Institut National de la Recherche Agronomique, 78350 Jouyen-Josas and Rennes-St Gilles, 35590 l'Hennitage, France ABSTRACT

Little information is available on the effects of growth hormone (GH) and growth hormone-releasing factor (GRF and GHRH) treatment on bone metabolism in pigs. Thus, tibial bending moments and ash contents were studied in 12, 6-wk-old pigs weighing 13 f .2 kg. Six pigs (GRF group) were injected S.C. twice daily with 75 pg GRF (hGRF [1-29]NH2)/kg BW for 52 d and six remained untreated (control group, C). Average daily gain was slightly (5%; P e .lo) increased in treated pigs. At slaughter, plasma measurements related to calcium homeostasis, such as concentrations of Ca, inorganic P, and vitamin D metabolites (25-OH and 1,25-(0H)2 vitamin D3), were not changed by GRF injection. At slaughter, plasma GH levels were 3.3 times greater in treated (11.3 rt 3 ng/ml) than in untreated pigs (3.4 f .5 ng/ml, P < .02), whereas those of insulin-like growth factor I were increased by approximately 38%. No difference was observed between the two groups at slaughter in tibial weight, density, bending moment, ash relative to bone volume (29 f 1 vs 30 f 2 g/lW cm3, GRF vs C), total ash content, or ash relative to dry matter in cortical or medullary bone. Our GRF treatment did not affect bone and mineral metabolism in young, growing pigs. Key Words: Pigs, GRF, Bones, Mineralization, Vitamin D J. Anim. Sci. 1991. 69:1454-1460


Bone is a target organ for growth hormone (GH; Isaksson et al., 1987). Most of the effects of GH on bone metabolism have been studied in vitro. Very few data are available conceming in vivo effects of hormonally stimulated growth on pig bones. Most suggest that pGH treatment has little impact on bone ash and Ca contents (Chung et al., 1985; Goff et al., 1988; Capema et al., 1989). Recently a pGH

'We wish to acknowledge B. Cayron, C. Colin, P. Camus, F. Giovanni and J. P. Oudin for technical

assistance. 2Laboratoire de Nuhition et S&uritd Alimentaire, Jou -en Josas. %;tion de Recherches Porcines, Rennes-St. GUS. %nit6 de Recherches sur I'EndoCrinologie du Placenta, Labratoire de Biologie Cellulaire, Jouy-en-Josas. Received May 7, 1990. Accepted Septemk 25. 1990.

preliminary report (Komegay and Wood, 1989) described heavier and larger bones with unchanged mineral content or bone strength in finishing pigs, whereas heavier, but also stronger, bones with unchanged density have been reported for growing pigs (Prunier et al., 1990) treated with pGH over 8 w k Human growth hormone-releasing factor (GRF) is a potent stimulator of somatotropin (GH) release in pigs (Etherton et al., 1986), but the effects of GRF on bones have not yet been investigated. The hGRF (1-29)NH2 fragment has the same first 29 amino acid sequence as the porcine GRF ( 1 4 l ) N H ~molecule (Bohlen et al., 1983) with full biological activity on GH release in vitro (Spiess et al., 1982) and the same potency in vivo (Della-Fera et al., 1986; Petitclerc et al., 1987). We used hGRF (1-29)NH2 to stimulate endogenous GH secretion in growing pigs and to study the consequences of a 7-wk treatment on several


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A. PointillartZ, M. Bonneau3 and G. Kann4


bone criteria and associated concentrations of minerals and vitamin D metabolites.

Animals and Treatment. Twelve 6-wk-old male Large White pigs (13 kg BW) were divided into two groups of six each. One grou (GRF) was treated with hGRF (1-29)NHz , whereas the control group (C) was treated with vehicle for 52 d by S.C. injection in the retroauricular area of the neck These animals were similar in age to the pigs that we used to test bone responses to jGH treatments (Prunier et al., 1990). Pigs were fed a commercial diet (based on cereals and soybean meal) formulated to contain 20% protein (1.1% lysine), 1.1% Ca, .7% total P, and 2,000 IU vitamin D&g. They were housed individually. Pigs were weighed every week and feed intake was adjusted every 3 to 4 d on the basis of average daily consumption (semi-ad libitum feeding). Animals were pair-fed to obtain similar intakes in Ca, P, and vitamin D throughout the experiment. Mean intake was 1.6 f .01 kg/d. Pigs were injected twice each day between O900 and 1O00, and between 1600 and 1700 with either 75 pg GRF/kg BW or vehicle. This dose was selected based on work by Etherton et al. (1986). These authors observed an effect on area under the GH curve of 100 pgikg BW daily of intramuscularly injected hpGRF but no change in growth rate. Thus, we increased the daily dosage to 150 pg/kg BW divided into two injections to yield a more sustained effect. Syringes containing GRF were prepared weekly after weighing the pigs and were stored at -20'C before use (stock solution was 1 mg GRF/ml; GRF was dissolved in sterile physiological saline). Plasma Analysis. Growth hormone responses to GRF injections were analyzed in samples taken four times (on d 1, 3, 43, and 50) during the experiment; blood was obtained


%atch SR95752, generously donated by SANOFI (courtesy of F. Deletang), Montpellier, France. 6vcB bioproducts, BNSS~S,Belgium. 7A gift from the National Hormone and Pituitary Program (Univenity of Maryland School of Medicine) and the National institute of Diabetes and digestive and Kidney Diseases. 'A slft from L.Underwood and J. J. Van Wyk, North

Carolina. %atch 7 4 2 4 , a gift from B. D. Burleigh, Life Sciences Division, Pitmau-Moore, Northbrook, IL.

from the anterior vena cava over 3 h following S.C. administration of 75 pg of GRF. Blood samples were collected at 0, 15, 30, 45, 60, 120, and 180 min postinjection. Plasma insulin-like growth factor I (IGF-I) was determined at three periods over the last 10 d of the experiment. At slaughter, the following plasma concentrations were analyzed: Ca, inorganic P, 25 hydroxy (25-OHD3) and 1,25 dihydroxy (1,25-(O&D3) vitamin D3 (Pointillart et al., 1987), GH, and IGF-I. Plasma pGH concentration was determined using a specific homologous double antibody radioimmunoassay. Antiserum6 was used at a final dilution of 1:30,000. USDA-pGH-1-1 (AFp-G400)7 was used for iodination and standard curve. Sensitivity was 1 ng/ml. Crossreactions with pPRL, pLH, and pFSH were less than .4%. Coefficients of variation for plasma samples containing 3.8, 12, and 25 ng/ ml were as follows: intra-assay, 9.2, 5.9, and 8.6%, respectively; interassay, 9.4, 12.7, and 14.7%, respectively. For IGF-I assay, binding protein was removed from plasma samples by acid-ethanol extraction described by Daughaday et al. (1980). Measurement of recovery revealed some variation among samples. Recovery of IGF-I was determined by adding labeled IGF-I to plasma before extraction. Recovery percentages varied from 75 to 99%. Double antibody radioimmunoassay was conducted according to recommendations of the National Hormone and Pituitary program (NIDDK, Baltimore, h4D) with the antiserum UBK 4878. Recombinant N-Met IGF-I9 was used a standard preparation and as source of radioiodinated tracer. This procedure allowed detection of 6.25 pg IGF-Utube; the intra-assay CV was 8%. All IGF-I determinations for this experiment were run in the same assay. Bones. At slaughter, the tibia was excised from the left hind leg. Ash contents were measured on the whole tibia, the midshaft, and the proximal and distal epiphyses of the tibia. Thus, epiphyses were taken to represent spongy and diaphyses were taken to represent cortical samples. This tibia was used previously to measure bone bending moment, density and ash relative to bone volume. All these bone measurements have been described previously (pointillart et al., 1987). Statistical Analysis. Student's t-test was used to compare the means from C and GRF groups.

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Materlals and Methods





Initial wt, kg

12.6 50.2 725 2.23

g Feed/aainratio

GRF .3 f .7 f 16 f .05


12.6 52.2 762 2.09

f .2 f .3* f gt f .02*

aSix pigs/treatment. tP < .lo.




10.3 8.7 28.3 87

P, mgfdl 25-OH D3,ng/mt 1.2540HhD2, ~ a / m l


f .2 10.2 f .3 f .4 8.1 f .2 f .6 27.9 rt .9 f 33 78 f 24

'Means f SEM (n = 6). There was no significant difference between control and GRF-treated groups.

*P< .05.


Administration of GRF increased (P < .05) feed efficiency and final weight by 6 and 4%, respectively; overall average daily gain (ADG) tended (P c .lo) to increase (approximately 5%; Table 1). Average daily gain was increased significantly only from d 25 to slaughter in the treated pigs (836 f 19 vs 746 f 20 g/d, GRF vs C, P < .05); from the beginning to d 25, ADG was equal for both groups (683 vs 689 g/d f 12 for GRF vs C). Treatment with GRF did not alter any plasma minerals or vitamin D metabolites measured (Table 2). Administration of GRF increased plasma GH concentration, as shown by individual values collected in four pigs (Figure l), by the average response curve (Figure 2) over a 3-h postinjection period, and by slaughter values in treated pigs (Table 3). Maximal pGH release was observed 15 min after the S.C. GRF injection (47 f 3 ng/ml, n = 4), and the GH response (P < .Ol) remained for 90 min (10 f 2.3 ndml); the basal value in the control pigs was 3.9 (f .3) n g / d (overall mean, n = 28) and did not change between the beginning (5 f 1 ng/ml, n = 6) and the end (3.6 f .2 ng/ml, n = 14) of the experiment. In GRF-treated pigs, plasma levels of IGF-I (Tables 3 and 4) were increased (P < .10 to .01) by 25 to 77%; the average increase from overall means during the trial was 51%. At slaughter, GRF-treated pigs had 38% greater (P < .lo) IGF-I than control pigs. There was no effect of the GRF treatment on any of the bone parameters analyzed (Table 5). The mineral content of tibias from GRFtreated pigs as well as tibial breaking strength as reflected by bending moment value were unchanged.


Administration of GRF over 7 wk slightly stimulated growth performance without changing bone and mineral metabolism. The unchanged total bone ash, ash relative to bone dry matter, and ash to bone volume ratio also indicate that bone mineralization, at tissue or at organ levels, was not affected. These are the first detailed results conceming the effects of GRF on bone metabolism. The data described in literature essentially concern the effects of GH on carcass ash percentage, which is slightly increased in GH-treated pigs (Steele et al., 1987; Goff et al., 1988; Capema et al., 1989). However, at least in growing pigs, ash percentage relative to bone dry matter or bone density generally is unchanged (Goff et al., 1988; Capema et al., 1989; Prunier et al., 1990). Thus, an increased ash percentage in the empty body of pigs might result from an increase in carcass bone percentage alone (i.e., an overall increase in bone weights). Indeed, heavier bone in finishing (Komegay and Wood,1989) and growing pigs (prunier et al., 1990) treated with pGH and some heavier bones in finishing pigs treated with GRF (Pommier et al., 1988) have been observed. However, these results are not completely conclusive because heavier and stronger bones (+15% compared with control values) also have been described in growing pigs treated with pGH over 8 wk without any significant change in growth performance or carcass bone percentage (Prunier et al., 1990). Moreover, Chung et al. (1985) did not observe any alteration in femur weight or length in pGHtreated growing pigs exhibiting a growth response. Campbell et al. (1988) and Goff et al. (1988) have suggested that changes in the rate

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Item GH, ngiml IGF-I, n g h l


3.4 f 5 374 f 32


GRF 11.3 f 2.9** 515 f 62t

%ix pigs/treatment. Pigs were slaughtered 60 to 160 or saline solutions. tP < .lo. **P < .u2.

min after the last injection of GRF

at which ash was deposited were associated with an accelerated skeletal development in growth-stimulated pigs. In this situation, heavier and stronger bones would have been expected. Therefore, the unchanged bone weight and strength in the GRF-treated pigs might be related to our changes in growth performance being too small (5 to 6%) to involve bone mineralization rate when contrasted with the 16 to 26% gain responses noted in pGH-treated pigs by Campbell et al. (1988) and Goff et al. (1988). Some detrimental effects on bone such as osteochondrosis or decreased ash percentage, especially in finishing pigs treated with pGH, have been noted (Bryan et al., 1987; Evock et al., 1988; Komegay and Wood, 1989); such effects were not observed in our GRF-treated pigs. Administration of GRF has been reported to have less effect on pig performance than on pGH itself (Etherton et al., 1986). In fact, the treated pigs exhibited a higher GH response to S.C. GRF administration (75 p&g BW) than those of Etherton et al. (1986), which were given a higher i.m. or i.v. dose of GRF (100 pgkg BW) and did not exhibit a growthperformance response. Our GRF-treated pigs exhibited an elevated plasma concentration of GH lasting at least 3 Wd. if we assumed that GH release described in Figures 1 and 2 can be considered as representative of the whole experiment. Nevertheless, elevations are more prolonged with pGH injections. For instance, this elevation of plasma GH was maintained for 4 to 5 h in pigs administered 22 to 44 pgekg BW-l-d-l (Chung et al., 1985). This also might explain the smaller effect of GRF than GH on growth rate and bone parameters. In a previous study, pigs treated from 25 to 55 kg live weight with 100 pg pGH/kg daily

fig P U P



Control GRF Sigmficance

478 f 32 434 f 12 434 & 17 595 k 55 767 f 28 655 f 32 P < .IO P < .01 P < .001

"43 d and 50 d: means (fSEM) of pooled samples collected during GH-release studies (control: n = 1 X 8; GRF: n = 2 x 8). bMleans of all values collected/group during the experiment including slaughter values.

had circulating concentrations of 1,25-(0H)2 vitamin D 41% higher and plasma 25-OH vitamin D 21% lower than control pigs (Goff et al., 1988). These changes were absent in our pigs both at slaughter and 10 d before (overall mean for 1,25-(OH)2D3 on d 43 and d 5 0 74 k 9 vs 68 & 4 pg/ml, C (n = 11) vs GRF (n = 14), P e .lo). There was no need for an increased rate of vitamin D metabolism because increased mineralization rate was absent in our pigs, in contrast to those of Goff et al. (1988). Effects of GRF treatment on IGF-I plasma levels in pigs have not yet been fully described, although levels have been reported to increase in pGH-treated pigs (Chung et al.,



110 f Fresh wt, g Apparentdensity,g/cm3 1.23 f Bendingmoment, Nxm 37.4 f Totill ash, g 27.9 f Ash,% dry bonea Whole tibia 48.3 f Diaphyses 57.8 f Epiphyses 44.7 f Ash content relative to bone volumeb, g/100 0 3 30.2 f

GRF 3 .005

1.4 1

115 f 2 1.22 f .01 35.9 f 2.1 27.6 f .7


49.0 f 1.1

.6 .7

58.5 f 1.4 45.0 f 1


29.2 f .9

aExpressed on a marrow-free dry bone basis. Diaphysis: whole diaphysis; epiphyses: mean of both proximal and distal epiphyses. bI'otal a ~ content h relative to apparent total bone volume. No s i w c a n t difference between the two groups of six pigs each.

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serum IGF-I concentration was elevated linearly in response to dose of pGH, supporting the well-established role of somatomedins in stimulating growth in mammals. Plasma concentrations of IGF-I in control and GRFtreated pigs are in agreement with the values given by Etherton et al. (1987). Consequently, the absence of bone changes in the GRFtreated group cannot be attributed to a lack of

PIG 67.C

h -Q-


* PIG 86.GRF


* PIG 39.GRF










O+ tO



























TIME POST-INJECTION (minutes) Figure 1. Plasma growth hormone (GH)protile following S.C. injection of saline (control) or growth hormonereleasing factor (GRF,75 -). Individual responses in two control and four treated pigs sampled on d 43 (T4gure la) and d 50 (Figure lb).

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1985; Etherton et al., 1987; Evock et al., 1988; Walton and Etherton, 1989). The greater plasma IGF-I values observed in the GRFtreated group seems to be a permanent feature because it was present at time 0 in the pigs that were chronically sampled (on d 43 and 50, data not shown) as well as for the overall mean value of sampling over the last 10 d and at slaughter. Etherton et al. (1987) also found that





















TIME POST-INJECTION (minutes) Figure 2. Plasma growth hormone (GH)profile following S.C. injection of saline (control) or growth hormonereleasing factor (GFW,75 pgkg). Average response c w e s from all sampled pigs including valFes collected on d 1 and d 3 (individual curves not shown; means f SEM). Number of samples is given in parentheses. P < .01, GRP vs control at a given time.

response of IGF-I; IGF-I is considered a major mediator of hormonal action on skeletal cells (Canalis et al., 1989). In fact, bone metabolism probably was not stimulated enough by the levels of GEW used in this experiment to elicit a detectable change in vitamin D metabolites or the other bone-related parameters mentioned. lrnplicatlons

In vivo relationships between growth hormone and bone metabolism need further study because bone development could limit extensive use of porcine growth hormone in pig husbandry. Growth hormone-releasing factor, a potent growth hormone stimulator, had no detrimental effect on bones in growing pigs treated during several weeks at a high dose. In this study, effects of growth hormone-releasing factor on growth performance were small, and no effects on bone parameters were detected. Literature Cited

Bryan,K. A., D. E. Carbaugh, A. M. Clark, D. R. Hagen and J. M. Hammond. 1987. Effect of porcine growth hormone @GH)on growth and carcass compositionof gilts. J. Anim. Sci. 65(Suppl. 1):244 (Abstr.). Biihlen, P., P. Esch, P.Brazeau. N. Ling and R. Guillemin. 1983. Isolation and characterization of the porcine hypothalamic growth hormonsreleasing factor. Biochem. Biophys. Res. Commun. 116:726.

Campbell,R. G., N. C. Steele, T.J. Capema, I. P.McMurtry, M. B.Solomonand A. D. Mitchell. 1988.Interrelationships between energy intake and endogenous porcine growth hormone administration on the performance, body compositionand protein and energy metabolism of growing pigs weighing 25 to 55 kilograms live weight. J. Anim. Sci. 66:1643. Canalis, E., T.L.McCarthy and M.Centrella 1989. Growth factors and the skeletal system. J. Endocrinol. Invest. 12577. Capema, T. J., R G. Campbell and N. C. Steele. 1989. Interrelationships of exogenous porcine growth hormone administration and feed intake level affecting various tissue levels of iron, copper, zinc and bone calcium of growing pigs. J. Anim. Sci. 67:654. Chung, C. S., T. D. Etherton and J. P. Wiggins. 1985. Stimulation of swine growth by porcine growth hormone. J. Anim. Sci. 60:118. Daughaday, W. H.,I. K. Mariz and S. L. Blethew. 1980. Inhibitor of access of bound somatomedin to membrane receptor and immuno-bending sites: a comparison of radioreceptor-and radioimmunoassay of somatomedin in native and acid-ethanol extracted serum. J. Clin. Endocrinol. & Metab. 51:781. Della-Fera, M. A., F. C. Buonomo and C. A. Baile. 1986. Growth hormone releasing factors and secretion of growth hormone in sheep, calves and pigs. Domest. Anim. Endocrinol. 3165. Etherton,T. D., J. P.Wiggins, C. S. Chung, C. M.Evock, J. F. Rebhun and P. E. Walton. 1986. Stimulation of pig growth pexformance be porcine growth hormone and growth hormone-releasing factor. J. Anim. Sci. 63: 1389. Etherton,T. D., J. P.Wiggins,C. M. Evock, C. S.Chung,J. P. Rebhu, P. E. Walton and N. C. Steele. 1987. Stimulation of pig growth performance by porcine growth hormone: determination of the dose-response relationship. J. Anim. Sci. M433. Evock, C. M., T. D. Etherton, C. S. Chung and R. E. Ivy.

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POINTILLART ET AL. triticale or corn J. Nutr. 1 1 7 9 7 . Pommier, S., P. Dubred, P. Gaudreau, G. Pelletier, C. Fanner,D. Petitclerc, H. Lapierre, Y. Couture and T. Mowles. 1988. Effect of a potent analog of human growth hormonereleasing factor (hGRF) on the carcass composition of market pigs. J. Anim Sci. 66(Suppl. 1):295 (Abstr.). Runier, A., A. Pointillart and M. Bomeau. 1990. Influence de l'injection de somatotropineporcine (F'ST) sur les d6veloppements coxporel, osseux et sexuel de jeunes femelles de race Meishan. Journ. Rech. Porcine Fr. 22: 77. Steele, N. C., R G. Campbell and T. J. Caperna. 1987. Update of porcine growth hormone researck practical and biological implications. In: Proc. Cornell Nutr. Cod. of Feed Manufacturers, Oct. 2628, 1987. pp 15-22. Syracuse, NY. Spiess. J., J. Rivier, M. "homer and W. Wale. 1982. Sequence analysis of a growth hormone-releasing factor from a human pancreatic islet tumor. Biochemistry 215037. Walton, P. E. and T. D. Ethexton. 1989. Effects of porcine growth hormone and iosulinelike growth factor-I (IGF-I) on immunoreactive IGPbinding protein concentration in pigs. J. Endocnno ' 1. 120153.

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1988. Pituitary porcine growth hormone @GH)and a recombinant pGH analog stimulate pig growth performance in a similar manner. J. Anim. Sci. 66:1928. Goff, J. P., T. J. Capema, R. G. Campbell and N.C. Steele. 1988. Interaction of porcine growth hormone @GH) administrationand dietary energy intake on circulating vitamin D metabolite concentrations in growing pigs. J. Anim. Sci. 66(Suppl. 1):291 (Abstr.). Isalrsson,O.G.P., A. Lindahl A. Nilson and J. Isgaard. 1987. Mechanism of the stimulatory effect of growth hormone on longitudinalbone growth. Endm. Rev. 8: 426. Komegay, E. T. and C. M. Wood. 1989. Dimensional and strength characteristics and mineral composition of metacarpal and metatarsal bones from control and porcine somatotropininjected finish& pigs. J. Anim. Sci. 67(Suppl. 2):39 (Abstr.). Petitclerc, D., G. Pelletier, H. Lapierre, P. Gaudreau, Y. Couture, P.Dubreuil, J. MorissetandP. Brazeau. 1987. Dose effect of two synthetic human growth homonereleasing factors on growth hormone release in heifers and piglets. J. Anim. Sci. 6 5 9 6 . Pointjkut, A., A. Fourdin and N. Fontaine. 1987. Importance of cereal phytase activity for phytate phosphom utilization by growing pigs fed diets containing

Effect of human growth hormone-releasing factor on bone and mineral metabolism in growing pigs.

Little information is available on the effects of growth hormone (GH) and growth hormone-releasing factor (GRF and GHRH) treatment on bone metabolism ...
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