DGAT and LEP gene polymorphisms and their association with carcass characteristics and the lipid profile of meat from Nellore cattle

ABSTRACT The objective of this study was to evaluate the association of polymorphisms in the diacylglycerol acyltransferase ( DGAT ) and leptin ( LEP ) genes with the performance, carcass characteristics, meat quality, and lipid profile of Nellore cattle. A total of 100 intact male Nelore cattle were used to analyze the performance, carcass, physicochemical and centesimal composition, and fatty acid profile of beef. To identify the polymorphisms, the PCR–single-strand conformation polymorphism (SSCP) technique was applied to genomic DNA extracted from muscle tissue. The SSCP technique revealed the presence of four band patterns for the DGAT gene (AC, AD, AE and BB) with five alleles (A, B, C, D and E). For the LEP gene, five band patterns (AA, AB, AC, BB and BC) with three alleles (A, B and C) were observed. For the LEP gene, the AB genotype was associated with higher backfat thickness and ribs weight, while the BB genotype was associated with lower ribs yield; higher hindquarter yield was associated with AC and BB genotypes. Higher contents of C17:0, C18:0 and lower contents of C18:2ω6C, total polyunsaturated fatty acids, total ω6 and ratio of polyunsaturated and saturated fatty acids (PUFA/SFA) were verified for the AC genotype of the LEP gene. The AC and AA genotypes of the LEP gene were associated with higher means of C15:0 and C18:1ω9t. For the DGAT gene, the highest C24:0 content was associated with the AE genotype and the lowest with the AD and BB genotypes. Polymorphisms in the DGAT and LEP genes influence carcass parameters and the lipid profile of the meat of Nellore cattle.


ABSTRACT
The objective of this study was to evaluate the association of polymorphisms in the diacylglycerol acyltransferase (DGAT) and leptin (LEP) genes with the performance, carcass characteristics, meat quality, and lipid profile of Nellore cattle.A total of 100 intact male Nelore cattle were used to analyze the performance, carcass, physicochemical and centesimal composition, and fatty acid profile of beef.To identify the polymorphisms, the PCR-single-strand conformation polymorphism (SSCP) technique was applied to genomic DNA extracted from muscle tissue.The SSCP technique revealed the presence of four band patterns for the DGAT gene (AC, AD, AE and BB) with five alleles (A, B, C, D and E).For the LEP gene, five band patterns (AA, AB, AC, BB and BC) with three alleles (A, B and C) were observed.For the LEP gene, the AB genotype was associated with higher backfat thickness and ribs weight, while the BB genotype was associated with lower ribs yield; higher hindquarter yield was associated with AC and BB genotypes.Higher contents of C17:0, C18:0 and lower contents of C18:2ω6C, total polyunsaturated fatty acids, total ω6 and ratio of polyunsaturated and saturated fatty acids (PUFA/SFA) were verified for the AC genotype of the LEP gene.The AC and AA genotypes of the LEP gene were associated with higher means of C15:0 and C18:1ω9t.For the DGAT gene, the highest C24:0 content was associated with the AE genotype and

INTRODUCTION
Molecular genetics tools have been effecting at identifying polymorphisms at loci associated with the production of enzymes involved in the synthesis of fatty acids and study their possible associations with performance, carcass characteristics, and lipid profile in ruminants.Some polymorphisms affecting these characteristics have been described in the genes encoding the enzymes diacylglycerol acyltransferase (DGAT) and leptin (LEP) (Grisart et al., 2002;Taniguchi et al., 2002;Casas et al., 2005).Diacylglycerol acyltransferase catalyses the final phase of triglyceride synthesis that occurs in the endoplasmic reticulum membrane, affecting fat metabolism, including its production and percentage in milk (Cases et al., 1998(Cases et al., , 2001;;Silva et al., 2011).This enzyme is found in several tissues but has greater activity in the liver, adipose tissue, and lactating mammary gland (Fang et al., 2012).Polymorphisms in the DGAT gene have been associated with production characteristics, milk composition, subcutaneous fat deposition, and marbling in beef cattle (Casas et al., 2005;Wu et al., 2005;Tantia et al., 2006;Schennink et al., 2007).Leptin is a hormone encoded by the obesity gene and is expressed in adipocytes, mainly in white adipose tissue, but also in skeletal muscle, mammary epithelium, gastric epithelium, and placenta in smaller proportions (Chilliard et al., 2005).In cattle, LEP gene polymorphisms have been associated with carcass fat deposition (Lien et al., 1997;Pomp et al., 1997;Buchanan et al., 2002), ribeye area (Oprzadek et al., 2003), and growth characteristics and finishing precocity (Yang et al., 2007).Thus, due to its relevance, LEP has been studied as a candidate gene in studies related to characteristics of economic interest (Oprzadek et al., 2003;Shin & Chung, 2007;Corva et al., 2009), such as sexual precocity, percentage of milk fat, milk production, weight gain, carcass fat deposition, and meat quality (Lusk, 2007;Kulig & Kmiec, 2009;Lara et al., 2012).The objective of this study was to evaluate the association of polymorphisms in the DGAT and LEP genes with the performance, carcass characteristics, meat quality, and lipid profile of the meat of Nellore cattle.

MATERIAL AND METHODS
The experiment was conducted in the feedlot of the Frialto group, located 15 km from the city of Sinop, state of Mato Grosso (MT), Brazil.To perform this study, a population of 100 intact male Nellore animals was used, with an average initial weight of 386.19 ± 4.48 kg and slaughtered at a weight of 527.82 ± 17.96 kg at age 13-36 months.Cattle population numbers were selected from the contemporaneous group of animals, based on environmental conditions and feedlot management practices.The ingredients were supplied as complete feed for 88 days and were offered ad libitum.The diets were formulated to meet the requirements of 1.5 kg of daily weight gain, according to the NRC (2000).This study was approved by the Ethics Committee on Animal Use (CEUA) of UFLA under number 040/12.
To evaluate performance, the daily weight gain of the animals was measured, which corresponded to the difference between the initial and final weight divided by days in the feedlot (88 days).After slaughter and cooling of the carcasses for 24 hours at ± 1°C, cuts and linear evaluations of the carcasses were performed, and the following parameters were determined: backfat thickness (BF), ribeye area (Striploin) (REA), cold carcass weight, cold carcass yield, and weights and yields of cuts (forequarter, ribs, and hindquarter).The REA and BF measurements were taken in the left half carcass through a cut between the 10th and 11th ribs after 24 hours of cooling.To determine the REA, the contour of the longissimus thoracis muscle was drawn on tracing paper.This paper with the drawing of the muscle area was scanned in a scanner, and the area was analysed by ImageJ® software.The area was determined in cm 2 .The subcutaneous fat area was determined in millimetres (mm) using a digital calliper (DIGIMESS, China).After slaughter and cooling of the carcass for 24 hours at ± 1°C, samples of the longissimus thoracis muscle (Striploin) were collected to determine the physicochemical parameters and proximate composition.The final pH was determined 24 hours after slaughter in the Striploin cut of the left half-carcass using a pH metre with a penetration probe (Hanna Instruments,HI 99163,Romania).For this analysis, a colorimeter (Konica Minolta CM-700, Singapore) was used, operating in the CIEL*a*b* system, with a D65 illuminant, a 10° observer angle, and specular component excluded (SCE) measurement mode, to obtain the lightness (L*), redness (a*), and yellowness (b*) indices.The saturation index (C*) and hue angle (h*) values were determined according to Ramos & Gomide (2012).To determine the cooking loss (CL), the samples were weighed on a semi-analytical scale (METTLER M P1210, Toledo, Brazil), wrapped in aluminium foil, and cooked on an electric plate at 150°C until reaching 72°C inside of the sample, as described by Amasa (1978).The shear force was determined using a texture analyser with a Warner-Bratzler shear device (Chrystall & Devine, 1991).The proximate composition was determined by A.O.A.C. (1990).To analyse the fatty acid profile, samples of longissimus thoracis (Striploin) muscle tissue were collected from each animal.Lipid extraction followed the method of Folch et al. (1957), and the fatty acids were esterified according to Hartman & Lago (1973).Fatty acid analysis was performed by gas chromatography in a Shimadzu GC-2010 chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a flame ionization detector, split injector at a 1:50 ratio, and Supelco SPTM-2560 capillary column, 100 m × 0.25 mm × 0.20 μm (Supelco Inc., Bellefonte, PA, USA).The chromatographic conditions were an initial column temperature of 140°C/5 min, an increase of 4°C/min to 240°C, and a hold of 30 minutes, totalling 60 minutes.The injector and the detector temperature were 260°C.The carrier gas used was helium.The fatty acids were identified by comparing the retention times observed against those of the chromatographic standard SupelcoTM37 standard FAME Mix ® (Supelco Inc., Bellefonte, PA, USA) and are expressed as a percentage (%) of the total fatty acids identified.
Genomic DNA was extracted from samples of the longissimus dorsi muscle (Striploin) muscle following CTAB protocol for DNA extraction (Catonichexadecyl trimethyl ammonium bromide) described by Stefanova et al. (2013).The concentration and purity of the extracted DNA were quantified by reading the absorbance at 260 nm and 280 nm in a NanoDrop ND-1000 UV/Vis Spectrophotometer.The samples were diluted with sterile water to obtain the desired final concentration of 10 ng DNA/μL.Next, the integrity gel was prepared to ensure we only took excellent-quality genomic DNA for experiments.
To perform the DNA analysis, primers were designed for the evaluated genes DGAT (exons 16 and 17) (427 bp): forward (5'TCTTCCACGAGGTCAGTGC3') and reverse (5'GGCAAAGCAGTCCAACACC3'); and LEP (exon 2) (500 bp): forward (5'CTCTAGGGAAAGGCGGAGTC3') and reverse (5'CAGCCAGAAGCTCAGGTTTC3') using online software (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi)).First, the genetic sequences homologous to the selected genes were analysed using the Basic Local Alignment Search Tool software (https://blast.ncbi.nlm.nih.gov/Blast.cgi)against the sequences available in GenBank (http://www.ncbi.nlm.nih.gov/).Next, Oligo Perfect and Oligo Analyser software were used to design the primer pairs for each gene.Conventional polymerase chain reaction (PCR) was performed on 50 ng of genomic DNA in a final volume of 25 μl, containing 1× reaction buffer, 200 µM dNTPs, 1.25 U Taq DNA polymerase, 2.5 mM MgCl2, 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5 μM of each primer, and H2O.The PCRs were performed in a thermocycler (Mastercycler Eppendorf, USA) with the following thermal cycling profile: 5 minutes for initial denaturation at 95°C, 30 seconds for denaturation at 95°C, 30 seconds for annealing of primers (61°C and 60°C for DGAT and LEP, respectively), and 1 minute for extension at 72°C.The final extension was 2 minutes at 72°C.The PCR products were visualized by electrophoresis in a 1% agarose gel using TAE buffer and 1× staining with 200 ng/ml ethidium bromide.The amplified products from the different primers were subjected to electrophoresis in a 1.0% agarose gel in 1× TBE (45 mM Tris-borate, pH 8.0, and 1 mM EDTA) with 0.8 μg/mL ethidium bromide, and the resulting electrophoretic profiles were visualized in the gel and photo documented (Spectroline Ultraviolet Transilluminator).Gene mutations were analysed using the single-strand conformation polymorphism (PCR-SSCP) comparative method, in which 1 µL of each PCR product was added to 10 µL of denaturing buffer (98% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylenocyanol).After denaturation at 95°C for 5 minutes, the samples were immediately placed on ice and then loaded into a 12.5% acrylamide: bisacrylamide (37.5:1) gel.The samples were subjected to polyacrylamide gel electrophoresis in a horizontal vat (35×15) at 20°C in 0.5× TBE buffer for 6 hours for each gene, at 180 V, 150 mA, and 100 w.The gels were stained according to Byun et al. (2009).
Genotypic frequency and allele frequency were performed by direct counting in relation to the total polymorphisms identified in each gene (Ramalho et al., 2012).The data were analysed using the GLM procedure of the Statistical Analysis System (SAS 9.3, 2011, SAS Inst.Inc., Cary, NC).The age of the animals was considered a blocking variable, and the means were compared by Tukey's test (P=0.05) to evaluate the genotypes of each gene in relation to performance variables, carcass traits, physicochemical parameters, proximate composition, and fatty acid profile.

RESULTS
In this study, the DGAT and LEP genes were evaluated in Nellore cattle.The PCR products of these genes were subjected to gel electrophoresis, separating the DNA fragments according to size, i.e., DNA fragments of the same length formed a single band in the gel and were put directly into PCR-SSCP analysis.Four different genotypes of the DGAT gene were found: AE (n=12), AD (n=21), BB (n=58), and AC (n=7), with five different alleles (A, B, C, D, and E).Five different genotypes of the LEP gene were found: AA (n=12), AB (n=36), BB (n=36), BC (n=10), and AC (n=4), with three different alleles (A, B and C).The frequency of the genotypes and alleles of the DGAT and LEP genes are shown in Table 1.Statistical analysis revealed significant differences in lignoceric acid (C24:0) with DGAT genotype and in the pentadecanoic (C15:0), margaric (C17:0), stearic (C18:0), elaidic (C18:1ω9t), and linoleic (C18:2ω6c) fatty acids; PUFA; total ω6 fatty acids (∑ω6); and the PUFA/SFA ratio with LEP genotype (Table 3).The animals with the highest BF and RW had the LEP AB genotype, while the lowest values were found in the AA(BF) and BB(RW) genotypes.RY only differed under the BB genotype, which had lower means.The highest mean HY was found with the AC and BB genotypes, and the AB genotype had the lowest mean, Table 4.
The animals with the AA and AC genotypes of the LEP gene showed higher levels of pentadecanoic (C15:0) and elaidic (C18:1ω9t) fatty acids, while the animals with the AB and BB genotypes had lower values of margaric (C17:0), stearic (C18:0), and elaidic (C18:1ω9t) fatty acids.The animals with the AC genotype showed higher values of margaric (C17:0) and stearic (C18:0) fatty acids and lower values of conjugated linoleic acid (C18:2ω6c), PUFA, total ω6 fatty acids, and the ratio of PUFA/SFA.The BB and BC genotypes of the LEP gene had the highest values of conjugated linoleic acid (C18:2ω6c), PUFA, total ω6 fatty acids, and PUFA/SFA.In the DGAT gene, the animals with the highest lignoceric fatty acid values (24:0) had the AE genotype, while the lowest values were found in the AD and BB genotypes (Table 4).

DISCUSSION
The different genotypes of the DGAT gene showed no relationship with the carcass parameters of Nellore cattle.In fact, no associations between DGAT gene polymorphisms and any of the performance and carcass characteristics analysed in cattle have been reported (Casas et al., 2005;Fortes et al., 2009;Ardicli et al., 2019).The physicochemical parameters and proximate composition related to meat quality were also not associated with the polymorphisms in the LEP or DGAT gene.The same trend was observed by Casas et al. (2005) andFortes et al. (2009).This corroborates the results found in the present study, suggesting that the lack of association of the genes with carcass composition parameters and meat quality characteristics in Bos indicus cattle may be due to the genetic composition of the breed or to the polygenic effects associated with the expression of each trait (Ardicli et al., 2017).For LEP, the AB genotype showed higher BF and RW, while the BB genotype had lower RY.The AC and BB genotypes had higher HY.In the study by Nkrumah et al. (2004), evaluating polymorphisms in the LEP gene and their association with fat content in bovine carcasses using the PCRrestriction fragment length polymorphism (RFLP) technique, the authors observed that polymorphisms in the LEP gene showed associations with carcass fat content and carcass and lean meat yield, where the animals with the TT genotype had higher carcass fat content while the CT animals showed greater BF.However, TT animals had lower lean meat yield and yield grades  2016) observed that the CC genotype showed higher SFA and lower C18:1ω9 and C22:5ω3 fatty acids, while the CT genotype showed higher C22:5ω3 fatty acids and lower C18:1ω9 and MUFA.Conversely, the TT genotype showed higher C18:1ω9 and monounsaturated fatty acids and lower C22:5ω3 and SFA.The present study showed that animals with the AC, BB, and BC genotypes may provide meat with a better fatty acid profile for consumption.Animals with the AC genotype had a high amount of stearic acid (C18:0), which represents 43% of the SFA in meat and has a neutral function or even leads to lower cholesterol levels, since the animal metabolically transforms it into oleic acid (C18:1ω9).Likewise, the animals with the BB and BC genotypes had high levels of PUFA, the majority of which plays an important role in decreasing blood cholesterol (Spector, 1999).Similarly, these same animals showed higher amounts of omega-6 fatty acids, which are essential fatty acids and precursors of a set of eicosanoid substances, including thromboxanes, prostaglandins (which have hypotensive effects), prostacyclins (which inhibit platelet aggregation and increase highdensity lipoprotein), and leukotrienes (Wynder et al., 1997;Din et al., 2004;Barbosa et al., 2007).For the DGAT gene, an effect of genotype was only observed on the value of lignoceric acid (24:0).Urtnowski et al. (2011) evaluated the association between polymorphisms in the DGAT gene and characteristics related to meat production and quality in 156 Holstein cattle and found a relationship between DGAT exon 8 and lipid composition, with genotypes AA and GA showing higher values of conjugated linoleic acid (C18:2ω6t) and lower values of lauric acid (C12:0), while the animals with the GG genotype showed the opposite behaviour, higher lauric acid and lower conjugated linoleic acid.Tabaran et al. ( 2015) evaluated the occurrence of polymorphisms of this gene in 550 Holstein cattle and buffaloes in Romania and their effects on the fat percentage and fatty acid profile in milk, finding that DGAT gene polymorphisms resulted in increased production of C16:0 fatty acids and saturated/unsaturated fatty acid ratios and a reduction in the C14:0 and C18:0 values.However, there was no association of the DGAT gene polymorphisms with the other parameters of the lipid profile in the present study.This may be due to the genetic profile of the animals.The authors who found associations with DGAT gene polymorphisms studied dairy animals, and this effect may be less expressive in zebu beef cattle such as Nellore.
In spite of the positive results in this study, we recognize one limitation which was the small sample size and more studies should be carried out to confirm these results and to determine possible associations between these markers and other phenotypic traits in larger populations of Nelore Cattle.

CONCLUSIONS
The LEP gene polymorphisms affected carcass parameters and composition in relation to backfat thickness, ribs weight and yield, and hindquarter yield.DGAT and LEP gene polymorphisms influence beef lipid composition differently, with LEP polymorphisms having a greater effect on the fatty acid profile than DGAT polymorphisms.The performance, physicochemical and centesimal composition of the meat from nelore cattle, was not associated with polymorphisms of the LEP or DGAT genes.
made it possible to conduct the study by providing financial aid for projects and scholarships.v. 28, n. 3 p. 245, 1997.

REFERENCES
05); DGAT = genotype effect of the DGAT gene; LEP = genotype effect of the LEP gene; SFA = sum of total saturated fatty acids; MUFA = sum of total monounsaturated fatty acids; PUFA = sum of total polyunsaturated fatty acids; ∑ω3 = sum of total omega-3 fatty acids; ∑ω6 = sum of the total omega-6 fatty acids; SEM = standard error of the mean.
than CT and CC animals.Ardicli et al. (2017) investigated the effects of LEP gene polymorphisms on the fattening performance parameters and carcass traits of 81 Simmental cattle in Turkey and found LEP SNPs that had a significant effect on hot carcass weight, chilled carcass weight, and carcass yield (P<0.05).The animals with the TT genotype had higher hot carcass weight and chilled carcass weight than animals with the CC or CT genotype.The present study found an association between the LEP gene polymorphisms of Nellore cattle and their yield and fat deposition traits.Similar results were found by Carvalho et al. (2012), who evaluated allele and genotype frequencies of polymorphisms in the LEP gene (exon 2) in 201 crossbred cattle and observed that they had a direct association with backfat thickness.Regarding the lipid profile and its association with the LEP gene, two genotypes showed the opposite behaviour: The AC animals showed higher values of SFA (C 15:0, C17:0 and C18:0) and monounsaturated (C18:1ω9t) fatty acids and lower values of PUFA, ω6 (C18:2ω6c and ∑ω6), and PUFA/SFA ratio than the BB genotype.Kawaguichi et al. (2020) found an association of the CC genotype with higher values of C16:0 and SFA and lower values of C16:1, C18:1ω9, and MUFA.Conversely, the animals with the CT genotype showed higher values of C16:1, C18:1ω9, and MUFA and lower values of C16:0 and SFA (exon 2).Papaleo Mazzucco et al. (

Table 1 -
Frequency of the genotypes and polymorphism alleles according to the PCR-

Table 2 -
Statistical analysis of performance, carcass parameters, physicochemical and chemical composition parameters of the meat from Nellore cattle associated with the DGAT and LEP genes.

Table 3 -
Statistical analysis of the lipid profile of meat from Nellore cattle associated the DGAT and LEP genes.

Table 4 -
Breakdown of the parameters measured in the carcasses and the lipid profile of the meat (Striploin) of Nellore cattle according to the genotypes of the DGAT and LEP genes.