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ANIMAL NUTRITION |
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* Department of Animal Science and Interdepartmental Nutrition Program;
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Department of Food Science; and
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Department of Poultry Science, North Carolina State University, Raleigh 27695-7621
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Pork Quality • Swine • Vitamin C • Water
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Consequently, the NRC (1998) suggested that the inclusion of vitamin C to swine diets is not required. However, the modern pig may not be able to synthesize adequate amounts of ascorbic acid to meet its need during periods of adverse environmental conditions, disease, or exposure to other stressors (Wariss, 1984).
Several investigators have reported improvements in growth (Cromwell et al., 1970; Yen and Pond, 1981) and meat quality (Mourot et al., 1990; Kremer et al., 1999) when supplementing swine with vitamin C. Effects of vitamin C on pork quality may be the result of altered glucose and glycogen metabolism (Mourot et al., 1990). Specifically, the ascorbic acid metabolite, oxalic acid, has been shown to function as a glycolytic inhibitor (Tonon et al., 1998), which in turn may decrease lactic acid production postmortem and diminish the rapid drop in pH associated with poor meat quality. Vitamin C has further been reported to decrease the severity of a preslaughter stress response (Lauridsen et al., 1996) by inhibiting glucocorticoid synthesis (Thaxton and Pardue, 1984) and thereby decreasing the amount of glucose and glycogen available for lactic acid production.
Vitamin C is water-soluble and can easily be supplemented through drinking water for short periods of time and at critical time periods preslaughter. However, vitamin C is rapidly excreted in the urine once the plasma concentration exceeds the renal threshold and the half-life decreases as consumption increases (Tsao, 1997). Thus, understanding how rapidly vitamin C is metabolized and how soon after ingestion it is excreted may be crucial to obtaining a positive effect on meat quality. Therefore, two experiments were conducted to investigate the effects of vitamin C supplementation through the water on plasma ascorbic acid and oxalate concentrations and their effects on meat quality in swine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Experiment 1
The first experiment was conducted at the North Carolina State University Swine Educational Unit. Twenty-four crossbred pigs (87.8 ± 2.13 kg BW) were fitted with jugular catheters and housed in individual pens (0.76 x 2.74 m) to properly determine individual water and vitamin C intake. Pigs were blocked by sex and weight and randomly assigned within block to one of three treatments. Treatments consisted of 0, 1,000, or 2,000 mg/L of vitamin C (L-ascorbic acid; Roche Vitamins, Inc., Nutley, NJ). Pigs were fed a corn–soybean meal basal diet formulated to meet or exceed NRC (1998) nutrient requirement estimates. The diet was calculated to contain a total of 0.63% lysine, 0.73% Ca, and 0.50% P (on an as-fed basis). Total calculated concentrations of vitamin E and Se, which like vitamin C have an antioxidant role, were 41.2 and 0.38 mg/kg, respectively. Eight pigs were removed from the experiment due to failed catheter function, resulting in a total of four, six, and six observations for the 0, 1,000, and 2,000 mg of vitamin C/L treatments, respectively.
Water was supplied through individual water systems, using a 1.2-m piece of polyvinyl chloride pipe, 20.3 cm in diameter. The pipe was closed off at the bottom, and a commercial swine nipple was fitted to the water system 2 cm from the bottom. The water device was elevated on a concrete block to a height of 40 cm for easy access to water by the pigs. Vitamin C treatments were supplemented for a 48-h period. The supplemented drinking water was changed every 12 h during the 48-h supplementation period, and fresh vitamin C water was prepared to minimize vitamin C degradation during the treatment period. We have previously determined (our unpublished data) that vitamin C remained stable in the water for at least 12 h (recovery after 14 h was 90%).
Water supplied to pigs was weighed and recorded in order to determine individual vitamin C intake from the quantity of water that disappeared. Individual water disappearance was measured at 0, 2, 4, 6, 12, 24, and 48 h after supplementation was initiated. Furthermore, blood samples were collected from all pigs at 0, 2, 4, 6, 12, 24, and 48 h after supplementation, and then repeated again at 0, 2, 4, 6, 12, 24, and 48 h after vitamin C supplementation ceased. Approximately 12 mL of blood was collected in tubes containing sodium heparin. Immediately after collection, all samples were tightly sealed and placed on crushed ice. Blood samples were then centrifuged within 40 min after each collection time for 10 min at 1,200 x g. Plasma was collected and divided into three aliquots of 2, 4, and 2 mL for analysis of ascorbic acid, oxalate, and cortisol concentrations, respectively. Samples for cortisol analysis were obtained at 0 and 48 h of supplementation and at 48 h after supplementation ceased. All samples to be analyzed for ascorbic acid were immediately frozen and stored in liquid nitrogen (–196°C) for subsequent analysis within 7 d. Storage in liquid nitrogen has been reported to minimize losses in ascorbic acid concentrations in plasma for at least 7 d (Frappier, 1989). All samples for the analysis of oxalate and cortisol concentrations were immediately placed in a –20°C conventional freezer and stored for subsequent analysis.
Experiment 2
The second experiment was conducted in late spring at the North Carolina State University Swine Educational Unit. Thirty crossbred pigs (107.5 ± 0.54 kg BW) were housed in individual pens to properly determine individual water and vitamin C intake. Water was supplied to the pigs through individual water systems, which were constructed using 18.9-L plastic containers hanging from the ceiling. The containers were connected via a rubber tube to a commercial nipple waterer located in each individual pen. Pigs were blocked by sex and weight and assigned to one of three treatments, consisting of 0, 500, or 1,000 mg of vitamin C/L (L-ascorbic acid; Roche Vitamins, Inc.). Vitamin C was supplemented for a 48-h period before slaughter and was started at 0700. The supplemented drinking water was changed every 12 h during the 48-h supplementation period and fresh vitamin C water was prepared in order to minimize vitamin C degradation during the treatment period. Pigs were fed a corn–soybean meal basal diet calculated to contain a total of 0.63% lysine, 0.73% Ca, 0.50% P, 41.2 mg of vitamin E/kg, and 0.38 mg of Se/kg (on an as-fed basis).
Individual water supplies from all pigs were weighed and recorded to determine individual vitamin C intake from the quantity of water consumed. Efforts were made to minimize water waste; however, wastage was not quantified. At the end of the 48-h period, water supplementation was ceased and pigs were transported to a commercial slaughter plant. Once at the slaughter plant, pigs were randomly stunned and exsanguinated in groups of four, which occurred between 4 and 5 h after the end of vitamin C supplementation. All pigs were dehaired by scalding, eviscerated, and chilled for 24 h. Approximately 12 mL of blood was collected at the time of exsanguination into tubes containing sodium heparin. All samples were processed within 15 min of collection, as described in Exp. 1.
Muscle samples of approximately 4 g were removed from the loin at exsanguination, and 90 min and 24 h after slaughter. Each sample was divided in two subsets for the analysis of ascorbic acid and lactic acid concentrations. Fat and skin were removed from the samples before immediate freezing in liquid nitrogen (–196°C). Samples to be analyzed for ascorbic acid concentration were stored in liquid nitrogen (–196°C) for subsequent analysis. Muscle samples to be analyzed for lactic acid concentration were frozen in liquid nitrogen and then stored at –80°C for subsequent analysis.
Meat Quality Measurements
Carcass temperature was measured at 1 h after slaughter, and both initial (1 h after slaughter) and ultimate (24 h after slaughter) loin pH measurements were determined (Sentron Red-Line LanceFET pH probe; Sentron, Inc., Gig Harbor, WA). After chilling for 24 h, carcasses were processed into primal cuts and three loin chops (longissimus dorsi) were removed from the left loin of each carcass. The first loin chop of each pig was cleaned of bone debris and allowed to bloom for 15 min. Subsequently, the degree of fluid loss, visual color scores, and Minolta color measurements were determined on each chop. The water-holding capacity was assessed using the filter paper method as described by Kauffman (1986). Visual color scores were assessed using NPPC Japanese color scoring cards and given a score of 1 through 6. Minolta L*, a*, and b* color values were read with a Minolta chromameter (CR-200, Minolta USA, Ramsey, NJ). The same loin chop of each pig was subsequently deboned and placed in oxygen-impermeable bags (Cryovac Sealed Air Corp., Saddle Brook, NJ), vacuum packaged, and heat-sealed. They were then stored at –20°C for thiobarbituric acid reactive substances (TBARS) analysis as a measure of oxidation.
The remaining loin chops (two from each carcass) were weighed, placed individually onto polystyrene trays with Dri-Lock pads (Cryovac Sealed Air Corp.), and covered with oxygen-permeable polyethylene wrap. Loin chops were displayed in conditions similar to retail display in a refrigerated cooler at temperatures of 4°C for either 4 or 8 d. At the end of the display time, the loin chops were weighed and the degree of fluid that was lost was calculated as a percentage of the original chop weight. In addition, both visual and Minolta L*, a*, and b* color scores were assessed as described earlier. Each chop was then placed into an oxygen-impermeable bag (Cryovac Sealed Air Corp.), vacuum packaged, heat-sealed, and stored at –20°C for subsequent TBARS analysis.
Analytical Procedures
Ascorbic acid concentrations in plasma and muscle were determined within 7 and 5 d of collection, respectively, by the spectrophotometric micromethod of Zannoni et al. (1974). This method does not measure dehydroascorbic acid concentrations, which have been reported to be very low in tissues and biological fluids (Moser, 1990). However, water samples were analyzed following reduction of any dehydroascorbic acid that had been formed to ascorbic acid by dithiothreitol. Plasma oxalate concentrations were determined spectrophotometrically using the method of Rolton et al. (1989) and Sigma kit No. 591 (Sigma Chemical, St. Louis, MO). Plasma cortisol concentrations were assayed using a RIA kit (Coat-A-Count Cortisol; Diagnostic Products Corp., Los Angeles, CA). Muscle tissue samples were analyzed for lactic acid concentrations using lactate kit No. 735 (Sigma Chemical).
Oxidation of loin chops was measured by TBARS analysis as described previously by Witte et al. (1970) and Salih et al. (1987). Values are expressed as milligrams of malondialdehyde (MDA) equivalents per kilogram of sample.
Statistical Analyses
Plasma ascorbic acid and plasma oxalate concentrations from Exp. 1 were compared statistically as repeated measures using the mixed model procedure of SAS (SAS Inst., Inc., Cary, NC). The model included block, time, treatment, and the time (treatment interaction. Time effects were considered as repeated measures in the analysis. Remaining data were analyzed by ANOVA using the general linear models procedure of SAS. The model included block, sex, treatment, and the sex x treatment interaction. Linear and quadratic orthogonal contrasts were used to determine effects of vitamin C levels. Least squares means are presented and an alpha level of P < 0.05 was used for determination of significance.
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Results |
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Supplementation of vitamin C through the drinking water did not affect water disappearance over the entire 48-h period (Table 1). Vitamin C supplementation of drinking water at 1,000 mg/L resulted in increased plasma ascorbic acid concentrations (P = 0.071) compared with controls within 6 h after supplementation began (19.5 vs. 11.6 µg/mL, respectively; Figure 1). Furthermore, plasma ascorbic acid concentrations continued to remain elevated above the control levels 12 (P = 0.095), 24 (P = 0.100), and 48 h (P = 0.046) after vitamin C supplementation was initiated. Similarly, supplementing pigs with 2,000 mg of vitamin C/L increased plasma ascorbic acid concentrations (P = 0.008) within 6 h (23.4 vs. 11.6 µg/mL, respectively), and levels remained elevated for 12 (P = 0.057) and 48 h (P = 0.104) after starting supplementation. No differences in plasma ascorbic acid concentrations were observed between the two levels of vitamin C. When supplementation ceased at 48 h, plasma ascorbic acid concentrations from the supplemented pigs remained elevated, but did not differ statistically (P = 0.30) from those of control pigs (13.7, 18.2, and 18.6 µg/mL for the 0, 1,000, and 2,000 mg of vitamin C/L levels, respectively) within 2 h. Plasma oxalate concentrations did not differ between treatments during any of the time periods measured (Figure 2). Supplementing vitamin C through the drinking water did not affect plasma cortisol concentrations at 0 and 48 h of supplementation, but plasma cortisol increased quadratically (P = 0.077) with increasing vitamin C level at 96 h, which was 48 h after supplementation had ended (Table 2).
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Water disappearance tended to be affected in a quadratic manner when measured from 24 to 36 h of supplementation (P = 0.064), with water intake being least for pigs receiving 500 mg/L of vitamin C (Table 3). From 36 to 48 h, water disappearance decreased linearly (P = 0.013) with increasing vitamin C supplementation; however, water disappearance for the overall 48 h supplementation period did not differ across treatments. Supplementing vitamin C through the drinking water 48 h before slaughter did not affect plasma ascorbic acid or cortisol concentrations at slaughter (Table 4). However, plasma oxalate determined at slaughter tended to increase quadratically (P = 0.074) by vitamin C supplementation. Muscle ascorbic acid concentration at slaughter and lactic acid concentration in muscle tissue at 0, and 90 min after slaughter were not affected by vitamin C supplementation (Table 4). When measured 24 h after slaughter, muscle lactic acid concentration increased quadratically (P = 0.048) with vitamin C supplementation.
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). However, Minolta L* color scores increased quadratically at d 0 (P = 0.038), 4 (P = 0.010), and 8 (P = 0.051) of retail display, indicating paler meat in carcasses from pigs supplemented with 500 mg of vitamin C/L compared with control pigs or pigs supplemented with 1,000 mg of vitamin C/L. Minolta a* color scores at d 0 increased quadratically (P = 0.021), indicating increased redness in loin chops from pigs supplemented with 500 mg of vitamin C/L compared with controls or the 1,000 mg of vitamin C/L level. No further differences were observed in Minolta a* scores on d 4 and 8 of retail display. Vitamin C supplementation resulted in a quadratic increase in Minolta b* color scores, indicating increased yellowness on d 0 (P = 0.006), d 4 (P = 0.035) and 8 (P = 0.017) of retail display for pigs supplemented with 500 mg of vitamin C/L. In spite of the differences in Minolta color values, no differences in visual color scores were observed between the three treatments on d 0 or 4; however, visual color scores decreased quadratically (P = 0.047) when vitamin C was supplemented after 8 d of display storage (Table 5).
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Discussion |
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observed increased plasma ascorbic acid concentrations in broilers within 4 h when supplemented with 1,000 ppm of vitamin C for 24 h and reported that 80% of the total increase in plasma ascorbic acid occurred within the first 8 h. When supplementation ended, plasma ascorbic acid concentrations decreased by more than 50% within 4 h but remained elevated for up to 16 h after supplementation ceased (Pardue et al., 1984).
Ascorbic acid and its oxidized counterpart dehydroascorbic acid are degraded through a series of reactions into oxalic acid and other metabolites, including L-xylonic acid, L-lyxonic acid, L-xylose, L-threonic acid, and CO2 (Levine, 1986; Tsao, 1997). Therefore, it seems likely that by increasing ascorbic acid consumption, plasma oxalic acid concentrations would increase. However, supplementing vitamin C in the drinking water had no effect on plasma oxalate concentrations. In contrast, we have previously shown (Pion, 2001) that an i.v. injection of 22 mg of ascorbic acid/kg BW increased oxalic acid in plasma above basal concentrations (P < 0.001) at 15 min after the injection was given and remained elevated (P < 0.04) from the basal concentrations through 4 h after injection. However in that study, ascorbic acid concentration in plasma at 15 min after injection was 73.6 µg/mL—much greater than the maximum concentration of 23.4 µg/mL in the present study when vitamin C was supplemented through the water. In addition, ascorbic acid concentrations decreased by 52 and 76%, respectively, 1 and 6 h after the i.v. injection was administered. Pardue et al. (1984) demonstrated a decrease in plasma ascorbic acid of more than 50% within 4 h of the removal of vitamin C-supplemented drinking water. Therefore, it may be suggested that the possible lack of increased plasma oxalate concentrations in response to vitamin C supplementation could be due to the only moderate increase in ascorbic acid concentrations in plasma we observed and a relatively fast turnover rate of ascorbic acid pools.
Results of previous research (Wariss, 1979; Pardue et al., 1985; and Mourot et al., 1992), suggests reduced plasma cortisol levels due to vitamin C supplementation. However, in the current study, supplemental vitamin C had no effect on plasma cortisol concentrations. Pigs in Exp. 1 may not have been stressed enough to allow supplemental vitamin C to affect cortisol concentrations in the blood. Pigs in Exp. 2 were likely more stressed as indicated by their much greater cortisol levels, yet no effect of vitamin C on cortisol concentration was observed. Pardue et al. (1985) reported that the decline in plasma corticosteroid concentrations in response to 1,000 ppm of supplemental vitamin C was diminished when heat stress was removed from chicks.
Studies evaluating the effects of vitamin C supplementation on pork quality have yielded inconsistent results. Several investigators have reported improved pork quality characteristics (Mourot et al., 1990; 1992; Kremer et al., 1999), whereas several reports have shown no effects (Rajic, 1971) or negative effects of vitamin C supplementation (Rajic, 1971; Cabadaj et al., 1983). In the present study, we supplemented drinking water with vitamin C in an effort to provide an easy method of administration that could be implemented for a short period of time. The levels of vitamin C supplementation were less than those used in Exp. 1 because no differences were observed between the two levels in Exp. 1, and a lower level would be more cost effective. Supplementing swine with 500 and 1,000 mg of vitamin C/L in the drinking water did not affect pH values, visual color scores, or oxidative stability. These results are in contrast to the findings of Kremer et al. (1999) and Mourot et al. (1990, 1992). Kremer et al. (1999) observed increased muscle pH, decreased L* color scores, and decreased water loss when pigs were supplemented with 738 and 2,348 mg/kg of vitamin C for 4 h before stunning. In addition, Mourot et al. (1990, 1992) observed similar improvements in measures of pork quality (pH and color) when supplementing finishing swine with 250 mg of vitamin C/kg of feed from 35 to 100 kg of BW. However, effects of long-term supplementation with vitamin C on other processes, such as collagen synthesis, may impact meat quality and thus comparisons with studies using short-term supplementation should be conducted with caution. Injecting swine with vitamin C at levels of 200 mg s.c. (Rajic, 1971) and 1 g i.m. (Cabadaj et al., 1983) immediately before slaughter decreased the frequency of PSE carcasses. An improvement in oxidative stability or shelf life might have been expected given that vitamin C can regenerate vitamin E radicals (Doba et al., 1985; Mitsumoto et al., 1991), which in turn could decrease lipid oxidation in meat (Monahan et al., 1992; Monahan et al., 1993; and Cannon et al., 1996). However, the lack of an effect of vitamin C supplementation on oxidative stability of pork in the current study is in agreement with Tsai et al. (1978), who reported that supplementing swine with 2,000 ppm ascorbic acid in the feed from 10 to 91 kg BW failed to improve the oxidative stability of pork muscle. Although TBARS measurements can vary significantly between laboratories and studies, Gray and Pearson (1987) suggested a threshold value of 1 mg of MDA/kg of tissue for organoleptic detection of rancid flavor, which is well above the values observed in the present study.
The failure of vitamin C to improve pork quality in this experiment may be related to the fact that plasma and tissue ascorbic acid or oxalic acid concentrations were not affected by supplementation at the time of slaughter. It has been hypothesized that oxalate influences pork quality through its inhibition of glycolysis and lactic acid production in the muscle post-mortem (Mourot et al., 1990). However, it is not known whether vitamin C or oxalic acid concentrations in plasma are indicators of pork quality or whether elevated concentrations are required to be able to positively affect pork quality.
The inability to increase ascorbic acid concentrations in muscle and plasma may be explained, in part, by the water intake patterns of the pigs. Even though vitamin C supplementation did not affect water intake for the 48-h period, water intake seemed to be affected by the time of day. Water intake was greatest during the day and decreased substantially in the evenings and night. In fact, water intake decreased by 77% during the evenings in comparison to daytime water consumption. Pigs were slaughtered between 1100 and 1200, with the vitamin C water source withdrawn at 0700. A relatively low water consumption level was observed during the 12 h directly preceding slaughter. Therefore, it can be concluded that pigs consumed limited amounts of vitamin C before slaughter. In addition, pigs were slaughtered 4 to 5 h after water supplementation was ceased, which was a sufficient time period for ascorbic acid concentrations to return to basal levels (Exp.1).
The negative effects of vitamin C supplementation on pork quality at 500 mg/L, but not at 1,000 mg/L, were unexpected. However, decreases in pork quality with vitamin C supplementation have been reported previously (Rajic, 1971; Cabadaj et al., 1983). Cabadaj et al. (1983) observed an increase in the frequency of pork carcasses with PSE characteristics by supplementing swine with 20 mg of vitamin C/kg BW through the feed for 5 d before slaughter. Cabadaj et al. (1983) further reported declines in pork quality characteristics relative to the control group when 200 mg of vitamin C was injected s.c. on the farm, before transport to the slaughter plant. In addition, Rajic (1971) observed higher incidences of PSE carcasses from swine supplemented with 300 mg of vitamin C/kg of feed, 5 to 10 d preslaughter. In the current experiment, supplementing swine with 500 mg of vitamin C/L through the drinking water resulted in higher, more undesirable Minolta L* values and increased fluid loss in loin chops from supplemented pigs compared with the control group. In fact, the carcasses from pigs supplemented with 500 of vitamin C/L possessed a mean Minolta L* value of 54.4, which, according to PIC (2003) places those carcasses in the PSE category of pork quality.
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Footnotes |
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2 Present address: Michigan State University Extension, 201 E. State St., Cassopolis 49031.
4 Present address: Graduate School, 205 Peele Hall, North Carolina State Univ., Raleigh 27695.
3 Correspondence: Box 7621, Dept. of Anim. Sci. (phone: 919-513-1116; fax: 919-515-6316; e-mail: Eric_vanHeugten{at}ncsu.edu).
Received for publication July 21, 2003. Accepted for publication March 3, 2004.
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) increased plasma ascorbic acid concentrations at 6, 12, 24 (P < 0.10), and 48 h (P < 0.05) compared with controls (
). Supplementation with 2,000 mg of vitamin C/L (•) increased plasma ascorbic acid concentrations at 6, 12 (P < 0.05), and 48 h (P < 0.10) compared with controls.