Nutritional influences on muscle glycogen recovery following exercise in sheep and cattle.

Abstract

145 Nutritional influences on muscle glycogen recovery following exercise in sheep and cattle G.E. Gardner1, B.L. McIntyre2, G.D. Tudor3 and D.W. Pethick 1 1 2 3 School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch WA 6150 Present address: Animal Science, University of New England, Armidale NSW 2350 Agriculture Western Australia, South Per th WA 6151 Agriculture Western Australia, Bunbury WA 623 ggardner@pobox.une.edu.au Summary This paper investigates the nutritional regulation of glycogen concentration in skeletal muscle of sheep and cattle following exercise, with particular emphasis on a comparison between these species. Four experiments are summarized, indicating a clear relationship between metabolizable energy (ME) intake and rate of muscle glycogen repletion following exercise. Sensitivity to ME intake appeared to differ between species, with sheep demonstrating higher rates of glycogen repletion on lower levels of ME intake than cattle. Roughage diets appeared to produce maximal rates of muscle glycogen repletion in sheep. The use of a glycerol and propylene glycol supplement to enhance muscle glycogen repletion following exercise had marginal success as compared with normal rations, but the efficiency of conversion of the ME of the supplement into muscle glycogen was far higher. Keywords: metabolisable energy, glycerol, propylene glycol, hyperglycaemia, fluid intake different levels of nutrition on rate of recovery. The aim of this paper is to describe our recent experiments on the effect of nutrition on the recovery of muscle glycogen in the skeletal muscle of cattle and sheep following exercise stress. Particular emphasis is placed on between species comparisons of the nutritional responses. A model to study the metabolism of muscle glycogen To study the regulation of muscle glycogen in live animals a simple muscle biopsy procedure was developed (Gardner et al. 2000); samples are taken from the M. semimembranosis (SM, topside) and M. semitendinosis (ST, eye round). These muscles were chosen for ease of sampling and representation of a range in fibre types; the SM is classified as a fast red muscle, and the ST a fast white muscle (Braind et al. 1981). The SM has high levels of glycogen and is less sensitive to stress_induced depletion of glycogen, and the ST has lower glycogen levels and is more sensitive (Monin 1981). For the work described in this paper, glycogen concentration was measured enzymatically and represents total muscle glucose plus lactate. Further details of methodology and sampling procedures are described in Gardner et al. (2000). To study glycogen repletion, a model based on exercise was utilised to elicit significant levels of glycogen depletion (approximately 50%), from which rates of muscle glycogen repletion could be determined. The exercise regimen involved trotting cattle at 9 km/h for 5 x 15 min periods, or sheep at 8 km/h for 4 x 15 min periods, in both cases with a 15 min rest between each period and with an exercise intensity equivalent to about 65% VO2 max (Gardner et al. 2000; Pethick and Rowe 1996). Muscle biopsies were taken immediately before and after exercise, and 36 and 72 h post exercise, enabling both muscle glycogen depletion and the subsequent rates of repletion to be determined. Introduction Following slaughter, muscle glycogen fuels the gradual acidification of muscle via anaerobic production of lactic acid, reducing the pH from about 7.2 to an ultimate pH (pHu) of about 5.5. Factors such as poor nutrition or stress can reduce muscle glycogen at slaughter resulting in elevated pHu which, when exceeding pH 5.8_5.9, can lead to a problem of meat quality known as dark, firm and dry (DFD). DFD causes significant financial loss to cattle and sheep industries globally (Fabiansson et al. 1989; Warriss 1990), and work is in progress to identify animals potentially at risk of DFD before they are slaughtered. The `at risk' animals could be withheld from slaughter and fed to allow recovery of muscle glycogen. Thus nutritional regimens are required to enhance muscle glycogen recovery in depleted animals. Previous work has investigated the rates of recovery of muscle glycogen following stress but, to date, there is little information on the effect of Recent Advances in Animal Nutrition in Australia, Volume 13 (2001) 146 Gardner et al. The aims of this study were to use repeated muscle biopsy and the exercise model to determine the ability of cattle and sheep on (i) roughage and cereal grain based diets and (ii) glycerol and propylene glycol water supplements, to achieve repletion of muscle glycogen. Glycogen repletion on roughage/ grain rations A total of 40, 18 month old Angus steers (average liveweight 376 kg) were allocated randomly to 4 dietary treatments: pasture hay, fresh silage, barley, and maize (Table 1). In the sheep experiment 90, 6 month old Merino wethers (average liveweight 29.4 kg) were allocated randomly to three dietary treatments: hay, maize, and barley (Table 1). In both experiments sheep and cattle were housed individually and had ad libitum access to their dietary treatments for 6 weeks prior to, and immediately after being subjected to the exercise regimen. Feed intakes were measured daily. In cattle both the SM and ST of the animals on the hay diet, and the ST of the animals on the silage, barley, and maize diets, showed very little repletion of glycogen 72 h following exercise, but the higher energy grain rations produced marked levels of glycogen repletion in the SM (P<0.001) (Table 2). In sheep there was significant glycogen repletion in both the SM and ST for all diets, although the level of repletion in the ST was only half that seen in the SM (Table 2). This emphasises the importance of muscle type and confirms that muscle groups classified as fast white are relatively unresponsive to short term nutritional change. Given this contrast between muscles, the assessment of Table 1 Composition of diets for cattle and sheep exercise/glycogen repletion experiments. Component of diet (%) Hay Lupin Pasture hay Lucerne hay Silage Maize Barley Urea Molasses Minerals + Vitamins 2.8 66.3 0.9 5 1 5 2.8 Ration analysis ME (MJ/kg DM) CP (%) Intake (kg DM/head 8.0 8.0 6.98 10.2 15.9 7.81 11.3 13.3 10.76 11.3 12.3 11.77 8.1 13.1 1.13 10.9 16.9 1.17 11.4 16.8 1.14 2 2 100 64.2 62.8 0.2 0.4 57.6 100 Cattle dietary treatments Silage Barley 10 15 Maize 12 15 45 55 Sheep dietary treatments Hay Barley 20 15 Maize 25 15 Table 2 Effect of experimental rations on change in muscle glycogen concentration (g/100 g) in cattle and sheep 72 h after exercise. Dietary treatment Muscle Cattle Repletion 72 h post_exercise Sheep Repletion 72 h post_exercise SM ST 1.01 � 0.100 0.41 � 0.057 _ _ 0.82 � 0.099 0.35 � 0.075 0.76 � 0.068 0.35 � 0.055 n.s. n.s. SM ST 0.10 � 0.083a 0.08 � 0.082 0.32 � 0.089b 0.04 � 0.053 0.49 � 0.052bc _0.05 � 0.109 0.57 � 0.084c _0.02 � 0.088 ** n.s. Hay Silage Barley Maize Significance of effect (P) Values are mean � SEM. Values within rows followed by different letters are significantly different at P = 0.05 n.s., not significant; ** P<0.01 Nutritional influences on muscle glycogen recovery following exercise in sheep and cattle 147 nutritional impacts on muscle glycogen repletion for the remainder of this paper will focus on the more nutritionally responsive SM. In contrast to the SM of cattle the level of muscle glycogen repletion in sheep did not differ between rations (Table 2). This difference is further highlighted in Figure 1 which demonstrates a positive linear relationship (P<0.001) between SM glycogen repletion after 72 h and metabolizable energy (ME) intake per kgW0.75 in cattle, whereas no such relationship was evident in sheep. Thus glycogen repletion in sheep following exercise appears to be independent of ME intake over the range studied, though the range was smaller than for cattle. Consequently the experimental rations in the sheep experiment may not have properly tested the effect of ME intake on rate of muscle glycogen repletion. Would glycogen repletion in sheep be independent of ME at much lower levels or across a broader range of ME intake? However, the results do clearly show that the absolute rate of muscle glycogen repletion in sheep appears to be higher than in cattle on similar ME intakes per kgW0.75. Therefore the conclusions from this initial experiment were that (i) glycogen repletion in cattle was dependent on ME intake, (ii) the absolute rate of glycogen repletion in sheep was higher, and (iii) the range of ME intakes fed to sheep was smaller when expressed in terms of metabolic body weight--that is we did not really test the effect of ME intake on rate of muscle glycogen repletion in sheep. exercise during the rapid post exercise repletion phase (Sherman 1991). Hence sports drinks containing soluble carbohydrate, such as Powerade� and Gatorade�, will enhance an athlete's recovery post exercise. Given that ruminants extensively ferment carbohydrates, these drinks are unlikely to promote hyperglycaemia in sheep and cattle. However there are some recognised hyperglycaemic agents, such as glycerol, which are used widely within the dairy industry to treat bovine ketosis. Thus studies were carried out to identify the best hyperglycaemic agent and to assess its impact on muscle glycogen repletion post exercise in cattle and sheep. If it promoted glycogen repletion in muscle, it might be of use during the curfew/lairage period immediately pre_slaughter. Hyperglycaemic response to substrate delivered as a drench The initial work compared the effect of a range of hyperglycaemic agents drenched directly into the rumen of sheep. These included glycerol, propylene glycol, glucose, and a combination drench of 70% glycerol plus 30% propylene glycol; water was used as a control drench. The glucose drench included 150 g of glucose dissolved in water and made up to 300 ml. The other drenches were diluted to the same volume, and contained an equivalent amount of total carbon (excluding the water control). Blood samples were collected from indwelling jugular catheters over a 9 h period following the initial drench, for the determination of plasma glucose concentrations. Area under curve analysis was carried out for each of the treatments (indexed against the water control), demonstrating that the 70% glycerol and 30% propylene glycol combination, and glucose drenches, produced the greatest hyperglycaemic responses (P<0.001, Figure 2). The glycerol/propylene glycol response confirms earlier observations (Buswell et al. 1986; Rodriguez Iglesias et al. 1996) that a mixture of these Glycogen repletion on glycerol and propylene glycol in water supplements Glycogen repletion in the muscle of human athletes is maximised by dietary induced hyperglycaemia, particularly when carbohydrates are ingested soon after Figure 1 Relationship between SM glycogen repletion 72 h after exercise and ME intake, MJ/day/kg metabolic body weight (kgW0.75), for cattle and sheep. Cattle demonstrated a positive linear relationship (R2 = 0.37, P<0.001). Figure 2 Effect of administration of gluconeogenic substrates on plasma glucose concentration. Values are mean area under cur ve � SEM indexed against H2O; water was given an index value of 100 and all other treatments were indexed propor tionately against this value. 148 Gardner et al. substances is a potent hyperglycaemic agent when drenched into the rumen of sheep. The hyperglycaemic effect of glucose may indicate the possibility of direct glucose transport across the rumen wall, although practical concerns associated with attracting insects preclude the use of glucose in drinking water. Additionally, the propylene glycol component of the glycerol/propylene glycol drench may have benefits as a preservative (Baurle et al. 1985; Kinnunen and Koskela 1991), potentially improving the keeping quality of water trough contents. Therefore the combination glycerol_propylene glycol treatment was chosen as a drinking water supplement for use in the subsequent muscle glycogen studies. Hyperglycaemic response to substrate delivered in drinking water Having established the effectiveness of the glycerol/ propylene glycol supplement, we tested its hyperglycaemic impact when offered in drinking water at the rate of 5%, mimicking our intended application of the product. Thus sheep maintained on either low energy (roughage) or high energy (pelleted) rations, were offered either water (control) or water containing 3.5% glycerol and 1.5% propylene glycol over a 24 h period. Blood samples were collected from indwelling jugular catheters over a 9 h period following commencement of water supplementation, with a final sample taken after 24 h, for the determination of plasma glucose concentrations. The low energy ration consisted of 50% lucerne hay and 50% oaten hay (ME 10.0 MJ/ kg, CP 15.3%, intake 0.69 kg DM), and the pelleted ration consisted of 7% straw, 46% barley, 15% wheat, 15% oats and 11% lupins (ME 12.1 MJ/kg, CP 15.5%, intake 1.08 kg DM). These rations were divided into 9 equal portions, with one portion given hourly immediately after each collection of blood, and consumed by the lambs within 10 min, and the final portion given after the 9 h sample. The rates of water intakes, L/h, appeared to be relatively constant over the full 24 h treatment/sampling period and did not differ between treatment groups (Figure 3). Plasma glucose concentrations demonstrated a steady increase over the first 9 h after water supplementation began, but had declined to the same levels as the control by 24 h. This increase was more pronounced in the roughage fed sheep than the pellet fed sheep (P<0.01) (Figure 4). Thus the hyperglycaemic response was still evident when the substrates were offered in diluted form in the drinking water, although this response appeared to decline at some point between 9 and 24 h after the initial supplementation commenced. The greater response in the roughage fed animals may indicate there would be a greater effect of these supplements in pasture fed than in feedlot animals. Muscle glycogen repletion response to substrate delivered in drinking water To test the effect of glycerol/propylene glycol inclusion in the water on muscle glycogen metabolism we again used the exercise depletion/repletion model in sheep and cattle. In the cattle study 40, 18 months old Hereford cross heifers (average liveweight 349 kg) were maintained on a high energy mixed ration ad libitum (similar to the diets used to generate the data shown in Figure 1) in individual pens for 6 weeks. On the day prior to exercise, half of these animals were randomly allocated to the treatment group and offered drinking water with inclusions of glycerol and propylene glycol at the rates of 3.5% and 1.5% respectively. The water for the control group did not contain either substance. The following day all animals were subjected to the exercise regimen, with biopsies taken pre and post exercise and 36 h post exercise. Cattle were maintained on their experimental water treatments throughout the repletion period. In the sheep experiment 30 Merino wether lambs, average liveweight 32 kg, were housed in individual Figure 3 Effect of supplementing drinking water with 3.5% glycerol and 1.5% propylene glycol on cumulative fluid intake (L). Values are means � SEM. Figure 4 Effect of supplementing drinking water with 3.5% glycerol and 1.5% propylene glycol on plasma glucose concentration (mM). Values are means � SEM. Nutritional influences on muscle glycogen recovery following exercise in sheep and cattle 149 pens and fed a high energy total mixed ration ad libitum. They were subjected to the exercise protocol, with biopsies taken pre and post exercise, and then 24 h and 48 h post exercise. During the post exercise repletion period, half of the animals were offered water, and half were offered water containing 3.5% glycerol and 1.5% propylene glycol. All animals in both sheep and cattle experiments were maintained on their rations prior to exercise. Following exercise feed was withheld for the duration of the repletion phase. The results from the control treatments of both sheep and cattle experiments demonstrate that during the post exercise phase there was no significant repletion of muscle glycogen in the absence of food, contradicting the previous indication that muscle glycogen repletion in sheep is independent of ME intake (Table 3). Furthermore, glycogen repletion in both the SM and ST of sheep was increased after 48 h by the supplementation with glycerol and propylene glycol (P<0.05). There was a similar yet smaller trend evident in cattle although the response was not significant (Table 3). The glycogen repletion promoted by the glycerol and propylene glycol supplement was quite small in comparison to the levels evidenced in these species when offered full rations, demonstrating that substrate supplied through the water is not an adequate replacement for a high energy ration. This is hardly surprising, when assessing the relative ME intakes. There are no values for the ME of glycerol and propylene glycol, but the gross energy (GE) of the supplement is 16.8 MJ/L (Blaxter 1962). If GE were taken to equal ME, the ME intake of each lamb offered the glycerol and propylene glycol supplement was equivalent to 1.05 MJ/day. In contrast, lambs maintained on the barley ration following exercise (described in the experiment above) consumed 12.7 MJ ME/head/ day, this difference facilitating faster rates of repletion in the barley fed animals. The effectiveness of the two energy forms also differed. The associated rate of muscle glycogen repletion for the glycerol plus propylene glycol substrate over 48 h was equivalent to 0.068 g/100g per h, for every MJ of ME intake per kg metabolic liveweight per day. The sheep offered the high energy barley ration repleted at 0.011 g/100 g per h, for every MJ of ME intake per kg metabolic liveweight per day, demonstrating a six_fold increase in the efficiency of conversion of the glycerol and propylene glycol supplement into muscle glycogen compared with high energy rations. Using the assumed ME intake values for the glycerol and propylene glycol supplement, ME intake per kg metabolic body weight per day correlates strongly with increased rates of glycogen repletion in both sheep (P<0.001) and cattle (P<0.01) (Figure 5), dispelling the indication from Figure 2 that glycogen repletion in sheep is not responsive to ME intake. Furthermore, the slope of the glycogen response in sheep, shown in Figure 5, was greater than that in cattle, suggesting that at the lower ME levels the glycogen repletion in sheep was more sensitive to ME intake than in cattle. Thus the amount of ME provided by the hay ration was sufficient to maximise the rate of repletion in sheep in the first experiment, and additional ME provided by the grain rations was surplus to the repletion demands of the muscle glycogen depot following exercise in the `red' type muscles. Therefore the conclusions from the hyperglycaemic substrate experiments were that (i) glycerol and propylene glycol produced the largest hyperglycaemic response, (ii) when administered Table 3 Effect of 3.5% glycerol + 1.5% propylene glycol (G+PG) on change in muscle glycogen concentration (g/100 g) after exercise in cattle and sheep. Treatment Muscle Control G + PG Significance of effect (P) Cattle Glycogen repletion 36 h post_exercise (g/100g) Liquid intake dur ing 36h post_exercise (L/day) Sheep Glycogen repletion 48 h post_exercise (g/100g) Total liquid intake during 48h post_exercise (L/day) Values are means � SEM n.s., not significant; * P<0.05; ** P<0.01 1.25 � 0.179 1.54 � 0.261 n.s . SM ST _0.02 � 0.061 _0.12 � 0.059 0.37 0.17 � 0.128 � 0.095 ** * 32.80 � 1.52 28.90 � 2.56 n.s . SM ST _0.02 � 0.076 _0.08 � 0.052 0.200 � 0.116 0.072 � 0.062 n.s . n.s 150 Gardner et al. in the drinking water the impact on rate of glycogen recovery following exercise was small although it did correlate with ME intake in both sheep and cattle, and (iii) when given no access to either food or water borne substrates both sheep and cattle demonstrated no muscle glycogen repletion following exercise. Conclusion This work suggests that glycogen repletion in sheep may be more sensitive to ME intake than cattle; sheep respond at lower levels of ME intake, and at the higher levels provided by grain rations there was no further increase in the rate of muscle glycogen repletion. Muscle type was also shown to affect rate of glycogen repletion following exercise, with the red type muscles repleting faster than white type muscles. Lastly, although the efficiency of glycerol and propylene glycol substrates for enhancing muscle glycogen repletion appears to be greater than for high energy ruminant rations, it does not provide the same level of ME intake and therefore is not an adequate replacement for normal rations when animals are repleting glycogen stores. The practical implications of this work are that glycerol and propylene glycol supplements may marginally enhance the repletion of muscle glycogen following a marked level of depletion, but they are no match for the much greater amounts of substrates supplied by normal rations which result in much higher rates of muscle glycogen repletion. Additionally, the results suggest that feeding roughage rations to sheep will maximise rates of glycogen repletion, but that cattle require high energy rations to potentiate maximal rates of recovery. Proposed model for muscle glycogen responses to energy intake A proposed model for the response in muscle glycogen repletion relative to ME intake in sheep and cattle is shown in Figure 6. Although the data are limited, the results from the sheep and cattle experiments described in this paper can be explained by an exponential response, with approximate estimates of energy supplied by the grain and hay rations and the glycerol and propylene glycol treatments indicated on the x_axis. Of less certainty is whether the absolute rate of muscle glycogen repletion (Vmax) is higher in sheep than in cattle, more data over the higher levels of intake in both species being required for elucidation. Although fluid intakes in the post exercise phase were not affected by the glycerol and propylene glycol treatments, in the lairage scenario prior to slaughter the fluid intakes were at least doubled in all then trials with cattle and sheep. Thus the inclusion of glycerol/ propylene glycol in the drinking water of sheep and cattle does not have adverse effects on fluid intake, and may actually increase intake. 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