EFFECT OF PRE-NATAL RUNTING ON THE POST-NATAL DEVELOPMENT OF SKELETAL MUSCLES IN SWINE AND RATS 1'2

EFFECT OF PRE-NATAL RUNTING ON THE POST-NATAL DEVELOPMENT OF SKELETAL MUSCLES IN SWINE AND RATS 1'2 P. V. J. Ilegarty 3 and C. E. Allen 4 University f Minnesta, St. Paul 55108 SUMMARY Pre-natal runt and high-weight (birth), littermate cntrl animals were selected at day f birth frm swine and rat litters. All animals were suckled nrmally and then fed an adequate diet. The runt pigs required a significantly lnger time (23 days) t reach 106 kg than their cntrl littermates. In swine, runts had significantly lwer bdy weights and, in general, smaller muscles but n difference in muscle fiber diameters at day f birth. Runt pigs at similar adult weights t the cntrl pigs had larger muscle fiber diameters in tw f fur muscles studied and larger quantities f intramuscular lipid in three f the fur muscles. In tw f the muscles where length (based n bne length) and fat-free weight were similar between runt and cntrl pigs, it was pssible t infer that the significantly larger muscle fiber diameter in the muscles f the runt pigs must have been assciated with fewer muscle cells. At a similar adult bdy weight, runt and cntrl pigs had similar lineye areas, average backfat thickness, quantity f perirenal fat, and weight r length f three bnes. The adrenal weight was significantly heavier in the adult cntrl cmpared t the runt pigs. Runt and cntrl rats studied at three different bdy weights had similar muscle weights, muscle fiber diameters and bne lengths. (Key Wrds: Pre-Natal Nutritin, Runts, Grwth, Skeletal Muscles, Swine, Rats.) INTRODUCTION Pre-natal runting prduces lw birth weight 1 Scientific Jurnal Series, Paper N. 9587, Minnesta Agricultural Experiment Statin. 2 The authrs thank Mr. Patrick Oguagha fr technical assistance. This wrk was supprted in part by a grant frm the Graduate Schl, University f Minnesta. 3 Department f Fd Science and Nutritin. animals in many species, due t a reductin in nutrient supply as a result f the lcatin f the fetus in the uterine hrn. This effect has been reprted fr rats (Wiggleswrth, 1964), mice (McLaren, 1965), pigs (Perry and Rwell, 1969) and rabbits (Rseahn and Greene, 1936). The bdy weights f the runts are significantly less than littermates f the same sex at birth (Widdwsn, 197 la; McCance and Widdwsn, 1974), with crrespnding differences in the weights f the sft tissue rgans (Widdwsn, 1971a), and bnes (Adams, 1971). Cmparisn f the runts 'at term' with nrmal fetuses f the same bdy weight indicated that liver, kidneys and small intestines weighed the same in bth grups; the heart, spleen, brain and stmach were heavier in the runt; and the weight f a skeletal muscle was cnsiderably less in the runt than in a fetus f the same weight (Widdwsn, 1971a). These muscle weight differences were accmpanied by less carbhydrate, prtein and DNA, but a higher rati f prtein t DNA in the runt animal when cmpared with the fetus f the same bdy weight. There is n infrmatin n the effect f pre-natal runting n the size f skeletal muscle fibers. Therefre, the effect f pre-natal runting n the diameter f fibers in a number f skeletal muscles during pst-natal grwth was cmpared in swine and in rats. The pig and rat were the experimental animals in this study because the pig is mre develped than the rat at birth (Widdwsn, 1970). Bth f these species are extensively used as test mdels in animal and human nutritin studies. MATERIALS AND METHODS Experiment 1..-Cnstant Age (Day Old): Swine. Sixteen male Yrkshire r Yrkshire crssbred runt and large (cntrl) pigs were slaughtered at day f birth. Animals weighing 1.0 kg r less and 65% f the birth weight f the large littcrmate cntrls were defined as runts. The muscles were allwed t enter rigr 4 Department f Animal Science. mrris, which ccurred 6 t 8 hr pstmrtem. 1634 JOURNAL OF ANIMAL SCIENCE, Vl. 46, N. 6, 1978

NUTRITIONAL STRESS ON SKELETAL MUSCLE GROWTH 1635 The biceps brachii, psas majr and semitendinsus muscles were dissected frm rigin t insertin, trimmmed f adhering fat and cnnective tissue, and weighed. A cmplete crsssectin, taken frm the mid-prtin f each muscle, was placed in 10% neutral frmalin. Islated fibers were btained by a hmgenizatin technique (Hegarty and Naude', 1970). It was impssible t islate unfixed fibers frm the muscles f these yung animals withut prducing extensive fragmentatin f the individual fibers. N damage was bserved in the islated fixed fibers. Fiber diameter measurements in this, and in the tw experiments utlined belw, were made n 100 randmly selected fibers in each sample. It is a cmmn bservatin that the utline f skeletal muscle fibers in crss-sectin is irregular. Unpublished results in ur labratry have demnstrated that the diameters f fibers, determined by the prcedures used in this study, represent the maximum diameter f the fibers. Measurements f maximum diameter is achieved because the islated fibers are flated in a liquid media. One advantage f the technique is that the results are precise and reprducible (Hegarty and Naude', 1970). Experiment II-- Cnstant Weight: Swine. A male runt and large (cntrl) littermate pig were selected frm each f 13 Yrkshire r Yrkshire crssbred litters. The criteria fr the selectin f the runt animals were the same as in Experiment I. After weaning (42 days f age), the runts and large littermate cntrls were separated and fed in grups f 3 t 4 per pen. All pigs were fed a standard crn-sybean diet ad libitum frm weaning t slaughter at apprximately 108 kg bdy weight. This diet, cnsisting f a crn-sybean meal mixture cntaining 16% prtein, 2 t 4% fat and all knwn essential nutrients, was fed frm weaning (42 days) t 45.5 kg bdy weight. The animals were then fed a crn-sybean meal diet cntaining 13% prtein and 3.2% fat until each pig reached its designated weight. The muscles were allwed t enter rigr mrtis. The fllwing measurements were then made: lineye area (crss-sectinal area at 10th rib), carcass length (anterir edge f aitch bne t anterir edge f first rib) average backfat thickness (mean f measurements at first and at last rib, and at last lumbar regin); weight f perirenal fat and adrenals; weight and length f the femur, tibia and fibula; the weight, mean fiber diameter f unfixed fibers, and the fat cntent f the fllwing muscles: biceps brachii, lngissimus, psas majr and semitendinsus. The semitendinsus muscle has a distinct red and white prtin that differs with respect t muscle fiber diameter (Hegarty et al. (1973). Bth prtins were separated and examined fr fiber diameter differences in the present study. Intramuscular lipid was determined by the methd f A.O.A.C. (1965). Experiment HI -- Cnstant Weight within Age Grups: Rats. Male, Sprague-Dawley derived rats (Hhzman Cmpany, Madisn, WI) were designated runt and large littermate cntrls at day f birth. The criteria used in the selectin f runt rats was: at least 11 animals per litter and a bdy weight n greater than 75% f the large littermate cntrl animals at day f birth. Litter size during lactatin was nt cntrlled. The animals were weaned at 21 days f age nt a stck labratry diet (Purina Labratry Chw, Ralstn Purina C., St. Luis, MO). Animals were killed at apprximately 60, 290 and 450 g bdy weight (Grups I, II and III in table 3). All animals in a weight grup were at the same age when killed, weight and mean fiber diameter values (n unfixed samples in rigr mrtis) were btained at each f the abve weights fr the biceps bracbii and sleus muscles and mean fiber diameter values nly fr the sternmastideus muscle. The lengths f the femur and humerus were measured. All bservatins were statistically analyzed by t-test as utlined by Steel and Trrie (1960). R ESU LTS Experiment 1 -- Animals at Birth: Swine. As expected, cntrl pigs were.55 kg heavier (P(.05) than runt pigs at day f birth. The weights f the psas majr (P(.05) and semitendinsus (P(.05) (table 1) were als heavier in the large littermate cntrls. The muscles in the runt animals had smaller mean fiber diameters but this difference was significant nly in the biceps brachii muscle (P(.001). The biceps brachii has been classified as an early develping muscle in cattle (Butterfield and Berg, 1966) and in swine (Davies, 1974). Experiment H--Cnstant Weight: Swine. The cntrl pigs were.76 kg heavier (P(.001) than runt pigs at day f birth (table 1). The pigs were grwn t a similar mature bdy weight f apprximately 107 kilgrams. The runt pigs

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NUTRITIONAL STRESS ON SKELETAL MUSCLE GROWTH 1637 tk a lnger (P<.001) time (23.3 days) t achieve this weight. The fat-free weight f the biceps bracbii, psas majr and semitendinsus muscles were nt significantly different in the runt and cntrl pigs. The fat-free weight f the lngissimus muscle was less (P<.05) in the runt pigs when cmpared with the littermate cntrls. Intramuscular lipid cncentratin was greater (P<.05) in the biceps bracbii, lngissimus and semitendinsus muscles f the runt when cmpared t cntrl pigs. Mean fiber diameter was larger in the muscles f the runt pigs, attaining significant levels f difference in the psas majr (P<.O01) and the white and red regin f the semitendinsus (P<.05) table 1. Fiber diameter distributins were mnphasic fr all muscles frm the runt and cntrl pigs. This bservatin is in agreement with a recent reprt n fiber diameter changes in muscles frm malnurished swine Strickland et al. (1975). N "significant difference was bserved fr cld carcass weight, lineye area, average backfat thickness, perirenal fat weight and the length and weight f the femur, tibia and fibula in runt and large littermate cntrl pigs at the end f the experiment (table 2). Similar bne lengths wuld imply similar skeletal muscle lengths fr muscles assciated with these bnes in the tw grups f pigs. The lack f difference in lineye area and backfat thickness between the runt and cntrl pigs cnfirms recent bservatins n similar animals (England,,1974). Adrenal weight was less (P<.O1) in the runt pigs. Experiment 111 - Cnstant Weight: Rats. The mean birth weights f cntrl rats was 135 t 156% f the runt rats (table 3). Hwever, with ne exceptin, there were n significant differences between the runt and cntrl rats in final bdy weight, weight f the biceps bracbii and sleus muscles, diameter f the fibers in the biceps bracbii, sleus and sternmastideus muscles, and the length f the femur and bumerus in the three bdy weight grups studied. Discussin The pig is mre develped than the rat at birth (Widdwsn, 1971a). Therefre, deprivatin f nutrients during fetal develpment shuld have a greater effect n the pig than n the rat. This effect culd be manifested by impaired hyperplasia f skeletal muscle fibers in the pig. The real number f skeletal muscle fibers in the pig is apparently fixed at birth r shrtly thereafter (Staun, 1963; Stickland and TABLE 2. CARCASS COMPOSITION AND BONE WEIGHTS AND LENGTHS IN RUNT AND CONTROL PIGS Animals f cnstant weight Measurements Cntrl Runt Bdy weight, kg 108.2.98 N.S. Cld carcass weight, kg 79.7.48 N.S. Carcass length, cm 80.8.33 N.S. Lineye area, sq cm 35.4.84 N.S. Avg backfat thickness, cm 3.63.10 N.S. Perirenal fat weight, g 1662.4 143.1 N.S. Adrenal weight, g 4.41.24 ** Bnes Femur Weight, g 335.9 5.34 N.S. Length, cm 19.7.14 N.S. Tibia Weight, g 207.8 4.59 N.S. Length, cm 17.9.21 N.S. Fibula Weight, g 32.2.93 N.S. Length, cm 17.1 +-.23 N.S. 106.6.84 78.9.81 79.5.38 33.61 1.04 3.53.10 1969.0 125.0 3.70.32 322.3 + 5.86 19.7.13 203.0 5.03 18.2 +.13 31.2 -+.88 16.6.16 Mean SEM. N.S. = P>.05; **P<.O1.

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NUTRITIONAL STRESS ON SKELETAL MUSCLE GROWTH 1639 Gldspink, 1973; Swatland, 1976). Thus, factrs which wuld limit r reduce fiber number during this critical prenatal perid f cell develpment wuld apparently nt be crrected by hyperplasia during pst-natal develpment. Therefre, fr muscles f the same weight and the same lngitudinal dimensins (based n bne length measurements, tables 1 and 2), hypertrphy f muscle fibers cmpensates fr an apparent decrease in the number f fibers due t the prenatal nutritinal stress during the perid f active hyperplasia. Accrding t Davies (1974), the lngissimus, psas majr and semitendinsus muscles have a higher pstnatal grwth rati than the biceps bracbii muscle f the pig. This suggests that the biceps bracbii muscle is earlier maturing and may be less respnsive than the ther three muscles t the effects f runting n muscle fiber diameter and number. The significant differences between the psas majr and bth the red and white prtin f the semitendinsus fr the cnstant weight cntrl and runt pigs supprt this cncept (table 1). Hwever, there is a physilgical limit t the area f skeletal muscle fibers (Steinhaus, 1933). Hence the grwth ptential in sme f the skeletal muscles f the runt pigs appears t have been limited by the apparent decreased number f muscle fibers and by the physilgical limits n fiber hypertrphy. This reduced muscle grwth ptential may have cntributed t the fact that the runt pigs required abut 23 days lnger t reach final weight than their cntrl littermates. Similar reasning was als used t explain the reduced size and grwth ptential f miniature pigs cmpared t cnventinal-size pigs (Hegarty et al., 1973). N attempt was made t try and quantify the real number f fibers in prcine muscles examined in the present study. Swattand (1976) has utlined the difficulties in making accurate determinatins f real fiber number and the relatinship between real and apparent fiber number in prcine muscle. The significantly greater quantity f intramuscular fat in three f the fur skeletal muscles f the runt pigs (table 1) als supprts the idea that these muscles were mre mature in runt than cntrl pigs. Lee and Kauffman (1974) reprted that the majr increase in intramuscular lipid f the trapezius and semitendinsus muscles f barrws ccurred after 8 t 16 weeks f age and was paralleled by intramuscular adipcyte vlume and number increases between 8 and 24 weeks f age. The higher intramuscular lipid levels in muscles frm the runt pigs wuld nt be expected t ccur in the cntrl animals with an additinal 23 days f feeding when bth grups f animals wuld be the same age. Widdwsn (1977) reprts that muscles, particularly thse f the buttcks, n pigs rehabilitated after 2 and 3 years f undernutritin were s infiltrated with fat that muscle fibers were cmpletely embedded within the fat. Nrmally, in grwing swine, fllwing an initial perid f fast grwth, a transitin perid ccurs during which muscle prtein levels remain stable, muscle fiber grwth rate slws and intramuscular fat depsitin rate increases (Elsn et al., 1963). Pertinent t this discussin is the recent reprt that in human skeletal muscle there is a marked increase in adipcytes after the age f 60, and after the age f 80 the adipcytes cnstitute a majr prprtin f skeletal muscle mass (Inkvchi et al., 1975). One culd speculate that pre-natal undernutritin in pigs and humans may hasten the aging prcess in skeletal muscle with respect t fat accumulatin. Since the intramuscular lipid dept is nrmally the last f the majr lipid depts t develp in cattle and swine (Allen et al., 1976), it is smewhat unusual that there were n significant differences between runt and cntrl pigs fr either backfat thickness r weight f perirenal fat (table 2). Perhaps if the pigs had been grwn t heavier weights, differences in apparent muscle grwth ptential between runt and cntrl pigs wuld have been expressed by differences in fat depsitin at a cnstant bdy weight. The lack f any respnse in skeletal muscles in the rat t prenatal runting is apparently due t the fact that fiber hyperplasia cntinues after birth (Chiakulas and Pauly, 1965). Therefre, nutritinal stress during lactatin wuld prbably be mre critical in terms f subsequent develpment f skeletal muscles in the rat than in the pig. Fr example, litter size may be an imprtant variable in determining the number f muscle fibers, and therefre, subsequent grwth and bdy cmpsitin f the rat. This wuld explain the significant differences in prtein efficiency rati (PER) values when the same diet is fed t rats frm varying sized litters (Hegarty and Ahn, 1977). In the mature muse, nutritinal stress affects muscle fiber diameter but has n effect n muscle

1640 HEGARTY AND ALLEN fiber number (Rwe, 1968). This wrk may als be relevant t the effect f pre-natal nutritin n skeletal muscle develpment and aging in the human. The runt pig has been prpsed as a mdel fr pediatric research Cper (1975). The rat is nt a gd mdel fr pediatric research because newbrn rats are much less develped than newbrn infants (Widdwsn, 1971a,b). Hwever, the human baby grws much mre slwly than the rat r the pig (Widdwsn, 1971b). The present study indicates that in uter effects assciated with prductin f the runt pig result in pst-natal effects n the grwth and cmpsitin f sme prcine muscles. In uter runting in the rat did nt affect pst-natal muscle develpment. LITERATURE CITED Adams, P. H. 1971. Intra-uterine grwth retardatin in the pig. 2. Develpment f the skeletn. Bil. Nenate 19:341. Allen, C. E., D. C. Beitz, D. A. Cramer and R. G. Kauffman. 1976. Bilgy f fat in Meat Animals. Nrth Central Reginal Research Pub. N. 234. A.O.A.C. 1965. Official Methds f Analysis (loth Ed.). Assciatin f Official Analytical Chemists. Washingtn, DC. Butterfield, R. M. and R. T. Berg. 1966. A classificatin f bvine muscles based n their relative grwth patterns. Res. Vet. Sei. 7:326. Chiakupas, J. J. and J. E. Pauly. 1965. A study f pstnatal grwth f skeletal muscle in the rat. Anat. Rec. 152:55. Cper, J. E. 1975. The use f the pig as an animal mdel t study prblems assciated with lw birthweight. Lab. Anim. 9: 329. Davies, A. S. 1974. A cmparisn f tissue develpment in Pietrain and Large White pigs frm birth t 64 kg liveweight. 2. Grwth changes in muscle distributin. Anim. Prd. 19: 377. EIsn, C. E., W. A. Fuller, E. A. Kline and L. N. Hazel. 1963. Effect f age n the grwth f prcine muscle. J. Anim. Sci. 22:946. England, D. C. 1974. Husbandry cmpnents in prenatal and perinatal develpment in swine. J. Anim. Sci. 38:1045. Hegarty, P. V. J. and R. T. Naude. 1970. The accuracy f measurement f individual skeletal muscle fibers separated by a rapid technique. Lab. Pract. 19:161. Hegarty, P. V. J., L. C. Gundlach and C. E. Allen. 1973. Cmparative grwth f prcine skeletal muscle using an indirect predictin f muscle fiber number. Grwth 37: 333. Hegarty, P. V. J. and P. C. Ahn. 1977. Significance f bdy weight f three-week-ld rats in prtein efficiency rati (PER) measurements. J. Fd Sci. 42:255. Inkuchi, S., H. Ishikawa, S. iwamt and T. Kimura. 1975. Age-related changes in the histlgical cmpsitin f the rectus abdminis f the adult human. Human Bil. 47:231. Lee, Y. B. and Kauffman, R. G. 1974. Cellular and enzymatic changes with animal grwth in prcine intramuscular adipse tissue. J. Anim. Sci. 38: 532. McLaren, A. 1965. Genetic and envirnmental effects f fetal and placental grwth in mice. J. Reprd. Fertil. 9: 79. McCance, R. A. and E. M. Widdwsn. 1974. The determinants f grwth and frm. Prc. R. Sc. Lnd. (Sect. B) 185:1. Perry, J. S. and J. G. Rwell. 1969. Variatins in fetal weight and vascular supply alng the uterine hrn f the pig. J. Reprd. Fertil. 19:527. Rseahn, P. D. and H. S. N. Greene. 1936. The influence f intrauterine factrs n the fetal weight f rabbits. J. Exp. Med. 63:901. Rwe, R. W. D. 1968. Effect f lw nutritin n size f striated muscle fibers in the muse. J. Exp. Zl. 167:353. Staun, H. 1963. Varius factrs affecting number and size f muscle fibers in the pig. Acta. Agri. Scand. 13:293. Steel, R. G. D. and J. H. Trrie. 1960. Principles and Prcedures f Statistics. McGraw-Hill Bk C., New Yrk. Steinhaus, A. H. 1933. Chrnic effects f exercise. Physil. Revs. 13:103. Stickland, N. C. and G. Gldspink. 1973. A pssible indicatr muscle fr the fiber cntent and grwth characteristics f prcine muscle. Anita. Prd. 16:135. Sticldand, N. C., E. M. Widdwsn and G. Gldspink. 1975. Effects f severe energy and prtein differences n the fibers and nuclei in skeletal muscle f pigs. Brit. J. Nutr. 34:421. Swatland, H. J. 1976. Effect f grwth and plane f nutritin n apparent muscle fiber numbers in the pig. Grwth 40: 285. Widdwsn, E. M. 1970. Harmny f grwth. Lancet i:901. Widdwsn, E. M. 1971a. lntra-uterine grwth retardatin in the pig. 1. Organ size and cellular develpment f birth and after grwth t maturity. Bil. Nenate 19: 329. Widdwsn, E. M. 1971b. Fd intake and grwth in the newly-brn. Prc. Nutr. Sc. 30:127. Widdwsn, E. M. 1977. Undemutritin and retarded grwth befre and after birth. Nutr. Metabl. 21:76. Wiggleswrth, J. S. 1964. Experimental grwth retardatin in the fetal rat. J. Pathl. Bacteril. 88:1.