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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
School of Veterinary and Biomedical Sciences, James Cook University, Townsville 4811, Queensland, Australia
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: acid-base • Bos indicus • cattle • electrolyte • osmolyte • transport
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Tarrant et al., 1992; Parker et al., 2003b). Hydration strategies used by the livestock industries rely primarily on the replacement of lost body water and electrolytes at the completion of the journey, after the welfare of the animals may have been compromised (Wythes et al., 1980; Schaefer et al., 1990, 1997). The expansion of plasma volume before exposure to a dehydrating environment, such as the marketing process, is problematic for most mammalian species. Hyperhydrating with water alone is transitory because the kidney rapidly excretes any excess fluid.
Prophylactic hyperhydration with the osmolyte glycerol at 2 g/kg of BW has been used to prevent or delay dehydration in human athletes (Freund et al., 1995; Hitchins et al., 1999). Improved cellular water retention in cattle has been achieved by including betaine in the ration at 0.03 g/kg of BW for 7 d preslaughter (Bock et al., 2002). Glycerol is also a metabolic substrate for liver gluconeogenesis (Lehninger et al., 1993), whereas betaine may provide a source of ruminally available N or methyl groups to the rumen microbial population (Löest et al., 2002). Therefore, the administration of osmolytes before transportation of long duration may have merit in attenuating the deleterious effects of dehydration and promote glucose production while sparing muscle protein degradation.
We investigated the effects of 2 organic osmolytes, betaine and glycerol, on the total body water, electrolyte, glucose, and acid-base balance of Bos indicus steers subjected to transportation for 48 h. Our working hypothesis was that prophylactic treatment of steers with osmolytes would attenuate the loss of body water, enhance glucose production, and spare muscle protein degradation during long duration transportation.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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All experimental procedures were reviewed and approved by the animal ethics committee at James Cook University. Bos indicus steers (n = 24; 2.5-yr old; mean BW = 321 ± 5 kg) were sorted in ascending order of BW and allocated sequentially to 1 of 4 treatment groups: 1) control, feed and water deprived for 48 h (n = 6); 2) transported, deprived of feed and water and transported for 48 h (n = 6); 3) glycerol, dosed with glycerol (2 g/kg of BW, Biotech Pharmaceuticals Pty Ltd., Carole Park, Queensland, Australia), deprived of feed and water and transported for 48 h (n = 6); and 4) betaine, dosed with betaine (0.25 g/kg of BW, Betafin, Brisbane, Queensland, Australia), deprived of feed and water and transported for 48 h (n = 6). All transported steers were dosed using a nasogastric tube (7 mm o.d. x 5 mm i.d.) and all treatments had 500 mL of distilled water added to aid the flow of the product through the tube. The transported group received a placebo of 500 mL of distilled water. All groups had their water and feed withdrawn 12 h before departure of the transported groups.
The study was divided into 3 journeys of 48 h over a 2-wk period, with steers allocated to the journeys at random. The steers that were transported were conveyed in a rigid-body truck, equipped with an adjustable gate separating the holding compartment into 2 areas. The truck carried 2 steers from each of the transported groups per journey at a density of 1.20 m2/steer. The groups that were transported were trucked for 24 h, unloaded, sampled, and weighed, and then transported for a further 24 h before being unloaded, sampled, and weighed again. The sampling process, including the body water assays, took 2.5 h to complete. The steers did not have access to feed and water in the yards while waiting to be sampled and were immediately processed upon exiting the unloading ramp.
At sampling, all steers were gently coerced into a chute, where the steers were captured in a cattle head bail and restrained. A halter was placed on each individual animal and their head was then restrained to the side with an attendant holding the head while samples were taken. The steers used in the current study were accustomed to being handled and behaved in a quiet and amicable manner when sampled.
During journey 1, the blood gas analyzer developed a mechanical problem at the 24 h sampling. Subsequently, the samples for blood gas analysis could not be processed within the required time limit to yield reliable data. The data in Table 1 reflect this incident, where, for the 24 h analysis for blood gas measurements, n = 4/group, except the control, where n = 5.
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Sample Collection
After 24 and 48 h of transportation, 24.5 mL of blood was manually collected by jugular venipuncture from each animal from all groups; 20 mL into two 10-mL tubes containing lithium heparin (Disposable Products Pty Ltd., Adelaide, South Australia, Australia); one 2.5-mL tube containing fluoride oxalate (Sarstedt Australia, Technology Park, South Australia, Australia), and a 2-mL blood gas syringe containing lithium heparin (Sarstedt Australia) to sample venous blood gases. The samples containing fluoride oxalate were used for the analysis of plasma lactate and glucose. The tubes containing lithium heparin were used for all other analyses. Blood samples were immediately placed into an ice-water slurry until centrifugation at 200 x g for 15 min. Plasma was decanted within 2 h of the blood sample being taken and frozen (–20°C) for analysis at a later date. Blood gas syringes were capped and placed into an ice-water slurry for immediate analysis of blood gases. All blood gas assays were performed within 0.5 h of collection.
Blood Variable Measurement
Venous blood pH and partial pressures of carbon dioxide (pCO2) and bicarbonate (HCO3–) were measured using a blood gas analyzer (Ciba Corning Model 278, Bayer Diagnostics, Brisbane, Queensland, Australia). Plasma concentrations of Na and K were measured using ion-selective electrodes (Lablyte System 830, Beckman Instruments Inc., Brea, CA). Sodium and K analyses were completed on singular samples, and quality control samples (Liquichek controls, 16171 & 16172, BioRad Laboratories, Regents Park, New South Wales, Australia) were performed every 10 samples. Lactate, glucose, albumin, total protein, P, Ca, Mg, and Cl concentrations in plasma were measured using a Mira Autoanalyzer (Roche Diagnostics, Brisbane, Queensland, Australia) with standard enzymatic and spectrophotometric kits [Lactate, Roche Diagnostics; and P (TR30025), Albumin (TR36025), Total Protein (TR34025), Ca (TR29248), and Cl (TR38025), Trace Scientific Ltd. Noble Park, Victoria, Australia). Plasma urea N was analyzed with a Technicon AutoAnalyser 2 (Bran + Leubbe Pty Ltd., Homebush, New South Wales, Australia) according to the AutoAnalyser method SE40001FD4.
Anion gap (AG; Polancic, 2000) was obtained from the equation: AG, mEq/L = [(Na+ + K+) – (Cl– –HCO3–)], whereas strong ion difference (SID; Stewart, 1983) was obtained from the equation: SID, mEq/L = [(Na+ + K+) – (Cl– – lactate)]. Total weak acids (Atotal) were calculated from the equation of Figge et al. (1992): Atotal, mEq/L = {albumin x [1.23 x (pH – 6.31)]} + [({phosphorous x [0.309 x (pH – 0.469)]} x 10)/30.97].
Urea Space Measurements
Urea space was determined at the conclusion of each 24-h transit period for each animal using the technique described by Preston and Kock (1973). In brief, after catheterization of a jugular vein, a solution containing 20% (wt/vol) urea dissolved in 0.9% (wt/vol) saline was administered through the catheter over a 2-min period. The volume injected was calculated to provide 130 mg of urea/kg of BW. The catheter was immediately flushed with 15 mL of heparinized saline solution (35,000 IU/L of 0.9% saline) to prevent clotting between samples. Blood samples were collected through the catheter before infusion and at 15 min postinfusion.
Statistical Analysis
A 2-way ANOVA with the main effects of time (24 and 48 h) and group (control, transported, glycerol, or betaine) and an interaction effect of steers nested within treatments was used to analyze the data using SPSS 10 software package (SPSS, Chicago, IL). Quantitative variables (plasma electrolytes, albumin, total protein, glucose, hematocrit, osmolality, and blood gases) were independently sampled. Body weight and accumulated BW loss (Table 1
) were analyzed using a 2-way, repeated measures ANOVA using SPSS 10, with the main effects of time (24 and 48 h) and group (control, transported, glycerol, or betaine) and the interaction effect of group x time. Least squares means and SE are presented. Multiple comparison tests within the factors were performed using Tukey’s honestly significant difference test. Differences were considered significant for P < 0.05.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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There was a treatment group x time interaction (P = 0.01) between 24 and 48 h indicating that the control group lost less BW than the transported groups at 48 h (Table 1). A time effect for all treatment groups demonstrated a decrease in BW with increased time off feed and water. Loss of BW is similar to other reported values in long haul transportation studies (Camp et al., 1981; Fisher et al., 1999). Loss of BW from fasting and transport largely reflect changes in gut fill and defecation and urination rates (Wythes et al., 1980). There was no differentiation due to treatment group or transportation during the first 36 h of feed and water deprivation for BW, suggesting that transportation stress per se did not have a significant effect on BW shrink. This is in contrast to Cole et al. (1988), where transportation of 195-kg calves caused an increase in shrinkage, compared with feed and water deprivation alone.
Body Water
The glycerol group had a greater (P = 0.05 and 0.02, respectively) body water content at 24 h than the control and transported groups (Table 1). However, 1 animal contributed largely to the statistical difference seen in the glycerol group for body water. Therefore, the body water data should be interpreted with caution. Human trials with glycerol have demonstrated a greater retention of fluid within the body and a delay in the loss of body water in tropical and temperate environs (Riedesel et al., 1987; Freund et al., 1995; Hitchins et al., 1999). Glycerol increases fluid retention by reducing free water clearance (Freund et al., 1995). An increase in urination rate has been shown to be a contributing factor to dehydrating cattle under heat stress conditions (El-Nouty et al., 1980). In addition, stress-like concentrations of cortisol have induced a diuresis in sheep (Parker et al., 2003a). Therefore, it would be reasonable to hypothesize that osmolytes, such as glycerol, may attenuate the effects of dehydration in transported ruminants; however, our data appear equivocal. Although the body water data support a greater hydration effect for glycerol, BW does not reflect the expected greater value for the glycerol group. Our failure to concurrently demonstrate a greater BW for the glycerol group in support of the body water assay may lie in an increase in gut motility and hence a greater loss of gastrointestinal tract contents for the glycerol group. Indeed, the pharmacologic effects of glycerol in humans are known to include nausea, gastrointestinal cramps, and vomiting (Wagner, 1999). Further, the urea space in the ruminant is a measure of empty body water (total body water less the water in the gastrointestinal tract; Preston and Kock, 1973). It may be possible that the dose rate of glycerol used in this study maintained empty body water in the steers, but increased gastrointestinal emptying.
Hyperhydration with osmolytes may only slow the development of water loss from the body, as such. Perhaps a greater difference between both osmolyte treated groups would be seen with shorter transportation intervals or in carcass tissues. Further studies involving the use of osmolytes and subsequent carcass appraisal on ruminants are recommended.
Blood Acid-Base Status
There was no difference in the pH of venous blood between treatment groups at the specified sampling times (Table 2). This confirms other reports that it is unlikely that transportation stress results in differences in the arterial or venous acid-base status of transported compared with nontransported ruminants (Schaefer et al., 1988, 1992; Parker et al., 2003b).
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). No further differences were detected between groups at 48 h. The lower HCO3 concentration in control groups is consistent with our previous work showing a dehydration-linked mild acidosis during long haul transportation (Parker et al., 2003b). The explanation for the increased plasma HCO3 concentration in the glycerol-treated animals is complex and may involve respiratory or metabolic compensation, or both, which in turn leads to a greater HCO3 and a greater pCO2 for a constant [HCO3]/[CO2], and thereby maintaining blood pH (Walmsley et al., 1988).
There was no difference in anion gap between groups at 24 or 48 h (Table 2). The anion gap values for all groups at 24 and 48 h remained within previously reported values for penned Bos indicus steers that had access to feed and water (17.26 ± 3.33 mEq/L; Parker et al., 2003b). This is in contrast to the data presented by Schaefer et al. (1990), in which the AG of bulls transported for 6 h decreased by 23 mEq/L between pre-and posttransportation values. Similarly, the strong ion difference was not different between treatment groups at 24 or 48 h. Total weak acids were not different between groups and there was no effect due to time. The values reported were similar to previous results in steers subjected to long haul transportation of 48 h (Parker et al., 2003b).
Plasma Electrolytes
There was no difference between groups for plasma Na, and all groups remained within normal values for cattle (Table 3). There was a trend for plasma concentrations of K to increase (P = 0.06) with time. However, plasma K concentrations also remained within normal limits for cattle (Blood and Radostits, 1989).
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).
Plasma concentrations of Ca were greater (P = 0.04) in the control group than the glycerol group. Stress and an inadequate intake of minerals have been associated with episodes of transport tetany. Treatment protocols that include electrolyte solutions containing P, Mg, and Ca have been advocated in the sheep industry (Lucas et al., 1982). Despite differences between groups for Mg and Ca, all groups remained within physiological limits at 24 and 48 h (Blood and Radostits, 1989).
A time effect was demonstrated for Cl and P, increasing (P < 0.001 and P = 0.01, respectively) in concentration with time for all groups. The increase in concentrations of Cl and P may be the result of a loss of water from the extracellular pool.
Metabolites
The glycerol group maintained greater (P = 0.001) plasma glucose concentrations than the control group at 24 and 48 h. Increased glycerol is not only an important carbon source for gluconeogenesis in the liver, but the elevated blood glucose concentrations in the glycerol-treated animals may have an important protein-sparing effect in part due to 1) providing a preferential fuel for liver gluconeogenesis, and 2) increasing insulin secretion and thereby further inhibiting breakdown of muscle protein. In addition, carbohydrates have been found to have nitrogen-sparing effects in ruminants (Asplund et al., 1985), and cortisol has the opposite effect (Rang and Dale, 1991). Prophylactic glycerol administration may also antagonize effects of cortisol on the glucogenic and ketogenic amino acid pool of the body as the alternate and preferential substrate for glucose production. If the expected hepatic glucose production for a 320-kg steer was 200 mmol/h and the glycerol-treated animals were administered with 6.9 mol of glycerol before feed deprivation and transportation, then potentially 58 mmol/h of glucose were provided to the animals in the glycerol treatment group. This may explain the elevated plasma glucose concentrations for the glycerol group and further supports the proposed impact of glycerol on gluconeogenesis. This has possible implications for the preservation of carcass protein and a reduction in dark cutting beef. In contrast, the osmolyte betaine had little effect on blood glucose concentrations compared with glycerol.
It has been previously demonstrated that Bos indicus cattle rely to a greater extent on fat metabolism during fasting compared with Bos taurus cattle (O’Kelly, 1985). Orally ingested glycerol follows the same carbohydrate metabolic pathway as endogenous glycerol from the breakdown of triglycerides. The ultimate fate of glycerol is dependent upon the metabolic state of the individual; for example, in starvation glycerol is a primary fuel for liver gluconeogenesis (Freund et al., 1995). In addition, O’Kelly (1985) has suggested that Bos indicus animals utilized less muscle mass than Bos taurus animals during a 96-h fast. O’Kelly (1985) further argued that a high fat diet before fasting decreased the breakdown of muscle protein in both genotypes during fasting.
In a stress-induced gluconeogenic state, skeletal muscle protein supplies most of the carbon needed for net glucose synthesis (Lehninger et al., 1993). The resultant deleterious effects on carcass and meat quality have been well documented (Tarrant et al., 1992; Gardner et al., 1999; Knowles, 1999). All of the transported animals would have been in a gluconeogenic state in the current study, and the glycerol-treated animals had 64% greater cortisol concentrations at 48 h compared with the other groups (Table 4).
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transported Bos indicus x Bos taurus steers and heifers for 2 h, and demonstrated an elevation of 4.00 ± 2.20 mmol/L for plasma lactate in the transported animals over the control group. Similarly, Schaefer et al. (1990) transported bulls for 6 h and demonstrated serum lactate to range from 3.36 ± 0.60 to 5.00 ± 0.59 mmol/L between their treatment groups. We have previously reported no difference in lactate concentrations between penned control steers and those subjected to long duration transport for 48 h, and we hypothesized that this was due to animals resting in transit during the last 24 h period (Parker et al., 2003b). We noted that during the current study that individual animals started to lie down while the truck was in motion at 18 h. Resting in transit, presumably due to fatigue, has been reported to manifest itself in ruminants from 20 h in transit (Tarrant, 1990; Tarrant et al., 1992; Knowles et al., 1999). This resting behavior may have been sufficient to reduce plasma lactate concentrations in the transported steer groups at 48 h in the current study. Alternatively, plasma concentrations of lactate are a reflection of the ability of the animal to mobilize glycogen. At 48 h, the animal may have less glycogen to mobilize and therefore less lactate.
The deprivation of feed and water to all animals resulted in hematocrit values similar to those seen in other long-haul transportation studies (Tarrant et al., 1992; Knowles et al., 1999). However, a difference was detected between treatment groups. Although penned control animals had numerically greater hematocrit values than all transported groups at 24 and 48 h, hematocrit was only different (P = 0.04) from the betaine group. Broom et al. (1996) demonstrated similar phenomena in transported sheep, in which hematocrit was greater when the sheep were in a stationary, unstressed condition. Similarly, restraint and isolation stress in sheep induced a significant decrease in hematocrit compared with handling alone (Parrott et al., 1987). Stressor-induced changes in fluid compartments within the transported animals may be responsible for these changes (Broom et al., 1996). This perhaps highlights the inconsistency of utilizing hematocrit as an indicator of hydration status in animals placed under stress. Plasma cortisol concentrations are shown in Tables 4. There was no difference between groups at 24 or 48 h.
The prophylactic treatment of Bos indicus steers with the osmolyte glycerol attenuated the loss of body water during transportation for 24 h but did not appear to be effective at 48 h. However, the glycerol-linked enhanced gluconeogenic state persisted for 48 h. Elevated plasma glucose concentrations in the glycerol-treated animals would lead to greater insulin concentrations, which in turn would inhibit the breakdown of muscle proteins and also counter the amino-acid mobilizing effect of increased cortisol concentrations. In conclusion, glycerol treatment not only leads to hyperhydration, but also decreases the energy deficit by increasing plasma glucose concentrations that may lead to an insulin-linked sparing of muscle protein degradation during transportation of long duration. The implications of these findings would be the preservation of carcass protein and a reduction in dark cutting beef. Further studies are required to demonstrate the benefit and underlying mechanisms of glycerol treatment to minimize muscle wasting and promote the health of the animal during livestock transport.
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Footnotes |
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2 Current address: Ridley Agriproducts, Pakenham Victoria, Australia.
3 Corresponding author: lee.fitzpatrick{at}jcu.edu.au
Received for publication March 29, 2007. Accepted for publication May 21, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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