Productive Performance of the Dairy Cattle Girolando Breed Mediated by the Fat-Related Genes DGAT1 and LEP and Their Polymorphisms ⇑ S.R

Research in Veterinary Science xxx (2011) xxx–xxx

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Research in Veterinary Science

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Productive performance of the dairy cattle Girolando breed mediated by the fat-related genes DGAT1 and LEP and their polymorphisms ⇑ S.R. Cardoso, L.B. Queiroz, V. Alonso Goulart, G.B. Mourão, E. Benedetti, L.R. Goulart

Laboratório de Nanobiotecnologia, Universidade Federal de Uberlândia, Av. Amazonas, s/n, Bloco 2E, Uberlândia-MG, Brazil article info abstract

Article history: Candidate genes have been associated with milk production in bovines, such as the diacylglycerol Received 17 October 2010 O-acyltransferase 1 (DGAT1) and leptin (LEP); however, they have not been simultaneously investi- Accepted 12 February 2011 gated nor have been evaluated in the Brazilian Girolando breed (Gir Holstein, backcrossed to Available online xxxx Holstein). Our aim was to determine the influence of fat-related genes, DGAT1 and LEP, and their polymorphisms on performance traits of milk production in the Girolando breed. Results indicated Keywords: that the K allele of the DGAT1 gene showed a significant association with total and average daily milk Girolando breed production with additive effect. The LEP gene showed that the A allele and its homozygote are highly Bovine prevalent and almost fixed in this population and may have been favorably selected during backcross- DGAT1 LEP ing for the origin of this breed. The important impact of the K allele of the DGAT1 gene on milk Milk production production corroborates the initiative of performing marker-assisted selections with this gene in Polymorphisms breeding programs of the Girolando breed. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Selection of specific genotypes or alleles from candidate genes in bovines that possess economically favorable traits are advanta- Brazilian bovines breeds have shown low indices of productiv- geous (Williams, 2005) and among them, the diacylglycerol ity, which may be related to climate, nutrition or genetic inheri- O-acyltransferase 1 (DGAT1) and Leptin (LEP) genes have been con- tance. The increasing productive performance of bovines is a sidered of high interest for dairy cattle (Ripoli et al., 2006, 2011). common goal for many cattle breeding programs worldwide. In or- The DGAT1 gene expresses the protein Acyl-CoA Diacylglycerol der to promote a better adaptation of high yield bovine breeds to Acyltransferase, a microsomal enzyme integrating the membrane, adverse environments, Brazilian breeding programs have gener- which acts in the synthesis of triglycerides in animals (Kuhn ated new breeds from zebu and taurine crosses, such as, for exam- et al., 2004; Tantia et al., 2006). As stated by Smith et al. (2000), ple, the Brazilian breeds Girolando (Gir Holstein), Brangus the absence of the gene DGAT1 in some females, affects the synthe- (Nelore Angus) and Simbrasil (Guzera Simental). These breeds sis of Triacylglycerides, contributing to the reduction and/or have shown better adaptation to the hot and humid climate, boost- extinction of milk production in the animal. Furbass et al. (2006) ing dairy cattle productivity (Bicalho et al., 2006). have shown that the gene DGAT1, located in the terminal portion We have decided to investigate the variations of specific gene of the centromere referring to chromosome 14 in bovines, is prob- polymorphisms in the Girolando breed due to some extrinsic and ably a locus with a quantitative profile (QTL). Grisart et al. (2001); intrinsic factors linked to the environment and to the genetic back- Winter et al. (2002) and Roos et al. (2007) demonstrated that the ground, respectively. Javanmard et al. (2005) have explained that oscillation in the QTL profile is often caused by a substitution of both factors play a decisive role in the metabolism of the Girolando non-conservative dinucleotides ApA ? GpC in the exon 8 of the breed in terms of milk synthesis. Amongst these factors, it is worth gene DGAT1, changing from Lysine to Alanine in position 232 highlighting the feeding (Restle et al., 2005), type of management (mutation K232A) in the encoded protein. The increase in fat in (Cerdótes et al., 2004; Khatib et al., 2006; Oliveira and Nogueira, the milk in different breeds of bovine is intimately associated with 2006; López et al., 2007) and genotype (Smith et al., 2000; Lysine (K) in position 232 of the protein encoded by DGAT1 in Komisarek et al., 2004; Grisart et al., 2004; Khatib et al., 2006). bovines (Kaupe et al., 2004), while an Alanine (A) in this position is associated with a low fat content in milk (Winter et al., 2002; Kuhn et al., 2004). ⇑ Corresponding author. Address: Laboratório de Nanobiotecnologia, Instituto de The LEP gene expresses a 16-kDa protein synthesized by the Genética e Bioquímica, Universidade Federal de Uberlândia, Campus Umuarama, Av. Amazonas, s/n, Bloco 2E, Uberlândia-MG, Brazil. Tel./fax: +55 34 32182203. adipose tissue. Its function has been linked to energy homeostasis E-mail address: [email protected] (L.R. Goulart). (Bartha et al., 2005; Chilliard et al., 2005), fertility (Liefers et al.,

2002, 2005) and immunological properties of the herd (Fruhbreck mix contained 1 lL of genomic DNA, 0.5 U of Taq DNA polymerase et al., 1998; Lord et al., 1998). In bovines, the LEP gene is found on (Platinum, Invitrogen), 10 pmol of each primer, 200 lM of dNTPs, chromosome 4, which consists of 3 exons and 2 introns, although 1, 5 mM of MgCl2, 1X reaction buffer and water. The PCR reaction only 2 exons express the protein (Stone et al., 1996). Lindersson included an initial denaturation of 94 °C for 4 min, followed by et al. (1998) discovered a QTL for the production of milk near the 10 cycles of 60 s at 94 °C, 60 s at 66 °C(1°C.cycle1) and 1 min at LEP gene, at a distance of 82.8 centimorgans (cM). Various poly- 72 °C, and a further 25 cycles 1 min at 94 °C, 2 min at 56 °C and morphisms in the LEP gene, which influence milk production, 1 min at 72 °C, with a final extension of 10 min at 72 °C. reproduction and physiology of nutrients’ ingestion such as three For the LEP genotyping, a PCR product of 400 bp, located in the SNPs in exon 2 (Arg/Cy) and exon 3 (Ala/Val, Glu/Arg), other poly- intron 2 between exons 2 and 3, was used for PCR-RFLP and PCR- morphisms located in the promoter region and nine SNPs in the in- SSCP analyses. The sequences of primers were: sense 50-TGGAGTGGC tron 2, were also found in the bovine genome (Javanmard et al., TTGTTATTTTCTTCT-30 and antisense 50-GTCCCCGCTTCTGGCTACC 2010). Veerkamp et al. (2000), Alfonso (2005) and Williams TAACT-30 near the location of the polymorphism (Liefers et al., (2005) indicated the LEP as a candidate gene for marker-assisted 2002). A 20 lL reaction mix contained 1 lL of genomic DNA, selection, due to it is important physiological role, and its putative 0.5 U of Taq DNA polymerase (Platinum, Invitrogen), 10 pmol of association with the productive performance of dairy cattle, which each primer, 200 lM of dNTPs, 1, 5 mM of MgCl2, 1X reaction buf- was demonstrated elsewhere (Akers, 2006; Liefers et al., 2005). fer and water. The PCR reaction included an initial denaturation This investigation aimed determining the influence of polymor- step of 94 °C for 2 min, followed by 35 cycles of 94 °C, 55 °C and phisms of the two metabolic fat-related genes DGAT1 and LEP in 72 °C (1 min each) and finished with a final extension of 15 min the milk production and its components in the Girolando breed. at 75 °C. Two microlitres of each PCR product was added to 18 lL of Low Ionic Strength – LIS solution (10% sacarose, 0.01% Bromophenol 2. Materials and methods Blue and 0.01% Xylene cyanol) homogenized and heated at 95 °C for 12 min and immediately transferred to an ice bath and loaded 2.1. Sample collection onto a 12% polyacrylamide gel (49:1; acrylamide:bis-acrylamide). Electrophoresis was carried out at room temperature, 15 mA Blood samples were collected from 349 females Girolando (150 W, close to 1000 V) in 1X TBE buffer during 16 h. The PAGE breed (3/8 Gir + 5/8 Holstein). All the animals under analysis were was silver stained and dried, according to the Popescu (1993) milked twice a day. Due to differential frequencies of lactation, the adapted for a porous transparent cellophane paper fixation, per- lactation order greater than seven (8–11) were considered as sev- formed at room temperature for 12 h stretched on the surface of enth order, as registered by the Brazilian Association of Girolando a glass plate. Breeders (ABCG), the official dairy control program monitored by the Dairy Control Service of the Association (SCL). Feeding regimes were grouped into 1 = pasture with a regular mineralized salt sup- 2.3. Statistical analysis plement; 2 = pasture with greater mineralized supplementation and/or confined animals. For the total production, the linear effect The allelic and genotypic frequencies were calculated according of the lactation length (in days) as co-variable was also considered. to Weir (1996). Hardy–Weinberg equilibrium (HWE) for each gene. Information about the daily and annual production of milk, period The total milk production, daily milk production, lactation length of lactation of the cow, and birth intervals were provided by the and birth intervals were associated to the gene polymorphisms, SCL. using the following mixed model:

yijkðlÞmnop ¼ l þ Ri þ Gl þ dijkðlÞ þ SmðiÞ þ An þ Mo þ Op þ bðIijkðlÞmnop IÞþijkðlÞmnop 2.2. DNA extraction and genotyping

where: yijk(l)mnop = the breeding value; l = general mean; Ri = fixed Lymphocyte cells were isolated from PBMC samples by washing effects of the ith herd, com i = 1, 2, ..., 18; Gj = fixed effects of the them with 0.9% saline solution and precipitating them at 2500 rpm jth genotype; j =1,2,3;dijk(l) = random effect of the lth cow (with for 10 min. The lymphocyte cell pellet was washed three times l = 1, 2, ..., 350 to total milk yield; l = 1, 2, ..., 352 for daily milk yield with a red blood cell lysis buffer (5 mM Sucrose, 10 mM Tris–HCl and lactation length within kth bull (com k = 1, 2, ..., 136), in the jth 2 2 pH 7.5, 5 mM MgCl2, 1% Triton X-100) and once with nuclear lysis genotype and ith herd; assuming that dijk(l) N (0, Ir d), which Ir d buffer (10 mM Tris–HCl, 2 mM EDTA, pH 8.0, 400 mM NaCl). The is the variance and covariance matrices, considering residues’ inde- resulting pellet was incubated with a cellular lysis solution con- pendence; Sm(i) = fixed effect of the mth feeding regimes within the taining proteinase K (10 mg/mL) overnight at 37 °C. Lastly, DNA ith herd; m =1,2;An = fixed effect of the nth year of birth; n =1,2, was extracted from the cells using phenol–chloroform (Sambrook ..., 13 for the total production of milk and n = 1, 2, ..., 14 for the daily and Russel, 2001). milk yield and lactation length; Mo = fixed effect of the oth month of Genotyping of both DGAT1 and LEP was performed by conven- birth; o = 1, 2, ..., 12; Op = fixed effect of the pth birth order; p =1,2, tional PCR-RFLP, Polymerase Chain Reaction–restriction fragment ..., 7; b = linear coefficient (co-variable) associated with lactation length polymorphism as described elsewhere (Liefers et al., 2002) length (for the total production of milk); Iijk(l)mnop = lactation length and by PCR–SSCP (PCR – Single Strand Conformation Polymor- (for the total production of milk); I = lactation length mean (for the phisms) as described by Orita et al. (1989). The PCR–SSCP electro- total production of milk); eijk(l)mnop = random effect associated with phoretic patterns were compared with previous PCR–RFLP profiles lth cow within kth bull, in the jth genotype and ith herd in the pth 2 2 for both genes, from which genotypes and their frequencies were birth order; assuming eijk(l)mnop V (0, Vse ), which Vse is the vari- calculated. The PCR–SSCP was also used to verify if novel muta- ance and covariance matrices, considering residues’ dependence. tions were present in the same genomic region. The data were analyzed through a mixed model, adapted by For the DGAT1 genotyping, a 411-bp fragment located in the Buchanan et al. (2003), using the statistics software SAS version exon 8, which includes the K232A polymorphism, was used for 9.1.3 (System and SASÒ, 2005). This model also included the fixed PCR–RFLP and PCR–SSCP analyses. The sequences of primers effects of the year and the month in which lactation began, order of consisted of:sense 50-GCACCATCCTCTTCCTCAAG-30 and antisense lactation, feeding regimes on different cattle ranches and the geno- 50-GGAAGCGCTTTCGGATG-30 (Liefers et al., 2002). A 20 lL reaction type. Furthermore, the random effects of the bull (father of the

Figure 1. PCR–SSCP genotyping for genes DGAT1 (A) and LEP (B) performed in a 12% polyacrylamide (49:1, acrylamide:bis) gel electrophoresis carried out with 15 mA in 1X TBE buffer during 16 h at room temperature, followed by silver-staining. The genotypes are indicated at bottom of the gel.

Table 1 Estimated allelic and genotypic frequencies for the DGAT1 and LEP genes in Girolando cattle breed. Table 2 Genes Alleles Genotypes Additive and dominance effects of DGAT1 and LEP alleles on four productive performance traits in the dairy cattle Girolando breed. DGAT1 A K AA KA KK 0.46 0.54 0.19 0.54 0.27 Gene effects Total milk Daily milk Birth Lactation LEP A B AA AB BB production production intervals length 0.87 0.13 0.75 0.24 0.01 DF P > F DF P > F DF P > F DF P > F DGAT1 Additive 213 0.05⁄ 212 0.05⁄ 111 0.15 165 0.76 Dominant 239 0.41 227 0.52 133 0.35 167 0.34 cow), cow on the ranch and residue were taken into consideration. LEP Simultaneously, the structure of variable components, due to re- Additive 175 0.63 162 0.55 76.4 0.21 78.5 0.76 Dominant 169 0.23 155 0.19 79.8 0.23 76.6 0.56 peated measures on the same animal, was also taken into account. ⁄ For the total production, the linear effect of the lactation length (in DF = degrees of freedom; P > F (probability for the F test) = P < 0.05.

Figure 2. Regression analyses for allelic substitution for the DGAT1 gene polymorphisms (AA, KA, KK) considering the mean minimum squares of the total milk production and the average daily milk production in the Girolando breed.

3. Results Mean comparisons among genotypes for all four productive performance traits of the Girolando breed are presented in Table The PCR-SSCP technique was successful in substituting the 3. Mean values for the DGAT1 genotypes were significantly differ- PCR-RFLP analysis, and has easily discriminated all the genotypes ent for the total milk production and daily milk production average for both DGAT1 and LEP genes. The DNA banding pattern shown (p < 0.05). in the modified PAGE electrophoresis presented very specific In order to investigate possible interactions between genes, we profiles (Fig. 1). Genotypes were identified based on cross- have performed a simple combined analysis of frequencies, and matched profiles presented by their counterpart analysis with although individual genotypic frequencies for both genes follow PCR–RFLP, which evidenced the same banding patterns described the Hardy–Weinberg equilibrium, the combined genotypic fre- by Liefers et al. (2002, data not shown). No mutations or differ- quencies (Table 4) has shown a partial disequilibrium for specific ential band profiles were observed in PCR–SSCP; therefore, the genotypic interactions, specifically the AA (DGAT1) BB (LEP) that polymorphisms are the only ones within the genomic regions had no observations. Due to the very low frequencies of BB geno- investigated. However, our banding patterns for A and K alleles types, the sample size did not present statistical power to test did not match with profiles described elsewhere (Ripoli et al., genotypic interactions; however, it is interesting to emphasize that 2006), probably because of the many different parameters used, the most favorable DGAT1 genotypes (KA and KK) combined with such as: electrophoresis voltage, time, temperature, gel concen- the AA genotype of LEP presented a frequency of 0.62. tration, acrylamide:bis-acrylamide ratio, and the low-ionic strength buffer used for the amplicon denaturation (LIS–SSCP), which may have influenced in the conformational pattern of 4. Discussion and conclusions each single stranded DNA molecule. The allelic and genotypic frequencies for the DGAT1 and LEP The values found of DGAT1 gene were those that had been ex- genes are presented in Table 1. The DGAT1 gene frequency had a pected, as the Girolando breed results from the crossbreeding greater prevalence of the K allele (0.54), with KK and KA genotypes involving tropically adapted taurine and zebu breeds, with the lat- predominating in the population, with frequencies of 0.27 and ter presenting a high frequency of the K allele (Winter et al., 2002). 0.54, respectively. However, the LEP gene polymorphisms in this In the Holstein breed, a large spectrum of variations in the allelic breed was more restrict and due to the higher frequency of the A frequency of the DGAT1 gene in populations selected for dairy pro- allele (0.87), with very high frequencies for the AA (0.75) and AB duction can be found, with the frequency of the K varying from (0.24) genotypes. The genotypic frequencies of both genes (DGAT1 0.35 to 0.7, as reported by Grisart et al. (2001) and Spelman and LEP) are in agreement with the Hardy–Weinberg equilibrium (2002). Komisarek et al. (2004) found a frequency of 0.83 and for the population under study (p > 0.05). 0.71 for the K and A alleles respectively in the Jersey breed. A significant additive effect was observed for DGAT1 alleles, European breeds have high frequencies of the K allele, despite specifically for the total milk production and average daily milk being traditionally bred for meat production, and as such, this production (p < 0.05) (Table 2). It has also been shown that the ef- allele is considered an ancestral part of the haplotype of DGAT1. fect of A ? K allele substitution promotes an increase of 106.46 kg On the other hand, a low frequency of the K allele is found in pop- in the total milk production and 0.365 kg in the daily milk produc- ulations of the zebu breeds (Winter et al., 2002). In Indian Holstein tion average (Fig. 2). The LEP alleles and genotypes did not present Bulls the results indicated allelic frequency of K allele (0.59) was any significant impact in any of the four traits investigated higher compared to A allele (0.41) for DGAT1 (Patel et al., 2009). (p > 0.05). In relation to the frequency of alleles of the LEP gene similar results were related by Liefers et al. (2002) where only one homo-

Table 3 zygote animal for the B allele was found and Ripoli et al., (2011) Mean comparisons and their minimum mean square errors (MMSE) among DGAT1 where the frequencies among bovine breeds were 0.900, 0.100, and LEP genotypes for four productive performance traits in the dairy cattle Girolando and 0.00 for AA, AB e BB, respectively, (p < 0.21). Furthermore, breed. in the work of Liefers et al. (2002), cows with the genotype Average (MMSE)* Sau3IA-AB produced 1.32 kg/d more milk and consumed 0.73 kg/ Genotypes Total milk Daily milk Birth Lactation d more food compared to the SauAI-AA genotype, suggesting that production production intervals length the RFLP-B allele could obtain a greater production of milk without having a negative effect on the balance of energy and fertility of the DGAT1 AA 3621.2 (105.7)a* 13.2 (0.4)a 397.7 (12.7)a 278.9 (7.6)a animals. KA 3668.6 (79.5)ab 13.4 (0.3)ab 412.7 (10.2)a 285.1 (5.6)a This result stems from the exclusively additive effect of the KK 3834.1 (103.2)b 13.9 (0.3)b 413.5 (11.8)a 281.3 (7.2)a DGAT1 gene in the studied population. A number of studies have LEP associated the K allele with high production of fat in the milk of AA 3708.2 (69.7)a 13.5 (0.2)a 406.5 (8.6)a 284.72 (5.0)a dairy cattle, a tendency that is supported by the high frequency a a a a AB 3583.0 (91.3) 13.1 (0.3) 407.3 (10.4) 281.61 (6.4) of this allele in individuals from the Holstein breed selected for this BB 3857.0 (310.3)a 14.2 (1.1)a 374.6 (25.5)a 290.77 (19.8)a end (Komisarek et al., 2004). This behavior of the K variant of the * Averages followed by different letters within columns are statistically different polymorphism of the DGAT1 gene, was also observed in other (p < 0.05), according to Student’s t-test. breeds of dairy cattle, such as: the Holstein–Friesian (Grisart

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