Growth Characteristics, Protein Synthesis, and Protein Degradation in Muscles From Fast and Slow-Growing Chickens

Growth Characteristics, Protein Synthesis, and Protein Degradation in Muscles From Fast and Slow-Growing Chickens K. C. KLASING1-2 and C. C. CALVERT3 University of California, Davis, CA 95616 and V. L. JARRELL3 University of Illinois, Urbana, IL 61801

ABSTRACT Developmental changes in muscle growth were studied in Single Comb White Leghorn (SCWL) and broiler-type (B) chickens. The extensor digiti communus (EDC) and ulnaris lateralis (UL) muscles were chosen for study because these muscles can be maintained in vitro, permitting the direct measurement of the fractional rate of protein synthesis (FSR) and the fractional rate of degradation (FDR). These muscles were removed from chicks at 1, 5, 10, and 20 days of age. Muscles for B were heavier, grew at a faster rate, and had greater fractional rates of growth than muscles from SCWL. Muscle protein concentrations were similar for SCWL and B. The deoxyribonucleic acid (DNA) concentrations were greater in EDC muscles from SCWL than B at all time periods. Concentrations of DNA were greater in UL muscles from SCWL than B after Day 1. The FSR and FDR were measured in muscles incubated in vitro. At 9 days of age, FSR in broiler and SCWL chicks was not significantly different in either muscle. The FDR was 12 and 19% lower in broiler EDC and UL muscles, respectively, demonstrating that broiler EDC and UL muscles accrete protein at a greater rate and more efficiently than SCWL muscles because of a slower rate of protein degradation. The FSR and FDR were also compared in B and SCWL chicks of 8 and 11 days, respectively, with equal DNA unit sizes. The FSR in B was 27 and 13% greater in EDC and UL muscles, respectively, demonstrating that protein synthesis per nucleus is greater in B chicks. (Key words: protein synthesis, protein degradation, muscle, growth, broilers, Leghorns) 1987 Poultry Science 66:1189-1196 INTRODUCTION

To produce economically lean poultry meat, it is important to maximize the rate and efficiency of protein deposition. Selection for the modern meat-type bird (broiler) has resulted in faster muscle protein accretion (Dawson et al., 1958) and increased feed efficiency. Because the rate of protein accretion depends on the relative rates of protein synthesis and degradation, broilers must have a greater ratio of protein synthesis to protein degradation than slower growing strains. An increased ratio may be achieved by an increased rate of protein synthesis or a decreased rate of protein degradation; however, the latter is energetically more efficient. Because protein synthesis and protein degradation require

Department of Avian Sciences. To whom correspondence should be addressed. 'Department of Animal Science.

2

energy, maximal efficiency of protein accretion occurs at minimal rates of protein synthesis and degradation (Swick, 1982). As broilers are more efficient at converting feed protein into muscle protein than egg-type chickens, it follows that broilers' increased rates of protein accretion should be a result of decreased protein degradation. This relationship, however, has not been positively demonstrated. Measuring rates of protein synthesis and particularly protein degradation are technically difficult, and results from experiments comparing fast and slow-growing strains are conflicting. Using an in vitro muscle culture system, Hentges et al. (1983) were unable to detect any differences in the rates of protein synthesis or degradation between White Leghorn and broiler chicks. Orcutt and Young (1982) demonstrated embryonic cells from broilers had similar rates of protein synthesis but lower rates of protein degradation than cells from White Leghorns. In vivo measurements are similarly in conflict.

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(Received for publication June 19, 1986)

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MATERIALS AND METHODS

Animals. Male Cornell K-Strain Single Comb White Leghorns (SCWL) and male Hubbard broiler (B) chicks were used in all experiments. Birds were neither vaccinated nor wingbanded. Chicks were housed in battery brooders with 24 h of constant light and fed a 24% protein, cornsoy ration ad libitum. Experimental chicks of both strains were selected from larger populations for uniform body weights. In Experiment 1, four chicks per strain were weighed and killed by cervical dislocation at 1, 5, 10, and 20 days of age. Muscles were removed and analyzed for DNA and protein content as described below. In Experiment 2, five chicks per strain were used for measurements of protein syntheses; all chicks were the same age. Experiment 3 was conducted similarly to Experiment 2 except chicks from each strain were of different ages so that rates of protein synthesis and degradation could be compared in muscles of the same DNA unit size. In all experiments, feed was removed 2 h prior to weighing or removing muscles. Measurement of Muscle Protein and Deoxyribonucleic Acid (DNA). Extensor digiti

communus (EDC) and ulnaris lateralis (UL) were blotted, weighed, and then homogenized in 10 mL distilled water (4 C). Aliquots were further diluted and solubilized in hot NaOH (1 N) for protein determination (Lowry et al., 1951). The DNA concentrations were determined fluorometrically after the addition of an equal volume of bisbenzimidazole (2 mg/mL; Sigma Chemical Co., St. Louis, MO) as described by Labarca and Paigen (1980). Measurement of Protein Synthesis and Degradation. In vitro rates of protein synthesis and degradation were determined by methods similar to those of Fulks et al.(1975). Chicks were anesthetized with ether and the EDC and UL muscles were separated from surrounding muscles by blunt dissection. Each muscle was removed intact without damaging the cells by severing the tendons at their points of insertion and origin. Muscles were placed in individual flasks containing 3 mL of Krebs-Ringer bicarbonate buffer (111.5 mAf NaCl, 5 mM KC1, 3 mM CaCl2, 1 mM MgS0 4 , 1 mAf KH 2 P0 4 and 25 mM NaHC0 3 , pH 7.4) with 100 units/mL penicillin, 100 |xg/mL streptomycin (GIBCO, Grand Island, NY), .1 unit/mL bovine insulin (Sigma Chemical Co., St. Louis, MO), 22 |xM valine, 25 \y.M leucine, 18 [iM isoleucine, and 25 mM glucose. This incubation medium minimizes rates of protein degradation in chick muscles (Klasing and Jarrell, 1985). Flasks were gassed with 0 2 :C0 2 (19:1) and incubated at 39 C for 60 min to allow adaptation. To measure rates of protein degradation, one muscle was weighed and homogenized (Polytron homogenizer, Brinkman Co., Westbury, CN) in 3 mL H 2 0 immediately following the adaptation period. One mL of 50% trichloroacetic acid was used to wash the homogenizer and the washings were added to the muscle homogenate. The contralateral muscle from each chick was transferred to a flask containing 3 mL fresh medium and cycloheximide (.5 mM). After 1.5 h of incubation, muscles and media were homogenized as before. The concentrations of free tyrosine in the acidsoluble supernatants were determined by the methods of Waalkes and Udenfriend (1957). The rate of tyrosine release was calculated by subtracting the quantity of tyrosine (muscle only) present at the beginning of the experimental period from the total quantity (muscle plus media) present at the end. Protein synthetic rates were determined following a 60 min adaptation period by transferring muscles to flasks containing fresh incuba-

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Studies with 2-wk-old birds indicated slightly higher fractional synthetic rates of breast muscle protein in fast growing Rock Cornish compared with rates in slower growing New Hampshire x Single Comb White Leghorns (Maruyama et al., 1978). Kang etal. (1985) compared several studies and suggested muscle protein accretion occurred at a greater rate in broilers than in laying hens because of lower fractional rates of degradation. Bergen et al. (1984) reported that meat-type birds have higher fractional rates of protein synthesis and protein degradation than layer-type birds. None of these in vivo studies directly measured protein degradation but arrived at values by comparing protein synthesis and protein accretion. Experiments were conducted to study the relationship between rates of protein synthesis and degradation in determining the growth rates of muscles from slow and fast-growing strains of chickens. Age (Maruyama et al., 1978), deoxyribonucleic acid (DNA) concentration (Waterlow, 1978), and muscle size (Klasing and Jarrell, 1985) can influence protein synthesis or degradation and thus were considered in these investigations. An in vitro technique was used in order to directly measure protein degradation.

PROTEIN TURNOVER IN CHICK MUSCLES

RESULTS

In Experiment 1, B chicks were larger than SCWL chicks and grew at a greater rate during

the 20-day trial (Table 1). Broiler chicks grew at an average rate of 19.0 g/day, which is 3.6 times greater than the rate of SCWL chicks (5.3 g/day). Fractional rates of growth in SCWL and B chicks, expressed as percent gain per day, were significantly different. The interaction between this parameter and chick age was significant. The significant interaction was due to greater FGR for broilers at Days 1 to 5 and 10 to 20 but not on Days 5 to 10. Individual muscles from B chicks grew faster than corresponding muscles from SCWL chicks (Table 2). During the 20-day experiment, EDC and UL muscles from B chicks grew at average rates of 4.46 and 8.49 mg/day, respectively, which were 3.7 and 4.8 times greater than the growth rate of corresponding SCWL muscles (1.19 and 1.77 mg/day, respectively). The FGR of EDC and UL muscles were also greater for broiler chicks. This difference was very large during the first 10 days and diminished during the last 10 days. Both EDC and UL muscles from broilers grew at faster rates than did the whole chick, as demonstrated by an increase over time of the muscle weight: body weight ratios. In SCWL chicks, EDC and UL muscles did not grow at a significantly faster rate than the whole chick until after Day 10. Muscle protein content (Table 3) was not significantly influenced by strain or age of birds. Protein bound-tyrosine concentrations, used to calculate FSR and FGR values, were not differ-

TABLE 1. Developmental changes in weight, growth rate, and fractional growth rates (FGR) of Single Comb White Leghorn (SCWL) and broiler (B) chicks1 Growth 4 ts

Weight2.3 SCWL (days)

(g)

Oto 1 1 to 5 5 to 10 10 to 20 Pooled SEM7

26.6 39.7 77.0 131.1 3.8

1

Broiler

SCWL

Broiler

(g/day) 51.7 85.3 145.1 430.8 13.6

3.3 7.5 5.4 .3

FGR 4 . 6 SCWL

Broiler

(%/day) 8.4 12.0 28.6 1.8

9.9 12.8 5.2 .8

12.3 10.4 9.9 1.0

Data represents means of 4 chicks/period.

2

End of period.

3

Chick weights between breeds are significantly different (P<.01).

4

Average of period.

s

Growth rates between breeds are significantly different (P<.01).

6 Fractional growth rates between breeds are significantly different (P<.05); fractional growth rate by age interaction is significantly different (P<.05). 7

SEM = Standard error of the mean.

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tion medium plus 14C tyrosine (Amersham, Arlington Heights, IL). The quantity of 14C tyrosine incorporated into protein and the specific activity of intracellular tyrosine were measured by techniques described by Fulks et al. (1975). The nanomoles of tyrosine incorporated into protein were calculated by dividing the 14C tyrosine counts in protein by the specific activity of intracellular tyrosine at the end of the incubation period. Because the muscle from each wing could be used to determine rates of protein synthesis, two independent observations per muscle type per bird were obtained. Fractional synthetic rates (FSR) and fractional degradative rates (FDR) were calculated from the quantity of tyrosine incorporated or released per day per milligram of tissue divided by the amount of protein-bound tyrosine per milligram tissue. Fractional growth rates (FGR) were calculated from the changes in muscle weight per day divided by muscle protein content. In addition to a direct estimate of FDR by measuring rates of tyrosine release, the rate of protein degradation was also determined indirectly by the relationship: FDRC = FSR - FGR. Means were compared by Student's t test and analysis of variance (Steel and Torrie, 1980).

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TABLE 2. Developmental changes in the growth of extensor digiti communis (EDO and ulnaris lateralis (UL) muscles in Single Comb White Leghorn (SCWL) and broiler chicks1 Muscle weight1'2 Muscle

EDC

UL

Age

SCWL

(days)

(mg)

Oto 1 1 to 5 5 to 10 10 to 20 Pooled SEM7

2.8 4.9 9.9 25.5 .9

3.1 9.1 30.4 87.8 2.1

10.5 12.3 12.9 19.4 1.1

6.0 10.7 21.0 20.4 1.3

.53 1.0 1.6 .08

Oto 1 1 to 5 5 to 10 10 to 20 Pooled SEM

4.4 6.6 13.1 38.0 .9

3.7 13.9 53.5 165.1 3.2

16.5 16.6 17.0 29.0 1.3

7.2 16.3 36.9 38.3 3.0

.55 1.3 2.5 .09

Broiler

SCWL

Broiler

FGR 1 ' 6

Gain'*,5 SCWL

Broiler

(mg/day)

(X 1,000)

SCWL

Broiler

(%/day) 1.5 4.3 5.7 .39 2.6 7.9 11.2 .5

13.6 13.5 8.8 .7

24.6 21.6 9.7 1.6

10.0 13.2 9.7 .8

29.0 23.5 10.2 1.8

End of period.

Muscle weights between breeds are significantly different (P<.01).

'Percent body weights between breeds are significantly different (P<.01); percent body weight by time (days) interactions were significant (P<.05). 4

Average of period.

5

Muscle weight gains between breeds are significantly different (P<.01).

6

Fractional growth rates (FGR) between breeds are significantly different (P<.05).

7

SEM = Standard error of the mean.

TABLE 3. Developmental changes in DNA, protein, and DNA unit size of extensor digiti communus and ulnaris lateralis (UL) muscles from (SCWL) and broiler chicks1

DNA unit size3

DNA2

Protein

(EDO

Broiler

SCWL

Broiler

SCWL

2.71 .68 .47 .23

2.60 ,59 .33 .14

69 288 402 786

70 318 585 1,364

.04

33

49

2.10 .61 .31 .13

86 216 381 827

81 270 577 1,415

35

52

Muscle

Day

SCWL

Broiler

EDC3

1 5 10 20 Pooled SEM"

187 196 189 181

181 188 193 191

15

16

.03

UL

1 5 10 20 Pooled SEM

166 173 179 182

171 165 179 184

1.92 .80 .47 .22

14

15

.05

.04

• (mg/g

(mg)

1

DNA = Deoxyribonucleic Acid; SCWL = Single Comb White Leghorn.

2

Muscle DNA concentrations between breeds are significantly different.

3

In: milligram protein/milligram DNA. Muscle DNA unit sizes between breeds are significantly different.

4

SEM = Standard error of the mean.

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1 2

% Body weight 3

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different ages and muscle weights (Table 5) with muscles of equal DNA unit sizes. For both muscle types, the FSR was significantly lower in muscles from SCWL chicks, whereas the FDR did not differ between strains. The ratio FSR:FDR was significantly lower for SCWL chicks in UL muscles and tended to be lower in EDC muscles. As in the previous experiment, the values obtained for FDRa were considerably higher than the indirectly determined FDRC.

ent between strains and averaged 3.3% (data not shown). Muscle DNA concentrations decreased as chicks' ages increased. Concentrations of DNA were significantly greater in EDC and UL muscles from SCWL than from B chicks; this difference was greatest at older ages. The concentration of DNA in EDC muscles was similar to that in UL muscles within each strain. The DNA unit size (milligram protein/milligram DNA) increased rapidly with age. At Day 1, the DNA unit sizes were similar between strains; however, the age-related increases was greater for B than for SCWL chicks. The rates of protein synthesis and degradation in B and SCWL and chicks were examined in the second and third experiments. In the second experiment, SCWL and B chicks of the same age but differing in muscle weight and DNA unit size were compared (Table 4). The FSR in EDC and UL muscles were similar between strains, but the FDR was significantly lower in B chicks, resulting in a higher synthesis: degradation ratio. When FDR was calculated from measurements of FSR and FGR (FDRC), values were between 40 and 50% lower than the corresponding experimentally determined FDR (FDRa). In the third experiment, comparisons were made between SCWL and B chicks of

DISCUSSION

-TABLE 4. Protein synthesis and degradation in muscles from Single Comb White Leghorn (SCWL) and broiler chicks of the same agel Ulnaris lateralis

Extensor digiti c o m m u n u s Variable 2

Broiler

Weight, m g DNA u n i t size 3 FSR, %/day F D R a , " %/day FSR/FDRa F G R , %/day F D R C , S %/day FSR/FDRC DNA activity 6

28.1 411 30.5 28.6 1.07 15.3 15.2 2.01 125

SCWL ± 1.9 ± 12 ± 1.8 t 1.1 ± .04 ± 1.1 .7 ± .06 ± + 3.0

9.8 367 28.8 32.4 .89 10.2 18.6 1.54 106

SCWL

Broiler ± 1.0* ±10* ± .9 ± .7* ± .04* ± .6* ± .5* ± .07* ± 5.1*

36.6 423 27.3 24.1 1.13 15.1 12.2 2.24 115

± 1.7 ± 12 ± 1.5 + .9 .03 ± ± .9 ± .8 .07 ± ± 3.3

14.4 369 27.2 29.6 .92 10.0 17.2 1.58 100

± .09* ±9* ± 1.2 ± .8* ± .04 ± 1.0* ± .8* ± .07* ± 5.1*

'Means ± standard error; chicks were 9 days of age with body weights averaging 136 and 70.8 g for broiler and SCWL chicks, respectively. 2

FSR = Fractional rate of protein synthesis; FDR = fractional rate of degradation; FGR = fractional growth

rate. 3

Milligram protein/milligram deoxyribonucleic acid (DNA).

4

FDR a = Actual values determined from measured rates of tyrosine released per day as described in materials and methods section. 5

FDR C = Values calculated from the relationship FDR=FSR-FGR.

6

Grams protein synthesized per gram DNA per day.

'Value is significantly different (P<.05) from the same measurement in broiler chicks.

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The main objective of this investigation was to determine the mechanism(s) by which protein accretion occurs at different fractional rates in muscles from B and SCWL chickens. A faster rate of protein accretion must result from a higher rate of protein synthesis relative to protein degradation. Because broilers have a higher feed efficiency for growth than SCWL chicks and because protein accretion is a major part of growth as well as an energetically expensive process, it follows that broilers may accomplish a greater growth velocity with lower rates of protein degradation. It would be energetically most efficient if rates of protein degradation were decreased sufficiently toward zero so that

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TABLE 5. Protein synthesis and degradation in muscles from Single Comb White Leghorn (SCWL) and broiler chicks of the same DNA unit size1 Extensor digit! c o m m u n u s Broiler

Weight, m g DNA unit size 3 FSR, %/day F D R a , 4 %/day FSR/FDRa F G R , %/day F D R C , S %/day FSR/FDRC DNA activity 6

24.2 428 31.4 30.3 1.03 14.4 17.0 1.85 134

± 1.7 ± 16 ± 1.3 ± 1.7 ± .08 ± 1.0 ± 1.1 .07 ± ± 5.1

Ulnaris lateralis

SCWL

Broiler

10.9 ± 1.1* 416 ± 15 24.7 ± 1.0* 27.4 ± 1.3 .90± .07 .7* 9.9 ± 14.8 ± 1.2 1.67± .07 103 ± 5.2*

32.4 434 28.6 24.2 1.18 13.0 15.6 1.83 124

SCWL ± 1.8 ± 15 ± .8 ± .9 ± .07 ± .9 ± 1.0 ± .08 ± 4.1

17.5 438 25.3 26.2 .96 9.3 16.0 1.58 111

± 1.0* ± 11 ± .9* ± 1.0 ± .08 ± .8* ± .9 ± .07* ± 6.3

'Means ± standard error; broiler and SCWL chicks were 8 and 11 days of age with average body weights of 123 and 80 g, respectively. 2

FSR = Fractional rate of protein synthesis; FDR = Fractional rate of degradation; FGR = Fractional Growth

rate. 'Milligram protein per milligrams (DNA). 4

Actual values determined from measured rates of tyrosine released per day.

S

FDR C = values calculated from the relationship FDR=FSR-FGR.

6

Grams protein synthesized per gram DNA per day.

*Value is significantly different (P<.05) from the same measurement in broiler chicks.

protein synthetic rates might also be reduced and thus result in higher rates of protein accretion with minimal energy expenditure. This study (Table 4) demonstrated that the B chick accomplishes a greater velocity of protein accretion primarily through lower FDR (an FDRa of 11.7 and 18.6% less in the EDC and UL, respectively); however, rates of protein synthesis tend to be slightly higher in the EDC. The opposing changes result in larger synthesis: degradation ratios in broilers, indicating a greater FGR. These results suggest some savings in energy due to a lower rate of protein turnover and consequently a greater efficiency of protein accretion in B chicks. Our results are similar to those of Orcutt and Young (1982) where in vitro cultures of embryonic muscles from broiler and layer chicks were found to have comparable rates of protein synthesis. The faster rates of protein accumulation by broiler muscle cells were primarily due to slower rates of protein degradation. Hentges et al. (1983), using similar in vitro techniques as reported in this paper, were unable to detect any differences between breeds in protein synthetic and degradative rates of EDC or extensor carpi ulnaris muscles. Maruyama et al. (1978) measured rates of protein synthesis and degradation in vivo and

did not find significantly different rates of protein synthesis in either the leg or breast muscles of fast growing Cornish and slow growing New Hampshire x SCWL chicks. Protein degradation values could not be statistically analyzed because of inherent limitations of the technique utilized, but rates tended to be lower in leg but not breast muscles of Cornish birds. Kang et al. (1985) compared the results of two separate experiments conducted in their lab and concluded that broiler skeletal muscles accrete protein faster and with greater efficiency than White Leghorn muscles, primarily as a result of slower protein degradation. Again, protein degradation was arrived at indirectly from measurements of protein synthesis and protein accretion. Also, measurements of protein synthesis in broilers were made using a novel isotope emulsion method (Kang et al., 1985), whereas measurements in White Leghorns were determined by injecting a massive dose of the isotope (MacDonald and Swick, 1981). In rats, the larger FGR in a fast growing albino strain compared to a slow growing hooded strain was found to be a result of decreases in both the FSR and FDR, with a relatively larger decrease in the FDR (Bates and Millward, 1981). In these present experiments, rates of protein synthesis and protein degradation were esti-

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Variable 2

PROTEIN TURNOVER IN CHICK MUSCLES

In the present experiment, rates of protein synthesis were slightly higher than rates of protein degradation in B muscles, demonstrating protein accretion. In SCWL chicks, rates of protein synthesis tended to be lower than rates of degradation, demonstrating that either the FSR was underestimated or the FDR was overestimated. Passive stretch has been shown to retard protein degradation in isolated rat muscles (Goldberg et al., 1975; Goldspink, 1978). In chickens, the anterior and posterior latissimus dorsi muscles respond to stretch by increased protein accretion due to increased protein synthesis (Laurent et al., 1978). It is possible that stretching may be important in maintaining isolated chick muscles in positive protein balance. Thus, in vitro measurements of protein turnover should not be considered as a replacement for well designed in vivo experiments but as an adjunct, providing supportive information. Data from Experiment 2 can be used to compare the actual in vitro measurement of FDR (FDRa) with the method used for in vivo experiments in which FDR is calculated from actual measurements of FSR and FGR by the relationship: FDRC = FSR - FGR. This comparison demonstrates the lack of congruence of the two methods. For example, in EDC muscles from B chicks, FDRa was 28.6% per day, whereas FDRC was 15.2% per day. Nevertheless, both FDRa and FDRC values from this experiment indicated lower rates of protein degradation and higher synthesis:degradation ratios for B chicks. The use of FSR and FGR to determine FDRC has received much criticism for two reasons. First, measurements of FSR and FGR are made over different time periods, with FSR measurements taken over minutes to several hours and FGR measurements taken over several days. Because FSR is subject to diurnal variation (Garlick et al., 1973), the value obtained during any short period of measurement may not be representative of the average value during the entire FGR measurement period. Second, in vivo measurements of FSR suffer from imprecision due to the uncertainty of the true specific activity of the precursor pool. Thus, FDRC values may be inaccurate due to a compounding of these errors. In vitro estimates of FSR are less subject to large errors due to uncertaintly of the specific activity of the precursor pool because all amino acid pools are equalized by flooding with both cold and labeled tyrosine. However, as discussed previously, the in vitro measurement may not be an accurate measurement of in vivo rates

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mated in vitro. This technique has been used successfully to determine the effects of hormones and metabolites on rates of protein degradation in muscles from chicks (Klasing and Jarrell, 1985) and rats (reviewed by Libby and Goldberg, 1980). The application and interpretation of this in vitro technique have several limitations. First, only muscles which have discrete tendons on both ends can be used. Additionally, these muscles must be small enough for in vitro viability to be maintained through diffusion of nutrients. In our laboratory, we have found muscles larger than 40 mg have poor viability in vitro (unpublished results). Hentges et al. (1983) also reported poor application of this technique with large muscles. Consequently, muscles of major economic importance such as the pectoralis cannot be used and only immature birds may be studied with this in vitro technique. Extensor digiti communus muscles from broilers older than 10 days (180 g live body weight), SCWL chicks older than 24 days (160 g live body weight), UL muscles from B older than 9 days (160 g), or SCWL chicks older than 20 days (130 g) do not yield valid results. Hence, our studies used muscles from relatively young chicks to study protein synthesis and degradation. Because measurements were made under in vitro incubation conditions their relevance to in vivo conditions may be suspect. Rat muscles incubated in vitro undergo net protein catabolism unless glucose, insulin, and branched-chain amino acids are present (Fulks et al., 1975). Insulin, glucose, and branchedchain amino acids also minimize protein degradation in chick muscles incubated in vitro (Klasing and Jarrell, 1985). Goldspink (1978) demonstrated that additions of insulin, glucose, and amino acids to the incubation medium help maintain tissues in a positive nitrogen balance by increasing synthesis and decreasing degradation. These additions reflect the probable situation in vivo and, in rats, give rates which are in good agreement with in vivo measurements by other investigators (Millward et al., 1973; Turner and Garlick, 1974). Clark and Mitch (1983) found that rates of protein synthesis and degradation estimated in rat epitrochlearis muscle incubated in vitro were similar to rates measured in perfused hindquarter preparations. The in vitro technique has the advantage that rates of protein degradation are measured directly instead of indirectly by subtracting protein accretion rates from synthetic rates.

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as a result of different muscle tension as well as metabolite and hormonal milieu. In both strains of chicks, DNA concentration decreased and the DNA unit size increased with age. These changes were more rapid for B chicks, resulting in a large difference between strains at 20 days. Orcutt and Young (1982) showed that DNA unit size is also greater in B than SCWL embryonic muscle cells. Hentges et al. (1983) reported an age-related decline in muscle DNA concentrations, but these investigators did not observe a difference between strains. Waterlow et al. (1978) suggested that, within a tissue, protein synthesis per DNA unit (DNA activity) remains constant and that the FSR decreases with age, probably as a result of decreased DNA and, consequently, RNA concentrations. In Experiments 2 and 3, muscles from B chicks had greater DNA activity, demonstrating that each B nucleus can support a greater rate of protein synthesis than a nucleus from SCWL cells. This relationship held whether values were calculated for the two strains at equal ages but different DNA unit sizes (Table 4), or if DNA unit sizes were equalized by using chicks from each strain at different ages (Table 5). Interestingly, when the DNA unit size was equalized, rates of protein degradation were not significantly different between strains (Table 5). Because rates of both protein synthesis and degradation fall with age (Hentges et al., 1983; Kang et al., 1985; Lauterio etal., 1986), results of this last experiment may have been due to age differences, not DNA unit size similarities.