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J. Anim. Sci. 2003. 81:167-171
© 2003 American Society of Animal Science

Serum concentrations of leptin in six genetic lines of swine and relationship with growth and carcass characteristics1

E. P. Berg*, E. L. McFadin*, K. R. Maddock*, R. N. Goodwin{dagger}, T. J. Baas{ddagger} and D. H. Keisler*,2

* Animal Sciences Department, University of Missouri, Columbia 65211-5300; and {dagger} National Pork Board, Des Moines, IA 50306; and and {ddagger} Animal Sciences Department, Iowa State University, Ames 50011-3150

2 Correspondence:
160 Animal Science Research Center (phone: 573-882-7267; fax: 573-882-6827; E-mail:
KeislerD{at}missouri.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
The objective of this study was to evaluate the relationship between serum concentrations of the hormone leptin with growth and carcass traits in six distinct breeds of pigs entered into the 2000 National Barrow Show Sire Progeny Test. Breeds evaluated were Berkshire (n = 131), Chester White (n = 33), Duroc (n = 40), Landrace (n = 23), Poland China (n = 26), and Yorkshire (n = 41). Serum samples were collected and assayed for concentrations of leptin at entry into test (On-Test Leptin) at 34 ± 6.7 kg of live weight and again 24 h prior to harvest (Off-Test Leptin) at 111 ± 3.1 kg of live weight. Carcass measurements taken included hot carcass weight, carcass length, backfat, longissimus muscle area (LMA), longissimus pH, Hunter L-value, chemically determined intramuscular fat (IMF), and subjective color, marbling, and firmness scores. Average daily gain, IMF percentages, and water-holding capacity (WHC) were also determined. On-Test Leptin concentrations were not different (P > 0.10) between swine breeds; however, Off-Test Leptin concentrations did differ (P < 0.001) across genotype. Berkshire had the greatest Off-Test Leptin concentrations (6.58 ± 0.43 ng/mL), and Duroc and Yorkshire had the lowest (3.49 and 3.96 ± 0.68 ng/mL; respectively). In addition, Off-Test Leptin concentrations were correlated with average daily gain (r = 0.29; P < 0.001), last-rib fat thickness (r = 0.48; P < 0.001), 10th rib backfat (r = 0.52; P < 0.001), LMA (r = -0.33; P < 0.001), percent fat-free carcass lean (r = -0.51; P < 0.001), and WHC (r = 0.15; P < 0.05). Off-Test Leptin concentrations also differed by gender, with barrows having greater (P < 0.001) serum concentrations of leptin than gilts (6.55 ± 0.48 vs 3.35 ± 0.44). Differences exist between breeds of pigs in a manner consistent with breed-specific traits for growth, leanness, and quality; thus, leptin may serve as a useful marker for selection or identification of specific growth and carcass traits.

Key Words: Leptin • Meat Quality • Pigs


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
The protein hormone leptin is secreted from adipose tissue, circulates in the blood, and assists with appetite regulation and energy balance. As adipocytes increase in mass, peripheral concentrations of leptin increase (Considine, 1996). Thus, leptin serves as an endocrine messenger from the body to the brain and elsewhere to communicate nutritional status of the body, which in turn influences feeding behavior, metabolism, and energy balance in rodents, primates, livestock, and humans (Campfield et al., 1995; Keisler et al., 1999). Previously, Robert et al. (1998) reported that leptin messenger RNA levels were positively associated with backfat thickness in pigs.

Many genetic companies and breed associations have devoted considerable effort to identifying and marketing breeding stock possessing genetic potential for superior pork quality. These companies and associations have evaluated carcass lean components for such quality characteristics as water holding capacity, color, lean tissue firmness, and marbling (Meisinger, 2002). Aside from laboratory testing for the presence of the halothane gene, an accurate means of assessing quality differences between genotypes in the live animal has yet to be identified. Our objective was to determine if serum concentrations of leptin varied with breeds of pigs in a manner consistent with breed-specific traits for growth, leanness, and quality, and to determine the relationship between serum concentrations of leptin and growth and carcass traits.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Implications
 Literature Cited
 
Animals and Data Collection

Animal live weights and blood samples were collected from offspring of sires representing six distinct breeds of pigs entered into the 2000 National Barrow Show Sire Progeny Test (New Hampton, IA). All pigs were housed in modified open-front structures with ad libitum access to water and a traditional corn-soybean meal diet. Pigs were randomly allotted to pens upon arrival at the test site and were placed eight to a pen according to production system point of origin and breed until they reached the designated harvest weight of 111 kg. Breeds evaluated were Berkshire (n = 131), Chester White (n = 33), Duroc (n = 40), Landrace (n = 23), Poland China (n = 26), and Yorkshire (n = 41). Barrows (n = 168) and gilts (n = 126) were penned separately according to production system point of origin and breed, but both sexes received the same diet. Blood samples were collected upon entry (On-Test Leptin) into the test (average BW = 34 kg) using an optical bleed (Leman et al., 1992) and again via jugular venipuncture at the end of the test, 24 h prior to harvest (Off-Test Leptin). All blood samples were allowed to clot for 6 to 18 h at 4°C followed by centrifugation at 2,500 x g for 30 min. Serum was harvested and stored at -20°C until assayed as described subsequently for leptin by radioimmunoassay.

Pigs were transported for harvest at Quality Pork Processors (Austin, MN; approximately 124 km) in six slaughter groups. Pigs were transported on Mondays, kept in lairage overnight with ad libitum access to water, and humanely harvested as the first group on Tuesday mornings. Postharvest measurements taken were hot carcass weight, carcass length, and midline backfat depth at the last rib. Longissimus muscles were collected from the fabrication line with the outer skin surface and overlying subcutaneous fat remaining on the longissimus muscle (untrimmed) and dissected at the juncture of the 10th and 11th rib for measurement of 10th-rib backfat depth and longissimus muscle area. Longissimus muscle pH (pH-Star probe; SFK Technologies, Peosta, IA) and Hunter L-value (Minolta colorimeter model CR-310; Reston, VA; with a D65 illuminant and standardized on each day of data collection to a white and black tile) were recorded on (in) the cut lean surface adjacent the 10th rib at 24 to 28 h postmortem. Longissimus muscle water-holding capacity (filter paper method; Kauffman et al., 1986), subjective color, marbling, and firmness scores (NPPC, 2000) were evaluated approximately 48 h postmortem. Average daily gain [ADG = (off-test weight - on-test weight)/(off-test date - on-test date)] and chemically derived longissimus muscle (11th rib) intramuscular fat (IMF; official procedure AOAC 960.39; AOAC, 2000) percentages were calculated.

Leptin Radioimmunoassay

Serum concentrations of leptin were quantitated using the double-antibody leptin radioimmunoassay procedures described by Delavaud et al. (2000) with one modification consisting of the substitution of the reported primary antiserum with a different rabbit anti-ovine leptin primary antiserum (number 7105). Briefly, standard concentrations of both recombinant ovine leptin (Gertler et al., 1998) and porcine leptin (Raver et al., 2000; 0.1, 0.2, 0.3, 0.5, 0.8, 1.2, 2.0, 3.5, 5.0 and 7.5 ng/300 µL per tube) and increasing volumes of serum (25, 40, 60, 100, 175, 250, 300 µL) from a pool of serum collected from a fat gilt were added to assay tubes in quadruplicate and the total volume balanced to 300 µL per tube with buffer consisting of 0.1% gelatin, 0.01 M EDTA, 0.9% NaCl, 0.01 M PO4, 0.01% sodium azide, 0.05% Tween-20 (Fisher Scientific; Springfield, New Jersey), pH = 7.1 (PABET). Likewise, 200 µL of the serum samples to be quantified were added to assay tubes in triplicate and the volume balanced to 300 µL/tube with PABET. Immediately thereafter, 100 µL of rabbit anti-ovine leptin primary antiserum (7105; final tube dilution of 1:15,000 in PABET) was added to samples and standards incubated at 4°C for 24 h. After the initial incubation, 100 µL of [125I] ovine leptin (20,000 counts per minute) was added to each tube and incubation continued for an additional 24 h at 4°C. The antigen-antibody complex was then precipitated following a 15-min, 22°C incubation with 100 µL of a preprecipitated sheep-anti-rabbit second antiserum by centrifugation at 2,000 x g for 30 min, and the supernatant removed by aspiration. Assay tubes containing the pellets were counted for 1 min on a LKB1275 gamma counter (LKB Wallac; Turku, Finland).

Standards (ovine and porcine leptins) and pooled aliquots of serum from a single source of fat-gilt serum were linear (log/logit transformation; R2 > 0.98) and parallel over a mass of 0.1 to 7.5 ng/tube and a serum volume of 25 to 300 µL, respectively. Total specific binding was 35%, the minimal detectable concentration was 0.1 ng/tube, percentage recovery of mass was >97% across the range of 25 to 300 µL of sample, and the inter- and intraassay coefficients of variations were less than 10%.

Statistics

Data obtained from carcasses without corresponding Off-Test Leptin values were excluded from analysis. In addition, all carcasses less than 104 kg were excluded from statistical analysis. Data were analyzed using the GLM of SAS (SAS Inst., Inc., Cary, NC). Fixed effects of off-test date (off-date), breed, and gender (sex) were evaluated for differences associated with On-Test Leptin, Off-Test Leptin, carcass composition, and carcass quality parameters. Effects within the model included On-Test Leptin, Off-Test Leptin, and carcass data predicted by the class variables off-date, breed, sex, and breed x sex interaction. Off-date interactions with breed and gender were tested and found to be insignificant (P > 0.1) and consequently removed from the model. Relationships between concentrations of leptin and carcass traits were quantified by Pearson correlation coefficients. Least squares means were computed and statistically separated with the PDIFF option of SAS. All data are presented as least squares means ± SEM. Differences were designated as significant at P < 0.05 with trends established at P < 0.10.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Implications
 Literature Cited
 
Mean serum concentrations of leptin and carcass measurements across the swine breeds are provided in Table 1Go. At the beginning of the trial (34 kg BW), serum concentrations of leptin did not differ with respect to breed or sex of the pig. These observations were not unexpected, as peripheral concentrations of leptin in the 34-kg growing pigs were very low, likely due to the low total fat mass within these animals at this stage in their life. However, as an animal approaches maturity, growth is in the form of adipose tissue deposition; thus, it would seem reasonable that serum concentrations of leptin subsequently increased as the pigs matured. The simple correlation between On-Test and Off-Test Leptin (Table 2Go), however, did reveal a small (r = 0.152; P < 0.01) relationship between early development and mature leptin concentrations. At the end of the test and just prior to harvest of the animals at 111 kg, Off-Test Leptin concentrations differed (P < 0.001) with sex and breed of the animal.


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  		Table 1. Least square means (standard error) for concentrations of leptin and carcass traits between barrows and gilts (sex) and genetic type (GT)
 

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  		Table 2. Pearson correlation coefficients and probability > |r| under H0: Rho = 0 for preharvest serum concentrations of leptin and live swine and pork carcass parameters
 
As to sex-related differences in Off-Test Leptin concentrations, barrows had greater serum concentrations of leptin than gilts (6.55 ± 0.48 vs 3.35 ± 0.44; P < 0.001). This agrees with work done in horses in which geldings had greater serum leptin concentrations than did mares (Buff et al., 2002). In work done in beef cattle, steers were observed to have higher marbling scores and kidney, pelvic, and heart (KPH) weight without an increase in leptin, even though gender had no affect on serum concentrations of leptin, suggesting that leptin synthesis or degradation is different between steers and heifers (Geary et al., 2003). Sex differences in peripheral concentrations of leptin have been previously reported to exist in humans as well (Hellstrom et al., 2000), with females typically possessing greater fat mass and peripheral concentrations of leptin than males regardless of body mass. Our observations, however, were that barrows, which had greater fat mass (more 10th-rib backfat) and less muscle (lower longissimus muscle area and percentage of fat-free carcass lean) than gilts, had the greater serum leptin concentration. Unfortunately, no contemporary group of intact boars was available for sampling for comparison purposes.

Off-Test Leptin concentrations were associated with breed effects (P < 0.001). Berkshire pigs had greater (P < 0.05) serum concentrations of leptin at harvest (6.58 ng/mL) than did all breeds except for Poland China (6.45 ng/mL) and Landrace (4.77 ng/mL). These observations were not unexpected, as these breeds differed in body type and because breed differences in peripheral concentrations of leptin have been previously reported to exist in other livestock species noted for their differential ability to deposit fat. In beef cattle for example, Angus bulls had greater concentrations of serum leptin than did Brangus and Brahman bulls of similar ages (Thomas et al., 2002). Peripheral concentrations of leptin have been reported to increase as adipocytes increase in mass (Considine, 1996); therefore, it was not surprising that the Berkshire breed also has the lowest percentage of fat-free carcass lean (46.22%). Off-Test Leptin concentrations were also highly correlated with 10th-rib carcass fat depth (r = 0.518; P < 0.001) and last-rib backfat (r = 0.476; P < 0.001; Table 2Go). This agrees with work done in cattle (Minton et al., 1998; Geary et al., 2003), sheep (Delavaud et al., 2000), pigs (Estienne et al., 2000), and horses (Buff et al., 2002), in which subcutaneous fat thickness was correlated to circulating concentrations of leptin.

Correlations (P < 0.001) were also found between Off-Test Leptin and ADG (r = 0.293), carcass length (r = -0.330), longissimus muscle area (r = -0.326), and percentage of fat-free carcass lean (r = -0.508; Table 2Go). In beef cattle, two different results were found between longissimus muscle area and serum concentrations of leptin. In a group consisting of only steers, serum leptin concentrations was not found to be correlated to longissimus muscle area; however, in a separate group consisting of steers and heifers, serum concentrations of leptin were negatively correlated to longissimus muscle area (r = - 0.45, P < 0.001; Geary et al., 2003). Differences between gender and breed may have contributed to differences seen in the cattle study. Off-Test Leptin concentrations tended (P = 0.072) to correlate with subjective marbling scores (r = 0.106), but not (P = 0.23) with longissimus muscle percentage intramuscular fat as determined by chemical analysis (r = 0.070). Correlations between marbling scores and serum concentrations of leptin have also been found in cattle (Minton et al., 1998; Geary et al., 2003). A correlation (P = 0.012) was found between Off-Test Leptin concentrations and water-holding capacity (r = -0.147).


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
Literature Cited
 
These data provide evidence that serum concentrations of leptin differ between barrows and gilts and breeds of pigs in a manner consistent with breed-associated traits of growth, leanness, and quality. A highly positive correlation was observed between serum concentrations of leptin and subcutaneous fat measurements but no relationship was observed with marbling in the longissimus muscle. A strong negative correlation was observed between serum concentrations of leptin and carcass muscle content. Peripheral concentrations of leptin may ultimately become a useful live animal marker for selection or identification of specific growth and carcass traits in swine.


   Footnotes
 
1 This research was supported in part by the Missouri Agric. Exp. Stn. project number MOASSL0569. The authors thank C. Stahl, D. Newcomb, and M. Linville for assisting with blood collection, G. Miller of the NE IA Swine Improvement Association, and L. Rasch and the staff of the 2000 National Barrow Show. Back

Received for publication March 28, 2002. Accepted for publication September 3, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 


AOAC. 2000. Official Methods of Analysis. 17th ed. Vol. 2. Assoc. of Offic. Anal. Chem. Washington. DC.

Buff, P. R., A. C. Dodds, C. D. Morrison, N. C. Whitley, E. L. McFadin, J. A. Daniel, J. Djiane, and D. H. Keisler. 2002. Leptin in horses: Tissue localization and relation between peripheral concentrations and body condition. J. Anim. Sci. 80:2942–2948.[Abstract/Free Full Text]

Campfield, L. A., F. J. Smith, Y. Guisez, R. Devos, and P. Burn. 1995. Recombinant mouse OB protein: Evidence for a peripheral signal linking adiposity and central neural networks. Science (Wash, DC) 269:546–549.[Abstract/Free Full Text]

Considine, R. V., M. K. Sinha, M. L. Heiman, A. Kriaucinunas, T. W. Stephens, M. R. Nyce, J. P. Ohannesian, C. C. Marco, L. J. McKee, T. L. Bauer, and J. F. Caro. 1996. Serum immunoreactive-leptin concentrations in normal weight and obese humans. N. Engl. J. Med. 334:292–295.[Abstract/Free Full Text]

Delavaud, C., F. Bocquier, Y. Chilliard, D. H. Keisler, A. Gertler, and G. Kann. 2000. Plasma leptin determination in ruminants: Effect of nutritional status and body fatness on plasma leptin concentration assessed by a specific RIA in sheep. J. Endocrinol. 165:519–526.[Abstract]

Estienne, M. J., A. F. Harper, C. R. Barb, and M. J. Azain. 2000. Concentrations of leptin in serum and milk collected from lactating sows differing in body condition. Domest. Anim. Endocrinol. 19:275–280.[Medline]

Geary, T. W., E. L. McFadin, M. D. MacNeil, E. E. Grings, R. E. Short, R. N. Funston, and D. H. Keisler. 2003. Leptin as a predictor of carcass composition in beef cattle. J. Anim. Sci. 81:1–8.[Abstract/Free Full Text]

Gertler, A., J. Simmons, and D. H. Keisler. 1998. Large-scale preparation of biologically active recombinant ovine obese protein (leptin). FEBS Lett. 422:137–140.[Medline]

Hellstrom, L., H. Wahrenberg, K. Hruska, S. Reynisdottir, and P. Arner. 2000. Mechanisms behind gender differences in circulating leptin levels. J. Intern. Med. 247:457–462.[Medline]

Kauffman, R. G., G. Eikelenboom, P. G. van der Wal, G. Markus, and M. Zaar. 1986. The use of filter paper to estimate drip loss of porcine musculature. Meat Sci. 18:191–200.

Keisler, D. H., J. A. Daniel, and C. D. Morrison. 1999. The role of leptin in nutritional status and reproductive function. J. Reprod. Fertil. (Suppl.)54:425–435.

Leman, A. D. 1992. Diseases of Swine. Page 806 in Veterinary Practice. 7th ed. A. D. Leman, B. E. Straw, W. L. Mengeling, S. D’Allaire, and D. J. Taylor, ed. Iowa State Univ. Press, Ames.

Meisinger, D. J. 2002. A System for Assuring Pork Quality. Natl. Pork Board. Des Moines, IA.

Minton, J. E., D. J. Bidel, J. S. Drouillard, E. C. Titgemeyer, D. M. Grieger, and C. M. Hill. 1998. Serum leptin is associated with carcass traits in finishing cattle. J. Anim. Sci. 76 (Suppl. 1):231(Abstr.).

NPPC. 2000. Pork Composition and Quality Assessment Procedures. Natl. Pork Prod. Council, Des Moines, IA.

Raver, N., E. E. Gussakovshy, D. H. Keisler, R. Krishna, J. Mistry, and A. Gertler. 2000. Preparation of recombinant bovine, porcine and porcine W4R/R5K leptons and comparison of their activity and immunoreactivity with ovine, chicken and human leptins. Protein Exp. Purif. 19:30–40.[Medline]

Robert, C., M. F. Paulin, N. Coulombs, C. Roberge, F. G. Silversides, B. F. Bankel, R. M. McKay, and G. Pelletier. 1998. Backfat thickness in pigs is positively associated with leptin mRNA levels. Can. J. Anim. Sci. 78:473–482.

Thomas, M. G., R. M. Enns, D. M. Hallford, D. H. Keisler, B. S. Obeidat, C. D. Morrison, J. A. Hernandez, W. D. Bryant, R. Flores, R. Lopez, and L. Narro. 2002. Relationships of metabolic hormones and serum glucose to growth and reproductive development in performance-tested Angus, Brangus, and Brahman bulls. J. Anim. Sci. 80:757–767.[Abstract/Free Full Text]


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