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Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
Association Study Between a SNP in Bovine SCD1 Gene with Fatty Acid Composition in a Spanish Commercial Population Fed with Two Different Diets a
b
a
c
d
C. Avilés , O. Polvillo , F. Peña , A. Horcada , M. Juárez & A. Molina
a
a
MERAGEM Research Group, Veterinary Faculty , University of Cordoba , Cordoba , Spain
b
CITIUS , Sevilla , Spain
c
Department of Agroforestry Science , University of Sevilla , Sevilla , Spain
d
Agriculture and Agri-Food Canada , Lacombe Research Center , Lacombe , Canada Published online: 25 Aug 2014.
To cite this article: C. Avilés , O. Polvillo , F. Peña , A. Horcada , M. Juárez & A. Molina (2015) Association Study Between a SNP in Bovine SCD1 Gene with Fatty Acid Composition in a Spanish Commercial Population Fed with Two Different Diets, Animal Biotechnology, 26:1, 40-44, DOI: 10.1080/10495398.2014.880712 To link to this article: http://dx.doi.org/10.1080/10495398.2014.880712
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Animal Biotechnology, 26:40–44, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2014.880712
Association Study Between a SNP in Bovine SCD1 Gene with Fatty Acid Composition in a Spanish Commercial Population Fed with Two Different Diets C. Avile´s,1 O. Polvillo,2 F. Pen˜a,1 A. Horcada,3 M. Jua´rez,4 and A. Molina1 1
MERAGEM Research Group, Veterinary Faculty, University of Cordoba, Cordoba, Spain CITIUS, Sevilla, Spain 3 Department of Agroforestry Science, University of Sevilla, Sevilla, Spain 4 Agriculture and Agri-Food Canada, Lacombe Research Center, Lacombe, Canada
Downloaded by [Nipissing University] at 03:31 08 October 2014
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Stearoyl-CoA desaturase (SCD1 gene) is an enzyme responsible for the endogenous conversion of saturated fatty acid into monounsaturated fatty acids. The objective of this study was to assess the association of a single nucleotide polymorphism (SNP) in the SCD1 gene with the fatty acid composition of beef intramuscular fat of a Spanish commercial bull population (n ¼ 155) finished with two different diets. The results suggested that the marker could be used as a candidate gene to obtain a healthier final product. Keywords Association study; Bos taurus; Fatty acid composition; Molecular markers; SNP; Stearoyl CoA-desaturase
described. The objective of the present study was to assess this association with the fatty acid composition of the beef IMF of a Spanish commercial bull population finished with two different diets.
Fatty acid profile contribute importantly to various aspects of meat quality and are central to the nutritional value of meat (1). However, the concern regarding benefits and adverse effect of the dietary fat and fatty acid intake is a matter of current interest among the different population groups (age, sex, and different nutritional necessities). Several molecular mechanisms regulating the synthesis and assembly of fatty acids are still scarcely studied but many authors agree that the putative atherogenic saturated fatty acids (SFA) are the C12:0, C14:0, and C16:0 (2). Stearoyl-CoA desaturase (SCD1), also known as D9desaturase, plays a key role in the conversion of SFA into monounsaturated fatty acids (MUFA) as well as in the polyunsaturated fatty acid (PUFA) production: C18:2 cis-9, trans-11 from C18:1 trans-11 and C18:2 trans-7, cis9 from C18:1 trans-7 (3). A single nucleotide polymorphism (SNP) in the position 878 of a DNA fragment of this gene (Accession # AB075020) has been published to be associated with beef fatty acid profile (4). Although the diet used in the finishing period of animals is crucial for intramuscular fat (IMF) of the meat, the genetic component of the animal is involved (to a lesser extent) in the final expression of this kind of traits. The association of this marker previously described with the fatty acid composition of beef of the common cattle populations produced in the south-western Iberian Peninsula has not been thus far
MATERIAL AND METHODS To carry out the experiment 155 bulls were allocated to two different feedlots with two different finishing diets (Table 1): a Total Mixed Ration (TMR) composed by concentrate plus mineral-vitamins, maize silage and wheat straw (n ¼ 73) and a Conventional Ration (CR) made of concentrate and wheat straw provided separately (n ¼ 83). The animal management, blood sample collection, and DNA analysis have been previously described (5). In order to determine the fatty acid composition, longissimus dorsi muscle (between the 6th thoracic and the 6th lumbar vertebra) was removed from the left carcass side at 24 hours postmortem as per the procedure reported by Insausti et al. (6) and sliced into 2-cm thick steaks and trimmed of any external fat. Total fatty acids were extracted, methylated and analyzed by an adaptation of the method described by Aldai et al. (7). Separation and quantification of the fatty acid methyl esters was carried out using a gas-chromatograph (GC, Agilent 6890 N, Agilent Technologies Spain, S.L., Madrid, Spain) equipped with a flame ionization detector automatic sample injector HP 7683, and using a HP-88 J&W fused silica capillary column (100 m, 0.25 mm i.d.,0.2-mm film thickness, Agilent Technologies Spain, S.L., Madrid, Spain). Individual fatty acids were expressed as the percentage of total fatty acids
Address correspondence to C. Avile´s, MERAGEM Research Group, Veterinary Faculty, Dpto. de Produccio´n Animal, Campus de Rabanales, Universidad de Co´rdoba 14071, Cordoba, Spain. E-mail: [email protected]
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SNP IN BOVINE SCD1 LINKED TO FATTY ACID PROFILE
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TABLE 1 Chemical composition of the finishing diets
Ingredients Barley meal (g=kg) Maize meal (g=kg) Whole soybean (g=kg) Corn silage (g=kg) Wheat bran (g=kg) Hydrogenate oil (g=kg) Palm oil (g=kg) Wheat Straw (g=kg) Mineral-vitamin Chemical composition DM (%) Crude protein (g=kg DM) Crude fibre (g=kg DM) Ash (g=kg DM) Fat (g=kg DM) ME (MJ=kg)
Conventional ration
Total mixed ration
322.7 360.0 140.2
247.9 198.9 98.4 318.3
120.0 12.5 12.5 32.1
19.9 99.5 17.1
88.6 143.3 41.6 58.6 49.8 10.6
66.1 93.8 78.9 41.0 34.0 7.2
Abbreviations: DM, Dry matter; ME, Metabolizable energy.
identified and grouped as follows: SFA, MUFA, and polyunsaturated (PUFA). The C14, C16, and C18 indexes were calculated as follows: C14 index ¼ C14:1 cis-9=(C14:0 þ C14:1 cis-9) 100; C16 index ¼ C16:1 cis-9=(C16:0 þ C16:1 cis-9) 100; C18 index ¼ C18:1 cis-9=(C18:0 þ C18:1 cis-9) 100. Finally, the MUFA=SFA ratio was estimated and a Health index (HI) proposed by Li et al. (8) as HI ¼ (total MUFA þ total PUFA)=(4 C14:0 þ C16:0). The counts of the genotype for each diet and overall population were calculated as it was described previously (5). To assess the Hardy Weinberg equilibrium, the population was analyzed using the exact probability test of Genepop (9). A Chi-squared test was carried out to establish if there were significant differences (P < 0.05) between the allelic and genotypic frequencies of each diet. The data to compare the percentage of the main fatty acids in beef related to the apparent activity of the enzyme, ratio and indexes for each genotype of the SCD1 gene were tested using a general linear model through the GLM procedure of SAS (SAS Version 9.2, SAS Institute, Cary, NC, USA). The statistic model was as follows: yij ¼ l þ Di þ SCD1j þ ðD SCD1Þij þ bSW þ eij where yij ¼ phenotypic observation, l ¼ general mean of the trait, Dj ¼ fixed effect of the diet, SCD1j ¼ fixed effect of the genotype of the marker, bSW ¼ linear effect of slaughter weight as covariate, and eij ¼ residual error. Interaction
between diet genotype was included. Mean comparison was carried out using the LSMEANS option using F-test protected LSD (P < 0.05). Quadratic contrasts were conducted to further explore the additive effect of the genotype on the traits. A linear regression on the number of hypothetic favorable alleles (0, 1, and 2) with models including diet as a fixed factor and slaughter weight as a linear covariate was used to estimate the average allele substitution effects. The contribution of the marker to the variation of the individual and groups of fatty acids observed was estimated as the reduction of the residual variance when the full model was compared with the reduced model without the effect of the SCD1 genotype. RESULTS AND DISCUSSION As it was previously reported, the beef content of fatty acids is highly influenced by the composition of the diet. In the present study, the difference between diets in the experiment affected the fatty acid composition of the meat in the population studied (Table 2). Significant effects of diet (P < 0.05) was detected in three individual fatty acids: C18:0, C16:1 cis-9, and C18:1 trans-11; two groups of fatty acid: SFA and MUFA; MUFA=SFA ratio; and three indexes: C14, C16, and C18 index. The TMR was poorer in roughage and cereals than the CR (Table 1); consequently, the amount of unsaturated fatty acids of this second diet is higher than the quantity in the former (10). TABLE 2 F-test probability for diet, genotype effects, and interactions on each trait tested in the Spanish commercial beef population (P-values) Diet C14:0 C16:0 C18:0 C14:1 cis-9 C16:1 cis-9 C18:1 cis-9 C18:1 trans-11 CLA cis-9, trans-11 SFA MUFA PUFA C14 index C16 index C18 index MUFA=SFA Health index
Marker Feedlot Marker
1.77n.s. 2.82t 0.06n.s. 1.59n.s. 11.56 0.58n.s. 1.55n.s. 5.77 5.74 0.10n.s. t 3.74 1.88n.s. 16.45 0.00n.s. n.s. 0.19 0.95n.s. 9.63 3.02t 7.12 1.79n.s. n.s. 1.17 0.21n.s. 13.29 23.18 7.93 0.02n.s. 7.91 1.11n.s. 11.12 3.15 n.s. 1.97 3.27
1.20n.s. 3.56 0.06n.s. 0.95n.s. 0.92n.s. 1.50n.s. 1.93n.s. 0.09n.s. 1.80n.s. 1.47n.s. 2.95n.s. 0.04n.s. 0.38n.s. 0.92n.s. 0.50n.s. 2.44n.s.
Significance level: ns: P > 0.1; t: P < 0.1; P < 0.05; P < 0.001.
P < 0.01;
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In ruminants, the biohydrogenation of unsaturated fatty acids performed by the microorganisms of the rumen is responsible for the variations in fatty acid composition of the IMF. However, the PUFA proportion of phospholipids is strictly controlled by an endogenous complex enzymatic system (11) where the SCD1 is included. Therefore, the final composition of fatty acids reveals the action of SCD1 on some of the main saturated fatty acids stored in the fat depots such as C16:0 or C18:0 (12). In this study, the effect of the SCD1 polymorphism was significant (P < 0.05) in the C14:1 cis-9, in the C14 index, in the MUFA=SFA ratio, and in the HI. The contribution to the variance of the effects ranged from 0.20% for the HI to 2.13% for the C14 index. Moreover, a tendency toward significance (P < 0.1) was also observed in the effect of the marker on the C14:0 and SFA. No significant departures from Hardy–Weinberg equilibrium were identified for the SCD1 SNP. The counts of the genotypes (CC, CT, and TT) are in parenthesis in Table 3. The Chi-squared test did not indicate differences in the allelic or genotypic frequency distributions among diets and the overall population. The frequency distributions were not different to those previously reported by Avile´s et al. (5) in a larger population of the common breeds reared in the South-west of the Iberian Peninsula. Taniguchi et al. (4) initially described the association of the SCD1 c.878C>T SNP with MUFA percentage and melting point in fat tissue of Japanese Black cattle. In our results, the CR provided to the animals, contributed to the accumulation of unsaturated fatty acids such as
C16:1 cis-9, C18:1 trans-11, or the total amount of MUFA (Table 3) and this was reflected in the MUFA=SFA ratio; in contrast, levels of saturated fatty acids such as C18:0 and total SFA were higher in animals fed with the TMR. Regarding the effect of the SCD1 marker, the CC genotype of the SNP was particularly associated to differences related to the C14 fatty acid, presenting higher values of C14:1 cis-9 (P < 0.01) and C14 index (P < 0.001). The approximate estimation of the SCD1 activity made by this desaturation index suggested that in individuals with CC and CT genotype, the enzyme is more active compared with those with TT genotype. Moreover, this genotype seemed to be linked to lower amounts of SFA (P < 0.01) and, as a consequence, higher MUFA=SFA ratio. These results are consistent with those previously reported by Bartonˇ et al. (13) in Fleckvieh bulls, Li et al. (14) in two Spanish paternal breeds, Li et al. (8) in Canadian Angus and Charolais-based commercial crossbred beef steers and Ohsaki et al. (15) in Japanese Black cattle. Supporting this association, significant differences were also observed for the HI. The CC and CT genotypes presented higher values for the index than the TT genotype (P < 0.05) for individuals fed with the CR. This fact becomes important as the C14:0 acid was identified as the most atherogenic (16), with approximately four times the cholesterol-raising potential of C16:0. The only significant interaction (P < 0.05) between diet and genotype was observed for C16:0 (Fig. 1). Animals inheriting the TT genotype and fed with the CR presented higher levels (P < 0.05) of C16:0 regarding CC or CT genotype. This effect showed
TABLE 3 Least-square means of the marker and average allele substitution effect for each suggestive association in the Spanish commercial beef population tested Total mixed ration
C14:0 C16:0 C18:0 C14:1 cis-9 C16:1 cis-9 C18:1 cis-9 C18:1 trans-11 SFA MUFA C14 index C16 index C18 index MUFA=SFA Health index x
CC (23)
CT (34)
2.20 0.12 26.20 0.42 18.96 0.53 0.45 0.03 2.36 0.15 28.31 0.84 1.05 0.08 49.66 0.62 33.48 0.99 16.73 0.57a 8.20 0.42 59.63 1.24 0.68 0.02 1.46 0.05
2.21 0.07 26.10 0.25 19.19 0.31 0.42 0.02 2.40 0.09 29.32 0.50 1.15 0.05 49.66 0.37 34.61 0.59 15.65 0.34b 8.34 0.25 60.36 0.74 0.70 0.13 1.46 0.03
TT (16)
Conventional ration ax
2.34 0.10 26.05 0.35 19.20 0.44 0.37 0.02 2.38 0.12 27.64 0.70 1.20 0.06 49.87 0.52 32.80 0.83 13.54 0.48c 1.40 0.51 8.30 0.35 58.83 1.04 0.66 0.02 1.43 0.04
CC (16)
CT (42)
2.03 0.06b 2.14 0.08b 25.97 0.21b 25.61 0.16b 17.66 0.33 18.203 0.25 0.50 0.02a 0.42 0.02b 2.65 0.11 2.52 0.08 30.94 0.75 29.71 0.58 1.46 0.10 1.37 0.08 47.92 0.37b 48.18 0.29b 37.16 0.85 35.48 0.66 18.89 0.54a 17.09 0.42b 9.21 0.34 8.91 0.25 63.55 0.93 61.78 0.72 0.78 0.02a 0.74 0.02b a 1.52 0.03 1.54 0.02a
TT (24)
a
2.33 0.09a 0.09 0.06t 26.72 0.24a 0.40 0.17 18.27 0.37 0.40 0.03b 0.05 0.02 2.67 0.12 29.04 0.86 1.32 0.11 49.83 0.42a 0.98 0.30 34.79 0.97 14.58 0.62c 2.22 0.51 9.06 0.37 61.21 1.06 0.70 0.02c 0.03 0.01 1.39 0.03b 0.06 0.02
a : Average allele substitution effect SE. Means bearing different superscript (in row) differ significantly (P < 0.05) or present a tendency toward signification (P < 0.1). Significance levels: ns: P > 0.1. t: P < 0.1. P < 0.05. P < 0.01. P < 0.001.
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SNP IN BOVINE SCD1 LINKED TO FATTY ACID PROFILE
FIG. 1. Interaction between SCD1 genotype and diet for the C16:0 fatty acid. a-c: Different letters within diet indicate significant differences among genotypes. The amount of C16:0 is expressed as the percentage of total fatty acids. a different pattern when the animals were fed with the TMR. The C16:0 is the most abundant saturated fatty acid in meat and, together with C12:0 and C14:0, is considered unhealthy because of its atherogenic properties (2). Therefore, this interaction suggests a management strategy based in the feeding scheme avoiding the CR for animals that inherit the TT genotype in order to obtain a healthier beef product. With respect to the C18:0, C18:1 cis-9 and CLA cis-9, trans-11, no significant effects of the marker on the fatty acid composition were found. The C18:0 is used to assess the nutritional implications of IMF on human diet through the desirable fatty acids estimation (MUFA þ PUFA þ C18:0) described by Huerta-Leidenz et al. (17). The C18:1 cis-9 and CLA cis-9, trans-11 also play an important role on human health (18). However, according to our results the SNP was not useful to estimate the amount of these favorable fatty acids. To sum up we can conclude that, in spite of the population size, different genotypes of SCD1 c.878C>T SNP presented a demonstrated effect on the fatty acid composition of the IMF of beef in Spanish common cattle populations. The results suggested that this marker could be used as a predictor of the nutritional quality of this meat to obtain a healthier final product. FUNDING The authors would like to thank CICAP for the coordination work and COVAP, CTA, and IDEA agency for their financial support. REFERENCES 1. Wood JD, Enser M, Fisher AV, et al. Fat deposition, fatty acid composition and meat quality: A review. Meat Sci 2008; 78(4):343–358.
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2. Ulbricht TL, Southgate DA. Coronary heart disease: seven dietary factors. Lancet 1991; 338(8773):985–992. 3. Palmquist DL, Lock AL, Shingfield KJ, Bauman DE. Biosynthesis of conjugated linoleic acid in ruminants and humans. In: Steve LT, ed. Advances in Food and Nutrition Research. San Diego: Academic Press; 2005; 179–217. 4. Taniguchi M, Utsugi T, Oyama K, et al. Genotype of stearoyl-CoA desaturase is associated with fatty acid composition in Japanese Black cattle. Mammalian Genome 2004; 15(2):142–148. 5. Avile´s C, Polvillo O, Pen˜a F, Jua´rez M, Martı´nez AL, Molina A. Associations between DGAT1, FABP4, LEP, RORC, and SCD1 gene polymorphisms and fat deposition in Spanish commercial beef. J Anim Sci 2013; 91(10): 4571–4577. 6. Insausti K, Beriain MJ, Alzueta MJ, Carr TR, Purroy A. Lipid composition of the intramuscular fat of beef from Spanish cattle breeds stored under modified atmosphere. Meat Sci 2004; 66(3):639–646. 7. Aldai N, Osoro K, Barron LJ, Najera AI. Gas-liquid chromatographic method for analysing complex mixtures of fatty acids including conjugated linoleic acids (cis-9 trans-11 and trans-10 cis-12 isomers) and long-chain (n-3 or n-6) polyunsaturated fatty acids. Application to the intramuscular fat of beef meat J Chromatogr A 2006; 31:1–2. 8. Li C, Aldai N, Vinsky M, Dugan MER, McAllister TA. Association analyses of single nucleotide polymorphisms in bovine stearoyl-CoA desaturase and fatty acid synthase genes with fatty acid composition in commercial cross-bred beef steers. Anim Gen 2012; 43(1):5. 9. Raymond M, Rousset F. GENEPOP (Version 1.2): Population Genetics Software for Exact Tests and Ecumenicism. J Hered 1995; 86(3):248–249. 10. van Ackeren C, Steingaß H, Hartung K, Funk R, Drochner W. Effect of roughage level in a total mixed ration on feed intake, ruminal fermentation patterns and chewing activity of earlyweaned calves with ad libitum access to grass hay. Anim Feed Sci Technol 2009; 153(1–2):48–59. 11. Raes K, De Smet S, Demeyer D. Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: a review. Anim Feed Sci Technol 2004; 113(1–4): 199–221. 12. Kim YC, Ntambi JM. Regulation of stearoyl-CoA desaturase genes: role in cellular metabolism and preadipocyte differentiation. Biochem Biophys Res Commun 1999; 266(1):1–4. ˇ eha´k D, Zahra´dkova´ R, 13. Bartonˇ L, Kott T, Bures D, R Kottova´ B. The polymorphisms of stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes and their association with the fatty acid profile of muscle and subcutaneous fat in Fleckvieh bulls. Meat Sci 2010; 85(1):15–20. 14. Li C, Vinsky M, Aldai N, Osoro K, Dugan MER. Associations of a bovine SCD SNP with fatty acid composition in concentrate-finished yearling bulls of different muscular hypertrophy genotype. Proceedings of 9th World Congress on Genetics Applied to Livestock Production (WCGALP); 2010; Leipzig, Germany 505;2010.
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15. Ohsaki H, Tanaka A, Hoashi S, et al. Effect of SCD and SREBP genotypes on fatty acid composition in adipose tissue of Japanese black cattle herds. Anim Sci J 2009; 80(3):225–232. 16. Hegsted DM, McGandy RB, Myers ML, Stare FJ. Quantitative effects of dietary fat on serum cholesterol in man. Am J Clin Nutr 1965; 17:281–296.
17. Huerta-Leidenz NO, Cross HR, Lunt DK, Pelton LS, Savell JW, Smith SB. Growth, carcass traits, and fatty acid profiles of adipose tissues from steers fed whole cottonseed. J Anim Sci 1991; 69(9):3665–3672. 18. Pariza MW, Park Y, Cook ME. The biologically active isomers of conjugated linoleic acid. Prog Lipid Res 2001; 40(4):283–298.
Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
Association Study Between a SNP in Bovine SCD1 Gene with Fatty Acid Composition in a Spanish Commercial Population Fed with Two Different Diets a
b
a
c
d
C. Avilés , O. Polvillo , F. Peña , A. Horcada , M. Juárez & A. Molina
a
a
MERAGEM Research Group, Veterinary Faculty , University of Cordoba , Cordoba , Spain
b
CITIUS , Sevilla , Spain
c
Department of Agroforestry Science , University of Sevilla , Sevilla , Spain
d
Agriculture and Agri-Food Canada , Lacombe Research Center , Lacombe , Canada Published online: 25 Aug 2014.
To cite this article: C. Avilés , O. Polvillo , F. Peña , A. Horcada , M. Juárez & A. Molina (2015) Association Study Between a SNP in Bovine SCD1 Gene with Fatty Acid Composition in a Spanish Commercial Population Fed with Two Different Diets, Animal Biotechnology, 26:1, 40-44, DOI: 10.1080/10495398.2014.880712 To link to this article: http://dx.doi.org/10.1080/10495398.2014.880712
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Animal Biotechnology, 26:40–44, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2014.880712
Association Study Between a SNP in Bovine SCD1 Gene with Fatty Acid Composition in a Spanish Commercial Population Fed with Two Different Diets C. Avile´s,1 O. Polvillo,2 F. Pen˜a,1 A. Horcada,3 M. Jua´rez,4 and A. Molina1 1
MERAGEM Research Group, Veterinary Faculty, University of Cordoba, Cordoba, Spain CITIUS, Sevilla, Spain 3 Department of Agroforestry Science, University of Sevilla, Sevilla, Spain 4 Agriculture and Agri-Food Canada, Lacombe Research Center, Lacombe, Canada
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Stearoyl-CoA desaturase (SCD1 gene) is an enzyme responsible for the endogenous conversion of saturated fatty acid into monounsaturated fatty acids. The objective of this study was to assess the association of a single nucleotide polymorphism (SNP) in the SCD1 gene with the fatty acid composition of beef intramuscular fat of a Spanish commercial bull population (n ¼ 155) finished with two different diets. The results suggested that the marker could be used as a candidate gene to obtain a healthier final product. Keywords Association study; Bos taurus; Fatty acid composition; Molecular markers; SNP; Stearoyl CoA-desaturase
described. The objective of the present study was to assess this association with the fatty acid composition of the beef IMF of a Spanish commercial bull population finished with two different diets.
Fatty acid profile contribute importantly to various aspects of meat quality and are central to the nutritional value of meat (1). However, the concern regarding benefits and adverse effect of the dietary fat and fatty acid intake is a matter of current interest among the different population groups (age, sex, and different nutritional necessities). Several molecular mechanisms regulating the synthesis and assembly of fatty acids are still scarcely studied but many authors agree that the putative atherogenic saturated fatty acids (SFA) are the C12:0, C14:0, and C16:0 (2). Stearoyl-CoA desaturase (SCD1), also known as D9desaturase, plays a key role in the conversion of SFA into monounsaturated fatty acids (MUFA) as well as in the polyunsaturated fatty acid (PUFA) production: C18:2 cis-9, trans-11 from C18:1 trans-11 and C18:2 trans-7, cis9 from C18:1 trans-7 (3). A single nucleotide polymorphism (SNP) in the position 878 of a DNA fragment of this gene (Accession # AB075020) has been published to be associated with beef fatty acid profile (4). Although the diet used in the finishing period of animals is crucial for intramuscular fat (IMF) of the meat, the genetic component of the animal is involved (to a lesser extent) in the final expression of this kind of traits. The association of this marker previously described with the fatty acid composition of beef of the common cattle populations produced in the south-western Iberian Peninsula has not been thus far
MATERIAL AND METHODS To carry out the experiment 155 bulls were allocated to two different feedlots with two different finishing diets (Table 1): a Total Mixed Ration (TMR) composed by concentrate plus mineral-vitamins, maize silage and wheat straw (n ¼ 73) and a Conventional Ration (CR) made of concentrate and wheat straw provided separately (n ¼ 83). The animal management, blood sample collection, and DNA analysis have been previously described (5). In order to determine the fatty acid composition, longissimus dorsi muscle (between the 6th thoracic and the 6th lumbar vertebra) was removed from the left carcass side at 24 hours postmortem as per the procedure reported by Insausti et al. (6) and sliced into 2-cm thick steaks and trimmed of any external fat. Total fatty acids were extracted, methylated and analyzed by an adaptation of the method described by Aldai et al. (7). Separation and quantification of the fatty acid methyl esters was carried out using a gas-chromatograph (GC, Agilent 6890 N, Agilent Technologies Spain, S.L., Madrid, Spain) equipped with a flame ionization detector automatic sample injector HP 7683, and using a HP-88 J&W fused silica capillary column (100 m, 0.25 mm i.d.,0.2-mm film thickness, Agilent Technologies Spain, S.L., Madrid, Spain). Individual fatty acids were expressed as the percentage of total fatty acids
Address correspondence to C. Avile´s, MERAGEM Research Group, Veterinary Faculty, Dpto. de Produccio´n Animal, Campus de Rabanales, Universidad de Co´rdoba 14071, Cordoba, Spain. E-mail: [email protected]
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SNP IN BOVINE SCD1 LINKED TO FATTY ACID PROFILE
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TABLE 1 Chemical composition of the finishing diets
Ingredients Barley meal (g=kg) Maize meal (g=kg) Whole soybean (g=kg) Corn silage (g=kg) Wheat bran (g=kg) Hydrogenate oil (g=kg) Palm oil (g=kg) Wheat Straw (g=kg) Mineral-vitamin Chemical composition DM (%) Crude protein (g=kg DM) Crude fibre (g=kg DM) Ash (g=kg DM) Fat (g=kg DM) ME (MJ=kg)
Conventional ration
Total mixed ration
322.7 360.0 140.2
247.9 198.9 98.4 318.3
120.0 12.5 12.5 32.1
19.9 99.5 17.1
88.6 143.3 41.6 58.6 49.8 10.6
66.1 93.8 78.9 41.0 34.0 7.2
Abbreviations: DM, Dry matter; ME, Metabolizable energy.
identified and grouped as follows: SFA, MUFA, and polyunsaturated (PUFA). The C14, C16, and C18 indexes were calculated as follows: C14 index ¼ C14:1 cis-9=(C14:0 þ C14:1 cis-9) 100; C16 index ¼ C16:1 cis-9=(C16:0 þ C16:1 cis-9) 100; C18 index ¼ C18:1 cis-9=(C18:0 þ C18:1 cis-9) 100. Finally, the MUFA=SFA ratio was estimated and a Health index (HI) proposed by Li et al. (8) as HI ¼ (total MUFA þ total PUFA)=(4 C14:0 þ C16:0). The counts of the genotype for each diet and overall population were calculated as it was described previously (5). To assess the Hardy Weinberg equilibrium, the population was analyzed using the exact probability test of Genepop (9). A Chi-squared test was carried out to establish if there were significant differences (P < 0.05) between the allelic and genotypic frequencies of each diet. The data to compare the percentage of the main fatty acids in beef related to the apparent activity of the enzyme, ratio and indexes for each genotype of the SCD1 gene were tested using a general linear model through the GLM procedure of SAS (SAS Version 9.2, SAS Institute, Cary, NC, USA). The statistic model was as follows: yij ¼ l þ Di þ SCD1j þ ðD SCD1Þij þ bSW þ eij where yij ¼ phenotypic observation, l ¼ general mean of the trait, Dj ¼ fixed effect of the diet, SCD1j ¼ fixed effect of the genotype of the marker, bSW ¼ linear effect of slaughter weight as covariate, and eij ¼ residual error. Interaction
between diet genotype was included. Mean comparison was carried out using the LSMEANS option using F-test protected LSD (P < 0.05). Quadratic contrasts were conducted to further explore the additive effect of the genotype on the traits. A linear regression on the number of hypothetic favorable alleles (0, 1, and 2) with models including diet as a fixed factor and slaughter weight as a linear covariate was used to estimate the average allele substitution effects. The contribution of the marker to the variation of the individual and groups of fatty acids observed was estimated as the reduction of the residual variance when the full model was compared with the reduced model without the effect of the SCD1 genotype. RESULTS AND DISCUSSION As it was previously reported, the beef content of fatty acids is highly influenced by the composition of the diet. In the present study, the difference between diets in the experiment affected the fatty acid composition of the meat in the population studied (Table 2). Significant effects of diet (P < 0.05) was detected in three individual fatty acids: C18:0, C16:1 cis-9, and C18:1 trans-11; two groups of fatty acid: SFA and MUFA; MUFA=SFA ratio; and three indexes: C14, C16, and C18 index. The TMR was poorer in roughage and cereals than the CR (Table 1); consequently, the amount of unsaturated fatty acids of this second diet is higher than the quantity in the former (10). TABLE 2 F-test probability for diet, genotype effects, and interactions on each trait tested in the Spanish commercial beef population (P-values) Diet C14:0 C16:0 C18:0 C14:1 cis-9 C16:1 cis-9 C18:1 cis-9 C18:1 trans-11 CLA cis-9, trans-11 SFA MUFA PUFA C14 index C16 index C18 index MUFA=SFA Health index
Marker Feedlot Marker
1.77n.s. 2.82t 0.06n.s. 1.59n.s. 11.56 0.58n.s. 1.55n.s. 5.77 5.74 0.10n.s. t 3.74 1.88n.s. 16.45 0.00n.s. n.s. 0.19 0.95n.s. 9.63 3.02t 7.12 1.79n.s. n.s. 1.17 0.21n.s. 13.29 23.18 7.93 0.02n.s. 7.91 1.11n.s. 11.12 3.15 n.s. 1.97 3.27
1.20n.s. 3.56 0.06n.s. 0.95n.s. 0.92n.s. 1.50n.s. 1.93n.s. 0.09n.s. 1.80n.s. 1.47n.s. 2.95n.s. 0.04n.s. 0.38n.s. 0.92n.s. 0.50n.s. 2.44n.s.
Significance level: ns: P > 0.1; t: P < 0.1; P < 0.05; P < 0.001.
P < 0.01;
C. AVILE´S ET AL.
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In ruminants, the biohydrogenation of unsaturated fatty acids performed by the microorganisms of the rumen is responsible for the variations in fatty acid composition of the IMF. However, the PUFA proportion of phospholipids is strictly controlled by an endogenous complex enzymatic system (11) where the SCD1 is included. Therefore, the final composition of fatty acids reveals the action of SCD1 on some of the main saturated fatty acids stored in the fat depots such as C16:0 or C18:0 (12). In this study, the effect of the SCD1 polymorphism was significant (P < 0.05) in the C14:1 cis-9, in the C14 index, in the MUFA=SFA ratio, and in the HI. The contribution to the variance of the effects ranged from 0.20% for the HI to 2.13% for the C14 index. Moreover, a tendency toward significance (P < 0.1) was also observed in the effect of the marker on the C14:0 and SFA. No significant departures from Hardy–Weinberg equilibrium were identified for the SCD1 SNP. The counts of the genotypes (CC, CT, and TT) are in parenthesis in Table 3. The Chi-squared test did not indicate differences in the allelic or genotypic frequency distributions among diets and the overall population. The frequency distributions were not different to those previously reported by Avile´s et al. (5) in a larger population of the common breeds reared in the South-west of the Iberian Peninsula. Taniguchi et al. (4) initially described the association of the SCD1 c.878C>T SNP with MUFA percentage and melting point in fat tissue of Japanese Black cattle. In our results, the CR provided to the animals, contributed to the accumulation of unsaturated fatty acids such as
C16:1 cis-9, C18:1 trans-11, or the total amount of MUFA (Table 3) and this was reflected in the MUFA=SFA ratio; in contrast, levels of saturated fatty acids such as C18:0 and total SFA were higher in animals fed with the TMR. Regarding the effect of the SCD1 marker, the CC genotype of the SNP was particularly associated to differences related to the C14 fatty acid, presenting higher values of C14:1 cis-9 (P < 0.01) and C14 index (P < 0.001). The approximate estimation of the SCD1 activity made by this desaturation index suggested that in individuals with CC and CT genotype, the enzyme is more active compared with those with TT genotype. Moreover, this genotype seemed to be linked to lower amounts of SFA (P < 0.01) and, as a consequence, higher MUFA=SFA ratio. These results are consistent with those previously reported by Bartonˇ et al. (13) in Fleckvieh bulls, Li et al. (14) in two Spanish paternal breeds, Li et al. (8) in Canadian Angus and Charolais-based commercial crossbred beef steers and Ohsaki et al. (15) in Japanese Black cattle. Supporting this association, significant differences were also observed for the HI. The CC and CT genotypes presented higher values for the index than the TT genotype (P < 0.05) for individuals fed with the CR. This fact becomes important as the C14:0 acid was identified as the most atherogenic (16), with approximately four times the cholesterol-raising potential of C16:0. The only significant interaction (P < 0.05) between diet and genotype was observed for C16:0 (Fig. 1). Animals inheriting the TT genotype and fed with the CR presented higher levels (P < 0.05) of C16:0 regarding CC or CT genotype. This effect showed
TABLE 3 Least-square means of the marker and average allele substitution effect for each suggestive association in the Spanish commercial beef population tested Total mixed ration
C14:0 C16:0 C18:0 C14:1 cis-9 C16:1 cis-9 C18:1 cis-9 C18:1 trans-11 SFA MUFA C14 index C16 index C18 index MUFA=SFA Health index x
CC (23)
CT (34)
2.20 0.12 26.20 0.42 18.96 0.53 0.45 0.03 2.36 0.15 28.31 0.84 1.05 0.08 49.66 0.62 33.48 0.99 16.73 0.57a 8.20 0.42 59.63 1.24 0.68 0.02 1.46 0.05
2.21 0.07 26.10 0.25 19.19 0.31 0.42 0.02 2.40 0.09 29.32 0.50 1.15 0.05 49.66 0.37 34.61 0.59 15.65 0.34b 8.34 0.25 60.36 0.74 0.70 0.13 1.46 0.03
TT (16)
Conventional ration ax
2.34 0.10 26.05 0.35 19.20 0.44 0.37 0.02 2.38 0.12 27.64 0.70 1.20 0.06 49.87 0.52 32.80 0.83 13.54 0.48c 1.40 0.51 8.30 0.35 58.83 1.04 0.66 0.02 1.43 0.04
CC (16)
CT (42)
2.03 0.06b 2.14 0.08b 25.97 0.21b 25.61 0.16b 17.66 0.33 18.203 0.25 0.50 0.02a 0.42 0.02b 2.65 0.11 2.52 0.08 30.94 0.75 29.71 0.58 1.46 0.10 1.37 0.08 47.92 0.37b 48.18 0.29b 37.16 0.85 35.48 0.66 18.89 0.54a 17.09 0.42b 9.21 0.34 8.91 0.25 63.55 0.93 61.78 0.72 0.78 0.02a 0.74 0.02b a 1.52 0.03 1.54 0.02a
TT (24)
a
2.33 0.09a 0.09 0.06t 26.72 0.24a 0.40 0.17 18.27 0.37 0.40 0.03b 0.05 0.02 2.67 0.12 29.04 0.86 1.32 0.11 49.83 0.42a 0.98 0.30 34.79 0.97 14.58 0.62c 2.22 0.51 9.06 0.37 61.21 1.06 0.70 0.02c 0.03 0.01 1.39 0.03b 0.06 0.02
a : Average allele substitution effect SE. Means bearing different superscript (in row) differ significantly (P < 0.05) or present a tendency toward signification (P < 0.1). Significance levels: ns: P > 0.1. t: P < 0.1. P < 0.05. P < 0.01. P < 0.001.
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SNP IN BOVINE SCD1 LINKED TO FATTY ACID PROFILE
FIG. 1. Interaction between SCD1 genotype and diet for the C16:0 fatty acid. a-c: Different letters within diet indicate significant differences among genotypes. The amount of C16:0 is expressed as the percentage of total fatty acids. a different pattern when the animals were fed with the TMR. The C16:0 is the most abundant saturated fatty acid in meat and, together with C12:0 and C14:0, is considered unhealthy because of its atherogenic properties (2). Therefore, this interaction suggests a management strategy based in the feeding scheme avoiding the CR for animals that inherit the TT genotype in order to obtain a healthier beef product. With respect to the C18:0, C18:1 cis-9 and CLA cis-9, trans-11, no significant effects of the marker on the fatty acid composition were found. The C18:0 is used to assess the nutritional implications of IMF on human diet through the desirable fatty acids estimation (MUFA þ PUFA þ C18:0) described by Huerta-Leidenz et al. (17). The C18:1 cis-9 and CLA cis-9, trans-11 also play an important role on human health (18). However, according to our results the SNP was not useful to estimate the amount of these favorable fatty acids. To sum up we can conclude that, in spite of the population size, different genotypes of SCD1 c.878C>T SNP presented a demonstrated effect on the fatty acid composition of the IMF of beef in Spanish common cattle populations. The results suggested that this marker could be used as a predictor of the nutritional quality of this meat to obtain a healthier final product. FUNDING The authors would like to thank CICAP for the coordination work and COVAP, CTA, and IDEA agency for their financial support. REFERENCES 1. Wood JD, Enser M, Fisher AV, et al. Fat deposition, fatty acid composition and meat quality: A review. Meat Sci 2008; 78(4):343–358.
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