DIFFERENTIAL GROWTH RETARDATION AND MYOFIBRILLAR FRAGMENTATION IN RATS SUBMITTED TO FEED RESTRICTION AND REALIMENTATION

Feed regimens alter muscle growth rate, hence they might impact the proteolytic system involved in tenderization during meat conditioning. The aim of this project was to verify the effects of feed restriction regimens on muscular and animal growth and their impact on postmortem myofibrillar fragmentation. The regimens were: 1) Feeding ad libitum for 11 d (Al/2); 2) Feed restriction (60% of Net Energy for maintenance – NEm) for 11 d (Rt/2); 3) Ad libitum for 22 d (Al); 4) Ad libitum for 4 d and feed restriction (60% NEm) for 18 d (Rt); 5) Ad libitum for 19 d and 3 d of fast (Ft); 6) Feed restriction (60% NEm) for 11 d and ad libitum until 22 d (Ral). The regimens Al/2 and Rt/2 had different intestine weights (19.3 ± 1.1 and 15.8 ± 1.9 g, respectively; P < 0.07). At 22 d, Al animals had higher (P < 0.07) intestine weight (21.8 ± 3.8). Moreover, Ral animals had heavier intestine (19.9 ± 1.5) as compared to Rt (16.6 ± 1.6) or Ft (12.8 ± 1.9). The intestine/live weight percentage ratio was lower (P < 0.05) for Ft (6.3%) as compared to Al (8.4%) and to Ral (9.2%), but it was similar to Rt (7.6%). Liver weight (g) in the Ral (9.5 ± 1.1) did not differ from Al (10.7 ± 2.5) or Rt (8.5 ± 1.1), although the two latter were different (P < 0.05). There was an effect of feed restriction over muscle protein degradation verified by Myofibrillar Fragmentation Index (MFI). The animals at Rt, Ft or Ral showed the lowest MFI 0d (42 ± 1.9; 40 ± 2.7; 40 ± 3.6; respectively) and MFI 5d (77 ± 2.7; 74 ± 3.0; 74 ± 2.9; respectively) as compared to Al, whose indexes were 54 ± 3.0 and 82 ± 3.3. Even though the MFI 5d were lower for the restricted animals, the rates of fragmentation postmortem were higher. Feed restriction altered myofibrillar protein degradation, reflected in lower extended fragmentation of the myofibrils.


INTRODUCTION
The effect of feed restriction on growth and final weight of different animal organs is well established (Pethes et al., 1985;Hicks et al., 1990;Murphy & Loerch, 1994;Cardoso & Stock, 1996;Wertz et al., 2001;Choat et al., 2002;Farmer et al., 2004).Some of the authors showed that liver, intestine and other non-carcass tissues are the first to respond to feed restriction and realimentation.On the other hand, skeletal muscles give little response, being spared until higher levels of tissue nutrients are imposed to mobilization due to food deprivation (Brandstetter et al., 1998 in bovine;Potokar-Candek et al., 1999 in swine;Maxwell et al., 1992 in rats).The depletion of muscle mass and proteins are linked to changes in the protein degradation systems, which are important contributors to the growth rate of animals (Huang & Forsberg, 1998;Kristensen et al., 2002;Owens et al., 1993).
The growth rate immediately before slaughter is especially interesting due to its possible impact on the postmortem muscle proteolytic process associated to the regulation of endogenous protease systems.The postmortem proteolysis has been known to be a key effector on tenderization during meat conditioning under refrigeration (Koohmaraie, 1992).The enzymatic system involved in the postmortem proteolysis and muscle protein turnover is known as calpain system (Koohmaraie et al., 1990), and its effects on myofibrillar degradation are correlated to Myofibrillar Fragmentation Index (MFI) (Whipple et al., 1990b).There are evidences that feeding regimens may alter the calpain system and myofibrillar fragmentation (McDonagh et al., 2001).However, there are no reports on the effect of different schemes of feed restriction and realimentation in the postmortem myofibrillar fragmentation process.
The aim of this study was to verify whether the changes in animal growth elicited by different feeding restriction regimens during the muscle growth phase (protein accretion) would have an impact on the rate and extension of postmortem proteolysis evaluated by the myofibrillar fragmentation index.

Animals and feeding regimens
Thirty-six growing Wistar rats (Rattus novergicus), right after weaning (14 days of age), were kept 22 days of experimental period (d.e.p.).Six Polytron homogenizer (15000 rpm) with MFI buffer and under cooled conditions according to Culler et al. (1978).

Statistical Analysis
The data were analyzed using the procedures of GLM of the SAS package (SAS Inst., Cary, NC, 2001).The basic model used live weight (LW), carcass weight (CW), intestine weight (IW), liver weight (LrW), visceral weight (VW) and total weight of thigh muscles (MW) as dependent variables.The kinetics of postmortem myofibrillar fragmentation resulting from lengthening the time of refrigerated conditioning was evaluated by the following exponential function (Riley et al., 2003): where: κ0 represents the maximum MFI after the last day of maturation, κ1 represents the difference between the initial MFI and the maximum MFI, κ2 represents the increase rate, "di" is the day postmortem and εi is the error associated with the observation of the data (assumed independent and distributed identi-cally).This analysis was made using the procedure NLIN of SAS.The data of each treatment were separated in individual curves.The parameters were compared to evaluate potential differences in the treatment.

Growth and organ weight
LW and CW of the animals that were under Al (i.e., Al/2 and Al) regimens were higher (P < 0.001) than all restricted regimens (i.e., Rt/2 and, Ral, Rt and Ft) at both evaluation times (Table 2).The results were expected and confirmed the undernutrition of the animals that were submitted to different feeding restriction regimens.
IW was higher (P ≤ 0.07) for animals fed Al for the two evaluation times (i.e., Al/2 and Al).This same variable differed (P ≤ 0.07) among the restricted regimens at the end of the period (i.e., Ral, Rt and Ft).In the Ft regimen, the internal organs which normally demand high energy, especially the intestine, presented lower weight as compared to other regimens.Fasting for 72 hours causes hypoplasia in intestinal villi Table 2 -Live weight (LW), carcass weight (CW), intestine, liver, visceral and thigh muscle weights of young rats submitted to different feeding regimens.
Table 3 -Intestine live weight ratio (Int/LW), liver live weight ratio (Liv/LW), visceral live weight ratio (Visc/LW) and thigh muscle live weight ratio (Musc/LW) for young rats submitted to different feeding regimens.
Each value represents the mean±standard error.Means followed by different letters in the same column differ (P < 0.001).and crypts, and decreases activity of several enzymes from epithelial cells (Holt et al., 1986).Although Rt did not show such an acute case in terms of compromising the final weight of internal organs, it was the regimen that showed the lowest (P < 0.001) intestine/ body weight ratio (Table 3).On the other hand, Ral reverted partially the slow growth rate during restriction, which confirms that animals which had undergone severe food deprivation are able to counteract the downward regulation of intestinal villi height and crypt depth as well as in enterocyte enzymatic activity, when they were allowed to ad libitum feeding (Holt et al., 1979).
Rats under Al regimen also presented higher (P < 0.05) liver weight, at older age, than those under feed restriction, except for Ral (Table 2).In order to allow lower maintenance energy expenditure and increased feed efficiency under feed restriction regimens, the liver is one of the first organs to reduce its growth (Hicks et al., 1990;Levin et al., 1993).In severe feed restriction, there is a reduction of the liver growth hormone mRNA receptor (Brameld et al., 1996) and IGF-I gene expression (Straus & Takemoto, 1990), which may be correlated to reduced liver growth.On the other hand, in the Ral regimen liver might have experienced compensatory growth, which may have been a result of increased liver cell (hepatocytes) sizes that result from refeeding animals under feed restriction (Sainz & Bentley, 1997).The smaller liver observed for the rats submitted to Ft, which were 29% lighter than those under Al, agreed with a 23% reduction in liver size reported in rats fasting for 48 hours (Anand & Gruppuso, 2005).The Rt rats also had their liver weight decreased, even though the effect was more moderate, in the order of 20%.
Differences observed in liver and intestines were expected to reflect in the visceral weight.Furthermore, in animals under feed restriction other visceral organs were supposed to present growth decrease due to their high energy requirement.Indeed Al rats presented higher (P < 0.05) visceral weight than the restricted rats on both observation times (Table 2).However, Rt and Ral did not differ in visceral weight.This result suggests that even though Ral was sufficient to promote compensatory liver and intestine growth rate, there were other visceral organs that had significant contribution to total visceral weight, also affected by restriction, which did not recover its size after the refeeding period.Besides liver, there was a reduction in visceral weight especially in organs such as heart, gonads and kidney in rats under severe protein restriction (Reichling & German, 2000).The lowest visceral weight observed for the Ft regimen could be explained by lower RNA and protein contents fol-lowed by decreases in cell size that resulted from long fasting periods in rats, which have drastic impact in the total visceral weight (Burrin et al., 1988).
At the first observation time, restricted animals (Rt/2) already showed lower (P < 0.05) muscle weight (Table 2), which would be expected since young rats under feeding restriction had been shown to decrease rapidly muscle protein synthesis and increase degradation (Ogata et al., 1978).The animals from the Rt, Ral and Ft groups had lower (P < 0.05) muscle weight as compared to Al, but did not differ among them (Table 2).Although skeletal muscle may be spared for some time under feeding restriction, there was a negative impact on thigh muscles.These muscles are mostly phasic muscles, which could be the explanation for the observed results considering the reported differences in response of phasic and tonic muscles to feed restriction, phasic muscles having been more sensitive to undernutrition (Goldspink, 1978).Dirks & Leeuwenburgh (2006) shows that experimental animals (e.g., rat) under feed or caloric restriction experience a significant decrease in both fat mass and lean body mass, even though the decrease in muscle mass/body weight ratio is attenuated with age (Dirks & Leeuwenburgh, 2004).

Myofibrillar Fragmentation Index and its kinetics
The spectrophotometric (540 nm) readings were higher on the muscle samples from 0 day postmortem and decreased with time postmortem under refrigeration.There is a possibility that very small myofibril fragments in later times postmortem might have scattered more light forward which ended up reaching the photo cell detector and so resulting in lower absorbance readings (lower turbidity).The phase-contrast microscopy showed an increased myofibrillar fragmentation with time postmortem (Figure 1).The increased fragmentation was also verified by the appearance of bands less than 90 kDa, especially in the range of 30 kDa, in SDS-PAGE (Figure 2).In order to better interpret these observations and not allow misinterpretations such as a decrease in fragmentation with time postmortem, The MFI (Table 4) was calculated, subtracting the standardized MFI (200 X Absorbance reading) from 100.
The feeding regimens had impact on the rate and extent of postmortem myofibrillar fragmentation on muscles under refrigeration (Figure 3).The estimated parameter κ0, indicator of maximum MFI (Table 5), were greater (P < 0.05) for animals under Al regimen at the two observation times (Table 4).Among the restricted regimens there were no differences in κ0.The lower extent of fragmentation associated with lower initial MFI (Table 4) in restriction and/or realimentation Table 4 -Myofibrillar Fragmentation Index (1) of muscles kept under refrigerationat for 0, 1 and 5 days postmortem for young rats submitted to different feeding regimens. (1) Means ± SD followed by different letters in the same column differ (P < 0.05);    regimens may be related to a possible increase of calpastatin activity of muscles, which has been observed for animals submitted to undernutrition (Goicoechea & Conde, 1997;McDonagh et al., 2001).
Although there were no differences in the extent of myofibrillar fragmentation between Rt and Ral regimens, there are reports showing trends of increased activity of µ-calpain, higher MFI values, and lower shear force in pigs that presented compensatory growth (Kristensen et al., 2002;Therkildsen et al., 2002).The lack of differentiation between restricted and compensating animals might be related to a rather slow response of the in vivo proteolytic system going from restricted to ad libitum feeding, which would require several days before an effect could be expected on myofibrillar fragilization (Therkildsen et al., 2002).
Another possibility for higher initial and final MFI for non-restricted animals (Al) may be related to fast glycolytic fiber (FG) transformation towards fast oxidative-glycolytic (FOG) and oxidative fibers (SO), observed in underfed animals.Solomon et al. (1988) reported smaller SO and FG as well as higher proportions of FOG and lower proportions of FG fibers in restrictively fed pigs as compared to pigs with ad libitum access to diet.The percentage area of FOG is negatively correlated to the myofibril fragmentation index (Whipple et al., 1990a).On the other hand, the parameter κ1 was lower for Al in relation to the Rt, Ral and Ft regimens (Table 5), which suggests that most of the fragilization occurred within 24 hours postmortem in the muscle of the animals from the Al regimen and resulted in a greater extent of myofibrillar fragmentation.The restricted regimens did not differ among them.
The results for the κ2 parameter were higher for restricted regimens as compared to Al. Considering the idea that higher calpastatin would be elicited for the restriction of feeding it would have had a negative impact on proteolysis.The problem would be to explain why the difference between initial MFI and maximal MFI (κ1) was higher and why there were higher postmortem proteolytic rates (κ2) on the restricted regimens.One hypothesis would be an increased amount of µ-calpain in vivo as result of feed restriction and muscle mass loss (Brooks et al., 1983;Thomson et al., 1997;Du et al., 2004).In case of greater amounts of calpain spared from autolysis due to calpastatin binding during the first postmortem hours, this would allow a more stable enzyme to proceed with myofibrillar protein proteolysis afterwards (Koohmaraie et al., 1991;Dransfield, 1993;Geesink & Koohmaraie, 1999a;Geesink & Koohmaraie, 1999b).The first postmortem hours are recognized as important for the extent of postmortem myofibrillar fragmentation (Taylor et al., 1995), probably because of optimal pH and ionic strength conditions for calpain activity which becomes limiting as time postmortem proceeds (Kendall et al., 1993).
In summary, in the feed restricted animal muscles there would be a limitation in the rate and extent of initial myofibrillar proteolysis and therefore a lower initial MFI.On the other hand, a faster rate of proteolysis may occur after 24 hours compared to non-restricted animal muscles.However, the suboptimal conditions for proteolytic activity would limit the compensation for lower initial fragmentation, re-Table 5 -Estimated parameters (1) of the myofribillar fragmentation index kinetics (2) for muscles for young rats submitted to different feeding regimens.
(1) Means ± standard error followed by different letters in the same column, differ (P < 0.05); (2) MFI = κ0 + κ1 exp{κ2 di} + εi, where κ0 = maximum MFI; κ1 = difference between initial MFI and the maximum MFI; κ2 = increase rate, di = day postmortem; εi = error associated with the observation of the data; sulting in a lower final MFI.Muscular growth is affected by alimentary restriction in a differentiated way, according to the animal growth phase the occurred changes have impact on the postmortem myofibrillar fragmentation indicating that a proteolytic system is involved.

Figure 3 -
Figure 3 -Myofibrillar Fragmentation Index kinetics as a function of postmortem days for different feeding regimens.Al/ 2 -Feeding ad libitum for 11 d; Rt/2 -Feed restriction (60% of Net Energy for maintenance -NEm) for 11 d; Al -Ad libitum for 22 d; Rt -Ad libitum for 4 d and feed restriction (60% NEm) for 18 d; Ft -Ad libitum for 19 d and 3 d of fast; Ral -Feed restriction (60% NEm) for 11 d and ad libitum until 22 d.

Table 1 -
Dry matter and nutrients of the diet.