calved, farm o represents the farm effect and e is the residual error.Dominance effects were estimated as the deviation of mean values of the studied traits in heterozygotes from the mean of homozygotes, using the least square means.Additive effect was calculated as the half of the difference between the two homozygotes.Significant level of these factors was detected by the method of the least square difference (LSD).Variance in tables below represents the proportion of variation in the dependent variable explained by the regression model itself.

Results and discussion
With regard to herd genetic structure analyses, differences between the observed and expected genotype frequency values of DGAT1 genotypes were significant only in the Jersey breed (Table 1).The calculated χ 2 values for the leptin and TG genotypes indicated Hardy-Weinberg equilibrium in the population.The expected values are presented in brackets (df=2).
Concerning TG genotypes there was a good agreement in all breeds between the observed and expected frequency values (Table 3).The calculated least square means (LSM) and standard errors for milk production traits of the three breeds included in this study are presented in Table 4, 5 and 6.The expected values are presented in brackets (df=2).In all breeds DGAT1 GC/GC cows had the highest and AA/AA cows the lowest 305-day milk yield values.Contrasting the AA/AA genotype vs. GC/GC, the difference proved to be significant (P<0.05).There was a negative correlation between milk yield and milk fat percentage, so in case of 305-day milk fat percent a decreasing tendency could be observed from genotype AA/ AA to GC/GC.Differences between genotypes were significant (P<0.05).In respect of milk fat content, similar results were obtained by Winter et al. (2002) in Holstein-Friesian, Simmental and Braunvieh breeds, by Strzałkowska et al. (2005) in Polish Black-and-White (Friesian) cattle, and by Schennink et al. (2007) in Dutch Holstein-Friesian cows.Concerning 305-day milk fat percentage, estimated variances within dependent variables caused by DGAT1 genotypes in the Holstein-Friesian, Jersey and Hungarian Simmental breeds were 11.5, 8.2 and 9.2 %, respectively.Considering 305-day milk protein percent, AA/AA animals showed significantly higher values (P<0.05)than other genotypes.Effects of the DGAT1 K232A polymorphism on milk yield, fat and protein percentage are consistent with previous studies made by Grisart et al. (2002) and Spelman et al. (2002).As for leptin locus there was no demonstrable effect of the genotype on milk yield.Differences of means could not be proved to be significant in Holstein-Friesian and Jersey breeds, due to the small number of TT cows and the large standard error.Concerning Hungarian Simmental breed, leptin CC animals showed significantly higher 305-day milk protein percent values (P<0.05)than other genotypes.In this breed leptin TT cows realised the highest milk fat percentage values; the difference between genotypes was significant (P<0.05).Some previous studies have focused on associations between polymorphisms in the coding region of the leptin gene and milk production traits (Liefers et al. 2002, Buchanan et al. 2003).However there are no former results concerning the effect of promoter region UASMS2 polymorphism (present study) on milk production traits.
Referring to TG locus, TT cows produced in all breeds the highest 305-day milk fat percent values; in case of Jersey and Hungarian Simmental breeds differences between CC and TT genotypes were significant (P<0.05).Former analysis of 29 Holstein sire families showed no significant association between TG variants and milk production traits (Khatib et al. 2007).
Our previous research in Angus cattle (Anton et al. 2011) revealed that leptin and thyroglobulin TT animals could be associated with the highest intramuscular fat content values, similarly to the milk fat content in the current study.
The present study was carried out to test the possible association of three specific polymorphisms with milk production traits.An eventual marker assisted selection (MAS) approach -such as selecting for AA/AA genotypes at the DGAT1 locus -might be performed, if higher milk yield is preferred.In this case, simultaneous selection for leptin and thyroglobulin TT genotypes could increase milk fat and protein percentage as well.However, from public health point of view Schennink et al. (2007) recommend to increase alanin variant (GC/GC) of DGAT1 in the population by selective breeding (because of its association with more unsaturated milk-fat), reducing in this way saturated fatty acid intake of humans.

Table 1
Frequencies of DGAT1 genotypes in the studied breeds Referring to leptin genotypes, only the Jersey breed fit the Hardy-Weinberg equilibrium, in case of Holstein-Friesian and Hungarian Simmental breeds the calculated χ 2 values did not indicate Hardy-Weinberg equilibrium in the population (Table2).

Table 2
Frequencies of leptin genotypes in the studied breeds

Table 4
Least square means (LSM) and standard errors, variance, additive effect and dominance for 305-day milk yield, milk fat percent and milk protein percent in Holstein-Friesian cows Variance: percentage of variance due to the studied loci in the total phenotypic variance, a,b,c Different letters indicate significant difference between genotypes, *confidence level of the predicted factors (P<0.05)a,b different letters indicate significant difference between genotypes, *confidence level of the predicted factors (P<0.05),# percentage of variance due to the studied loci in the total phenotypic variance

Table 6
Least square means (LSM) and standard errors, variance, additive effect and dominance for 305-day milk yield, milk fat percent and milk protein percent in Hungarian Simmental cows Different letters indicate significant difference between genotypes, *confidence level of the predicted factors (P<0.05),# percentage of variance due to the studied loci in the total phenotypic variance