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Muscle fibre growth in undernourished reindeer calves (Rangifer tarandus tarandus L.) during winter
Comparative Biochemistry and Physiology Part A 129 Ž2001. 495᎐500
Muscle fibre growth in undernourished reindeer calves ž Rangifer tarandus tarandus L./ during winter A.R. Poso ¨ ¨ a,U , U. Heiskari b, M. Lindstrom ¨ a , M. Nieminenb, T. Soveri a a
Department of Basic Veterinary Sciences, Uni¨ ersity of Helsinki, P.O. Box 57, Fin-00014 Helsinki, Finland b Finnish Game and Fisheries Research Institute, Reindeer Research, Fin-99910 Kaamanen, Finland Received 14 April 2000; received in revised form 9 January 2001; accepted 15 January 2001
Abstract To study whether moderate under-nutrition causes muscle wasting, reindeer Ž Rangifer tarandus tarandus L.. calves were fed either pelleted reindeer feed ad libitum Ž n s 8. or restricted amounts of lichens Ž n s 8.. The restricted amount was 60% of ad libitum intake of lichens, and the feeding period was 6 weeks preceded by a 2-week adjustment period. Biopsy samples from the middle gluteal muscle Ž M. gluteus medius. for the analysis of fibre composition and area, as well as for the activity of cathepsin B were taken before the restriction period in November and January, and after the restriction period in April. In all calves the muscle fibre composition remained unchanged during the winter. In the lichen group, the fibre size also remained unchanged, whereas in control calves the cross sectional area of type I and type IIA fibres increased significantly from November to April. Cathepsin B activity decreased in all calves from November to January and remained at that low level for the rest of the study period, which suggests an attenuated rate of protein degradation. These results can be taken as an indication that moderate under-nutrition causes no muscle wasting in reindeer calves, and the decreased availability of nitrogen is partially compensated for by adaptive decrease in protein degradation. Interestingly the adaptive changes in protein metabolism are equally well seen in the well-fed controls as in the undernourished lichen-fed reindeer. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Cathepsin; Middle gluteal muscle; Muscle fibre; Protein degradation; Reindeer; Under-nutrition
1. Introduction The metabolism of the semi-domesticated reindeer Ž Rangifer tarandus tarandus L.. is well adapted to limited availability of feed during the arctic winter. For the freely grazing reindeer the
U
Corresponding author. Tel.: q358-9-19149522; fax:q 3589-19149799. E-mail address: [email protected] ŽA.R. Poso ¨ ¨..
main source of energy is carbohydrates from lichens ŽNieminen and Heiskari, 1989.. For additional energy production the reindeer mobilises triglycerides from adipose tissue, as indicated by the increased concentrations of serum nonesterified fatty acids ŽNEFA. and glycerol ŽSoveri et al., 1992.. This increased mobilisation of fat is also indicated by a moderate increase in the concentrations of ketone bodies ŽSoveri et al., 1992.. Such adaptation of fat metabolism appears to be regulated by seasonal changes, because similar
1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 2 8 6 - 0
496
A.R. Poso ¨ ¨ et al. r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 495᎐500
increases in ketone-body concentrations are also seen in racing reindeer fed a commercial reindeer feed ad libitum throughout the winter ŽPoso ¨ ¨ et al., 1994.. As a ruminant reindeer has a low blood glucose concentration ŽNieminen, 1980., and its liver and kidneys are able to synthesise the needed glucose from glycerol, lactate, and amino acids. Previous reports show that during winter no major changes occur in blood glucose concentration ŽSoveri et al., 1992; Poso ¨ ¨ et al., 1994.. The situation is more complex in the case of protein and nitrogenous compounds. Lichens are low in nitrogen ŽNieminen and Heiskari, 1989., and the need to save nitrogen is indicated by more efficient recycling of urea into the rumen during winter ŽPoso ¨¨ et al., 1994.. Amino acids formed in protein degradation are used for gluconeogenesis, but also for protein synthesis. Most of the body protein mass is in the muscles, but what happens to this muscle protein during winter remains unknown. It has been suggested that increased plasma urea concentrations in starving reindeer are an indication of increased utilisation of protein as an energy source ŽValtonen, 1979.. Furthermore, Kiessling and Kiessling Ž1984. have demonstrated that the Svalbard reindeer, which lives in the most harsh conditions, may use its muscles as an additional energy source during winter. This is seen in the decreased fibre area, especially that of type IIB fibres, which are the most glycolytic fibres and are recruited mainly during intense exercise ŽLindholm et al., 1974.. The aim of this study was to investigate whether similar changes in fibre area occur also in the semi-domesticated reindeer that encounter moderate under-nutrition during winter. Because winter is especially stressful for growing reindeer calves, and their need for amino acids for protein synthesis can be assumed to be higher than in adults, reindeer calves were used in the present study.
2. Material and methods
the ambient temperature and light of northern Finland Žlatitude 60⬚ 10⬘N.. From November to the end of January all the calves freely grazed on natural winter pastures. At the end of January, the reindeer were randomly divided into two groups: controls Ž n s 8. were fed commercial reindeer feed ŽPoronHerkku, Raisio Feed Ltd., Finland. ad libitum throughout the study. The experimental group was switched to lichens Ž Cladina spp.. that was offered ad libitum for the first 5 weeks with the amount of lichens consumed recorded. During the following 2 weeks the amount of lichen was reduced to 60% of the ad libitum amount, and this restricted feeding was continued for an additional 6 weeks. Thereafter, the calves were allowed to recover and were fed lichens supplemented with the commercial reindeer feed. Metabolisable energy in lichen was 10.2 MJrkg dry matter and that in the commercial feed 10.8 MJrkg dry matter. On dry-matter basis lichen and commercial feed contained 2.6 and 10.2% crude protein, and 2.5 and 3.9% of crude fat, respectively. Average energy and crude fat intake of the same reindeer calves have been reported separately ŽSoppela et al., 2000.. 2.2. Samples During the study the reindeer were weighed with an electronic balance in November, January, and April. Needle biopsy samples Ž5-mm diameter, ArS Mortensen, Bjaereskov, Denmark. from the middle gluteal muscle Ž M. gluteus medius. were taken under local anaesthesia as previously described in detail by Lindholm and Piehl Ž1974.. The samples were taken from the left side of the animal approximately 8 cm dorso᎐caudally to the tuber coxae. The window of the biopsy needle was inserted to a depth of 4 cm. The samples were rapidly blotted dry of blood, and the samples for histochemistry were rolled in talcum powder before being frozen in liquid nitrogen, whereas samples for biochemistry were frozen directly. All samples were stored at y80⬚C until analysed.
2.1. Animals and diets Male reindeer calves Ž n s 16., 7 months of age, from the experimental herd of the Reindeer Herders Association were, during this study kept in large fenced feeding grounds and exposed to
2.3. Analytical Half of each muscle sample was lyophilised and dissected free of blood, visible fat, and connective tissue; 1᎐2 mg of dry muscle was homogenised in
A.R. Poso ¨ ¨ et al. r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 495᎐500
a phosphate buffer and used for the determination of the activity of lysosomal cathepsin B ŽBarrett, 1980.. The rest of the muscle sample was used for histochemistry. Transverse thin sections Ž10 m. were cut and stained for myosin ATPase at pH 4.6 by use of a modification ŽEssen´ Gustavsson and Rehbinder, 1984. of the original staining method ŽBrooke and Kaiser, 1970.. On the basis of the intensity of the stain the cells were classified into types I, IIA and IIB. The fibre type composition and the fibre areas were measured with a computerised image analyser ŽBioRad Scan-Beam, Hadsund, Denmark.. 2.4. Statistics Within the control and the experimental groups the values were compared to each other by repeated measures analysis of variance. For the between-group comparisons at each sampling occasion, Student’s t-test was used. The difference was regarded as significant at a P-level of 0.05. The results are shown as means " standard error of the mean ŽS.E.M...
497
Fig. 1. Body weight Žmean " S.E.M.. of reindeer calves fed either commercial reindeer feed ad libitum Žopen bars. or lichen Žhatched bars.. Amount of lichen was restricted for the last 6 weeks of the experiment. UU P- 0.01 and UUU P- 0.001 from the respective body weight in November.
gained on average 5.8 kg. At the end of April, difference in body weight between the two groups was highly significant Ž P- 0.001.. 3.2. Fibre composition The fibre composition of the middle gluteal muscle in both groups was similar at the beginning and end of the study ŽTable 1.. The winter period had no effect on fibre composition ŽTable 1..
3. Results 3.1. Body weight From November to January when all the calves were equally fed ŽFig. 1., there were no differences in mean body weight between the groups. From January to April average weight loss was 8.8 kg in the lichen group, but the control calves
3.3. Fibre area In samples taken in November and January, i.e. before the experiment and at the beginning of the lichen-feeding period, the fibre area of all fibre
Table 1 Fibre-type composition and fibre areas in reindeer calves fed commercial feed ad libitum ŽControl. and reindeer calves fed lichen from January to April ŽLichen. a Control
Fibre type, % I IIA IIB Fibre area, m2 I IIA IIB a
Lichen
November
January
April
November
38.0" 3.5 20.9" 1.7 40.9" 4.1
38.4" 2.1 28.7" 2.1 32.9" 2.3
36.6" 1.9 24.3" 1.4 39.1" 1.7
1573 " 90 1595 " 114 1689 " 113
1663 " 75 1651 " 80 1691 " 76
1954 " 126 U 1873 " 117 1938 " 114
U
January
April
34.4" 3.3 26.8" 3.5 38.8" 2.3
34.4" 2.9 26.8" 1.5 38.8" 2.7
34.7" 1.4 26.0" 2.0 39.4" 2.0
1563 " 69 1490 " 53 11678 " 54
1620 " 37 1561 " 62 1656 " 50
1615 " 72 UU 1575 " 64 1648 " 97
Amount of feed intake was reduced to 60% of ad libitum level for the last 7 weeks. UU P- 0.05 from the corresponding value in November; P- 0.05 from the corresponding value in the control group.
U
UU
498
A.R. Poso ¨ ¨ et al. r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 495᎐500
Fig. 2. Cathepsin B activity in the middle gluteal muscle of control Žopen bars. and lichen-fed Žhatched bars. reindeer calves in November, January, and April. U P- 0.05, UU P- 0.01 and UUU P- 0.001 from the respective value in November.
types ŽI, IIA and IIB. in the middle gluteal muscle in the two groups was similar ŽTable 1.. In samples at the end of the experiment the transverse fibre area of type I and IIA fibres was significantly larger in the control group. Moreover, within the control group the areas of type I and IIA fibres were higher in April than in November or January, whereas no such increase appeared in the lichen group. 3.4. Cathepsin B Cathepsin B activity was similar Žno statistical differences . in the two groups throughout the study ŽFig. 2.: a significant decrease in cathepsin B activity from November to January, with activity thereafter remaining at this low level. In the control group there was, however, an increasing tendency from January to April Ž P- 0.055..
4. Discussion The main finding of this study was to show at the cellular level that reindeer calves’ muscles did not waste due to moderate under-nutrition during their first winter. Equally interesting was the finding that the control calves fed the commercial diet ad libitum continued to grow and actually increased the size of their muscle fibres. From these results it can be concluded that the reindeer calves during their first winter were already well adapted to the under-nutrition that is well tolerated by freely grazing reindeer. Our finding
differs from that of Kiessling and Kiessling Ž1984., who found a significant decrease in the transverse areas of the type IIB fibres in Svalbard reindeer during winter. This difference may be due to the degree of under-nutrition, which in our study was moderate in comparison to the extreme environmental conditions of the Svalbard reindeer. Furthermore, in reindeer calves that are starved more severely and lose 25% of their weight, changes in blood chemistry differ from those in moderately undernourished reindeer ŽSoveri et al., 1992; Soppela et al., 2000.. In the present study the body weight loss may have resulted from reduction of body fat and also reduction of rumen microbial mass and liquid due to the change in diet. Our findings are important also from the practical point of view, because they suggest that moderate under-nutrition does not cause drastic reductions in the weight of the valuable meat. Equally important is that the reindeer in the control group continued their growth throughout the winter. This is unexpected, because reindeer reduce their feed intake during winter, and when fed lichens show a negative energy balance both in captivity and on natural pastures ŽNieminen, 1985; Soveri et al., 1992.. In late winter, even the reindeer fed concentrate ad libitum show signs of negative energy balance as indicated by the increased concentrations of serum NEFA and glycerol ŽPoso ¨ ¨ et al., 1994; Soppela et al., 2000.. It has been suggested that insulin, which in reindeer appears to be regulated by the light᎐dark conditions, may contribute to the regulation of the seasonal changes in appetite and body growth ŽStimmelmayer and White, 1999.. Our results, however, show that additional factors, such as availability of nitrogen in the form of amino acids, are involved in regulation of body growth. In the present study, protein content of the concentrate was four times as high as in lichens. It is tempting to speculate that the anabolic effect of amino acids is mediated via insulin, because some amino acids, such as leucine, glycine, and arginine, are potent stimulators of insulin release from the pancreas ŽKuhara et al., 1991.. Other anabolic signals may be involved, as well. From a practical point of view, it is interesting to note that the low protein content of lichens is sufficient to maintain muscle fibre size, which is consistent with the well-accepted view that reindeer are adapted to low protein feed. To further evaluate the nitrogen balance during winter, it will be important to
A.R. Poso ¨ ¨ et al. r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 495᎐500
determine the minimum protein content that will allow the growth of the muscle fibres also during winter. Irrespective of growth, there was a significant decrease in the activity of one of the major lysosomal proteolytic enzymes, cathepsin B, in muscle, a decrease which occurred early in the winter. This indicates that a decrease in lysosomal activity is one of the adaptive changes occurring in reindeer during winter. Because muscle protein mass is determined by the balance between rate of protein synthesis and of protein degradation, it can be speculated that the decrease in proteolytic activity contributes to the maintenance of muscle protein mass, and thus reduces the need for amino acids for protein synthesis. On the other hand, the lower use of amino acids for protein synthesis allows a higher use of amino acids for gluconeogenesis. The finding that the transverse fibre area in the lichen group did not change during winter, together with the decrease in the proteolytic activity, suggests that a parallel decrease occurs in the rate of protein synthesis. In the control group, however, protein synthesis seems to be maintained throughout the winter, because the fibre areas were increased, and the proteolytic activity was decreased. Interestingly a similar increase was apparent in the transverse areas of all fibre types, although this increase was statistically significant only for type I and IIA fibres. The fibre type composition was similar to that reported earlier ŽPoso ¨ ¨ et al., 1996.. Thus, the growth of the slow-twitch fibres, which are recruited during grazing and low intensity exercise, did not differ from that of the fast-twitch fibres recruited mainly during intense exercise ŽLindholm et al., 1974.. Reindeer is extremely well adapted to large variations in the nitrogen content of its feed and may have a greater potential to recycle urea than the domestic ruminants ŽWales et al., 1972.. During winter, when the protein content of the feed is low, also the conservation of nitrogen is more effective due to a decrease in glomerular filtration rates and an increase in fractional urea reabsorption ŽValtonen, 1979.. High serum urea concentrations have been measured in severely starved reindeer ŽNieminen, 1980. and deer ŽDeCalesta et al., 1977. which has been taken as an indication of increased protein degradation and use of body protein for energy production. The observed decrease in the activity of cathepsin B together with the maintenance of muscle fibre
499
size suggests that during winter the turnover of muscle protein is slow and in part contributes to the conservation of nitrogen. In conclusion, the results of this study show that muscle fibre areas are well maintained in moderately undernourished reindeer calves. The decrease in activity of cathepsin B suggests that the rates both of protein synthesis and degradation are attenuated during winter. In the control calves, the increase in fibre area, together with the decrease in cathepsin B activity, indicates a positive difference between the rates of protein synthesis and protein degradation. It can thus be concluded that additional feed intake may allow growth also during winter.
Acknowledgements We wish to thank the staff of the Reindeer Herders Association for the care of the reindeer and Ms Pirjo Puroranta, Ms Ritva Ristilahde and ¨ Ms Marja Miskala for technical assistance. This project was supported by a grant from the Ministry of Agriculture and Forestry in Finland. References Barrett, A.J., 1980. Fluorimetric assays of cathepsin B and cathepsin H with methylcoumaride substrates. Biochem. J. 187, 909᎐912. Brooke, M.H., Kaiser, K.K., 1970. Three ‘myosin adenosine triphosphatase’ systems: the nature of their pH lability and sulphydryl dependence. J. Histochem. Cytochem. 18, 670᎐672. DeCalesta, D.S., Nagy, J.G., Bailey, J.A., 1977. Experiments on starvation and recovery of mule deer. J. Wildl. Manage. 41, 81᎐86. Essen-Gustavsson, B., Rehbinder, C., 1984. The influ´ ence of stress on substrate utilization in skeletal muscle fibres of reindeer Ž Rangifer tarandus L... Rangifer 4, 2᎐8. Kiessling, K.-H., Kiessling, A., 1984. Fibre composition and enzyme activities in five different muscles from the Svalbard reindeer. Comp. Biochem. Physiol. 77A, 75᎐78. Kuhara, T., Ikeda, S., Ohmeda, A., Sasaki, Y., 1991. Effects of intravenous infusion of 17 amino acids on the secretion of GH, glucagon, and insulin in sheep. Am. J. Physiol. 260, E21᎐E26. Lindholm, A., Piehl, K., 1974. Fibre composition, enzyme activity and concentrations of metabolites and electrolytes in muscles of Standardbred horses. Acta Vet. Scand. 15, 287᎐309.
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Lindholm, A., Bjerneld, H., Saltin, B., 1974. Glycogen depletion pattern in muscle fibres of trotting horses. Acta Physiol. Scand. 90, 475᎐484. Nieminen, M., 1980. Nutritional and seasonal effects on the haematology and blood chemistry in reindeer Ž Rangifer tarandus tarandus L... Comp. Biochem. Physiol. 66A, 399᎐413. Nieminen, M., 1985. Hirvielainten kunto ja kunnon ¨ ŽMethods for assessing the nutritiomaarittaminen ¨¨ ¨ nal status of Cervidae; English summary.. Suomen Riista 32, 90᎐110. Nieminen, M., Heiskari, U., 1989. Diets of freely grazing and captive reindeer during summer and winter. Rangifer 9, 17᎐34. Poso, ¨ ¨ A.R., Nieminen, M., Sankari, S., Soveri, T., 1994. Exercise-induced changes in blood composition of racing reindeer Ž Rangifer tarandus tarandus L... Am. J. Physiol. 267, R1209᎐R1216. Poso, L.A., ¨ ¨ A.R., Nieminen, M., Raulio, J., Rasanen, ¨¨ Soveri, T., 1996. Skeletal muscle characteristics of racing reindeer Ž Rangifer tarandus.. Comp. Biochem. Physiol. 114A, 277᎐281.
Soppela, P., Heiskari, U., Nieminen, M., Salminen, I., Sankari, S., Kindahl, H., 2000. The effects of prolonged undernutrition on serum lipids and fatty acid composition of reindeer calves during winter and spring. Acta Physiol. Scand. 168, 337᎐350. Soveri, T., Sankari, S., Nieminen, M., 1992. Blood chemistry of reindeer calves Ž Rangifer tarandus. during the winter season. Comp. Biochem. Physiol. 102A, 191᎐196. Stimmelmayer, R., White, R.G., 1999. Photoperiodassociated changes in serum insulin levels in reindeer. Rangifer 4, 39. Valtonen, M., 1979. Renal responses of reindeer to high and low protein diet and sodium supplement. J. Sci. Agr. Soc. Finland 51, 387᎐419. Wales, R.A., Milligan, L.P., McEwan, E.H., 1972. Urea recycling in caribou, cattle and sheep. Proc. 1st Int. ReindeerrCaribou Symp. Fairbanks, Alaska, 297᎐307.
Muscle fibre growth in undernourished reindeer calves ž Rangifer tarandus tarandus L./ during winter A.R. Poso ¨ ¨ a,U , U. Heiskari b, M. Lindstrom ¨ a , M. Nieminenb, T. Soveri a a
Department of Basic Veterinary Sciences, Uni¨ ersity of Helsinki, P.O. Box 57, Fin-00014 Helsinki, Finland b Finnish Game and Fisheries Research Institute, Reindeer Research, Fin-99910 Kaamanen, Finland Received 14 April 2000; received in revised form 9 January 2001; accepted 15 January 2001
Abstract To study whether moderate under-nutrition causes muscle wasting, reindeer Ž Rangifer tarandus tarandus L.. calves were fed either pelleted reindeer feed ad libitum Ž n s 8. or restricted amounts of lichens Ž n s 8.. The restricted amount was 60% of ad libitum intake of lichens, and the feeding period was 6 weeks preceded by a 2-week adjustment period. Biopsy samples from the middle gluteal muscle Ž M. gluteus medius. for the analysis of fibre composition and area, as well as for the activity of cathepsin B were taken before the restriction period in November and January, and after the restriction period in April. In all calves the muscle fibre composition remained unchanged during the winter. In the lichen group, the fibre size also remained unchanged, whereas in control calves the cross sectional area of type I and type IIA fibres increased significantly from November to April. Cathepsin B activity decreased in all calves from November to January and remained at that low level for the rest of the study period, which suggests an attenuated rate of protein degradation. These results can be taken as an indication that moderate under-nutrition causes no muscle wasting in reindeer calves, and the decreased availability of nitrogen is partially compensated for by adaptive decrease in protein degradation. Interestingly the adaptive changes in protein metabolism are equally well seen in the well-fed controls as in the undernourished lichen-fed reindeer. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Cathepsin; Middle gluteal muscle; Muscle fibre; Protein degradation; Reindeer; Under-nutrition
1. Introduction The metabolism of the semi-domesticated reindeer Ž Rangifer tarandus tarandus L.. is well adapted to limited availability of feed during the arctic winter. For the freely grazing reindeer the
U
Corresponding author. Tel.: q358-9-19149522; fax:q 3589-19149799. E-mail address: [email protected] ŽA.R. Poso ¨ ¨..
main source of energy is carbohydrates from lichens ŽNieminen and Heiskari, 1989.. For additional energy production the reindeer mobilises triglycerides from adipose tissue, as indicated by the increased concentrations of serum nonesterified fatty acids ŽNEFA. and glycerol ŽSoveri et al., 1992.. This increased mobilisation of fat is also indicated by a moderate increase in the concentrations of ketone bodies ŽSoveri et al., 1992.. Such adaptation of fat metabolism appears to be regulated by seasonal changes, because similar
1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 2 8 6 - 0
496
A.R. Poso ¨ ¨ et al. r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 495᎐500
increases in ketone-body concentrations are also seen in racing reindeer fed a commercial reindeer feed ad libitum throughout the winter ŽPoso ¨ ¨ et al., 1994.. As a ruminant reindeer has a low blood glucose concentration ŽNieminen, 1980., and its liver and kidneys are able to synthesise the needed glucose from glycerol, lactate, and amino acids. Previous reports show that during winter no major changes occur in blood glucose concentration ŽSoveri et al., 1992; Poso ¨ ¨ et al., 1994.. The situation is more complex in the case of protein and nitrogenous compounds. Lichens are low in nitrogen ŽNieminen and Heiskari, 1989., and the need to save nitrogen is indicated by more efficient recycling of urea into the rumen during winter ŽPoso ¨¨ et al., 1994.. Amino acids formed in protein degradation are used for gluconeogenesis, but also for protein synthesis. Most of the body protein mass is in the muscles, but what happens to this muscle protein during winter remains unknown. It has been suggested that increased plasma urea concentrations in starving reindeer are an indication of increased utilisation of protein as an energy source ŽValtonen, 1979.. Furthermore, Kiessling and Kiessling Ž1984. have demonstrated that the Svalbard reindeer, which lives in the most harsh conditions, may use its muscles as an additional energy source during winter. This is seen in the decreased fibre area, especially that of type IIB fibres, which are the most glycolytic fibres and are recruited mainly during intense exercise ŽLindholm et al., 1974.. The aim of this study was to investigate whether similar changes in fibre area occur also in the semi-domesticated reindeer that encounter moderate under-nutrition during winter. Because winter is especially stressful for growing reindeer calves, and their need for amino acids for protein synthesis can be assumed to be higher than in adults, reindeer calves were used in the present study.
2. Material and methods
the ambient temperature and light of northern Finland Žlatitude 60⬚ 10⬘N.. From November to the end of January all the calves freely grazed on natural winter pastures. At the end of January, the reindeer were randomly divided into two groups: controls Ž n s 8. were fed commercial reindeer feed ŽPoronHerkku, Raisio Feed Ltd., Finland. ad libitum throughout the study. The experimental group was switched to lichens Ž Cladina spp.. that was offered ad libitum for the first 5 weeks with the amount of lichens consumed recorded. During the following 2 weeks the amount of lichen was reduced to 60% of the ad libitum amount, and this restricted feeding was continued for an additional 6 weeks. Thereafter, the calves were allowed to recover and were fed lichens supplemented with the commercial reindeer feed. Metabolisable energy in lichen was 10.2 MJrkg dry matter and that in the commercial feed 10.8 MJrkg dry matter. On dry-matter basis lichen and commercial feed contained 2.6 and 10.2% crude protein, and 2.5 and 3.9% of crude fat, respectively. Average energy and crude fat intake of the same reindeer calves have been reported separately ŽSoppela et al., 2000.. 2.2. Samples During the study the reindeer were weighed with an electronic balance in November, January, and April. Needle biopsy samples Ž5-mm diameter, ArS Mortensen, Bjaereskov, Denmark. from the middle gluteal muscle Ž M. gluteus medius. were taken under local anaesthesia as previously described in detail by Lindholm and Piehl Ž1974.. The samples were taken from the left side of the animal approximately 8 cm dorso᎐caudally to the tuber coxae. The window of the biopsy needle was inserted to a depth of 4 cm. The samples were rapidly blotted dry of blood, and the samples for histochemistry were rolled in talcum powder before being frozen in liquid nitrogen, whereas samples for biochemistry were frozen directly. All samples were stored at y80⬚C until analysed.
2.1. Animals and diets Male reindeer calves Ž n s 16., 7 months of age, from the experimental herd of the Reindeer Herders Association were, during this study kept in large fenced feeding grounds and exposed to
2.3. Analytical Half of each muscle sample was lyophilised and dissected free of blood, visible fat, and connective tissue; 1᎐2 mg of dry muscle was homogenised in
A.R. Poso ¨ ¨ et al. r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 495᎐500
a phosphate buffer and used for the determination of the activity of lysosomal cathepsin B ŽBarrett, 1980.. The rest of the muscle sample was used for histochemistry. Transverse thin sections Ž10 m. were cut and stained for myosin ATPase at pH 4.6 by use of a modification ŽEssen´ Gustavsson and Rehbinder, 1984. of the original staining method ŽBrooke and Kaiser, 1970.. On the basis of the intensity of the stain the cells were classified into types I, IIA and IIB. The fibre type composition and the fibre areas were measured with a computerised image analyser ŽBioRad Scan-Beam, Hadsund, Denmark.. 2.4. Statistics Within the control and the experimental groups the values were compared to each other by repeated measures analysis of variance. For the between-group comparisons at each sampling occasion, Student’s t-test was used. The difference was regarded as significant at a P-level of 0.05. The results are shown as means " standard error of the mean ŽS.E.M...
497
Fig. 1. Body weight Žmean " S.E.M.. of reindeer calves fed either commercial reindeer feed ad libitum Žopen bars. or lichen Žhatched bars.. Amount of lichen was restricted for the last 6 weeks of the experiment. UU P- 0.01 and UUU P- 0.001 from the respective body weight in November.
gained on average 5.8 kg. At the end of April, difference in body weight between the two groups was highly significant Ž P- 0.001.. 3.2. Fibre composition The fibre composition of the middle gluteal muscle in both groups was similar at the beginning and end of the study ŽTable 1.. The winter period had no effect on fibre composition ŽTable 1..
3. Results 3.1. Body weight From November to January when all the calves were equally fed ŽFig. 1., there were no differences in mean body weight between the groups. From January to April average weight loss was 8.8 kg in the lichen group, but the control calves
3.3. Fibre area In samples taken in November and January, i.e. before the experiment and at the beginning of the lichen-feeding period, the fibre area of all fibre
Table 1 Fibre-type composition and fibre areas in reindeer calves fed commercial feed ad libitum ŽControl. and reindeer calves fed lichen from January to April ŽLichen. a Control
Fibre type, % I IIA IIB Fibre area, m2 I IIA IIB a
Lichen
November
January
April
November
38.0" 3.5 20.9" 1.7 40.9" 4.1
38.4" 2.1 28.7" 2.1 32.9" 2.3
36.6" 1.9 24.3" 1.4 39.1" 1.7
1573 " 90 1595 " 114 1689 " 113
1663 " 75 1651 " 80 1691 " 76
1954 " 126 U 1873 " 117 1938 " 114
U
January
April
34.4" 3.3 26.8" 3.5 38.8" 2.3
34.4" 2.9 26.8" 1.5 38.8" 2.7
34.7" 1.4 26.0" 2.0 39.4" 2.0
1563 " 69 1490 " 53 11678 " 54
1620 " 37 1561 " 62 1656 " 50
1615 " 72 UU 1575 " 64 1648 " 97
Amount of feed intake was reduced to 60% of ad libitum level for the last 7 weeks. UU P- 0.05 from the corresponding value in November; P- 0.05 from the corresponding value in the control group.
U
UU
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Fig. 2. Cathepsin B activity in the middle gluteal muscle of control Žopen bars. and lichen-fed Žhatched bars. reindeer calves in November, January, and April. U P- 0.05, UU P- 0.01 and UUU P- 0.001 from the respective value in November.
types ŽI, IIA and IIB. in the middle gluteal muscle in the two groups was similar ŽTable 1.. In samples at the end of the experiment the transverse fibre area of type I and IIA fibres was significantly larger in the control group. Moreover, within the control group the areas of type I and IIA fibres were higher in April than in November or January, whereas no such increase appeared in the lichen group. 3.4. Cathepsin B Cathepsin B activity was similar Žno statistical differences . in the two groups throughout the study ŽFig. 2.: a significant decrease in cathepsin B activity from November to January, with activity thereafter remaining at this low level. In the control group there was, however, an increasing tendency from January to April Ž P- 0.055..
4. Discussion The main finding of this study was to show at the cellular level that reindeer calves’ muscles did not waste due to moderate under-nutrition during their first winter. Equally interesting was the finding that the control calves fed the commercial diet ad libitum continued to grow and actually increased the size of their muscle fibres. From these results it can be concluded that the reindeer calves during their first winter were already well adapted to the under-nutrition that is well tolerated by freely grazing reindeer. Our finding
differs from that of Kiessling and Kiessling Ž1984., who found a significant decrease in the transverse areas of the type IIB fibres in Svalbard reindeer during winter. This difference may be due to the degree of under-nutrition, which in our study was moderate in comparison to the extreme environmental conditions of the Svalbard reindeer. Furthermore, in reindeer calves that are starved more severely and lose 25% of their weight, changes in blood chemistry differ from those in moderately undernourished reindeer ŽSoveri et al., 1992; Soppela et al., 2000.. In the present study the body weight loss may have resulted from reduction of body fat and also reduction of rumen microbial mass and liquid due to the change in diet. Our findings are important also from the practical point of view, because they suggest that moderate under-nutrition does not cause drastic reductions in the weight of the valuable meat. Equally important is that the reindeer in the control group continued their growth throughout the winter. This is unexpected, because reindeer reduce their feed intake during winter, and when fed lichens show a negative energy balance both in captivity and on natural pastures ŽNieminen, 1985; Soveri et al., 1992.. In late winter, even the reindeer fed concentrate ad libitum show signs of negative energy balance as indicated by the increased concentrations of serum NEFA and glycerol ŽPoso ¨ ¨ et al., 1994; Soppela et al., 2000.. It has been suggested that insulin, which in reindeer appears to be regulated by the light᎐dark conditions, may contribute to the regulation of the seasonal changes in appetite and body growth ŽStimmelmayer and White, 1999.. Our results, however, show that additional factors, such as availability of nitrogen in the form of amino acids, are involved in regulation of body growth. In the present study, protein content of the concentrate was four times as high as in lichens. It is tempting to speculate that the anabolic effect of amino acids is mediated via insulin, because some amino acids, such as leucine, glycine, and arginine, are potent stimulators of insulin release from the pancreas ŽKuhara et al., 1991.. Other anabolic signals may be involved, as well. From a practical point of view, it is interesting to note that the low protein content of lichens is sufficient to maintain muscle fibre size, which is consistent with the well-accepted view that reindeer are adapted to low protein feed. To further evaluate the nitrogen balance during winter, it will be important to
A.R. Poso ¨ ¨ et al. r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 495᎐500
determine the minimum protein content that will allow the growth of the muscle fibres also during winter. Irrespective of growth, there was a significant decrease in the activity of one of the major lysosomal proteolytic enzymes, cathepsin B, in muscle, a decrease which occurred early in the winter. This indicates that a decrease in lysosomal activity is one of the adaptive changes occurring in reindeer during winter. Because muscle protein mass is determined by the balance between rate of protein synthesis and of protein degradation, it can be speculated that the decrease in proteolytic activity contributes to the maintenance of muscle protein mass, and thus reduces the need for amino acids for protein synthesis. On the other hand, the lower use of amino acids for protein synthesis allows a higher use of amino acids for gluconeogenesis. The finding that the transverse fibre area in the lichen group did not change during winter, together with the decrease in the proteolytic activity, suggests that a parallel decrease occurs in the rate of protein synthesis. In the control group, however, protein synthesis seems to be maintained throughout the winter, because the fibre areas were increased, and the proteolytic activity was decreased. Interestingly a similar increase was apparent in the transverse areas of all fibre types, although this increase was statistically significant only for type I and IIA fibres. The fibre type composition was similar to that reported earlier ŽPoso ¨ ¨ et al., 1996.. Thus, the growth of the slow-twitch fibres, which are recruited during grazing and low intensity exercise, did not differ from that of the fast-twitch fibres recruited mainly during intense exercise ŽLindholm et al., 1974.. Reindeer is extremely well adapted to large variations in the nitrogen content of its feed and may have a greater potential to recycle urea than the domestic ruminants ŽWales et al., 1972.. During winter, when the protein content of the feed is low, also the conservation of nitrogen is more effective due to a decrease in glomerular filtration rates and an increase in fractional urea reabsorption ŽValtonen, 1979.. High serum urea concentrations have been measured in severely starved reindeer ŽNieminen, 1980. and deer ŽDeCalesta et al., 1977. which has been taken as an indication of increased protein degradation and use of body protein for energy production. The observed decrease in the activity of cathepsin B together with the maintenance of muscle fibre
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size suggests that during winter the turnover of muscle protein is slow and in part contributes to the conservation of nitrogen. In conclusion, the results of this study show that muscle fibre areas are well maintained in moderately undernourished reindeer calves. The decrease in activity of cathepsin B suggests that the rates both of protein synthesis and degradation are attenuated during winter. In the control calves, the increase in fibre area, together with the decrease in cathepsin B activity, indicates a positive difference between the rates of protein synthesis and protein degradation. It can thus be concluded that additional feed intake may allow growth also during winter.
Acknowledgements We wish to thank the staff of the Reindeer Herders Association for the care of the reindeer and Ms Pirjo Puroranta, Ms Ritva Ristilahde and ¨ Ms Marja Miskala for technical assistance. This project was supported by a grant from the Ministry of Agriculture and Forestry in Finland. References Barrett, A.J., 1980. Fluorimetric assays of cathepsin B and cathepsin H with methylcoumaride substrates. Biochem. J. 187, 909᎐912. Brooke, M.H., Kaiser, K.K., 1970. Three ‘myosin adenosine triphosphatase’ systems: the nature of their pH lability and sulphydryl dependence. J. Histochem. Cytochem. 18, 670᎐672. DeCalesta, D.S., Nagy, J.G., Bailey, J.A., 1977. Experiments on starvation and recovery of mule deer. J. Wildl. Manage. 41, 81᎐86. Essen-Gustavsson, B., Rehbinder, C., 1984. The influ´ ence of stress on substrate utilization in skeletal muscle fibres of reindeer Ž Rangifer tarandus L... Rangifer 4, 2᎐8. Kiessling, K.-H., Kiessling, A., 1984. Fibre composition and enzyme activities in five different muscles from the Svalbard reindeer. Comp. Biochem. Physiol. 77A, 75᎐78. Kuhara, T., Ikeda, S., Ohmeda, A., Sasaki, Y., 1991. Effects of intravenous infusion of 17 amino acids on the secretion of GH, glucagon, and insulin in sheep. Am. J. Physiol. 260, E21᎐E26. Lindholm, A., Piehl, K., 1974. Fibre composition, enzyme activity and concentrations of metabolites and electrolytes in muscles of Standardbred horses. Acta Vet. Scand. 15, 287᎐309.
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Lindholm, A., Bjerneld, H., Saltin, B., 1974. Glycogen depletion pattern in muscle fibres of trotting horses. Acta Physiol. Scand. 90, 475᎐484. Nieminen, M., 1980. Nutritional and seasonal effects on the haematology and blood chemistry in reindeer Ž Rangifer tarandus tarandus L... Comp. Biochem. Physiol. 66A, 399᎐413. Nieminen, M., 1985. Hirvielainten kunto ja kunnon ¨ ŽMethods for assessing the nutritiomaarittaminen ¨¨ ¨ nal status of Cervidae; English summary.. Suomen Riista 32, 90᎐110. Nieminen, M., Heiskari, U., 1989. Diets of freely grazing and captive reindeer during summer and winter. Rangifer 9, 17᎐34. Poso, ¨ ¨ A.R., Nieminen, M., Sankari, S., Soveri, T., 1994. Exercise-induced changes in blood composition of racing reindeer Ž Rangifer tarandus tarandus L... Am. J. Physiol. 267, R1209᎐R1216. Poso, L.A., ¨ ¨ A.R., Nieminen, M., Raulio, J., Rasanen, ¨¨ Soveri, T., 1996. Skeletal muscle characteristics of racing reindeer Ž Rangifer tarandus.. Comp. Biochem. Physiol. 114A, 277᎐281.
Soppela, P., Heiskari, U., Nieminen, M., Salminen, I., Sankari, S., Kindahl, H., 2000. The effects of prolonged undernutrition on serum lipids and fatty acid composition of reindeer calves during winter and spring. Acta Physiol. Scand. 168, 337᎐350. Soveri, T., Sankari, S., Nieminen, M., 1992. Blood chemistry of reindeer calves Ž Rangifer tarandus. during the winter season. Comp. Biochem. Physiol. 102A, 191᎐196. Stimmelmayer, R., White, R.G., 1999. Photoperiodassociated changes in serum insulin levels in reindeer. Rangifer 4, 39. Valtonen, M., 1979. Renal responses of reindeer to high and low protein diet and sodium supplement. J. Sci. Agr. Soc. Finland 51, 387᎐419. Wales, R.A., Milligan, L.P., McEwan, E.H., 1972. Urea recycling in caribou, cattle and sheep. Proc. 1st Int. ReindeerrCaribou Symp. Fairbanks, Alaska, 297᎐307.