HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peeters, E. Articles by Geers, R. Search for Related Content PubMed PubMed Citation Articles by Peeters, E. Articles by Geers, R.

Influence of supplemental magnesium, tryptophan, vitamin C, and vitamin E on stress responses of pigs to vibration¹

E. Peeters^*,2, A. Neyt, F. Beckers, S. De Smet, A. E. Aubert and R. Geers^*

^* Laboratory for Quality Care in Animal Production, Zootechnical Centre, K.U.Leuven, B-3360 Lovenjoel, Belgium; and Laboratory of Experimental Cardiology, University Hospital Gasthuisberg O/N, B-3000 Leuven, Belgium; and and Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, UGent, B-9090 Melle, Belgium

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Literature Cited

 
Our objectives were to investigate and compare the effects of supplemental Mg, Trp, vitamin E (vit E), and vitamin C (vit C) on stress responses of pigs undergoing transport simulation. In this study, 126 pigs (25.1 ± 4.4 kg BW) were allocated to one of the six following treatments: 1) negative control (no supplementation); 2) positive control (i.m. injection with 0.5 mg of carazolol/20 kg BW 12 h before vibration, ß-blocker); 3) Trp (additional amount of 6 g/kg of feed for 5 d, as-fed basis); 4) Mg (3 g/L drinking water for 2 d); 5) vit E (additional amount of 150 mg/kg of feed for 21 d, as-fed basis); 6) or vit C (additional amount of 300 mg/kg of feed for 21 d, as-fed basis). Pigs were treated in groups of three, and each treatment was replicated seven times. Feed and water intake were not different among treatments. Heart rate variables (mean, peak, and minimum heart rate, ventricular ectopic beats, and ST elevation of Channels A and B) and heart rate variability were registered from the night before vibration. Pigs were subjected to vibration in a transport simulator (8 Hz, 3 m/s ) for 2 h and allowed to recover for 2 h. Generally, the positive control pigs had the lowest heart rate values (mean, peak, minimum heart rate, ST elevation of Channel A; P < 0.05), whereas Mg and Trp decreased ventricular ectopic beats and ST elevation of Channel B, respectively. The effect of vit C and E as vagal stimulators was clearly visible, whereas carazolol and Mg clearly blocked the sympathetic pathways of the autonomic nervous system. During vibration, the negative control pigs lay the least, and Mg pigs the most (P < 0.05). Salivary cortisol concentrations (taken before and after vibration and after recovery) showed that vit E pigs produced the least cortisol during stress periods. Intermediary metabolites (glucose, lactate, creatine kinase, and NEFA) were analyzed in plasma from blood taken before and after vibration. At the two sampling points, the vit E and Mg pigs had the lowest NEFA concentrations (P < 0.05), and the vit E pigs also had the lowest lactate concentrations before vibration. Urine samples were collected before and after vibration to determine catecholamine concentrations; only negative control pigs had an increase (P = 0.04) in epinephrine concentration, despite large individual variation. In general, these results indicate that the supplementation of Trp, Mg, vit E, or vit C improved coping ability of pigs during vibration comparison with the negative control treatment. A muscular injection of carazolol influenced only the heart rate variables.

Key Words: Magnesium • Pig • Stress • Tryptophan • Vitamin C • Vitamin E

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Literature Cited

 
Consumers now pay more attention to origin of meat, meat quality, and health of slaughtered animals. Animal welfare is another issue gaining more attention. During transport to an abattoir, pigs are exposed to different stressors (e.g., change of temperature, noises, and movements; Lambooij and van Putten, 1993). The stress caused by this transport may affect animal welfare and increase economic losses related to mortality, carcass damage, and decreased meat quality. In the past, pigs were sometimes injected with pharmacological tranquilizers to diminish stress. Because of the risk of food residues, the use of these sedatives has been banned in the European Community. Thus, there is a need for worthy alternatives to calm pigs during transport. One possible solution is the use of legal feed additives, to be added to feed or drinking water, which have a well-known effect on stress and meat quality. In this regard, we considered, Mg, Trp, vitamin E (vit E), and vitamin C (vit C) as possible alternatives.

The addition of Mg may decrease the concentration of plasma cortisol and catecholamines. The main effect of Mg is to decrease the neuromuscular stimulation (Kietzmann and Jablonski, 1985) due to the antagonistic effect of Mg on Ca (D’Souza et al., 1998). Tryptophan increases brain serotonin concentration (Henry et al., 1992), resulting in a sedative effect. Vitamin C and vit E are two supplements that are regularly investigated in relation to meat quality (Raskin et al., 1997; Eichenberger et al., 2004), but these supplements also can diminish stress (Satterlee et al., 1989; Sahin et al., 2003; Peeters et al., 2004). Therefore, the aim of this study was to determine the effects of supplementary Mg, Trp, vit E, and vit C on stress responses of pigs undergoing a transport simulation. The measured stress responses were heart rate (HR) variables, behavior, stress hormones, and intermediary metabolites (Broom, 1995).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Literature Cited

 
Animals and Housing
Crossbred (Piétrain x Hypor) pigs, heterozygous for the halothane gene, were kept at the Zootechnical Centre of Katholieke Universiteit Leuven in standardized housing conditions (1.70 m width x 2.05 m length pen with a plastic floor). Pigs were housed in groups of 12 from a weight of approximately 15 kg, and groups were formed at random with both familiar and unfamiliar pigs. The supplementation of vit E, vit C, and Trp started in these commercial pens and the pigs’ feed intake was measured. Two days before being subjected to vibration, three animals from the same pen (but no littermates) with an average BW of 25.1 ± 4.4 kg were moved to a smaller group pen (0.83 m width x 2.00 m length). This weight was chosen to allow for easy handling of the animals. It is assumed that pigs of this BW have physiological stress responses comparable to those of market pigs (Dantzer and Mormède, 1983). In the small pens, the measurement of feed and water intake by the three animals was possible. To avoid damage to the attached HR device, they were housed individually in metabolic cages 1 d before the vibration (after anesthesia; see "Preparation of the Animals") without visual but with auditory contact. The dimensions of these cages were 0.90 m wide x 1.00 m long x 0.92 m high, and again the feed and water intake of each individual was measured.

The animals were subjected to vibration in groups of three (with the same treatment) to avoid the effect of social isolation, which was taken into account in the statistical analysis (see subsequent section on statistical analyses). Each group had at least one representative from each sex (barrow or gilt). The vibration for each of six treatments was performed seven times for each treatment using 126 pigs (giving 21 pigs per treatment).

Treatments
The animals were given free access to drinking water (via a nipple) and feed. All pigs received a commercial feed (20% corn, 15% soybean meal) (17.0% CP and 9.61 MJ of DE/kg, as-fed basis), with 120 mg of vit E/kg, 100 mg of vit C/kg, 1.7 g of Mg/kg, and 2.3 g of Trp/kg (as-fed basis). In the experiment, six treatments were tested: Mg, Trp, vit E, vit C, a negative control treatment (without supplementation), and a positive control treatment (injection with ß-blocker). The dose, duration, and route of administration of the supplementation are presented in Table 1. The L-Trp (98%; Ajinomoto Eurolysine, Orffa, Londerzeel, Belgium), vit E (DL--tocopheryl acetate; Rovimix E-50 SD, DSM Nutrition, Deinze, Belgium), and vit C (L-ascorbic acid; Rovimix C-EC, DSM Nutrition) were mixed into the feed for 30 min in a feed mixer. These feed samples and a control sample were analyzed for Trp (colorimetric method, Directive 1998/64/EEC), -tocopherol acetate (HPLC; Manz and Philipp, 1981), and ascorbic acid (titration with 2,6-dicholophenolin-dolphenol). Magnesium (Mg acetate; Magac, Verdugt, Tiel, The Netherlands) was dissolved in water. Carazolol (0.5 mg/mL; Suacron, Pfizer, Puurs, Belgium) was injected i.m. behind the ear.

Transport Simulation
After a group of three pigs was transferred (a procedure lasting 3 min) by trolley from their pen to an air conditioned (average temperature = 24.3°C; SD = 2.2°C) vibration room, they were manually lifted into the vibration crate (a procedure taking 2 min). This crate was designed and assembled by the Silsoe Research Institute (Bedford, U.K.) with the following dimensions: 0.77 m wide x 1.22 m long x 0.64 m high. The simulation started at 1000. The pigs were subjected to vibrations in the vertical axis at a frequency of 8 Hz and root mean square acceleration of 3 m/s² for 2 h (Perremans et al., 1998). During vibration, the dry air temperature was registered (Hobo H8 Logger, Onset Computer Corp., Bourne, MA). Subsequently, the animals were moved to their individual pens, where they could rest for another 2 h (1200 until 1400). The first and second hour of vibration were labeled V1 and V2, respectively, whereas the first and second hour of the recovery period were labeled R1 and R2, respectively.

Behavioral Measurements
During transport simulation (1000 until 1200), the experimental animals were observed by a camera (KP-143; Hitachi, Brussel, Belgium) connected to a monitor. Their behavior (i.e., the time in seconds that each animal spent lying down) was noted for the purpose of evaluating their level of restlessness as a measure of stress during transport (Lambooij, 1988).

Saliva Sampling and Analysis
Saliva samples were collected with cotton swabs at three points in time: 1) a day before vibration at 1200 (reference sample); 2) after vibration (1200); and 3) after the recovery period (1400). After the cotton swabs were spun (Laborefuge 400R, Heraeus Instruments, Brussel, Belgium) in salivettes for 10 min at 932 x g, the saliva was frozen and stored at –20°C. Afterwards, cortisol in the saliva was analyzed by using a commercial RIA (ImmuChem cortisol assay, ICN Biomedicals, Asse, Belgium). The detection limit was 0.15 µg/dL, and the cross-reactivity was a maximum of 37% with prednisolone. The intra- and interassay CV were 7.0 and 7.9% for low (4.4 and 4.8 µg/dL), 5.8 and 6.5% for medium (22.3 and 23.1 µg/dL), and 5.1 and 6.0% for high (35.1 and 36.6 µg/dL) concentrations of cortisol.

Blood Sampling and Analyses
Blood was collected in two 4-mL tubes (Venoject, Terumo, Haasrode, Belgium) containing 9.00 mg of NaF + 9.00 mg of K₂Oxalate and 60 USP U of lithium heparin, respectively, by venopuncture of the vena jugularis and immediately stored on ice. This handling occurred before (reference sample) transport simulation (0900) and after transport simulation (1200). The samples were centrifuged for 15 min at 713 x g, and the plasma was frozen and stored at –20°C. Enzymatic colorimetric methods were used to analyze these samples to determine the concentrations of glucose (GOD/POD Trinder’s method; Instrumentation Laboratory, Zaventem, Belgium), lactate (NAD/LD; Sigma-Ald-rich, Bornem, Belgium), and NEFA (NEFA C; Wako Chemicals GmbH, Neuss, Germany) in the plasma of the blood collected in the NaF-K₂Oxalate tubes and creatine kinase (CK-NAC, Instrumentation Laboratory) in the plasma of blood collected in the heparin tubes. If animals were supplemented with vit C, vit E, Trp, or Mg, blood also was taken just before the beginning of the supplementation. These samples and the reference samples of the same animal were used to investigate the possible increase of the supplement in the blood, whereby Mg (colorimetric method with chlorophosphonazo III, Roche Diagnostics, Basel, Switzerland), vit C (colorimetric method with dinitro-phenylhydrazine and thiourea), and vit E (HPLC method described by Desai, 1984) were determined. The plasma of one pig of each Mg group also was analyzed for Ca (method according to Schwarzenbach [1955] with cresolphtalein complexone; Roche Diagnostics). For the Trp treatment, the plasma of one animal in each negative control and Trp-supplemented group was selected and analyzed for Trp and large neutral amino acids (LNAA; Tyr, Phe, Val, Leu, and Ile) by colorimetric analysis (Commission Directive 1998/64/EC; OJEU, 1998).

Urine Collection and Analyses
In the metabolic cages, pigs were housed on metal slats, so urine flowed off and was collected in boxes. Urine was collected from 1700 to 0900 (reference sample, before vibration); and 2) from 1200 to 1400 (after vibration). One milliliter per 10 mL of 0.1 M EDTA was added, and the sample was frozen and stored at –20°C. Afterward, samples were analyzed for creatinine concentration (Creatinine Kinetic Method; Bio-labo, Maizy, France). Epinephrine and norepinephrine were determined with solid-phase extraction and HPLC according to Hay and Mormède (1997) and were expressed per milligram of creatinine.

Statistical Analyses
The feed and water intake data per three pigs during housing in the commercial pen were subjected to AN-OVA using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC), with group as the experimental unit. The effect of treatment was calculated and sex and average group weight were covariates. Other data (HR variables, cortisol concentrations, behavior, intermediary metabolites, catecholamines, and plasma concentration of Trp and LNAA) were analyzed as a mixed model (Littell et al., 1996) to account for treatment, period, sex, weight, and the treatment x period interaction. Sex and weight were added to complete the model, so results on sex and weight are not reported. No interactions between treatment and period were significant. Group and individual number were used as random factors, thereby taking into account the relationship between the measurements from the same group. Individual pig served as the experimental unit. Feed and water intake in the metabolic cage were analyzed similarly, but without the main effect "period." For the behavioral observation data, the total time each pig spent lying during a given hour was calculated as a percentage of that hour. Least mean squares were calculated, and differences between treatments or periods were separated using orthogonal contrasts. If necessary, a transformation to a normal distribution was performed (VEB, STE of Channels A and B, spectral power of HRV, cortisol, lactate, creatine kinase, and NEFA). A paired t-test was used to investigate the possible increase of Mg, vit E, or vit C in the blood, with pig as experimental unit. The means of the transformed data were retransformed, and the standard errors were calculated using the delta method (Serfling, 1980).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Literature Cited

 
Feed and Water Intake
Feed samples analyses for Trp, -tocopheryl acetate, and ascorbic acid revealed 2.48 g/kg, 100 mg/kg, and 48 mg/kg feed (as-fed basis) in the control feed and 7.76 g/kg, 241 mg/kg, and 224 mg/kg (as-fed basis) in the supplemented feed. Feed intake did not differ among treatments (vit E, vit C, and Trp, data not shown; P = 0.23) during housing in the commercial pen; feed (P = 0.27) and water intake (P = 0.89) did not differ during housing in groups of three. Water intake in the metabolic cages differed (P = 0.05; data not shown) among the treatments: the negative control pigs drank the most (2.0 ± 0.2 L) and the vit C pigs least (1.2 ± 0.2 L). Moreover, during this period, the feed intake of the vit C treatment (469 ± 66 g; as-fed basis) was significantly lower than that by pigs in all other treatments (overall mean = 695 ± 65 g).

Concentration of Supplements in Plasma
Plasma Mg concentrations did not change after Mg supplementation of drinking water (1.75 ± 0.09 vs. 1.75 ± 0.06 mg/dL; P = 1.0). Moreover, the supplementation of Mg did not affect the Ca concentration in the plasma (9.28 ± 0.83 vs. 6.63 ± 1.44 mg/dL; P = 0.18). Although Mg and Ca act as antagonists, the correlation between Mg and Ca was not significant (P = 0.84). Analysis of plasma samples of pigs fed diets supplemented with Trp showed that Trp concentration and Trp/LNAA increased significantly due to a supplementation of 6 g of Trp/kg for 5 d (Table 2), resulting in differences (P = 0.01) from the negative control treatment on the day of vibration. The concentration of the other LNAA (Tyr, Phe, Val, Leu, and Ile) decreased significantly during the supplementation with Trp, but no differences between the negative control and Trp treatment were found on the day of the vibration for these analyzed LNAA. Supplementing diets with vit C for 3 wk did not affect the plasma vit C concentration (before supplementation = 0.42 ± 0.05 mg/dL; after supplementation = 0.43 ± 0.03 mg/dL; P = 0.81), whereas a supplementation with vit E for 3 wk increased the plasma vit E concentration (before supplementation = 16.6 ± 1.5 mg/dL, after supplementation = 34.8 mg/dL; P < 0.001).

For most treatments, the highest STE of Channel A was noted at R1 (Table 4). The negative control treatment had higher STE of Channel A compared with the other treatments (except vit E) at night (rest). From the onset of the vibration, the positive control pigs showed the smallest STE, resulting in a lower level (P = 0.02) in R2 than when at rest.

The peak in R1 also was recognizable for the STE of Channel B and was even more pronounced (Table 4) than the peak of STE of Channel A. The elevation of the Trp treatment was smaller than that of the Mg treatment in R1 (P = 0.02).

Heart rate variability was analyzed during the night, during the vibration, and during the recovery period. Table 5 shows the ratio of the power in the low frequency range to the power in the high frequency range (LF/HF), LF, and rMSSD. The rMSSD data are not shown because these data were strongly correlated with HF (P < 0.001; r = 0.81). From rest to vibration, the LF/HF ratio tended to decrease, which was significant for the pigs fed Mg (P = 0.001). Compared with the preceding rest period, LF power tended to increase during vibration for Trp, vit C, and vit E treatments, whereas the other treatments had the opposite tendency. Only the Mg treatment had a decrease (P = 0.004) in LF power. After the vibration period, all values returned to resting values. The rMSSD had only increased significantly in the vit C group (P = 0.04) during the vibration, and after vibration all values were similar to the resting values.

Intermediary Metabolites
No differences in plasma glucose concentrations among treatments or before and after vibration were found (Table 8). The lactate concentration, however, decreased during the transport simulation for the negative control (P < 0.001), positive control (P = 0.04), Trp (P < 0.001), and Mg (P = 0.04) treatments. The Mg pigs had a higher lactate concentration than the vit E pigs (P = 0.04) and the negative control pigs (P = 0.02) before and after the vibration, respectively. For most treatments (with the exception of Mg and vit C), the NEFA concentration increased. Vitamin C had the highest concentration before vibration, whereas for the positive control group, it was greatest after vibration. For all treatments, the creatine kinase concentration showed a tendency to increase during vibration, but this increase was only significant for the Mg treatment (P = 0.005).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Literature Cited

Magnesium
Pigs in the Mg treatment received 3 g of Mg acetate/L of drinking water for 2 d to decrease stress. The length of the supplementation also is critical for the meat quality. Frederick et al. (2004) concluded that adding 900 mg of elemental Mg from MgSO4•7H20 per liter of drinking water for 2 d was more efficacious for some meat quality traits (fluid loss, lipid oxidation, and color) than supplementation for 6, 4, or 0 d. In our experiment, supplementation of Mg did not affect the concentration of plasma Mg. Frederick et al. (2004) also reported a 9% increase in plasma Mg concentration of slaughter pigs after 2 d of supplementation (900 mg/L), but this difference was not significant. An increase of the plasma Mg concentrations by 14% was found after 5 d of supplementation with 3.6 g of Mg oxide/(pig•d) (Lahucky et al., 2004); and by 10% after 5 d of supplementation with 3.2 g of Mg sulfate/(pig•d) (D’Souza et al., 1999). In contrast, Geesink et al. (2004) did not find an increase in plasma Mg after a 5-d supplementation with 6.8 g of Mg acetate/kg feed. The different results could be due to different Mg sources. D’Souza (1999) demonstrated that plasma Mg concentrations of pigs that were fed MgSO₄ were higher than those of pigs that were fed MgCl₂ or Mg aspartate. In the current study, a decrease in plasma Ca concentration was noticed after Mg supplementation, but this was not significant. Only one plasma sample of each Mg group was analyzed for Ca; hence, the limited sample size might have decreased the ability to detect a significant differnce. Supplementation of Mg resulted in the lowest VEB during the registered period. The number of VEB reflects the anomalous beats not generated in the pacemaker of the heart but elsewhere in the myocardium, and is considered a measure of stress (Ellestadt, 1987). The ability of the mechanisms of Mg to decrease the incidence of ventricular arrhythmias is well-studied (Steurer et al., 1996; Delva, 2003), and our results confirmed the findings of decreased arrhythmias after Mg supplementation. The Mg pigs also had a small STE of Channel A, but during the first hour of the recovery period, the Mg pigs had a greater STE of Channel B, which is in contrast with the ability of Mg to stabilize heart functions. The decrease in sympathetic modulation was evidenced by the significant decrease of LF power during the vibration. The LF/HF ratio also indicated the vagal dominance during the vibration. In contrast, the decrease in LF power in the negative control group could be attributed to an overstimulation of sympathetic tone, causing the sinus node saturation and inability to respond. This is evidenced by the differences in epinephrine and norepinephrine concentration between these groups. In the negative control group, these concentrations increased after the test, whereas Mg treatment did not alter the concentrations. This could be because Mg pigs lay down more from the second half hour of the vibration. During the first half hour, pigs of all treatments explored the new environment of the vibration crate. Kuhn et al. (1981) also reported visually calmer pigs after long-distance transportation due to Mg supplementation and studies with normal Mg-sufficient rats, mice, and guinea pigs showed that intra-peritoneal injections with Mg decreased motility and explorations (Kaemmerer and Kietzmann, 1984). The vibration caused an increase in saliva cortisol concentration, which did not return to the rest value after recovery. In contrast to the negative control animals, the Mg pigs had no significant increase of epinephrine after vibration. Great variances for the catecholamines were observed to have been caused by great individual variations. The greater variances of the urine samples taken after the vibration are due to the fact that not all pigs urinated during the 2-h recovery period. The advantages of this method are the noninvasive and the integrative collection of hormones. D’Souza et al. (1999) took blood samples at slaughter and did not find differences in catecholamine concentration between negative control and Mg-supplemented pigs. Assuming that Mg plays an essential role in lowering stress effects resulting in decreased muscle glycogenolysis (D’Souza et al., 1998), a lower lactate concentration was expected in the plasma of the Mg pigs, but this was not the case. Moreover, the Mg group had a higher lactate concentration than the negative control group (P < 0.05) after vibration. In contrast, Geesink et al. (2004) found no significant difference in lactate concentration of negative control and Mg supplemented pigs at slaughter. The leakage of creatine kinase into the plasma is related to the injury of the membranes of the muscular tissue and Mg serves as a physiologic regulator of membrane stability (Lukaski, 2004). The vibration caused a doubling of creatine kinase in pigs of the Mg treatment, but it has to be mentioned that the level before vibration was relatively low, and the level after vibration was average relative to the other treatments. One explanation for the increase in injured membranes may be the fact that these animals lay more on the floor of the vibration crate. Thus, when lying, additional vibration energy enters directly through the body, whereas standing, the legs may offer some protection from the vibration originating from the floor (Randall et al., 1995). Nonesterified fatty acids, used as energy source during stress, did not increase significantly during vibration after Mg supplementation, and concentrations were at a minimum before and after vibration, which may indicate that the rate of lipolysis remained in the same range during the experiment.

Tryptophan
In this experiment, Trp did not influence feed or water intake, suggesting no imbalance of Trp because an excess of essential AA could affect the appetite of animals (Harper et al., 1970). In a preliminary experiment, Adeola and Ball (1992) found that the hypothalamic serotonin concentration peaked 5 d after introducing feed fortified with Trp (5 g of Trp/kg of feed), a precursor of brain serotonin. Increasing dietary Trp causes increases in plasma and brain Trp and brain serotonin (Fernstrom and Wurtman, 1971), and the transport of Trp over the blood–brain barrier depends on the concentration of Trp and LNAA (Fernstrom, 1983). In our experiment, 6 g of Trp/kg of feed was supplemented for 5 d. Analyses showed a significant increase in Trp and Trp/LNAA over this period, resulting in higher values (16.3 g/L and 0.17, respectively) compared with the negative control treatment (5.3 g/L and 0.07, respectively). Adeola and Ball (1992) observed a Trp/LNAA-ratio of 0.141 and 0.073 for the Trp and negative control treatments, respectively, but also found higher concentrations of some LNAA (Tyr, Phe, and Leu) after Trp addition. In our experiment, these differences were rather small. Moreover, when focused on the change over time, the concentration of LNAA decreased significantly for the Trp pigs. Because no plasma sample was collected from control pigs 5 d before vibration (because they also served as control for the other treatments), it is not certain that this decrease was due to the supplementation of Trp. The effect of Trp on the HR variables was relatively minor. The values of the mean, peak, and minimum heart rate followed the pattern of the negative control treatment. During the second hour of vibration, more VEB were recorded than in the Mg treatment, but no differences were found with the negative control treatment. The more VEB, the greater the deficiency of blood supply to the heart when it is overloaded, a condition leading to arrhythmicities (Ellestad, 1987). The time-dependent pattern of STE of Channel A was approximately the same as in our previous similar experiment (Peeters et al., 2004). The low STE of Channel B for the Trp pigs is remarkable because it is contrary to the results of our previous experiment. In that study, 5 g of Trp/L of drinking water was supplemented for 3 d and resulted in a higher STE of Channel B than in the negative control treatment; therefore, 6 g of Trp/kg of feed for 5 d seemed to have better results. Tryptophan did not change the LF/HF ratio. Both the sympathetic and the vagal indices did not change significantly during vibration, so this treatment had the smallest effect on HRV. The lying time of Trp pigs was intermediate between that of the negative control and the Mg pigs, but differences were not significant. This result confirms the findings of the previous study (Peeters et al., 2004), where Trp pigs also lay down more than negative control pigs. Due to the vibration the salivary cortisol level doubled in the Trp treatment, to return to the basal level at the end of the recovery period. This finding suggests that Trp could influence the cortisol release. Concentrations of salivary cortisol are, as with plasma cortisol, greatest in the early morning hours and least around midnight (Kirschbaum and Hellhammer, 1989). To exclude the effect of cortisol circadian rhythmicity, the first (reference sample) and second (after vibration) sampling points were collected at the same time of day. The influence of Trp on the cortisol concentration is in contrast to the results of the study by Meunier-Salan et al. (1991), which revealed that the plasma cortisol of young pigs was not affected by an excess of Trp.

Vitamin E
The surplus of vit E in the feed had no influence on daily feed intake. Likewise, Cannon et al. (1996) did not observe a difference in feeding characteristics between a negative control and vit E-supplemented group for a period of 84 d. In the present study, an application period of 3 wk was chosen because this period is required to transfer vit E from the liver to the muscles (Flachowsky, 2000). Analyses of vit E in plasma revealed that the vit E concentration doubled after 3 wk of supplementation. Flachowsky et al. (1998) also found a doubling of plasma vit E concentration after administering 1 g of -tocopheryl acetate/(pig•d) over a period of 3 wk before slaughtering. Vitamin E affected the mean HR because the general pattern of an increase at the beginning of the vibration was not found for this treatment; thus, vit E may allow for the stabilization of the mean HR. The low minimum HR and the significant decrease during recovery also revealed that animals supplemented with vit E could recover easily from vibration. Vitamin E caused a slight increase in LF power during the vibration, indicating augmented sympathetic activity; however, whereas the negative control group was oversaturated with sympathetic impulses, the vit E group still maintained its ability to modulate HR accordingly. Moreover, at the same time the stimulatory effect on vagal modulation of vit E is evidenced by a significant decrease in the LF/HF ratio and the higher rMSSD values, although the latter did not reach the significance level. The low HR in this group and the low epinephrine and norepinephrine concentrtions also support these findings. The effect of vit E on the working of heart is known chiefly for patients with heart diseases (Hoffman and Garewal, 1995; Clark and Armitage, 2002), but no literature on its effect on healthy beings was found. For one possible explanation of the working of vit E on the heart rate measurements, the authors referred to Peeters et al. (2004). In that experiment, the vit E pigs were more relaxed than the negative control treatment. The vit E treatment showed the best results for saliva cortisol with the lowest concentrations after vibration and recovery, confirming previous results (Peeters et al., 2004). The steady state of the concentration of lactate and creatine kinase with the vit E supplementation is remarkable, whereas other treatments resulted in decreases in lactate of at least 4 mg/dL or increases in creatine kinase of at least 500 IU/L. There is a relationship between the leakage of creatine kinase into the plasma and the injury of the membranes of the muscular tissue, so it may be concluded that vit E stabilizes the membranes, especially during stress situations. Lauridsen et al. (1999) and Asghar et al. (1991) did not find an effect of vit E on plasma or serum creatine kinase concentration of pigs, but in both experiments, blood was not taken after a stress situation, and therefore, results are similar to those in the present experiment in that before vibration an equal concentration was observed in vit E-supplemented and negative control pigs.

Vitamin C
During housing in commercial pens and in groups of three, water and feed intake by the pigs in the vit C treatment were comparable to that by pigs in the other treatments. When the pigs were individually housed, however, they ate and drank less during 1 d of the 3 wk treatment, which can be considered as unimportant. After supplementation of 300 mg of L-ascorbic acid/kg of feed for 3 wk, an increase in plasma concentration of vit C was expected, but this was not the case. In contrast, Yen and Pond (1987) and de Rodas et al. (1998) reported such an increase. An explanation for the different results might be the unstable properties of vit C in mixed foods (McDowell, 1989). In our experiment, the concentration of vit C of the supplemented and control feed also was analyzed, indicating a difference between the two feeds of 193 mg of vit C/kg of feed instead of 300 mg/kg (as-fed basis). It should be noted that this feed was analyzed 1 mo after the end of the experiment, so further oxidation was probable. Moreover, the vit C treatment influenced some of the investigated response criteria, so the lack of difference in plasma vit C concentration before and after 3 wk of supplementation is unexplained. The minimum HR of this treatment was relative low, with exception of the high rest levels. After vibration, the number of VEB returned to their rest level, but with 30 VEB at rest, this level was the highest of all treatments. Thus, there was only a small, negligible effect of vit C on the heart rate variables. In contrast, HRV indices clearly demonstrated the vagal predominance caused by the vit C treatment. This is evidenced by the significant increase in rMSSD and significant decrease in LF/HF ratio during vibration. Numerous studies have been published indicating the effect of vit C on endothelial function, more specifically in its antioxidant function (Drexler and Hornig, 1999; Richartz et al., 2001). This function provides an improvement for the vascular tone, which also is beneficial for autonomic control (Piccirillo et al., 2003). In addition, the salivary cortisol concentrations of pigs of this treatment were in the same range as those of the negative control group and remained high during recovery. The vit C pigs also lay more than the negative control ones. Compared with all treatments, the pigs supplemented with vit C had the highest concentration of NEFA. Vitamin C supplementation did not decrease muscle damage because plasma creatine kinase values were not related to vit C supplementation (Thompson et al., 2001; Dawson et al., 2002).

Positive Control
A withdrawal period of 12 h between the last administration of carazolol (positive control) and slaughter should be used for food safety, so with this in mind, the product was injected the evening before vibration. Our results indicated that the product was still effective after this period in producing low levels for mean, peak, minimum HR, STE of Channel A, low power, and rMSSD during rest, vibration, and recovery. The benefits of beta-blockers for the treatment of heart failure (Goldstein, 2002) and for decreasing HR (Petzold et al., 1999) are well-known. One negative aspect of carazolol was the high number of VEB at the start of vibration. As expected, the beta-blocker caused a decrease in LF power, presenting the lowest values before, during, and after the vibration. Sympathetic heart rate modulation was almost completely absent. Considering the behavior, intermediary metabolites, cortisol, and epinephrine concentrations, rather modest and higher values or concentrations were reached, so no meat quality amelioration of slaughter pigs should be expected.

This study investigated the effect of several feed supplements on pigs’ stress responses to vibration. A multidisciplinary approach was used, and when considered together, is an advantage because animals vary in their strategies to cope with adversity and in the susceptibility to adverse conditions (Broom, 1995). Moreover, this experiment allows for the comparison of the influences of the additives on the described variables.

Overall, these results indicated that supplementation of Trp, Mg, vit E, or vit C improved the coping ability of pigs during transport simulation compared with the negative control treatment. Vitamin C supplementation had the least effect, whereas vit E acted most effectively; however, the effect of vit C as a vagal stimulator was visible in this study. Pigs on the Mg treatment were the least physically active, but this was not reflected in the other measured stress variables. Further research will investigate the influences of the additives used in the present study on the stress responses and meat quality of slaughter pigs.

	Footnotes

 
¹ This project was funded by IWT-Vlaanderen (Institute for the Promotion of Innovation through Science and Technology in Flanders) and Katholieke Universiteit Leuven. Thanks are due to D. Henot and the staff of the Zootechnical Centre for taking care of the animals and the assistance. We also thank Trouw Nutrition, Gent, Belgium, DSM Nutrition, Deinze, Belgium, and Orffa, Londerzeel, Belgium/Ajinomoto Eurolysine, Paris, France for the analysis of plasma or feed.

² Correspondence: Bijzondere weg 12 (phone: +32-16-46 81 37; fax: +32-16-46 81 59; e-mail: Ester.Peeters{at}biw.kuleuven.be).

Received for publication October 21, 2004. Accepted for publication April 4, 2005.

	Literature Cited

Top Abstract Introduction Materials and Methods Results Discussion Literature Cited

 


Adeola, O., and R. O. Ball. 1992. Hypothalamic neurotransmitter concentrations and meat quality in stressed pigs offered excess dietary tryptophan and tyrosine. J. Anim. Sci. 70:1888–1894.[Abstract]

Asghar, A., J. I. Gray, E. R. Miller, P. K. Ku, A. M. Booren, and D. J. Buckley. 1991. Influence of supranutritional vitamin E supplementation in the feed on swine growth performance and deposition in different tissues. J. Sci. Food Agric. 57:19–29.

Aubert, A. E., D. Ramaekers, F. Beckers, R. Breem, C. Denef, F. Van de Werf, and H. Ector. 1999. The analysis of heart rate variability in unrestrained rats. Assessment of methods and results. Comput. Meth. Programs Biomed. 60:197–213.

Broom, D. M. 1995. Quantifying pigs welfare during transport using physiological measures. Pages 3–9 in Proc. EU Seminar "New information on welfare and meat quality of pigs as related to handling, transport and lairage conditions," Mariensee, Germany.

Broom, D. M., and K. G. Johnson. 1993. Stress and Animal Welfare. 1st ed. Chapman and Hall, London, U.K.

Cannon, J. E., J. B. Morgan, G. R. Schmidt, J. D. Tatum, J. N. Sofos, G. C. Smith, R. J. Delmore, and S. N. Williams. 1996. Growth and fresh meat quality characteristics of pigs supplemented with vitamin E. J. Anim. Sci. 74:98–105.[Abstract]

Dantzer, R., and P. Mormède. 1983. Stress in farm animals—A need for reevaluation. J. Anim. Sci. 57:6–18.

Dawson, B., G. J. Henry, C. Goodman, I. Gillam, J. R. Beilby, S. Ching, V. Fabian, D. Dasig, P. Morling, and B. A. Kakulus. 2002. Effect of vitamin C and E supplementation on biochemical and ultrastructural indices of muscle damage after a 21 km run. Int. J. Sports Med. 23:10–15.[Medline]

Delva, P. 2003. Magnesium and heart failure. Mol. Aspects Med. 24:79–105.[Medline]

Desai, I. D. 1984. Vitamin E analysis-methods for animal tissues. Methods Enzymol. 105:138–147.[Medline]

Drexler, H., and B. Hornig. 1999. Endothelial dysfunction in human disease. J. Mol. Cell. Cardiol. 31:51–60.[Medline]

D’Souza D. N., R. D. Warner, F. R. Dunshea, and B. J. Leury. 1999. Comparison of different dietary magnesium supplements on pork quality. Meat Sci. 51:221–225.

D’Souza, D. N., R. D. Warner, B. J. Leury, and F. R. Dunshea. 1998. The effect of dietary magnesium aspartate supplementation on pork quality. J. Anim. Sci. 76:104–109.[Abstract/Free Full Text]

Eichenberger, B., H. P. Pfirter, C. Wenk, and S. Gebert. 2004. Influence of dietary vitamin E and C supplementation on vitamin E and C content and thiobarbituric acid reactive substances (TBARS) in different tissues of growing pigs. Arch. Anim. Nutr. 58:195–208.[Medline]

Fernstrom, J. D. 1983. Role of precursor availability in control of monoamine biosynthesis in brain. Physiol. Rev. 63:484–546.[Free Full Text]

Fernstrom, J. D., and R. J. Wurtman. 1971. Brain-serotonin content: Physiological dependence on plasma tryptophan levels. Science 173:149–152.[Abstract/Free Full Text]

Flachowsky, G. 2000. Vitamin E-transfer from feed into pig tissues. J. Appl. Anim. Res. 17:69–80.

Flachowsky, G., T. Langbein, H. Bohme, A. Schneider, and K. Aulrich. 1998. Effect of false flax expeller combined with short-term vitamin E supplementation in pig feeding on the fatty acid pattern, vitamin E concentration and oxidative stability of various tissues. J. Anim. Physiol. Anim. Nutr. 78:187–195.

Frederick, B. R., E. van Heugten, and M. T. See. 2004. Timing of magnesium supplementation administered through drinking water to improve fresh and stored pork quality. J. Anim. Sci. 82:1454–1460.[Abstract/Free Full Text]

Geers, R., G. Parduyns, V. Goedseels, L. Bosschaerts, and J. De Ley. 1990. Skeletal muscularity and heart function in growing piglets. Ann. Rech. Vet. 21:231–236.[Medline]

Geesink, G. H., R. G. C. van Buren, B. Savenije, M. W. A. Verstegen, B. J. Ducro, J. G. P. van der Palen, and G. Hemke. 2004. Short-term feeding strategies and pork quality. Meat Sci. 67:1–6.

Goldstein, S. 2002. Benefits of beta-blocker therapy for heat failure—Weighing the evidence. Arch. Intern. Med. 162:641–648.[Abstract/Free Full Text]

Harper, A. E., N. J. Benevenga, and R. M. Wohlhueter. 1970. Effect of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 11:428–558.

Hay, M., and P. Mormède. 1997. Determination of catecholamines and methoxycatecholamines excretion patterns in pig and rat urine by ion-exchange liquid chromatography with electrochemical detection. J. Chromatogr. B 703:15–23.

Henry, Y., B. Sève, Y. Collèaux, P. Ganier, C. Saligaut, and P. Jégo. 1992. Interactive effects of dietary levels of tryptophan and protein on voluntary feed intake and growth performance in pigs, in relation to plasma amino acids and hypothalamic serotonin. J. Anim. Sci. 70:1873–1887.[Abstract]

Hoffman, R. M., and H. S. Garewal. 1995. Antioxidants and the prevention of coronary heart disease. Arch. Intern. Med. 155:241–246.[Abstract/Free Full Text]

Kaemmerer, K., and M. Kietzmann. 1984. Studies on magnesium. Zentralb. Veterinarmed. Reihe A 31:321–339.

Kietzmann, M., and H. Jablonski. 1985. On the blocking of stress by magnesiumaspartate hydrochloride in the pig. Prakt. Tierarzt. 66:328–335.

Kirschbaum, C., and H. Hellhammer. 1989. Salivary cortisol in psychobiological research: An overview. Neuropsychobiology 22:150–169.[Medline]

Kuhn, G., A. Nowak, E. Otto, V. Albrecht, B. Gassmann, E. Sandner, H. Przybilski, and L. Zahn. 1981. Studies on the control of meat quality by special treatment of swine. 1. Effects of stress and preventative magnesium feeding on selected parameters of carcass value and blood serum. Arch. Tierzucht 24:217–225.

Lahucky, R., U. Kuchenmeister, I. Bahelka, D. Vasicek, T. Liptaj, and K. Ender. 2004. The effect of dietary magnesium oxide supplementation on postmortem P-31 NMR spectroscopy parameters, rate of Ca2+ uptake and ATPase activity of M. longis-simus dorsi and meat quality of heterozygous and normal on malignant hyperthermia pigs. Meat Sci. 67:365–370.

Lambooij, E. 1988. Road transport of pigs over a long distance: Some aspects of behaviour, temperature and humidity during transport and some effect of the last two factors. Anim. Prod. 46:257–263.

Lambooij, E., and G. van Putten. 1993. Transport of pigs. Pages 213–231 in Livestock Handling and Transport. T. Grandin, ed. CABI Int., Wallingford, U.K.

Lauridsen, C., S. Hojsgaard, and M. T. Sorensen. 1999. Influence of dietary rapeseed oil, vitamin E, and copper on the performance and the antioxidative and oxidative status of pigs. J. Anim. Sci. 77:906–916.[Abstract/Free Full Text]

Littell, R. C., G. A. Milliken, W. W. Stroup, and R. D. Wolfinger. 1996. SAS System for mixed models. 3rd ed. SAS Inst., Inc., Cary, NC.

Manz, U., and K. Philipp. 1981. A method for the routine determination of tocopherols in animal feed and human foodstuffs with the aid of high performance liquid chromatography. Int. J. Vitam. Nutr. Res. 51:342–348.[Medline]

McDowell, L. R. 1989. Vitamins in Animal Nutrition: Comparative Aspects to Human Nutrition. Academic Press, San Diego, CA.

Meunier-Salaün, M. C., M. Monnier, Y. Colléaux, B. Sève, and Y. Henry. 1991. Impact of dietary tryptophan and behavioral type on behavior, plasma cortisol, and brain metabolites of young pigs. J. Anim. Sci. 69:3689–3698.[Abstract]

OJEU. 1986. Council Directive 86/64/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Off. J. Eur. Union L 358:0001–0028.

OJEU. 1998. Commission Directive 98/64/EC of 3 September 1998 establishing Community methods of analysis for the determination of amino-acids, crude oils and fats, and olaquindox in feedingstuffs and amending Directive 71/393/EEC. Off. J. Eur. Union L 257:0014–0028.

Peeters, E., B. Driessen, R. Steegmans, D. Henot, and R. Geers. 2004. Effect of supplemental tryptophan, vitamin E, and a herbal product on responses by pigs to vibration. J. Anim. Sci. 82:2410–2420.[Abstract/Free Full Text]

Perremans, S., J. M. Randall, L. Allegaert, M. A. Stiles, G. Rombouts, and R. Geers. 1998. Influence of vertical vibration on heart rate of pigs. J. Anim. Sci. 76:416–420.[Abstract/Free Full Text]

Petzold, T., P. Feindt, M. D. Menger, and E. Gams. 1999. Beta-adrenoceptor inhibition for the induction of acute cardiac failure in pigs. Lab. Anim. 33:366–371.[Abstract/Free Full Text]

Piccirillo, G., M. Nocco, A. Moise, M. Lionetti, C. Naso, S. di Carlo, and V. Marigliano. 2003. Influence of vitamin C on baroreflex sensitivity in chronic heart failure. Hypertension 41:1240–1245.[Abstract/Free Full Text]

Randall, J. M., J. A. Duggan, and M. A. Alami. 1995. Influence of motion and vibration of animals. Fleischwirtschaft 75:201–204.

Raskin, P., A. Clinquart, C. Marche, and L. Istasse. 1997. Vitamin E et qualité de viande. Ann. Med. Vet. 141:113–126.

Richartz, B. M., G. S. Werner, M. Ferrari, and H. R. Figulla. 2001. Reversibility of coronary endothelial vasomotor dysfunction in idiopathic dilated cardiomyopathy: Acute effects of vitamin C. Am. J. Cardiol. 88:1001–1005.[Medline]

Sahin, K., N. Sahin, and O. Kucuk. 2003. Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32 degrees C). Nutr. Res. 23:225–238.

Satterlee, D. G., I. Aguileraquintana, B. J. Munn, and B. A. Krautmann. 1989. Vitamin-C amelioration of the adrenal stress response in broiler-chickens being prepared for slaughter. Comp. Bioch. Physiol. A Physiol. 94:569–574.

Schwarzenbach, G. 1955. The complexones and their analytical application. Analyst 80:713–729.

Serfling, R. J. 1980. Chapter 3: Transformations of given statistics. Pages 117–137 in Approximation Theorems of Mathematical Statistics. 1st ed. John Wiley and Sons, New York, NY.

Steurer, G., P. Yang, V. Rao, W. Mohl, D. Glogar, and R. Smetana. 1996. Acute myocardial infarction, reperfusion injury, and intravenous magnesium therapy: Basic concepts and clinical implications. Am. Heart J. 132:478–482.[Medline]

Task Force. 1996. Heart rate variability: Standards of measurement, physiological interpretation and clinical use. Circulation 93:1043–1065.[Free Full Text]

Thompson, D., C. Williams, S. J. McGregor, C. W. Nicholas, F. McArdle, M. J. Jackson, and J. R. Powell. 2001. Prolonged vitamin C supplementation and recovery from demanding exercise. Int. J. Sport Nutr. Exerc. Metab. 11:466–481.[Medline]

Yen, J. T., and W. G. Pond. 1987. Effect of dietary supplementation with vitamin C or carbadox on weanling pigs subjected to crowding stress. J. Anim. Sci. 64:1672–1681.

This article has been cited by other articles:

				S. K. Park, K. L Tucker, M. S O'Neill, D. Sparrow, P. S Vokonas, H. Hu, and J. Schwartz Fruit, vegetable, and fish consumption and heart rate variability: the Veterans Administration Normative Aging Study Am. J. Clinical Nutrition, March 1, 2009; 89(3): 778 - 786. [Abstract] [Full Text] [PDF]

				E. Peeters, B. Driessen, and R. Geers Influence of supplemental magnesium, tryptophan, vitamin C, vitamin E, and herbs on stress responses and pork quality J Anim Sci, July 1, 2006; 84(7): 1827 - 1838. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peeters, E. Articles by Geers, R. Search for Related Content PubMed PubMed Citation Articles by Peeters, E. Articles by Geers, R.