J. Anim. Sci. 2005. 83:927-932
© 2005 American Society of Animal Science
The effect of a leptin single nucleotide polymorphism on quality grade, yield grade, and carcass weight of beef cattle
P. J. Kononoff*,1,
H. M. Deobald
,
E. L. Stewart,
A. D. Laycock and
F. L. S. Marquess
* Department of Animal and Nutritional Sciences, University of New Hampshire Ritzman Laboratory, Durham 03824; and
and
Quantum Genetics Research Inc., Saskatoon, SK, Canada S7N 4N1
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Abstract
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Feedlot producers could optimize the value of cattle in a given market grid if they were able to improve the uniformity of the body composition between cattle among loads. Allelic variation due to a single nucleotide transition (cytosine [C] to thymine [T] transition that results in a Arg25Cys) has been demonstrated to be associated with higher leptin mRNA levels in adipose tissue and increased fat deposition in mature beef, but the effect on economically important carcass traits has not been investigated in either market-ready steers or heifers. Therefore, the objective of this study was to determine the effects of a leptin SNP on the quality grade (QG), yield grade (YG), and weight of beef carcasses. A slaughter trial was conducted using 1,435 crossbred finished heifers and 142 crossbred finished steers as they entered the slaughter facility. Canada QG tended (main effect of genotype P = 0.16, but P < 0.01 for both CC vs. TT and CT vs. TT) to be affected by leptin genotype. Specifically, 7.6 and 7.1% more TT carcasses graded Canada AAA or higher than the CT and CC carcasses, respectively, which supports the suggestion that the leptin SNP is associated with carcass fat. The proportion of carcasses grading Canada YG 1, 2, or 3 was affected (P < 0.01, P = 0.05, and P = 0.02 for YG 1, 2, and 3) by leptin genotype. The proportion of TT carcasses of Canada YG 1 was 12.5 and 15.1% lower than that of CT and CC carcasses, respectively, indicating that rearing animals under the same management and feeding system may result in greater carcass fat and a lower probability of the proportion of carcasses grading YG 1 within certain genotypes. The carcass weights of animals with the CC genotype tended (P = 0.07) to be higher than those of the TT genotype (365.5 vs. 362.3 kg). No significant difference was observed between the TT and CT genotypes in carcass weight. The observed associations between leptin genotype and carcass characteristics may represent an opportunity to genetically identify animals that are most likely to reach specific marketing groups.
Key Words: Beef • Carcass • Leptin • Marbling • Quality Grade • Yield Grade
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Introduction
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Two factors that act to affect growth rate are 1) genetic potential, which regulates a complex of hormone and growth factors and their associated interactions, and 2) environmental conditions such as nutrition, climate, disease, or management (Hermesmeyer et al., 2000
). Leptin is a 16-kDa protein hormone secreted by white adipocytes that acts on central and peripheral tissues to regulate feed intake, energy expenditure, and whole-body energy balance (Houseknecht et al., 1998). More specifically, the role of leptin in metabolic regulation has been demonstrated through its action on the hypothalamic-pituitary-adrenal axis, and results seems to suggest that leptin plays an integral role in the growth process (Delavaud et al., 2002). Recently, a cytosine (C) to thymine (T) transition that encodes an AA change of an arginine to a cysteine (Arg25Cys) was identified in exon 2 of the bovine leptin gene (Buchanan et al., 2002). Investigations into a group of 154 unrelated beef bulls demonstrated that the T allele is associated with increased fat deposition and higher leptin mRNA levels in adipose tissue. It is hypothesized that the additional cysteine in the T allele in the obese gene results in a functional effect in the leptin molecule for alteration of disulfide bonding, which in turn affects intake regulation and body fat deposition.
The value-based marketing system of the North American beef industry is designed to pay premiums for well-marbled carcasses without excessive fat cover (Block et al., 2001). As a response to this, feedlot operators seek to develop programs that produce animals that fit within this marketing group, while also seeking to optimize productive performance. Unfortunately, a major limitation to these objectives is the inability to properly sort animals on arrival at the feedlot into optimal feeding and marketing groups. For this to be successful, tools are needed to identify factors that may be used to predict feed consumption and carcass composition (Perry and Fox, 1996). Although the SNP has been described as associated with carcass fat level, its effect on specific economically important carcass traits has not been investigated in either market steers or heifers. As a result, the objective of this study was to determine the effects of a leptin SNP on the quality grade (QG), yield grade (YG), and weight of beef carcasses.
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Materials and Methods
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Animals and Genotyping
A slaughter trial was conducted using 1,435 crossbred finished heifers and 142 crossbred finished steers as they entered the slaughter facility. All cattle originated from Western Canadian feedlots and were of mixed breed, predominantly crossbred with cattle of either European or British descent, without any representation of Bos indicus bloodlines. The breeds in the test population were Hereford, Angus, Charolais, Simmental, and Limousin. All of the cattle entered the feed yard at approximately 385 kg BW, with most subjected to a period of backgrounding before arrival. Upon arrival at the feedlot, cattle were randomly assigned to pen lots and fed until deemed ready for slaughter. Cattle were finished on a high-grain ration containing approximately 75% barley grain and 24% barley or corn silage. All animals were slaughtered in one of 15 kill lots in a federally inspected plant, where carcass measurements were taken according to Canadian Beef Grading Agency standards (Agriculture Canada, 1992).
Venous blood samples were taken and placed in blood collection tubes containing EDTA. The DNA was extracted from whole blood as described in Buchanan et al. (2002). Genotyping was performed using real-time capillary PCR via the LightCycler 1 model (Roche Molecular Biochemicals, Mannheim, Germany). The primer sequence was as follows: forward primer 5'AAG GAA AAT GCG CTG T 3', and reverse primer 5'ACG GTT CTA CCT CGT C 3'. The anchor probe sequence was 5'GGC CCT ATC TGT CTT ACG GGA GG 3', and finally the sensor probe sequence was 5'GTG CCC ATC CGC AAG G 3'. The master mix reaction consisted of 5.6 µL of distilled water, 1.2 µL of 25 mM MgCl2, 0.4 µL of 10 pmol/µL to give a final concentration of 4 pmol per reaction of forward primer, 0.4 µL of 10 pmol/µL, resulting in 4 pmol per reaction of reverse primer, 0.5 µL of 10 pmol/µL, resulting in 5 pmol per reaction of anchor probe, 0.5 µL of a 10 pmol/µL, resulting in 5 pmol per reaction of sensor probe, and 0.7 µL of LightCycler FastStart DNA master hybridization probes (catalog No. 2 239 272; Roche Molecular Biochemicals). For each reaction, 1.0 µL of sample template DNA was used, for a final reaction volume of 10.3 µL. The PCR fluorescence resonance transfer energy conditions included an amplification program beginning with denaturation at 95°C for 10 min, followed by 42 cycles of 95°C for 2 s (denaturation), 59°C for 10 s (annealing), and 72°C for 9 s (extension). The melting program of the reaction involved heating to 95°C for 0 s, and then cooling to 40°C for 240 s, with a continuous temperature transition rate of 0.2°C/s until a temperature of 75°C was reached. The cooling portion of the reaction was lowering the temperature to 40°C for 10 s. The anchor probe was labeled with flourescein as the donor, and the sensor probe was labeled with LightCycler Red 640 (TIB Molbiol LLC, Adelphia, NJ) as the acceptor for the fluorescence resonance transfer energy reaction. Melting temperatures were derived from melting peaks using LightCycler software version 3.5. Each test batch contained a maximum of 28 samples plus three positive controls and one negative control (water).
Statistical Analyses
The Effect of Genotype on Quality Grade and Yield Grade.
The effect of leptin genotype on the binary response variable, QG, Canada AAA or higher (yes or no), and on YG (Canada 1, 2, or 3) was tested using the NLMIXED procedure of SAS (Version 8.2; SAS Inst., Inc., Cary, N.C.). The initial model included the fixed effects of leptin genotype, sex, and the interactions between genotype and sex. Kill lot was included in the analysis and considered a random effect. Model main effects for genotype and sex were tested using the CONTRAST statement, and comparisons between genotypes, and the interaction between genotype and sex were tested using the ESTIMATE statement of SAS. The interaction between genotype and sex was tested, with a resulting P-value of 0.93, suggesting that the genotype effect on QG was not observed to be different between heifers and steers; as a consequence, the interaction term was removed from the final model. The interaction between genotype and sex on YG was tested and resulted in P-values of 0.23, 0.60, and 0.75 for YG 1, 2, and 3, respectively, suggesting that the genotype effect on YG was not different between heifers and steers. As a consequence, the interaction term was removed from the final model. In testing the effect of genotype on QG within YG, the initial model included the fixed effects of leptin genotype, sex, and the interactions between genotype and sex. The random effect of kill lot was originally tested, but it was not observed to be significant and was therefore removed from the final model. The interaction between genotype and sex was tested and resulted in P-values of 0.31, 0.19, and 0.95 for YG 1, 2, and 3, respectively, suggesting that the genotype effect on QG was not different between heifers and steers; as a consequence, the interaction term was removed from the final model.
The effect of leptin genotype on the continuous response variable, carcass weight, was tested using the MIXED procedure of SAS. The model included the fixed effects of genotype and sex, and kill lot was considered a random effect. The interaction between sex and genotype was tested, but it was not significant (P = 0.89), suggesting that the genotype effect on carcass weight was not different between steers and heifers. As a consequence, the interaction term was removed from the final model. All significant effects were declared at P 
0.05, and trends were declared when P 0.10. Mean separation and differences between genotypes were determined using the PDIFF statement. All means presented were generated using the LSMEAN statement. Significance for pairwise comparisons was noted only when the overall effect of genotype also was significant, but in cases when P 0.05 was observed for pairwise comparisons only, the effect was described as a trend.
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Results and Discussion
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Previous research into this SNP has indicated a significant association with carcass measurements, suggesting a polymorphism with a functional effect (Buchanan et al., 2002). More recently, circulating leptin concentrations were reported to be positively correlated with marbling score, fat depth, and QG (Geary et al., 2003). As a result, the objective of our study was to determine how allelic variation might affect carcass measurements. Table 1 outlines the frequency of Canadian QG within each of three leptin genotypes and the genotype frequency of the current population. The observed genotype frequency (24.9, 50.5, and 24.6% for CC, CT and TT, respectively) is similar to that observed by Buchanan et al. (2002), indicating that individual allele frequencies are approximately 0.5 for each allele in the sampled population. Figure 1 illustrates the effect of leptin genotype on Canadian QG. The proportion of carcasses grading AAA (equivalent to USDA Choice QG) or greater was significantly affected by sex, and tended to be affected by genotype. More specifically, the proportion of carcasses grading AAA or higher in animals of the CT genotype was observed to be 7.6% less than the TT genotype animals (P = 0.03). Although not significantly different, the proportion of carcasses grading AAA or higher in animals of the CC genotype was 7.1% lower than TT genotype animals.
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Figure 1. The proportion of beef carcasses grading Canadian quality grade AAA or greater within three leptin genotypes. Leptin genotype tended to affect observed proportions, but the overall model effect for genotype was not observed to be significant (P = 0.16). The observed P-values for pairwise comparisons were as follows: CC vs. TT <0.01; CC vs. CT = 0.57; and CT vs. TT = <0.01 (C = cytosine, T = thymine). Bars that represent the same carcass grade but with different letters tend to differ.
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Yield grade is an index estimate of the percentage of red meat in the carcass, with higher grades associated with higher muscle score and lower grade fat. Table 2 outlines the frequency of Canadian YG within each of three leptin genotypes, and Table 3 outlines the proportion of carcasses grading Canada YG 1, 2, or 3 within the leptin genotypes. The observed proportion of carcasses for each YG was significantly affected by sex, and leptin genotype had a significant effect on the proportions of carcasses with YG 1, 2, and 3. Compared with TT genotype animals, 12.5 and 15.1% more carcasses graded YG 1 in animals of the CT and CC genotype, respectively. Thus, these results indicate that TT genotype carcasses have a lower percentage of red meat as estimated by the Canadian YG calculation. Practically ad libitum feeding is documented to decrease the percentage of carcasses grading YG 1 (Hermesmeyer et al., 2000); thus, as a consequence of this, and because the T allele is associated with increased fat deposition and presumably intake (Buchanan et al., 2002), these results support the idea that if used as an identification tool, earlier slaughter of animals homozygous for the T allele, might have resulted in a greater proportion of Canada 1 YG carcasses.
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Table 3. The percentage of individual Canadian yield grades within three leptin genotypes of beef cattle (n = 392, 799, and 389 for CC, CT, and TT, respectively)
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The fact that the statistical effects were stronger for YG measurements than for QG is not surprising, given that YG estimates have been demonstrated to be associated with less random variation and are more accurate than those for QG (Fox and Black, 1984). In acknowledgment of this effect, Fox and Black (1984) noted that the explanation lies in the fact that YG are directly related to carcass fat, whereas QG is a function of marbling score, which is subject to variation in the distribution of fat within the carcass. Practically, it is important to note that i.m. fat deposition is influenced by an array of factors, including nutrition, and it occurs at a slower rate than deposition of internal and external fat deposition (Sainz and Hasting, 2000). Given this knowledge, we speculate that the animals homozygous for the C allele may require a greater amount of time on finishing diets to allow for maximum rate of lean tissue gain and intramuscular fat development, and that employing this feeding practice might produce carcasses of similar YG and potentially decrease differences observed in QG.
Figure 2 illustrates the observed proportion of Canada QG of AAA or greater of the three leptin genotypes within each YG, which tended to be affected by leptin genotype. The percentage of carcasses grading Canada AAA or better in TT genotype animals was higher in YG 1 carcasses than in the CC and CT genotypes. These results indicate that the TT genotype animals that are YG 1, which is a function of a greater muscle score and/ or lower grade fat, have a greater probability of grading Canada AAA or greater. Compared with YG 1, the number of carcasses grading Canada AAA or greater was not different across genotypes in either Canada YG 2 or 3 categories. Because Canada YG categories are descriptive in nature as to the amount of grade fat as well as the muscle score, it can be understood from Figure 2 that within each YG, the major influencing factor is grade fat. Therefore, it seems that cattle of the CC genotype generally have a lesser genetic propensity to reach a QG of AAA or greater when their grade fat (back fat) is at optimal YG 1 levels compared with those cattle of the TT genotype.
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Figure 2. The proportion of Canadian quality grades of AAA or greater of three leptin genotypes within different Canadian yield grades of beef carcasses. Bars that represent the same carcass yield grade but with different letters tended to differ. Based on the overall model effect for leptin genotype (P = 0.07) the observed proportion of yield grade Canada 1 carcasses grading AAA or greater tended to be affected by leptin genotype. Pairwise comparison P-values were as follows: CC vs. TT, P = 0.06; CC vs. CT, P = 0.97; and CT vs. TT, P = 0.04 (C = cytosine, T = thymine).
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Generally, feedlot operators attempt to time the slaughter of commercial cattle at the point where the ratio of lean to fat carcass composition results in a high probability of optimal grades. Unfortunately, the weight at which cattle reach the same chemical composition differs depending on a number of factors (NRC, 1996), and as a consequence, animals under the same management system will vary in body composition and, as a result, carcass grade. For example, compared with steers, heifers fatten more rapidly (Galbraith and Topps, 1981); thus, rearing heifers under the same management and feeding system will result in greater carcass fat levels and a lower probability of grading YG 1. As expected, in the current experiment, the proportion of carcasses grading AAA or higher and YG 1 was greater in heifers than in steers (heifers: 44.3, 45.3, and 52.8%; steers: 19.2, 19.6 and 43.5% for CC, CT, and TT genotypes, respectively). The lower proportion of these carcasses observed in CC and CT steers not only demonstrates the effect of genetic differences, but it may also represent an opportunity to genetically identify animals most likely to reach specific marketing groups. While observing significant correlations between circulating leptin and carcass composition, Geary et al. (2003) noted that the understanding may provide a valuable indicator of fat content in live cattle and facilitate more appropriate feeding and marketing management strategies. In light of these observations and the apparent ability of identifying functional differences in the leptin hormone, it could be suggested that identification of the leptin genotype may be an important component in such strategies.
Based on the observed main model effects, carcass weight was not significantly affected by genotype as listed in Table 4. Although a significant main model effect of genotype on carcass weight was not detected, differences tended to be observed using a pairwise comparison, suggesting a tendency of genotype to affect carcass weight. Specifically, the carcass weight of animals with the CC genotype tended (P = 0.07) to be higher that those of the TT genotype (365.5 vs. 362.3 kg). In comparison, and although lower (363.7 kg), no significant differences were observed between the TT and CT or CC and CT genotypes. Kulig et al. (2001) reported similar numerical trends when evaluating the effect of a polymorphism of the porcine leptin gene on the BW of Polish Landrace pigs. Although the current experiment was designed to determine the effect of leptin genotype on carcass characteristics, the potential effect on growth should be investigated further. Recently, it has been demonstrated that leptin acts at the anterior pituitary level to modulate growth hormone release (Zieba et al., 2003). Because modulation in circulating growth hormone level is known to affect both growth rate and final adult size, the observed tendency for differences in carcass weight supports the suggestion that the AA change from argentine to cysteine imparts a functional difference in the leptin molecule (Buchanan et al., 2002) that may include the link between the somatotropic axis and the neuroendocrine system, which may ultimately affect growth.
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Implications
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These results support the suggestion that the leptin polymorphism is associated with carcass fat level. In addition, these results suggest that the identification of the leptin genotype may improve methods that seek to allocate feed or to predict market suitability of feedlot animals to target specific value-based endpoints and superior quality and yield grades. Of the three leptin genotypes observed in this study, animals homozygous for the T allele were observed to have a higher probability of carcasses with Canadian quality grade AAA. Although this observation suggests that leptin genotype may affect marbling score, this difference might be minimized if the remaining genotypes were fed longer, so as to reach maximum compositional development.
1 Correspondence and current address: Dept. of Anim. Sci., P.O. Box 830908, Univ. of Nebraska, Lincoln 68583 (phone: 402-472-6442; fax: 402-472-6362; pkononoff2{at}unl.edu).
Received for publication April 28, 2004.
Accepted for publication October 15, 2004.
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Abstract
Introduction
