The aim of this work was the determination of the myofibrillar protein profiles in the fed and the mildly underfed rat. Sixteen male rats were divided into 2 groups: CR (control) fed ad libitum and MR (mildly undernourished) fed 75% of energetic maintenance needs. The animals were sacrificed at day 23 and the gastrocnemius muscle was taken for myofibrillar protein characterisation. The myofibrillar protein profiles were found to be very similar in the two groups revealing the lack of preferred catabolism of myofibrillar proteins and consequently that the muscle structure is maintained even in situations of mild undernutrition

Almeida, A M

Cardoso, L A

van Harten, S

English

Journals from University of Tartu

IntroductionLivestock production in tropical areas is seriouslyaffected by undernutrition caused by the season’spoor nutritive value of pastures during the dry sea-son. In fact this is frequent, in non-intensive sys-tems due to the lack of supplementation such that aseasonal weight loss of up to 40 % in beef and mut-ton herds can be expected (Clariget et al, 1998).Undernutrition is consequently a serious limitationto animal production in tropical areas significantlyreducing productive performances (Wilson, 1987).The above-mentioned weight loss is characterisedby the mobilisation of body depots, especially fatand to a lesser extent protein (Belkhou et al, 1991).Muscle, and therefore protein, breakdown occurs inorder to use the resulting amino-acids as an energysource, which tends to cause a various physiologi-cal responses at muscle level (depending on severalfactors such as the species studied or the severity offood restriction). Undernutrition significantly affects several musclecharacteristics (Bedi et al, 1982; Crouse et al, 1984;Mosoni et al, 1999; Ameredes et al, 1999) mainly asa result of the increase of degradation levels(Wassner et al, 1977; Ogata et al, 1978) but also thedecrease of protein synthesis (Fong et al, 1989; VanEanaeme et al, 1998). Increasing whole-proteindegradation levels are expected to influence notonly total myofibrillar protein breakdown but alsoeach one of the myofibrillar proteins (Fong et al,1989). This paper aims to characterise the physio-logical components of undernutrition, usingmyofibrillar protein profiles in order to establish, inrats, a model for draught-induced undernutritionperiods previously mentioned. Depending on thecharacteristics of the climate, seasonal undernutri-tion caused by lower rainfalls can be relatively mildas seen for instance in the Mediterranean climatesor in sub-tropical regions. Results obtained withthis experiment can hence be extrapolated tospecies of domestic animals such as sheep or goatsthat undergo relatively mild undernutrition periodsbelow maintenance levels.Material and MethodsSixteen adult male rats (380 g) were obtained fromthe Wistar Colony maintained at the GulbenkianScience Institute (Oeiras, Portugal) and divided intoScand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 2Published in the Scandinavian Journal of Laboratory Animal Science - an international journal of laboratory animal science101Myofibrillar Protein Status of the Gastrocnemius in Male Rats:Effect of Mild Undernutritionby A.M. Almeida*, S. van Harten & L.A. CardosoCentro de Veterinária e Zootecnia, Faculdade de Medicina Veterinária, Lisboa, PortugalSummaryThe aim of this work was the determination of the myofibrillar protein profiles in the fed and  the mildlyunderfed rat. Sixteen male rats were divided into 2 groups: CR (control) fed ad libitum and MR (mildlyundernourished) fed 75% of energetic maintenance needs. The animals were sacrificed at day 23 and thegastrocnemius muscle was taken for myofibrillar protein characterisation. The myofibrillar protein profileswere found to be very similar in the two groups revealing the lack of preferred catabolism of myofibrillarproteins and consequently that the muscle structure is maintained even in situations of mild undernutrition.*Correspondence: André Martinho de AlmeidaCentro de Veterinária e Zootecnia, Faculdade de MedicinaVeterinária, Rua Prof. Cid dos Santos, 1300-477 Lisboa,PortugalTel: +351213652800Fax: +351213652869E-mail: dealmeyda@hotmail.comJD 81 Myofibrillar Protein  28/06/06  13:59  Side 101Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 2102two weight-matched groups (n = 8): CR (Control)and MR (mildly underfed). Group CR animals werefed ad libitum, while MR group animals wererestricted to 75 % of the energetic maintenancerequirements at the beginning of the experiment (5g / day). Both groups were fed on milled standardCRM Interfauna Ibérica chow (16401 J / g, 17.5 %total protein, 5.3 % ash, 4 % rude fibre and 2.7 %fat). The animals were housed in a sound-proof room at24ºC with 8 hours dark per 16 hours of light usingindividual metabolic cages that allowed separationof faeces and urine (Cardoso & Stock, 1998).Nitrogen balances were conducted (Cardoso &Stock, 1998) at days 0 and 23 of the experiment inorder to characterize the level of undernutrition andhence estimate body depletion conditions. Afterquantification, faeces were dried to constant weightand the dry matter percentage determined. A sam-ple of the dried faeces was homogenized and usedfor crude protein and gross energy determinationsusing standard methodologies (Cardoso & Stock,1996). Briefly, crude protein was determined in aTecator Digestion System 40 in concentrated sul-phuric acid. Distillation was performed in a TecatorKjeltec System Distilling Unit using 13N SodiumHydroxide. tritation was conducted using 0.0714NHCl for Nitrogen content determination. Crude pro-tein was determined by multiplying N conten by6.25. Urine samples were processed using similarmethodology. Nitrogen balances were calculated bysubtracting total protein of the faeces and urinesfrom protein ingested. Animals were weighed every two days and feedintake registered daily. After 23 days of experimen-tal period, the animals were anaesthetised(intraperitoneal injection of Sodium Pentothal, 0.5%) and sacrificed.The eviscerated carcass was cleaned and “dressed”(decapitation, amputation at the tibio-tarsal, radio-cubit-carpal and sacro-coccigenal joints), and thegastrocnemius muscle collected and kept at -75ºC for further analysis. Gastrocnemius muscle isconsidered a model to study muscle plasticity andcharacteristics under stresses (Alford et al., 1987).Additionally it is a muscle of easy access, whichrenders subsequent manipulations easier.The remaining carcass was frozen, freeze-dried toconstant weight and milled and the resulting pow-der was used for the crude protein content determi-nation as previously described. Total fat was deter-mined by subtracting moisture and crude proteinfrom carcass fresh weight as described (Carter etal, 1991). Gross energy was conducted using a Parr1261 calorimetric bomb (Parr, Moline, Illinois).Total quantities of fat, protein and energy weredetermined by multiplying the contents by total car-cass weight.Gastrocnemius muscles were prepared using themethods described by Parrish Jr. et al (1973) for thedetermination of myofibrillar protein extract.Electrophoresis of myofibrillar proteins was doneon SDS-PAGE electrophoretic gels at 160 volts.Gels were fixed with methanol and acetic acid for30 min., washed with distilled water and colouredwith 1.0g per litre Coomassie R350. Gels were digi-tised and analysed for band areas of the followingmyofibrillar proteins: Myosin Heavy Chains,Protein C, α-Actinin, Tropomyosin + Troponin T,Actin and also the injected pattern Bovine SeraAlbumin, in a Kodak Digital Science Gel analyser,according to methods described by (Claeys et al1995).Results of both groups were compared by ANOVASingle factor. Results were considered significantlydifferent when p<0.05. Data are presented as meanvalues +/- SEM (Standard Error of Mean). This experiment and laboratory animal handlingprocedures were approved by the Scientific Councilof the Faculty of Veterinary Medicine (TechnicalUniversity of Lisbon, Portugal).Results and DiscussionTable 1 refers to the initial and final live weight ofrats of both groups, as well as growth and the rela-tive evolution of liveweight and cumulative feedintake. CR animals increased their live weight by78.80g (a 21 % increase relative to the beginning ofJD 81 Myofibrillar Protein  28/06/06  13:59  Side 102Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 2the experiment), while MR lost 99.01g (a 28 %decrease). Live weight at day 23, growth, as well aslive weight evolution registered, were significantlydifferent in both groups. Nitrogen balances are presented in Table 2. At thebeginning of the experiment, balances were positiveand similar for both groups. At the end of the exper-imental period, balances were negative for  the MRgroup as expected and as a consequence of theundernutrition experienced by this group. An evolution in live weight and negative nitrogenbalances, as experienced by the MR group  animals,led to subsequent alterations in the carcass compo-sition. As shown in Table 3, all the characteristicsstudied were significantly higher in the CR animals,with the exception of moisture percentage andcrude protein percentage.Based on previous results, animals in the MRgroup will be under a situation of undernutritionthat has consequences on the depletion of bodydepots, namely fat. The subsequent metabolicresponse of this will be depletion of the organism’smuscle proteins (Belkhou et al, 1991), a situationthat the MR animals are clearly experiencing asshown by the decrease in the total quantity of pro-tein. In such a context, it is interesting to know how mus-cle protein is affected by catabolism caused byundernutrition. We have conducted such research atthe level of the gastrocnemius muscle in the rat by103CR MRLive weight day 0 (g) 363.19 (4.77) 364.84 (7.98)Live weight day 23 (g) 441.99A (8.33) 269.84B (8.67)Growth (g) 78.80A (9.71) -99.01B (10.25)Live weight day 23 (as %LW day 0) 121 73Cumulated Feed Intake (g) 709.18A (20.95) 257.28B (0.48)A,B Rows with different superscripts indicate statistical significance (p<0.05); Standard Error of Mean values in parenthesisTable 1 - Growth and evolution of rat live weightCR MRNitrogen Intake day 0 (g) 0.89 (0.03) 0.83 (0.02)Nitrogen in the faeces day 0 (g) 0.15 (0.01) 0.17 (0.01)Nitrogen in the urine day 0 (g) 0.47 (0.02) 0.43 (0.04)Nitrogen Balance day 0 (g) 0.26 (0.03) 0.24 (0.04)Nitrogen Intake day 23 (g) 0.76A (0.07) 0.20B (0.01)Nitrogen in the faeces day 23 (g) 0.13A (0.01) 0.03B (0.01)Nitrogen in the urine day 23 (g) 0.38A (0.03) 0.27B (0.02)Nitrogen Balance day 23(g) 0.25A (0.06) -0.1B (0.03)A,B Rows with different superscripts indicate statistical significance (p<0.05); Standard Error of Mean values in parenthesisTable 2 - Nitrogen BalanceJD 81 Myofibrillar Protein  28/06/06  13:59  Side 103Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 2104electrophoresis. Band areas for the myofibrillarprotein Myosin Heavy Chains, Protein C, α -Actinin, Tropomyosin + Troponin T and Actin aredepicted in Figure 1. Such data were obtained fromgels such as the one depicted in Figure 2. Bothgroups registered very similar band areas for eachof the studied proteins. No significant differencewas registered between MR and CR animals. Suchresults are in accordance with previous results fromour experiments (Almeida et al, 2002) regardingmyofibrillar protein profiles in the rat gastrocne-mius muscle under severe undernutrition (34 %weight loss) and contrasts with the results from(Bates et al 1983) and (Clark & Wildenthal 1986)that point to a decrease in myosin synthesis in situ-ations of more severe undernutrition than that men-tioned in this work. The results seem to indicate that in the conditions ofthis experiment, there is no preferred protein catab-olism as a response to undernutrition, thus main-taining myofibrillar protein proportions and alsothe structure of the gastrocnemius muscle.Characteristics CR MRFresh Weight (g) 218.91A 145.33B(4.19) (3.86)Dry Matter (%) 30.32A 26.05B(1.09) (.45)Humidity (%) 69.68A 73.95B(1.09) (.45)Crude Protein 55.67A 75.90B(% of dry matter) (1.89) (1.22)Fat Content 44.33A 24.10B(% of dry matter) (1.89) (1.22)Gross Energy 25.3A 19.4B(kJ / g MS) (.032) (.08)Total Protein (g) 36.76A 28.78B(1.42) (1.20)Total Fat (g) 29.86A 9.12B(2.39) (.50)Total Energy (kJ) 1686.15A 738.05B(90) (36)A,B Rows with different superscripts indicate statistical significance (p<0.05); Standard Error of Mean values in parenthesisTable 3  Carcass CompositionFigure 1 – Myofibrillar Protein Profiles in the gas-trocnemius of laboratory rats following mild under-nutrition. MHC – Myosin Heavy ChainsJD 81 Myofibrillar Protein  28/06/06  13:59  Side 104Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 2105However, an increase in the level of undernutritionwould probably lead to a rupture in muscle struc-ture, with changes in the levels of proteins such asMyosin Heavy Chains, as reported (Bates et al.,1983; Clark & Wildenthal, 1986).Interestingly, alterations in the myofibrillar proteinprofiles have been reported in the semimembranousmuscle of male Capra hircus under mild undernu-trition of 20 % weight loss (Almeida et al, 2004).Such results might indicate that the responses toundernutrition, regarding myofibrillar proteins,vary between species and muscle studied. It seemstherefore interesting to extend this research to othermuscles in order to have a full picture of the influ-ence of undernutrition on myofibrillar protein pro-files.AcknowledgementsThe authors would like to thank the Science andTechnology Foundation of Portugal (Fundação paraa Ciência e a Tecnologia - PRAXIS XXI/BM/17921/98) for financial support, to JM Prates andGF Dias (Technical University of Lisbon, Portugal)for technical assistance, and to GM Almeida(University of Leicester, UK) for English editing ofthe manuscript.ReferencesAlford EK, RR Roy, JA Hodgson & VR Edgerton.Electromyography of rat soleus, medial gastroc-nemius and tibialis anterior during high limbsuspension. Experimental Neurology, 1987, 96,635-649.Almeida AM, S Van Harten & LA Cardoso. Serumamino acid and myofibrillar protein profiles offed and underfed laboratory rats. NutritionReseach, 2002, 22, 1453-1459.  Almeida AM, LM Schwalbach, HO deWaal, JPCGreyling & LA Cardoso. 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Myofibrillar Protein Status of the Gastrocnemius in Male Rats: Effect of Mild Undernutrition

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Myofibrillar Protein Status of the Gastrocnemius in Male Rats: Effect of Mild Undernutrition

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