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Assessment of chromium tripicolinate supplementation and dietary protein level on growth, carcass, and blood criteria in growing pigs^1,2

Department of Animal Sciences, University of Kentucky, Lexington 40546

⁴ Correspondence:
611 W.P. Garrigus Bldg. (phone: 859-257-7524; fax: 859-323-1027; E-mail:
mdlind1{at}uky.edu).

	Abstract

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This study was conducted to evaluate potential interactive effects of supplemental Cr and dietary protein levels in growing pigs. Thirty-six individually penned barrows, 22 to 63 kg, were used in a 2 x 3 factorial arrangement of supplemental Cr (0 or 200 ppb from chromium tripicolinate) and protein level (76, 83, or 90% of lysine requirement). A corn-soybean meal basal diet was designed to supply all mineral and vitamin needs, 90% of the estimated metabolizable energy need, and 76% of the estimated protein need at 70% of ad libitum feed intake. Additional protein to 83 or 90% of the lysine requirement was provided by a soy protein isolate supplement. Growth data were collected for a 50-d period, and pigs were killed at a mean of 63 kg BW. Increasing lysine levels linearly (P < 0.01) increased ADG and liver weight. Lysine level had a quadratic effect on 10th rib backfat thickness (P < 0.05) and cooler shrink (P < 0.01) with the highest responses at the 83% lysine level. Increasing lysine level linearly decreased (P < 0.05) carcass content of ash and lipid and quadratically increased the carcass water content (P < 0.01). Carcass accretion rate showed a linear increase for protein (P < 0.01) and water accretion (P < 0.01). Dry matter composition of the longissimus muscle showed linear increases of ash (P < 0.05) and protein (P < 0.01) and a linear decrease of lipid content (P < 0.01) resulting in a linear increase (P < 0.05) of the protein to lipid ratio based on the increasing lysine levels. Pre-feeding insulin levels were increased (P < 0.05) with increasing level of lysine. One hour post-feeding, a quadratic lysine response for plasma glucose (P < 0.05) was observed with the lowest concentration at 83% lysine. Cr addition increased 10th rib backfat thickness (P < 0.10). There was no Cr x lysine level interaction (P > 0.10) observed for any of the growth or carcass traits. Plasma glucose concentrations pre-feeding were lower for Cr-supplemented pigs (P < 0.01). As expected, increasing protein levels in protein-deficient diets increased protein accretion while decreasing lipid accretion in 22 to 63 kg growing pigs; however, these effects were more clearly seen in the longissimus muscle than in the entire carcass. Supplementation of Cr exerted only minor effects with few Cr x lysine interactions observed in this study.

Key Words: Carcasses • Chromium • Growth • Pigs • Protein

	Introduction

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The process of growth is one of primarily protein and fat accretion. Growing pigs up to 70 kg BW are still in the phase of increasing protein accretion with relatively little fat deposition (Uit den Bogaard et al., 1993). Protein accretion rate will continue to increase with increasing levels of dietary protein, when all other nutrients are in sufficient supply, until the genetic maximum for protein accretion rate is reached. The level of insulin, a hormone involved in both protein and fat accretion, has been shown to be affected by Cr supplementation (Mertz, 1997). Additionally, dietary addition of chromium tripicolinate (CrPic) has resulted in increased lean deposition in pigs in some studies (Page et al., 1993; Lindemann et al., 1995) although the results have not been consistent with other studies. Some studies have been conducted with Cr supplementation at varying levels of protein at or above NRC requirement estimates with varied results (Lindemann et al., 1995; Crow et al., 1997). But Kornegay et al. (1997) demonstrated in balance trials that CrPic supplementation of pigs scale-fed to BW below anticipated ad libitum intake resulted in positive effects on DM and CP digestibility.

This experiment followed a study where the potential interaction of ME level and supplemental Cr was assessed (Van de Ligt et al., 2002). Herein, diets with protein levels below the amount required for maximal growth rate were used to examine possible interactions between Cr supplementation and protein supply. The hypothesis tested was that CrPic supplementation to diets with protein levels inadequate for maximum protein accretion would result in an improved growth response due to either increased protein digestibility and/or increased efficiency of protein utilization.

	Materials and Methods

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This study was conducted under a protocol approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

Animals and Diets.
Fifty high-lean growth barrows (line 65 boar x Camborough sow; Pig Improvement Company Inc. [PIC], Franklin, KY) were obtained at 13 ± 2 d of age (mean BW of 5.1 kg) and raised on a common diet to 22 kg BW. A subset of 36 animals were then selected for the experiment, blocked by BW, and randomly allotted from within block to a 2 x 3 factorial arrangement of dietary treatments. Two levels of Cr supplementation (0 and 200 ppb Cr in the form of CrPic supplied as CHROMAX) and three lysine (Lys) levels (76, 83, and 90% of the estimated daily lysine requirement) were examined. Lysine levels were chosen to be in the linear portion of a lysine response curve. The pigs were individually housed in 1.1 x 1.2 m pens with totally slatted concrete floors. Pigs had ad libitum access to water. Feeding occurred in two equal meals per day during the periods 0730 to 0930 and 1500 to 1700. Feed was offered in meal form in stainless steel feeders. Pigs were weighed weekly. Six additional pigs similar to those used in the experiment were randomly selected and slaughtered for initial body composition measurements.

To eliminate variation in feed intake in the experiment, the daily nutrient allowances were supplied in a total of two meals that equated to 70% of estimated voluntary feed intake (VFI) and 90% of estimated ME requirement. The feeding concept employed was to supply all daily nutrient needs of the pigs, and 76% of the estimated lysine requirement, in a corn-soybean meal basal diet. Because of this being provided in 70% of VFI, additional protein to achieve the graded levels of lysine (83 and 90% of the estimated daily lysine requirement) could then be added to the basal diet without concern of feed refusal. This methodology standardized intake and eliminated orts.

Daily requirements of ME and lysine were derived from PIC (1996) and NRC (1988) requirement estimates for pigs weighing 18, 36, and 73 kg. Requirements of 76, 83, and 90% of lysine were then calculated from these derived requirements, and the 70% VFI was calculated from the VFI estimates. The data points of 70% VFI, 90% ME, and 76, 83, and 90% lysine were then regressed across the three BW to produce curves of VFI allowance, ME requirement, and lysine requirement over a continuous BW range. The regressions resulted in the following equations:

Three basal corn-soybean meal diets, with and without 200 ppb Cr from CrPic, were mixed for pigs with specific BW of 18, 36, and 73 kg (Table 1). A commercial product (CHROMAX; Prince Agri Products, Inc., Quincy, IL) was used to supply the CrPic to the diets. The basal diets provided 76% of the lysine and 90% of the ME requirements while meeting or exceeding vitamin and mineral requirement estimates (NRC, 1988) when fed at the 70% VFI for the respective BW. Amino acid levels other than lysine were set in relation to lysine based upon the Illinois ideal amino acid ratio for a 20- to 50-kg pig (Baker and Chung, 1992). Commercially available methionine and threonine sources were used to somewhat minimize the protein level and thus minimize excess amino acids that could be used as an energy source. Two soy protein isolate supplements were mixed (Table 2), one with CrPic to contain 200 ppb Cr and one with an equal amount of limestone as the control (0 ppb Cr).

The regression equations were then used with the BWestimate to calculate a new daily feed allowance, lysine requirement, and ME allowance for each pig. A blend of the basal diets for the 18, 36, and 73 kg weights was calculated to meet the 76% lysine requirement of the pig for the projected BW. The ME for the calculated blended basal diets were then checked to be 90% of the estimated ME requirement. The remainder of lysine required was determined and added via the appropriate soy protein isolate supplement. Then, the basal diets and protein supplement for that upcoming weekly period for each individual pig were mixed with an upright paddle mixer (HT 600; Hobart, Troy, OH).

Growth and Carcass Characteristics.
The experiment consisted of a 50-d growth trial with pigs of 25 kg expected to reach 65 kg mean BW in that time frame when fed the highest protein level. Final weight and ADG were the response parameters. After finishing the growth trial, pigs were kept on their respective feeding regimen to 65 kg mean slaughter weight to assess carcass characteristics.

Pigs were electrically stunned and then killed by exsanguination. Carcasses were dehaired, eviscerated, head removed, weighed (hot carcass weight), and washed before they were chilled at 1°C for 48 h. Backfat thickness (midline fat depth at first rib, last rib, and last lumbar vertebrae for both carcass sides) as well as carcass length (distance from first rib to aitch bone) were measured. Average midline backfat thickness was calculated by taking the average fat depth at the first rib, last rib, and last lumbar vertebrae of both the right and left side of the carcass. Dressing percentage was calculated as hot carcass weight divided by slaughter weight. Cold carcass weight was taken before further processing. Cooler shrink was calculated as the difference between hot carcass weight and cold carcass weight divided by hot carcass weight and expressed as a percentage. The right side of the carcass was split at the 10th rib, and the longissimus muscle was traced to determine standard 10th rib backfat thickness and longissimus muscle area (LMA). Liver and kidney weights were also recorded. Pigs selected for initial body composition were handled in the same manner but without the physical measurements after carcass chilling.

Composition Analysis.
Carcass and loin composition were both analyzed. The left half of the carcass was frozen and used for composition analysis. The right half was cut into wholesale cuts, weighed, and frozen. The frozen half was ground three times through a 6-mm plate on a whole body grinder (Autio Grinder, Astoria, OR) to ensure thorough grinding and homogeneity of the ground product. A representative sample of approximately 200 g was taken for further analysis. Dry matter was determined by drying a 1- to 2-g sample in a vacuum oven (AOAC, 1995). The remainder of the sample was lyophilized at -40°C in a freeze dryer (FTS Systems, Stone Ridge, NY).

A loin sample was taken from the right side of the carcass. The approximately 5-cm thick sample was taken anterior to the 10th rib and frozen until lyophilization. A rotary blade food processor (DLC-5; Cuisinart, East Windsor, NJ) was used for diminution of the lyophilized carcass and loin samples to a uniformly small particle size suitable for use in ash, N, and lipid determination. Ash was determined by muffle furnace using a 5-g sample (AOAC, 1995). Nitrogen content of 0.5-g carcass samples or 0.3-g longissimus muscle samples were analyzed with a N analyzer (Lecco FP-2000; Lecco Corp., St. Joseph, MO). To estimate the protein content of the samples, the analyzed N content was multiplied by 6.25. The lipid content of 5- and 3-g samples for carcass and longissimus muscle, respectively, were determined by a solvent extraction method adapted from AOAC (1995) using a Soxtec System HT (Tecator AB, Höganäs, Sweden).

For the initial slaughter pigs, the amounts of various chemical components from the carcass were regressed on initial BW to develop prediction equations for estimating initial body composition of the experimental pigs. Total gain of water, protein, lipid, and ash for each pig was estimated by subtracting the predicted initial weights of these components (based on its initial BW) from the final weights of these components for that same pig. The accretion rates of these components were determined by dividing the total gain of each component by the number of days on test.

Blood Sampling and Analysis.
Blood samples were taken by jugular venipuncture at d 10, 30, and 50 after a 16-h fast and 1 h after the morning meal. Blood samples were collected in plasma tubes with a glycolytic inhibitor (sodium heparin) and kept on ice until storage in a refrigerator at 6°C. Upon completion of each day’s collection, blood samples were centrifuged at 1,398 x g, and plasma was aliquoted into microfuge tubes, frozen, and stored at -25°C until analysis. Plasma insulin levels were determined by RIA (Coat-a-Count Insulin; Diagnostic Products Corporation, Los Angeles, CA). Plasma glucose concentrations were analyzed enzymatically with hexokinase (procedure no. 16-UV; Sigma Diagnostic, St. Louis, MO) on a Cobos Farra II (Roche Diagnostic Systems Inc., Montclair, NJ).

Statistical Analysis.
Collected data were analyzed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). Individual animals served as the experimental unit. The model for the growth trial, slaughter trial, and compositions was as follows:

where Y_jkl is the response parameter. With regard to the variables: k is a constant, b_j is block, Cr_k are the two Cr levels (0, 200 ppb), Lys_l represent the three lysine levels (76, 83, 90% of requirement), (Cr x Lys)_kl is the Cr x lysine interaction, and e_jkl is the error term for the model. Estimates for linear and quadratic components of the response due to graded levels of lysine were also determined.

Plasma metabolites were analyzed in a similar manner with day as a repeated measure added to the model:

where Y_jklm is the response parameter and the variable k is a constant, b_j is block, Cr_k is the two Cr levels (0, 200 ppb), Lys_l represents the three lysine levels (70, 80, 90% of requirement), (Cr x Lys)_kl is the Cr x lysine interaction, e_jkl is the error term of the main plot, d_m is day (10, 30, or 50), (Cr x d)_km is the Cr x day interaction, (Lys x d)_lm is lysine x day interaction, (Cr x Lys x d)_klm stands for the Cr x lysine x day three way interaction, and f_jklm is the error term for the subplot. Estimates for linear and quadratic components of the response were used with regard to lysine levels and day repetitions.

	Results and Discussion

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Analysis of the diets by mass spectroscopy for the specific CrPic compound confirmed that CrPic was appropriately mixed into the diets. Amino acid profiles of the diets were determined. Lysine was the first limiting amino acid in all basal diets. The four next limiting amino acids were in excess of their requirement relative to lysine according to the Illinois ideal ratio (Baker and Chung, 1992). The composition of the three diets and the protein supplement is reported in Tables 1 and 2, respectively. One animal was removed from the study because of a leg injury. The pigs were killed in groups according to their protein treatment on separate days, and BW was used as a covariate in the statistical models for slaughter measurements, carcass composition, carcass accretion, and loin composition.

Increasing lysine levels (76, 83, and 90%) resulted in a linear increase in ADG (P < 0.01) and, thus, final weight (P < 0.01; Table 3). Batterham et al. (1990) reported a similar response over a wider range of lysine allowance, 1.5 to 12.2 g/d, at slightly higher energy levels. In an experiment with energy levels similar to those in the present study, Van Lunen and Cole (1996) reported a higher mean ADG that increased with lysine level. Differences in ADG as observed among the studies may have resulted from differences in environment and/or type of animal in addition to the source and level of protein and energy. CrPic supplementation at 200 ppb Cr did not affect (P > 0.10) ADG or final weight in this study. No Cr x lysine interactions (P > 0.10) were observed; therefore only main effects are reported (Table 3).

CrPic supplementation resulted only in an increased 10th-rib fat depth (P < 0.05). The carcass characteristics of growing pigs may not be representative of finishing pigs because the fat accretion phase has not reached its peak in growing pigs. No Cr x lysine interactions were observed (P = 0.10) for carcass traits.

Lysine levels influenced percentage carcass composition in that increased lysine levels (76, 83, and 90% lysine requirement) resulted in a linear decrease (P < 0.05) of ash and lipid and a quadratic increase (P < 0.01) in water content (Table 4). The consequent result of the lipid and protein responses was a linear increase (P < 0.01) in the protein to lipid ratio. Accretion rate of protein and water increased (P < 0.01) with increasing lysine level with the response being totally dependent on the 90% lysine treatment. Differences in treatment responses between the carcass compostion and the carcass accretion rate are explained by the differences of the treatments on ADG. Batterham et al. (1990) observed a linear increase of protein and decrease of fat both in carcass content and accretion rate where the increase slowed at higher lysine levels. Van Lunen and Cole (1996) showed the same response for protein and fat accretion (lysine intake of 1.1, 1.5, 2.2, and 2.8 g/d) as Batterham et al. (1990) except for the highest lysine treatments (3.2 and 3.7 g/d) where the responses reversed, resulting in quadratic responses for protein and fat accretion rates. Carcass composition and accretion rate of ash, protein, lipid, and water were not affected (P > 0.10) by Cr supplementation, nor was a Cr x lysine interaction (P > 0.10) observed. These results are in agreement with Evock-Clover et al. (1993) although Mooney and Cromwell (1997) reported increased lipid accretion.

Glucose concentrations were approximately 5.2 mmol/L pre- and post-feeding which is in agreement with Van de Ligt et al. (2002), Amoikon et al. (1995), and fasting levels reported by Evock-Clover et al. (1993) and Matthews et al. (1998). Plasma glucose concentrations pre-feeding were unexplainably lower in pigs supplemented with Cr (Table 5, 4.9 vs 5.3 mmol/L across all days; P < 0.01). The reduced pre-feeding glucose levels with Cr supplementation were not observed by Van de Ligt et al. (2002) nor by Evock-Clover et al. (1993) or Amoikon et al. (1995). Plasma glucose levels 1 h post-feeding were similar to pre-feeding levels as was observed in Van de Ligt et al. (2002) and by Amoikon et al. (1995). Plasma glucose levels 1 h post-feeding were already reduced to normal levels which agrees with Steele et al. (1977) who concluded that pigs had excellent glucogenic regulation. With regard to the effect of the lysine treatments, post-feeding plasma glucose levels were affected quadratically (P < 0.05) by lysine treatment.

Insulin to glucose ratio (a crude indicator of tissue sensitivity to insulin) pre-feeding increased linearly (P < 0.05) with increasing lysine level. The day effects (P < 0.05) observed for pre-feeding insulin and pre- and post-feeding glucose levels may have been an artifact of analysis because plasma metabolites were analyzed by day, and these effects were not noted in quite similar studies conducted in the same facilities with pigs of a similar genetic background (Van de Ligt et al., 2002).

	Implications

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Dietary protein level clearly influences growth rate and carcass accretion rate of protein and water. Additionally, the ratio between dietary protein and energy affects growth and carcass leanness. The failure of chromium picolinate supplementation to affect growth or carcass criteria in growing pigs suggests that effects observed in market pigs result from accretion changes in the finishing stage. Therefore, based on this study, chromium picolinate supplementation to growing pigs, 22 to 63 kg, is not warranted for effects seen in that particular period. However, the degree to which body Cr status is maintained by supplementation in the grower phase and the degree to which that status contributes to any effects in the finishing stage can not be answered from this research.

	Footnotes

 
¹ This manuscript is based on research supported in part by the Kentucky Agricultural Experiment Station and is published by the Kentucky Agricultural Experiment Station as paper no. 00-06-194.

² Special appreciation is expressed to Prince Agri Products, Inc., Quincy, IL, for providing the chromium tripicolinate (CHROMAX) used in this study and for partial financial support of this research project. Appreciation is expressed to the Pig Improvement Company, Inc. (PIC), Franklin, KY, for donation of the animals. Appreciation is expressed to H. J. Monegue for help in care of the animals and to D. Higginbotham for help in diet preparation. Appreciation is also expressed to Archer Daniels Midland (ADM), Decatur, IL; to Carl S. Akey, Inc., Lewisburg, OH; to Griffin Industries, Inc., Cold Spring, KY; and to Heartland Lysine, Inc., Chicago, IL, for ingredients used in the experiments.

³ Current address: Cargill Animal Nutrition Center, P.O. Box 301, Elk River, MN 55330-0301.

Received for publication November 21, 2001. Accepted for publication May 9, 2002.

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