Protein Use and Muscle‐Fiber Changes in Free‐Ranging, Hibernating Black Bears

Studies of the metabolic and physiological changes that bears undergo during hibernation have, for the most part, supported the paradigm that bears use only fatty tissues as a metabolic substate during hibemation. This study was performed to document the extent of protein loss and alteration of muscle‐fiber characteristics of selected muscles in black bears during winter dormanc. Muscle biopises were removed from the gas‐trocnemius and biceps femoris from seven free‐ranging female black bears on the Uncompahgre Plateau in west‐central Colo‐rado. Six of the seven bears produced cubs during the hibernat‐ing season. Muscle samples were collected from the left hind limb shortly after bears entered their dens (fall), and additional samples were collected from the right hind limb Just prior to bears leaving their dens (spring). Protein concentration, fast‐and slow‐twitch muscle‐fiber rations and muscle‐fiber cross‐sectional areas, and citrate synthase activity were measured in the laboratory. While Protein concentration decreased in both muscles during the hibernation period, it was lower than pre‐dicted for lactating females, In addition, muscle‐fiber number and cross‐sectional area were unchanged in these muscles, sug‐gesting only limited muscle atrophy. In support of these obser‐vations, there were a moderate but significant increase in the proportion of fast‐twitch fibers only in the biceps femoris, with a concomitant decrese in citrate synthase activity, but no alteration of the fiber ratio in the gastrocnemius during hiber‐natio. The findings suggest that hibernating bears, particu

is, the cyclic degradation and synthesis of protein, may still be ever, no study has yet addressed muscle-fiber integrity during the unique circumstances experienced by black bears that re-high. For example, Lundberg et al. (1976) found that winter protein turnover increases three-to fivefold over summer levels main inactive for extended periods of time.
There have been two traditional models for studying muscle without a measurable net protein loss.
Even though it is generally thought that there is a marked disuse atrophy in mammals: plaster-cast immobilization and hind limb suspension. These conditions of disuse cause muscle reduction in protein catabolism during hibernation by small mammals (Bintz et al. 1979;Yacoe 1983;Krilowicz 1985), a atrophy characterized by a decrease in muscle-fiber protein, cell size and number, and altered ratio of muscle-fiber types, re-certain amount of protein use is required for all fasting or overwintering animals (Le Maho et al. 1981;Yacoe 1983). A sulting in an impaired muscle function (Musacchia et al. 1989).
However, the profile of muscle atrophy that results from these basal level of protein catabolism may be needed to sustain a models appears to be inconsistent, as evidenced by the broad continuous use of fat by providing a source of citric acid cycle range of findings. In addition, these models have limitations in intermediates from amino acid deamination (Bintz et al. 1979; that they are performed under unnatural conditions and they Yacoe 1983), to provide a source of water during periods of do not allow for repeated sampling of the same individuals over limited food and water intake, and to provide water, as well time. Recent studies using hibernating small mammals have as a nitrogen source, for milk production during lactation partially circumvented these concerns and have identified some (Bintz et al. 1979).
unique properties in what appears to be a more natural muscle Some researchers believe that the resulting metabolic water disuse model. But there are two major limitations to small mamproduced by fat catabolism can maintain a positive water balmal hibernators as a model to investigate effects of chronic ance in bears (Nelson et al. 1983). However, Bintz et al. (1979) immobility. First, because of their small size, the same individual calculated that catabolism of fat along with protein in a ratio cannot be repeatedly sampled, and different groups of animals of 2 : 1 would be necessary to maintain a water balance for must be killed and compared during stages of hibernation. Sechibernating ground squirrels. Under normal circumstances, the ond, small hibernators arouse with violent muscle shivering aprelease of water from protein is potentially negated by its loss proximately every 15-20 d, which may provide sufficient exerin urine to detoxify urea. However, bears, like some ground cise to retard changes in muscle-fiber-type ratios and even squirrels, recycle urea during winter hibernation (Nelson et al. enhance citrate synthase (CS) activity levels. Study of the black 1973,1975), which allows them to retain much of the bound bear allows researchers to circumvent these limitations; it is large water released as a result of protein breakdown. The problem enough to resample during the winter, and it does not undergo of maintaining a metabolic water balance is exacerbated for periodic arousal with violent shivering. hibernating female bears during gestation and lactation because Despite these advantages, relatively little work has been done of the extra requirements of both energy and water needed for on overwintering muscle physiology for this species. Koebel et milk and fetus production. The need for this additional water al. (1991) found that protein content as well as concentrations and nitrogen necessary for cub growth further taxes protein of glycogen, triglycerides, and other principal energy-supplying reserves (skeletal muscle and nonmuscle) of the lactating fesubstrates of muscles are unchanged in black bears during hibermale. These physiological challenges would seem to suggest nation, and that CS activity, an indicator of muscle oxidative that a net loss of protein during hibernation, especially by capacity, is also unchanged during the denning period. However, lactating females, is probably inevitable.
their study was conducted on three nonlactating bears held in captivity and does not necessarily reflect the dietary, thermal, Muscle Atrophy and Fiber-Type Alteration and activity profiles of naturally foraging and denning bears. The specific questions addressed in this study were: (1) How Another phenomenon typically associated with protein degramuch (if any) protein is catabolized from specific muscle areas dation during long periods of inactivity is the atrophy of skeleby lactating and nonlactating females? (2) Are muscle-fiber tal muscles. However, one of the most consistent observations type and cross-sectional area maintained during hibernation, made by biologists in the field is the apparent lack of impaired resulting in a retention of muscle integrity for spring emerlocomotor ability by bears aroused during hibernation (T. D. I. gence? and (3) Does CS activity during the denning period Beck, personal observation, over 140 denned bears). It remains become altered in a pattern similar to that of small mammal unclear how bears remain inactive, within a confined space, for hibernators or other disuse atrophy models? 4 -6 mo without any apparent impairment of muscle function. Many muscle disuse models have been described, developed Material and Methods primarily from studies of either rodent hibernators (see, e.g., Study Area Wickler et al. 1987Wickler et al. , 1991Musacchia et al. 1989;Steffen et al. 1991) or animals exposed to some type of acute limb immobili-The study population was located on the northwestern section of the Uncompahgre Plateau in Mesa County, Colorado. The zation (Musacchia et al. 1983;Desplanches et al. 1987). How-9g14$$jy12 05-28-98 11:49:40 pzas UC: PHYS ZOO elevation ranges from 6,000 to 9,000 ft, and the primary canopy had cubs and were therefore considered lactating. Each bear was weighed on a digital load scale (Dyna Link, model MSI-cover consists of ponderosa pine (Pinus ponderosa), quaking aspen (Populus tremuloides), and gambel oak (Quercus gambe-7200), accurate to within 0.1 kg. Snout/vent length was measured to the nearest centimeter. lii), while other canopy and understory species such as blue spruce (Picea pungens), serviceberry (Amelanchier alnifolia), All handling and surgical procedures were reviewed by Colorado Division of Wildlife and University of Wyoming animal and sagebrush (Artemesia sp.) were somewhat less abundant.
welfare committees. Two small (É400 mg) muscle-tissue samples were surgically removed from the hind limb of the bear, Study Population one from the lateral head of the gastrocnemius and one from the biceps femoris. A sterile field was established and main-Eighty-nine bears were ear-tagged, and all bears over 23 kg were fitted with radio-tracking collars transmitting in the 150 -tained in the areas surrounding each biopsy location.
Each biopsy sample was carefully cut into three equal por-152 mHz band. Of these, nine female bears believed to be in a breeding state with a high probability for winter parturition tions. One of the three pieces of tissue was immediately placed into a small, labeled Ziploc bag and quickly put into a liquid and lactation were selected as the study group. The average fall body mass of the study animals was 104 kg (SE Å 24.9).
nitrogen thermos, to be used later for assays of enzymatic activity. The second portion of tissue was mounted onto a small cork disc using a tissue-freezing matrix, with the muscle Field Methods fibers oriented perpendicular to the surface of the cork. This mounted tissue was placed into liquid isopentane cooled by All fieldwork was based out of the Cold Springs Ranger Station in the Uncompahgre National Forest. The fieldwork was di-liquid nitrogen and, once frozen, was put into a labeled Ziploc bag and into the liquid nitrogen thermos. The mounted tissue vided into two main efforts: the late fall, or early denning period (September -December 1994), and late winter/early was later used for fiber-type and histological analysis. The final piece of muscle tissue was then put into a labeled Ziploc bag spring, or late denning period (March -April 1995). Both aerial and ground radio tracking began in mid-September (aerial and placed into the liquid nitrogen for later analysis of protein content. All samples were transported frozen to the field sta-reconnaissance was provided by the Colorado Division of Wildlife and the Colorado State Patrol). In both instances, tion, where they were transferred to a large 35-L liquid nitrogen dewar flask for transport to the laboratory at the University of bears were located using a Telonics (Mesa, Ariz.) radio receiver and Yagi-type (Advanced Telemetry Systems, Isanti, Minn.) Wyoming. Immediately following the surgical biopsy, each bear was replaced in its den by sliding it on the plastic tarp to as antennas.
Bears were anesthetized using ketamine hydrochloride/xy-close to its original position as possible. The den entrance was then covered with vegetation (and snow, if snow was present lazine hydrochloride (200 mg ketamine and 50 mg xylazine mL 01 ) in a jab-stick at a dosage of 8.8 mg ketamine and 2.2 upon our arrival at the den).
This entire procedure was repeated on seven of the nine mg xylazine kg 01 bear weight. After the bear was immobilized, it was extracted from the den by placing it on a heavy plastic bears during the late denning season (March -April). At that time, we reweighed each bear and surgically removed two addi-tarp while still in the den and dragging the tarp out. The bear was then placed on an inflatable pad and heavy plastic tarp, tional muscle-tissue samples from the hind limb opposite the one sampled in the fall. As during the fall sampling, all bears which provided insulation from the snow and cold. If it was snowing or there was blowing snow, a shelter was constructed were replaced in their dens as close to the original position as possible before leaving the den site. over the bear, thereby keeping it dry.

Measurements and Surgical Procedures Laboratory Methods
Fiber-Type and Number Analysis. Thin sections (5 -6 mm) from All physical measurements and surgical procedures were performed on nine female bears during the fall field season and the frozen muscle biopsies were serially cut using an American Optical Histostat freezing microtome knife at 020ЊC, and then on seven of the same nine bears during the spring field season (bears 6 and 7, which were sampled during the fall season, had mounted on glass slides. Cutting continued until five consecutive high-quality slides for each bear muscle were obtained. left the den sometime during mid-to late March and were not resampled in the spring). Physical measurements and surgical These sections were air-dried overnight and then stained for myofibrillar ATPase activity and NADH activity (see Tinker procedures were identical during each season; that is, the surgical biopsies were all removed from the left hind limb during [1995] for complete protocol). The best slide, based on the quality and evenness of the staining, was selected from each the fall sampling and from the right hind limb during the spring sampling. Six of the seven bears sampled in the spring group of five for analysis. 9g14$$jy12 05-28-98 11:49:40 pzas UC: PHYS ZOO Using a compound microscope, a videoscope, a desktop interval was 152 d. Bears were resampled in the spring in approximately the same order as they were handled in the fall computer, and morphometry software known as Flexible Image Processing System (FIPS; Wirsam Scientific and Precision in an effort to maintain fairly equal intervals between samples. Fall measurements were made very soon after the bears entered Equipment, Ltd., Johannesburg, South Africa), each video image of the entire muscle cross section was visually divided their dens; spring measurements were made shortly before the bears left their dens. Change refers to the difference between into four subimages and enlarged at 6.3 magnification; these subimages were stored on a floppy disk for analysis. The diame-these two measurements during hibernation. Numbers in parentheses are standard errors of the mean. ter and cross-sectional area of approximately 300 -400 fibers was measured. All fibers from each subimage were classified as either Type I (slow oxidative) or Type II (fast glycolytic or fast, oxidative, glycolytic) according to the classification Body Mass Loss method of Peter et al. (1972). (The staining protocol used in this study does not differentiate between the various Type II The mean percent body mass loss for each bear during hibernafibers, e.g., fast glycolytic or fast, oxidative, glycolytic.) Fibertion was 24.3% ({6.1%) and ranged from 15% to 33% (Fig. type ratios (percentages of each fiber type) were calculated for 1). These values are consistent with expected body mass loss each muscle, and mean cross-sectional area and fiber diameter for bears during hibernation. were calculated for each muscle using the FIPS morphometry software. Additionally, total number of fibers for each muscle sample was determined by adding the number of fibers ana-Protein Use and Muscle-Tissue Water Content lyzed from each of the four subimages. Each subimage consisted of approximately 975 mm 2 ; therefore, the total area of Bradford Assay. Results from the Bradford assay of peptide all four subimages was approximately 3,900 mm 2 , or 3.9 mm 2 . bonds indicated that there was a significant decrease in skeletal Protein Concentration and Water Content. Protein concentramuscle protein concentration during hibernation in both the tion of each muscle was determined by a modified Bradford gastrocnemius muscle (mean change, 017.2 [{6.83] mg g 01 dry (Bradford 1976) colorimetric assay (see Tinker [1995] for complete protocol), and percent nitrogen of each muscle sample was measured by a carbon-hydrogen-nitrogen ignition method using a Carlo Erba model 4500 C/N analyzer. Total nitrogen (mg) was calculated based on the dry weight of the sample. Muscle water content was determined by weighing before and after drying in a Vertis freeze dryer for 24 h. CS Activity. CS activity was determined using a colorimetric assay (Shepherd and Garland 1969) on wet-weight samples weighing approximately 20 -40 mg (see Tinker [1995] for complete assay protocol).

Statistical Analyses.
A paired t-test with repeated measures design was used to detect significant differences in mean values of the measured parameter. A standard t-test was used for nonrepeatable measurements, for example, muscle-fiber crosssectional areas. One-tailed tests were performed when measured variables were expected to either increase or decrease, but not both; otherwise two-tailed tests were used. Percentage data were normalized before analysis using the arcsin -square root transformation. All alpha-levels are at 0.05 unless otherwise noted. Carbon/Hydrogen/Nitrogen Assay. Total nitrogen did not significantly change in either the gastrocnemius or the biceps femoris during hibernation (P Å 0.28 and P Å 0.15, respectively; Fig. 3).

Muscle-Fiber-Type Composition, Number, and Cross-Sectional Area
For muscle-fiber-type composition analysis, only six bears were sampled because one of the muscle biopsy samples was consid- during hibernation. However, there was no significant increase in percentage of fast-twitch fibers in the gastrocnemius (P Å 0.23; Fig. 5).
Cross-sectional areas of fast-twitch muscle fibers in both the gastrocnemius and the biceps femoris were not statistically significantly altered during hibernation (P Å 0.58 and P Å 0.48, respectively). Similarly, there was no significant change in crosssectional area of slow-twitch muscle fibers in either the gastrocnemius (P Å 0.18) or the biceps femoris (P Å 0.17; Fig. 6). There was also no significant change in the mean number of total fibers counted (per 3.9 mm 2 ) for either the gastrocnemius (P Å 0.58) or the biceps femoris (P Å 0.63; Fig. 7).

CS Activity
CS enzymatic activity decreased significantly in the biceps femoris muscles during hibernation (mean change, 08.41 mmol g 01 min 01 [{2.99], P Å 0.015). However, CS activity in the gastrocnemius muscle exhibited only a very slight and nonsig- hibernation. Notably, bear 7, the only bear sampled that was 9g14$$jy12 05-28-98 11:49:40 pzas UC: PHYS ZOO not identify any net loss in bear skeletal muscles during early and late hibernation (Nelson 1973(Nelson , 1980Lundberg et al. 1976;Koebel et al. 1991). For example, while Koebel et al. (1991) and Lundberg et al. (1976) did not observe a loss in muscle protein, Lundberg et al. (1976) found that protein turnover, a measure of both protein catabolism and protein synthesis, increases three-to fivefold in captive bears during hibernation. This statement seems to imply that net protein loss does not occur during hibernation in bears. Since it has been shown that bears do not eat or drink during hibernation (Nelson 1973), the only source of free amino acids that could be used for protein synthesis would have to be from protein breakdown. Therefore, if protein degradation increases during hibernation, it follows that protein synthesis would also have to increase in order to provide an exact protein replacement. Urea recycling could potentially assist in achieving this nitrogen balance. The recycling of nitrogen (from urea hydrolysis and hepatic amino acid synthesis) back into structural proteins has been offered as an explanation for the apparent lack of protein loss during hibernation (Lundberg et al. 1976). This scenario suggests, biceps femoris (BIFEM) during fall (open bars) and spring (hatched bars) for muscle samples from seven bears following freeze-drying for protein concentration analysis. Weights were obtained before drying and again after 24 h in the freeze-dryer for both fall and spring samples. Asterisk depicts a significant increase in water content in biceps femoris during hibernation. Vertical lines represent one standard error. without cubs and therefore not lactating during the study period, lost a smaller percentage of body mass (15%) than any of the bears with cubs and considerably less than the average of the seven bears.

Protein Use during Hibernation
There are conflicting reports of protein use during periods of inactivity by both hibernators and nonhibernators. Some studies (Musacchia et al. 1989;Steffen et al. 1991) suggest that no change in protein concentration occurs in hibernating ground squirrels (Spermophilus lateralis). Notably, these animals arouse and feed at regular intervals during hibernation, which may replenish much of the protein catabolized during torpor. However, other investigators have reported that protein concentration decreases during hibernation, as well as in specific muscles hibernation, they are contradictory to previous studies that did 9g14$$jy12 05-28-98 11:49:40 pzas UC: PHYS ZOO hibernation period. However, the magnitude of this loss was lower than that reported for other animals exposed to protracted immobilization (Desplanches et al. 1987;Thomason and Booth 1990). It is interesting to estimate the amount of protein lost during the winter based on the range of protein reduction observed during this study in the gastrocnemius and biceps femoris (065 mg g 01 to 0157 mg g 01 wet weight, respectively). On the basis of preliminary measurements (H. H. Harlow and D. B. Tinker, unpublished data) obtained by bioelectric impedance analysis (Farley and Robbins 1993), bears in this study (average weight Å 104 kg) had an average body fat content of approximately 39.2%. Assuming that their fat-free carcass contained approximately 53% skeletal muscle (Behnke et al. 1942), the losses in the gastrocnemius and biceps femoris would represent a whole-body seasonal loss of approximately 2.2 kg and 5.2 kg of protein, respectively. For sake of argument, if mixed skeletal muscle throughout the bear is used at a value intermediate to that of the gastrocnemius and biceps femoris Figure 6. Mean cross-sectional areas (mm 2 ) of both fast-and slowtwitch fibers for fall (open bars) and spring (hatched bars) in gas-(i.e., 3.7 kg), this use would account for about 13.3% of the trocnemius (GAST) and biceps femoris (BIFEM) muscles for seven total overwinter weight loss (100 1 3.7 kg protein/27.8 kg total bears. Neither fast-nor slow-twitch muscle exhibited a significant weight loss), assuming only minimal water loss from dehydrachange in fiber area during hibernation (P Å 0.18 and P Å 0.17 tion, as suggested in this study (Fig. 4). However, skeletal musfor gastrocnemius and biceps femoris, respectively). Vertical lines cle may not be the only source of protein used during hibernarepresent one standard error.
tion. For example, many vertebrates are believed to have labile however, that an extremely efficient (almost 100%) process of protein reserves (Le Maho et al. 1981) and use specific comnitrogen recycling is needed to achieve high protein turnover with no protein loss, a highly unlikely condition.
Our results from the carbon/hydrogen/nitrogen assay suggest that the mean percent nitrogen from both the gastrocnemius and biceps femoris was not significantly reduced during hibernation. Protein catabolism is characterized not just by a reduction in muscle protein but also by an increase in free amino acids, creatinine, ammonia, and urea as metabolic end products. A reduction of peptide bonds (the results of Bradford assay) without a concomitant reduction in total nitrogen (the results of carbon/hydrogen/nitrogen assay) may represent a high protein turnover. We identified these end products by carbon/hydrogen/nitrogen analysis in our biopsy of muscle and associated vasculature, but not the liver, where intermediates may accumulate. Our data suggest that some of the nitrogen from catabolized protein was being identified as other nitrogenous end products or intermediates because of a flux of protein and nitrogen at any point in time with a very rapid turnover in the liver. This is in partial agreement with Lundberg et al. (1976) in that it suggests an increase in muscle protein catabo-  (1991), we identified a significant loss of muscle protein over and biceps femoris (BIFEM) muscles for seven bears. There was this period, similar to protein losses during hibernation by no significant change in number of fibers per 3,900 mm 2 in either arctic ground squirrels (Citellus undulatus; Galster and Mormuscle during hibernation. Vertical lines represent one standard error. rison 1976), which, like bears, typically do not feed during the 9g14$$jy12 05-28-98 11:49:40 pzas UC: PHYS ZOO some amount of repartitioning of the proteins may be occurring; that is, proteins from skeletal muscles other than the ones we biopsied may be catabolized and some of the free amino acids synthesized into protein in the gastrocnemius and biceps femoris. If this in fact occurs, our sampling could underestimate actual muscle loss.

Muscle-Fiber Transformation, Cross-Sectional Area, and CS Activity
There was a significant decrease in percentage of Type I, slowtwitch, aerobic muscle fibers during hibernation in the biceps femoris but not the gastrocnemius, suggesting that some transformation from slow-to fast-twitch fibers in bears had occurred. However, the extent of this conversion was small compared with nonhibernator muscle disuse rodent models (Templeton et al. 1984;Thomason and Booth 1990) and certainly small compared with what would be expected of humans if confined for a comparable length of time (Appell 1990). mals subjected to some form of suspension or immobilization. Most studies using rodent hibernators have reported a signifipartments, including the skin and viscera, as well as blood cant increase in CS activity during hibernation (Wickler et al. albumins (Garcia-Rodrigues et al. 1987). Bintz et al. (1979Bintz et al. ( ) 1987Thomason and Booth 1990;Wickler and Hoyt 1990; determined that muscle (carcass) accounts for only about 52% Steffen et al. 1991). However, hind limb suspension or immobiof the protein catabolized by starving Richardson's ground lization models largely depict a decrease in CS activity in consquirrels (Spermophilus richardsonii), with the remainder procert with a loss of slow oxidative muscle fibers during periods vided from viscera, liver, skin, and fat. If these additional of muscle disuse (Booth 1977;Fell et al. 1985; Desplanches et sources of protein are available to bears in the same proporal. 1987). Unlike other hibernator models, CS activity actually tions reported for squirrels, the amount of protein, as catabodecreased in the biceps femoris from fall measurements in lized calculated from skeletal muscle, would approximately bears, which seems logical for the following reasons. First, as double (i.e., about another 3.7 kg), resulting in a total of about mentioned earlier, bears do not lower their body temperature 7.4 kg of catabolized protein, or a ratio of about 3 : 1 of fat to the same extent as more classic hibernators and do not to protein catabolism. This ratio is higher than the 2 : 1 ratio undergo the violent periods of shivering thermogenesis during proposed by Bintz and Mackin (1980) necessary to retain water periodic arousals. Shivering is achieved primarily by Type I balance by hibernating Richardson's ground squirrels, but fibers and is thought to be the main process that results in lower than the 5 : 1 ratio proposed by Cherel et al. (1995) the increase in CS activity in other rodent and insectivore on nonlactating hibernating hedgehogs (Erinaceus eruopaeus). hibernators (Wickler and Hoyt 1990;Wickler et al. 1991). Sec-Therefore, we believe that while overwintering black bears do ond, there is no supportive evidence that denning bears demindeed use skeletal muscle protein, they may have more robust onstrate periodic muscular activity such as violent shivering nonskeletal muscle protein reserves than those identified in bouts or isometric contractions (Barnes et al. 1994). Third, a other animals. These labile protein stores, together with skeletal decrease in overall CS activity would be anticipated on the muscle protein, would act as a source of water and nitrogen, basis of the observed decrease in slow, oxidative muscle fibers, especially during pregnancy and lactation. Alternately, the imwhich use CS in aerobic metabolic processes. pact of skeletal muscle catabolism on locomotor capacity may In the gastrocnemius, slow-/fast-fiber-type ratios and CS be minimized by using specific muscle groups (Le Maho et al. activity were essentially unchanged. These results agree with 1981) that are not as critical for producing body movement Koebel et al. (1991), who also found no significant alteration and were not among the ones tested in this study. It is possible in CS activity in postdenning biopsies from gastrocnemius of captive black bears. The alteration of CS activity by overwinter-that if protein is being continually degraded and synthesized, 9g14$$jy12 05-28-98 11:49:40 pzas UC: PHYS ZOO ing black bears was, therefore, more like that of animals artifi-in order to exhibit relatively normal muscle function following several months of inactivity. Monitoring of muscle activity of cially immobilized than that of rodent hibernators. A study by Booth (1977) made an interesting observation regarding mus-naturally hibernating bears using electromyogram telemetry and data recorders could be used to clarify this unique state. cle position and CS activity that may help explain this observed difference between large and small mammal hibernators. Booth (1977) found that rat gastrocnemius (as well as the soleus Conclusions muscles) exhibits an even greater decrease in CS activity when immobilized in a position that was flexed. It may be that the On the basis of the muscle biopsies obtained during early and late stages of denning and hibernation, black bears, and position assumed by rodent hibernators, and to a lesser extent bears during hibernation, helps retard muscle-fiber conversion particularly lactating female bears, experienced some amount of protein loss during hibernation. This catabolized protein and either stabilizes or, as in rodent hibernators, enhances CS activity.
was presumably used as a source of metabolic energy, Krebs cycle intermediates, and replacement water for insensible water Steffen and Musacchia (1984) have suggested that muscle atrophy results from a decrease in muscle cell size, rather than loss, as well as a source of nitrogen and water for cub production and growth. However, the magnitude of protein loss by a reduction in the number of fibers. This idea is supported by a number of studies on both hibernating small mammals and bears (a parameter of muscle atrophy) was not as great as predicted from small mammal hibernator and immobilization animals subjected to various types of immobilization or suspension. For example, hibernating ground squirrels (Spermo-disuse models. Hibernating bears do not seem to completely fit the charac-philus lateralis; Musacchia et al. 1989) and immobilized laboratory rats (Nicks et al. 1989) experience up to a 42% decrease terizations of any single muscle disuse model but apparently share some similarities of each of these models because of their in cross-sectional area of muscle fibers, but no change in fiber number. Similarly, the mean number of fibers per unit area unique, mild but continuous torpor period. In addition, when compared with human disuse models and rodent hind limb for black bear gastrocnemius and biceps femoris muscle in this study did not change during hibernation. However, unlike suspension models that exhibit profound atrophy in terms of muscle-fiber number, size, and reduction of Type I fibers, bears results from other disuse models, the mean cross-sectional area of the two muscles sampled from bears did not change over showed only limited muscle atrophy. There was no reduction in cross-sectional area or number of muscle fibers (indicators the 4-mo period, indicating no measurable atrophy of these skeletal muscles when muscle-fiber size is used as an index. It of muscle atrophy). The transformation of slow-twitch aerobic fibers to fast-twitch anaerobic fibers exhibited by these bears, is important to point out that muscle-tissue weights taken before and after freeze-drying for protein concentration analy-similar to that in rodent hibernators, was lower than that observed for the extended periods of inactivity reported for other sis showed that there was no change in water content of the gastrocnemius and only a small but significant increase in water disuse atrophy models, but the concomitant decrease in CS activity was more characteristic of nonhibernating immobile content of the biceps femoris sampled during hibernation. These data suggest that dehydration was not a factor affecting mammals. Clearly, bears employ a unique physiological strategy in order to maintain muscle tone during extended periods muscle cell shape or size during winter.
It appears that bears in this study did not experience any of inactivity while in hibernation by demonstrating only moderate skeletal muscle protein loss and slow oxidative muscle-significant muscle atrophy in terms of loss in muscle-fiber size or number during hibernation, and only a conservative loss in fiber transformation. protein and temperate alteration of fiber-type ratios. Perhaps this moderate alteration of protein content and fiber transition Acknowledgments and lack of reduction in cell size or number allowed bears to maintain muscle function for emergence in the spring. Possible This research was made possible by support from the Colorado Division of Wildlife (CDOW), as a part of their project no. explanations for this unique condition could involve biochemical processes of nitrogen recycling to retain skeletal muscle W-153-R, National Science Foundation grant EPS-9514105, and from the National Aeronautics and Space Administration protein and periods of mild shivering or some type of isometric activity during denning not previously identified in captive (NASA) and University of Wyoming Planetary and Space Science Center (NASA grant NGT-40050). The CDOW also pro-bears to maintain muscle strength. These explanations seem logical since, with the exception of a moderate decrease in vided invaluable aid by providing our crews with trucks, snowmobiles, all-terrain vehicles, and other bear-handling gear. We skeletal muscle protein concentration and moderately altered fiber type, bears apparently did not undergo the physiological owe our thanks to the managers of the Uncompahgre National Forest for the use of the Cold Springs Work Station for the changes common to other muscle disuse atrophy models. Hibernating bears must therefore be doing something different duration of our research. In addition, the Department of Zool-9g14$$jy12 05-28-98 11:49:40 pzas UC: PHYS ZOO