Growth, feed efficiency and body composition of transgenic mice expressing a sheep metallothionein 1a-sheep growth hormone fusion gene

Livestock Productwn Science, 31 (1992) 335-350 Elsevier Science Publishers B.V., Amsterdam

335

Growth, feed efficiency and body composition of transgenic mice expressing a sheep metallothionein 1z-sheep growth hormone fusion gene D a n i e l P o m p ' , Colin D. Nar, c a r r o w b, K e v i n A. W a r d b a n d J a m e s D. M u r r a y a "Department of Animal,Science; Universityof California, Davis, CA, US.x! bC.S.I.R.O., Division of Animal Production, N.S. W., Australia (Accepted 28 October 1991 )

ABSTRACT Pomp, D., Nancarrow, C,D., Ward, K.A. and Murray, J.D., 1992. Growth, feed efficiency and body composition of transgenic mice expressing a sheep metallothionein l a-sheep growth hormone fusion gene. Livest. Pred. Sol., 31: 33~-350. Growth, feed efficiency and body composition were studied in male mice possessing a sheep metalIothionein la-sheep growth hormone transgene (oMTla-oGH). The transgane was activated at weaning (2 ! d ) by 25 mM zinc sulfate provided in drinking water. Body weight and feed intake were measured at weekly intervals until 70 d. Transgenic males gained 53.3% more weight than controls during the test period (P<0.001) while consuming only 10.9% more feed (P<0.001). When adjusted for metabolic body weight, feed intake was similar for the two genotypes. Transgenics had more epididymal and subcutaneous fat at 70 d (P<0.001), but less fat at both depots (P<0.05) when adjusted to a commc n body weight. Lean tissue, estimated by trimmed hind carcass weight, was greater in transgenics when compared at a common age (P<0.01) but lower when compared at a common bedy weight (P< 0.001 ). If analyzed for a common weight gain, transgenics exhibit much higher efficiency of lean tissue production, due to a much lower intake required to produce lean. Visceral organs were heavier in transgenics than in controls, but only laver and spleen e.~hibited disproporlional enlargement relative to overall body size increases (P< 0.001 ). These results demonstrate that mice possessing an oMTia-oGH transgene exhibit increased efficiency of growth and of iean tissue production, especially whe,l considered over a standard weight gain period Keywords: transgenic mice; growth hormone

INTRODUCTION G e n e t i c i m p r o v e m e n t o f g r o w t h is a p r i m a r y goal in l i v e s t o c k b r e e d i n g . S i g n i f i c a n t p r o g r e s s has b e e n a c h i e v e d u s i n g t r a d i t i o n a l m e t h o d s s u c h as seCorrespondence to: D. Pomp, Department of Animal Science, Oklahoma State University, Stillwater, OK 74078-0425, USA.

0301-6226/92/$05.00 © 1992 Elsevier Science Publishers B.V. All r i o t s reserved.

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lection and crossbreeding. A new technique for improving growth characteristics utilizes transgenics. It is clear that body size can be significantly increased by integrating a growth hormone transgene into mice (Palmiter et al., 1982, 1983; Brem et al., 1989). However, increased growth p e r se is not in itself a desirable endpoint in animal agriculture, but rather increased efficiency of growth and particularly of lean tissue. A number of transgenic mouse lines have been established that carry a variety of growth promoting genes, including insulin.like growth factor I (Mathews et el., 1988), growth hormone releasing hormone (Hammer et al., 1985b) and various growth hormone coding regions (Palmiter et al., 1982, 1983; Brem et al., 1989; Orian et al., 1989). These fusion constructs have generally used the mouse metallothionein gene promoter (MT) to drive expression of the transgene. Transgenic mice utilizing this promoter are normally switched on shortly after birth, exposing the neonate to high levels of gene product throughout life. In all cases expression of the transgcae was correlated with significantly increased growth. Shanahan et al. (1989) described a growth hormone transgene in mice utilizing the sheep metallothionein I a (oMT I a) promoter that did not increase circulating levels of growth hormone unless activated by supplemental zinc. Induction of expression of the oMT I a--oGH transgene at weaning (21 d) resulted in increased growth to approximately 1.4 times the body size of nontransgenic controls. Upon removal of supplemental zinc, circulating growth hormone returned to basal levels within 24 h. While there are many reports of enhanced growth in mice possessing various growth hormone and related transgenes, there is a paucity of information regarding e f f i c i c ~ of conversion of feed intake into weight gain. In addition, little is known regarding the composition of weight gain in these transgenic mice. Recent information concerning transgenic pigs has been limited yet very promising, in that pigs possessing a growth hormone transgene may grow faster and more efficiently, and are leaner than littermate controls (Pursel et al., 1989). The present study utilized mice carrying an inducible sheep metallothionein I a-sheep growth hormone fusion gene. The objective was to analyze the growth promoting effects of transgene expression from day 21 to day 70, in terms of efficiency of growth relative to feed intake and components of growth, including lean, fat and visceral tissues. MATERIALS A N D M E T H O D S

Originand husbandry ofmice Mice analyzed in thisstudy were produced by mating wild-tyl~ FI (C57B I] 6 × C B A ) females with M G I 0 I males proven robe heterozygous for a sheep metallothionein la-sheep growth hormone fusion gene (Shanahan et al.,

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1989). There was a within-litter expectation of 50% heterozygous MGI01 progeny, which in the presence of zinc would fully express the transgene due to completely dominant gene action. The remaining 50% would be homozygous wild-type and would act as contemporary controls. At birth, litters were standardized to postnatal fraternity sizes of 7-9 pups. Litter size was not highly variable and crossfostering was minimal. At day 10, pups were weighed, individually identified by toe-notching, and characterized for presence of the transgene by polymerase chain reaction amplification of unique transgene sequence directly from toe-notch lysate (Pomp et al,, 1991 ). Mice were weaned at 21 days with feed (Purina Laboratory Chow 5001 ) supplied ad libitum. All mice received 25 mM zinc sulfate in the drinking water throughout the study, which stimulates expression of the transgene from the metallothionein I a promoter in the transgenic animals. Males were placed two per cage within genotype (transgenic or control) and, when possible, within litter. Females were randomly placed 6-8 per cage. Temperature (21°C), humidity (55%) and light cycle (12-h light:12-h dark) were controlled.

Growth and feed intake measurements Females were weighed at weekly intervals from weaning to 63 days of age. These were the only measurements for females in this study. Males were weighed weekly from weaning to 70 days of age. Feed intake of males was measured weekly during the same period, based on total intake per cage. This method was adopted due to large variations in intake and growth caused by stress when mice are caged individually. This could especially be relevant for transgenic animals whose transgene is regulated by a metallothionein promoter, since stress may be a stimulating factor in metallothionein synthesis (Bremner and Beattie, 1990). Feed efficiency was calculated weekly as the ratio of growth to intake per cage. Body composition and organ weight measurements At 70 days of age males were killed by cervical dislocation, and the following tissues and organs were immediately dissected and weighed: right hindlimb subcutaneous fat pad (SF), right epididymal fat pad (EF), trimmed hind carcass (HC), right testis (T), liver (L), spleen (S), heart (H) and right kidney (K). The remaining carcass was frozen for future skeletal an,aysis. Epididymal fat pad weight (as a percentage ofbody weight) was used as an indicator of total body fat since these traits have previously been found to be highly correlated (Eisen and Leatherwood, 1981; Rogers and Webb, 1980). Trimmed hind carcass weight (as a percentage of body weight) was used as an indicator of lean tissue content (Bhuvanakumar et al., 1985; Eisen, 1987 ).

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Statistical analyses All data were analyzed by least-squares procedures (SAS, I a88 ). For weekly (21-63 days) body weights and growth rates, the statistical model included fixed effects of sex and genotype and their interaction. For all other traits, measured exclusively in males, the model included only the fixed effect of genotype. For weekly feed intake and feed efficiency data, analyses were based on the average measurement per cage, with each genotype represented by 21 cages (replicates) of two males each. Weekly feed intake data were analyzed as raw weights and after adjustment by analysis of covariance for average metabolic body weight [ (body weight) °'Ts] per cage, to help adjust for size factors and differences in maintenance requirements. Simple estimates of weekly feed efficiencies adjusted for maintenance feed intake requirements were obtained by dividing means for weekly growth by least-squares means for weekly feed intake after adjustment by analysis of covariance for metabolic body weight. Fo~"body composition and organ weight data, in addition to the base analysis for raw unadjusted weights, data were analyzed as a percentage of 70-d body weight (%SF, %EF, %HC, %T, %L, %S, %H, %K) and as natural logarithms (LSF, LEF, LHC, LT, LL, LS, LH, LK) adjusted by analysis of covariance for the natural logarithm of 70-d body weight (LBW). With the e,~ception of LL, there were no significant interactions between genotype and LBW for any body composition or organ traits. Body composition data were also analyzed as percentages of total postweaning feed intake (%SH. %EFI, %HCI ). RESULTS

A total of 194 mice from 23 litters was produced for use in this study. Of these, 50.5% (98) were identified at 10 days of age as carriers of the oMTIaoGH transgene, which is not significantly different from the expected 50% ratio. Numbers of mice which contributed to the data were: 56 transgenic females, 54 control females, 42 transgenic males and 42 control males.

Body weigt:t and growth rate Body weights at 10 days and weekly weights from 21 to 70 days arc presented lrl "l'able 1. There were no diffelences (P> 0.05 ) among sexes or genotypes at 10 days or at weaning. Subsequently, males were heavier than females (P< 0.001 ) and ~ransgenic mice (pooled across sexes) were heavier than controls (P< 0.001 ). In addition, there were significant sex x genotype interaction effects on body weight from 28 to 63 days of age. This interaction was primarily due to female transgenic mice being relatively heavier than their respective controls than were male transgenic mice. At maturity, female

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TABLE I

Least-square~ means ( ± s,e ) of body weight (g) of male and female mice expressing ( + ) or not expressing ( - ) a sheep metaUothionein la-sheep growth hormone fusion gene Sex

Genotype

Age (days) 10

21

28

35

42

49

56

63

70 34.63 26.31 0.35

Male

+ s.e.

6.17 6.22 0.10 NS

! 1.04 10.91 0.17 NS

16.78 16.44 0.26 NS

21.81 20.61 0.28 "*

25.70 22.62 0.28 **"

29.05 23.94 0.31 *'*

31.23 24.86 0.34 "**

33.15 25.75 0.34 ***

Female

+ s.e.

6.25 6.24 0.09 NS

10.'8 10.69 0. i 5 NS

14.92 13.43 0.23 ***

19.55 15.81 0.25 ***

23.44 17£5 0.25 ***

26.17 18.30 0.27 ***

28.60 19.10 0.30 ***

29.'/5 19.47 0.30 ***

NS, means are not different ( P > 0.05 ). "*Me~,ns are different ( P < 0.01 ). " * ' M e a n s arc different (P < 0.001 ).

Genoty~Xsex interaction was significant ( P < 0 . 0 5 ) from 28 to 63 days,

transgenics were 53% heavier than controls (29.74 vs. 19.47 g at 63 days), while male transgenics were 32% heavier (34.62 vs. 26.31 g at 70 days). After 28 days, transgenic mice always gained more than controls (P< 0.001 ). Percent differences between transgenics and controls were 19.6, 47.4, 91.6, 244.3, 167.4, 142.9 and 162.5% for the periods 21-28, 28-35, 3542, 42-49, 49-56, 56-63 and 63-70 days, respectively. Males grew more than females from 21 to 28, 28 to 35, 42 to 49 and 56 to 63 days (P<0.001). Sex X genotype interaction effects were significant only at the eaJ'ly growth periods (21-28 and 28-35 days). This was again due to greater differences between genotypes in females than in males. Feed intake Weekly feed intakes from 21 to 70 days are presented iu Table 2. Feed intake increased with age. From 21 to 28 and from 28 to 35 days, intake was not different (P> 0.05) between transgenic and control males. In all subsequent periods, transgenic males ate more feed than controls (P< 0.001 ), the greatest differences occurring between 49 and 70 days. During the entire postweaning period (21-70 days), transgenic males consumed an average of 240.1 g while controls consumed an average of 216.5 g of feed (P<0.001). When adjusted for average metabolic body weight, there were no differences in feed intake between transgenic and control mice throughout the study (Table 2).

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TABLE 2 Least-squares weans ( :Ls,c,) of weekly feed intake (g) from 21 to 70 days of male mice expressing ( + ) or not expressing ( - ) a sheep metallothionein la-sheep growth hormone fusion gene, expressed as raw weightP (g) or afteradjustment by analysisof covariance 2 for average metabolic body weight Age (days) Genotype 21-28

28-35

R a w ~ Adj 2 R a w + s.c.

Adj

35--42

42--49

49-56

Raw

Adj

Raw

Adj

Raw

Adj

Raw

31.9 31.9 0.38 NS

36.0 31.8 0.35 ***

33.9 33.9 0.58 NS

38.1 32.9 0.33 ***

36.1 34.9 0.73 NS

38.8 36.8 32.7 34.7 0.31 0.72 *** NS

24.4 24.4 31.0 30.6 32.7 24.8 24,8 30.4 30.8 31.0 0,42 0.28 0.31 0.29 0,28 NS NS NS NS ***

56-63

63-70 Adir R a w

Adj

39.1 36,9 32.9 35,1 0.37 1.0 *** NS

SAverage feed intake in grams per cage, based on 21 cages of two mice each per genotype. 2Average feed mtake in grams per cage, after adjustment by analysis of covariancc for average metabolic body weight per cage for each weekly time period. NS, Means are not different (P> 0.05 ). ***Means are d,tYerent (P<0.001). TABLE 3 Least-squares means (+s.e.) of weeldy feed efficiencies (FE) from 21 to 70 days of male mice expressing ( + ) or not expressing ( - ) a sheep metallothionein la-sheep growth hormone fusion gene Age (days) Genotype

+ s.c.

21-28

28-35

35-42

FE j

Est 2

FE

Est

FE

24.5 24.5 1.4 NS

23.5 22.3

1 6 . 2 1 6 . 5 11.9 1 3 . 8 13.5 6.5 0.62 0.46 ** ***

42-49

Est

FE

12.2 9.3 6.3 4.1 0.39 ***

49-56

56-63

63-70

Est

FE

Est

FE

Est

FE

Est

9.9 3.9

5.7 2.8 0.36 ***

6.0 2.6

5.0 2.7 0.29 ***

5.2 2.6

3.8 1.7 0.34 ***

4.0 !.6

'Average feed efficiency [ (gain/intake) × 100 ] per cage based on 21 cages of 2 mice each per genotype. 2Simpl e estimate of feed efficiency, adjusted for maintenance requirements, calculated as the ratio of least-~uares means for weeldy growth and least-squares means for weekly feed intake following adjustment by analysis ofcovariance for metabolic body weight. NS, M,.an~ are not different (P> 0.05). **Mea is are different (P<0.01). ***Means are different (P< 0.001 ).

Feed efficiency Weekly feed efficiencies [ (gain/intake) X 100] from 21 to 70 days are presented in Table 3. Feed efficiency (FE) decreased with age. From 21 to 28 days, FE was the same for transgenic and control males. In all subsequent

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EFFICIENCY AND COMPOSITION OF TRANSGENIC MICE

periods, transgenic males had greater FE than controls, the greatest differences occurring from 35 to 42 ( 11.92 vs. 6.51 ) and from 42 to 49 (9.29 vs. 4.13) d~ys of age. Estimates of feed efficienc:: obtained after adjustment of feed *make for metabolic body weight slightly magnified the differences between transgenic and control males (Table 3).

Bodycomposition Body composition data were analyzed as raw weights, as percentages of body weight, as percentages oftotal feed intake and as adjusted natural logarithms. The latter analysis is appropriate when body components develop disproportionately relative to overall body weight, and also enables comparison ofgenotypes at a common body weight. Results are summarized in Table 4. Epididymal fat pad weight was greater in transgenic males than in controls when an."!~zcd as EF, %EF or %EFI (P < 0.01 ). However, the allometric analysis revealed that LEF was significantly lower in transgenics than in controls (?< 0.05). Subcutaneous fat pad weight followed the same pattern, with the exception that %SF was similar in the two genotypes (P> 0.05). Hind carcass weight was greater in transgenic males when considered as a raw weight (HC; P< 0.01 ), but was lower in transgenics when considered as %HC (P < 0.001 ), %HCI (P
÷ s.e.

Raw weight (g)

Adjusted weight'

% body weight 2

% feed intake ~

E l~

SF S

HC 6

EF

SF

HC

EF

SF

HC

EF

SF

HC

0.22 0.13 0.01

0.12 0.09 0.005

4.15 3.94 0.05

0.13 0.18 -

0.09 0.II -

3.74 4.34 -

0.62 0.48 0.03

0.35 0.33 0.01

12.0 15.0 0.I

0.13 0.06 0.01

0.05 0.04 0.002

l.g4 I 91 0.02

tin (fat pad or hind carcass weight) adjusted by analysis of covariance for In (70-day body weight), retransformed to grams. -'(Fat pad or hind carcass weight/70-day body weight) × ! 00. 3(Fat pad or hind carcass weight/21-70-day feed intake) x 100. 4Right epididymal fat pad. *Right hindlimb subcutaneous fat pad. 6Trimmed hind carcass. NS, means are not different (P> 0.05). *Means are different (P< 0.05 ). **Means are different (P
342

D. POMP ET At.

TABLE 5

Least-squares means ( _+s.e. ) of organ weights of 70-day-old male mice expressing ( + ) or not expressing ( - ) a sheep metallothionein [ a-sheep growth hormone fusion gene Genotype Raw weight (g) Ss

% body weight 2

Adjusted weighP

L3

K4

He

T7

L

K

S

H

+

2.9

0.33 0.13 0.19 0.I0

s.e.

1.5 0.25 0.06 0.16 0.09 1.9 0.29 0.07 0.18 0.09 5.7 0.96 0.21 0.05 0.01 0.01 0.01 0.002 . . . . . 0.09 0.01 0.01 *** *** *** *** *** NS *** NS NS *** NS ***

T

L

2.4 0.29 0.I0 0.17 0.09 8.5

K

S

H

T

0.95 0.37 0.56 0.28 0.60 0.33 0.01 0.01 *** ***

1In (organ weight) adjusted by analysis ofcovariance for In ( 70 day body weight ), retransformeO to grams. 2(organ weight/70 ,lay body weight)× I00. 'L!vcr. 4Right kidney.

SSpleen. ~Heart. 7Right testis. NS, means are not different ( P > 0.05 ). ***Means are differen! (P,-'0.001).

Organ weights Least-squares means from organ weight analyses are presented in Table 5. When considered as raw weights, all organs were significantly larger in transgenie males than in controls (P<0.01). The greatest differences were for spleen (0.13 vs. 0.06 g) and liver (2.94 vs. 1.5 g), while the smallest difference was for testis (0.1 vs. 0.09 g). As a percentage of body weight, only %L and %S were higher in transgenics than in controls (P<0.001). Transgenics actually had lower %H and %T than controls (P< 0.01 ), while %K was not different between the two genotypes. The allometric analysis revealed that liver and spleen weights were greater in transgenic males (P<0.001), while kidney, heart and testis weights were similar for transgenics and controls. DISCUSSION

A primary goal of transgenic manipulation of livestock is to provide animals which grow faster, produce more lean tissue and do not overcompensate for these changes with greatly increased maintenance and production costs. One of the first challenges which must be met is to determine which genes and accompanying regulatory elements will be most useful in attempting to achieve these goals. The present study has analyzed growth of mice (MGI01 ) which express a novel sheep metallothionein l a-sheep growth hormone fusion transgene (Shanahan et al., 1989 ). These transgenic mice grew faster and more efficiently than contemporary controls.

EFFICIENCY AND COMPOSITION (A: TRANSGENIC MICE

343

The increased growth rate of MG 101 mice confirms similar observations in other lines with the same transgene (Shanahan et al., 1989). Significant growth enhancement has also been observed in mice with other growth promoting transgenes (Palmiter et al., 1982, 1983; Hammer et al., 1985a,b; Morello et al., 1986; Mathews et al., 1988; Brem et al., 1989). It is increasingly evident that growth enhancement per s e can be achieved by a variety oftransgenic means in mice. Emerging data in livestock, especially pigs, indicates that this may hold true for other species as well. Treatment with exogenous porcine growth hormone has increased growth rates of pigs (Campbell et al., 1988, 1989; Evock et al., 1988). Production of pigs with growth hormone transgenes, however, has not always resulted in increased growth (Pmsel et al., 1990). Contradictory results may be in part due to transgenic × diet interaction effects, and significant growth enhancement was achieved in MT-bovine growth hormone transgenic pigs when this was taken into account (Pursel et al., 1989). In contrast, production of sheep with growth hormone transgenes has not resulted in increased growth (Rexroad et al., 1989; Nancarrow et al., 1991 ). Genotype × sex interaction effects on growth were an interesting ob~rvation in the present study. Transtenic females responded with greater relative growth increases than did transgenic males, especially during early postweaning periods. Campbell et al. (1989) found a similar interaction in pigs, whereby treatment with exogenous porcine growth hormone negated normal sex differences in growth performance and body composition. At present, however, enhanced g'owth in females due to transgenic manipulation has generally been accompanied by severe negative effects on reproduction in both mice (Hammer et al., 1985a; Bartke et al., 1988; Brem et al., 1989; Pomp, Nancarrow, Ward and Murray, in preparation ) and pigs (Pursel et al., 1990). Very little research has been carried out to ascertain the impetus for i~creased growth in transgenic animals. An animal which grows faster due mostly to increased appetite is not as valuable as one which grows faster as a result of increased efficiency of converting consumed food into growth. This is especially true for pigs, as approximately 60-70% of production costs are assc,ciated with feed costs. Transgenic mice in the present study did exhibit increased appetite relative to controls. However, this increase was greatest during the later stages of growth, when control mice essentially had ceased significant weight gain. During the first 4 weeks postweaning, which accounted for 76% and 84% of total postweaning growth for transgenics and controls, respectively, transgenics consumed only 5.2% more feed. When size differences and the resulting differences in maintenance requirements are accounted for in the statistical analysis the feed intake patterns of transgenic and control males become nearly identical. In terms of feed efficiency, transgenic males were much more efficient in

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D. POMP ET At.

converting intake to growth than were controls. During the first 4 weeks postweaning, transgenics converted 19% of feed consumed into growth, while controls converted only 13%. When feed intake is adjusted for size differences prior to calculation of feed efficiency, the differences between transgenics and controls become even greater. Similar results in terms of feed intake and feed efficiency were observed in female rats with growth hormone-secreting tumors (McCusker and Campion, 1986). There are inherent problems when comparing feed efficiency at fixed ages of two genotypes which differ significantly in growth rate. Bereskin and Steele (1985) reviewed a number of studies on feed efficiency in pigs, and among their conclusions was that efficiency should be measured from a starting age or weight to a standard final weight. Both transgenic and control males in the present study were weaned at the same age and at similar weights. While it is difficult to select a standard weight at which to conclude a test period for mice, transgenics had a mean weight of 25.7 g at 42 days while controls had a mean weight of 25.7 g at 63 days of age. To reach this weight, transgenics consumed less than half the feed consumed by controls. Thus, their efficiency of growth was over twice that achieved by controls to reach the same target weight. While this is more or less an arbitraril) chosen standard weight, other chosen weights would illustrate the same general picture, in that the transgenic males are more efficient at converting feed to gain to a weight-constant endpoint than are controls. Thus it would appear that over a fixed age range transgenic males exhibit slightly increased appetite, mainly due to increased maintenance requirements associated with a larger body size, while over a fixed weight range transgenics have greatly decreascd feed intake due to much more efficient conversion of feed into gain. Based on evidence in transgenic pigs, sheep and mice, as well as in rats with growth hormone-secreting tumors, transgenic mice in the present study would have been expected to have significantly more lean tissue at the expense of fat deposition. The increased levels of epididymal fat in transgenic males, both on a raw weight basis and as a percentage of body weight, are therefore surprising. Subcutaneous fat also increased on a raw weight basis, but not as a percentage of body weight. Comparing body parts as raw weights between groups of animals of different sizes does not present an accurate analysis of growth or size of that part relative to the whole body. A generally accepted alternative is to use the ratio of the body part to total body weight. While this does provide a more relative comparison, it still does not truly adjust for differences in body weight among different groups of animals (Huxley, 1932; Gould, 1966). Ideally, covariance analysis enables comparison between groups that is equivalent to adjusting groups to a common body weight. Additionally, log transformation prior to covariance adjustment provides an allometric analysis, which is appropriate when body parts do not increase in linear relationship with overall body size (Brody, 194:5). When the present fat data

EFFICIENCY AND COMPOSITION OF TRANSGENIC MICE

345

were adjusted for body weight, transgenic mice sht,,~ed significantly lower levels ofepididymal and subcutaneous fat. When compared at a common age, transgenic mice seemingl~ possess more fat than controls, but when compared at a common body weight they have less fat. These results are similar to those obtained for transgenic female ~nice from a related subline (Searle et al., 1992). Interestingly, they are also very similar to results obtained for mice which possess the 'high growth' major ggne (Calvert et al., 1985 ), which is characterized by low levels ofgrowth hormone and high levels of insulin-like growth factor I (IGF-I) (Medrano et al., 1991 ). Brem et al. (1989) reported a general finding of lower mesenteric fat levels in human growth hormone (hGH) transgenic mice. In contrast, Mathew~ et al. (1988) did not detect differences in weights ofthree fat pads in mice ~ith a human IGF-I transgene. Transgenic sheep possessing the same oMT I a--oGH fusion gene as used here in mice exhibited very little perirenal and mesenteric fat, and the deposition of backfat around the twelfth rib was greatly reduced (Nancarrow et al., 1991 ). Thus a very interesting species difference appears to exist, whereby oMTIa-oGH mice respond with escalated growth and little change in fat, while oMTIa-oGH sheep have dramatically leaner carcasses without an increase in growth. The present results could be a consequence of oGH being ineffective in inhibiting lipogenesis in mice, while stimulating growth via increased IGF-I synthesis. Data on plasma IGF-I levels as well as GH receptors in adipose tissue in these transgenic mice would be helpful in investigating this hypothesis. This could also help explain the species difference between mice and sheep, since lipogenesis in sheep should be depressed by an elevation in level of the native GH. Transgenic males in the present study had lower hind carcass weights and lower proportion of lean tissue than controls when compared at both a common age and a common body weight. This is in contrast to data from transgenic pigs, which show dramatic changes in the partitioning between fat and other carcass components, iuc!uding muscle (Steele and Pursel, 1990; Wieghart et al., 1990). Limited data exist from other growth hormone transgenic lines of mice. There were no differences in whole-body protein levels at various postweaning stages in oGH-transgenic females relative to controls (Searle et al., 1992), and similar results were reported by McCusker and Campion (1986) for female rats with growth hormone-secreting tumors. Alternatively, data from IGF-I transgenic mice (Mathews et al., 1988) suggest that muscle may be a primary contributor to the increased body weight observed in those mice. With the increasing emphasis being placed on lean tissue production in current meat-producing agricultural systems, it is useful to consider alternative methods for evaluating efficiency ofgrowth. It has been suggested that designating gain in terms of lean tissue growth may be more appropriate than a

346

D. POMP ET AL.

gain-to-feed ratio (Fowler ct al., 1976; Bereskin and Steele, 1985 ). When the present d~'a were considered as the ratio of hind carcass weight to feed intake, transgenic males exhibited less efficient lean growth than did controls. In addition, they 0.isplayed increased levels of fat relative to feed intake than controls. Hcwever, as with the feed efficiency data, looking at intake over the entire growth period may not accurately depict the efficiency of lean growth. If we were to consider this efficiency at a common body weight, or for gain to a standard body weight, transgenics would have slightly less lean tissue but they would have a greatly increased efficiency of lean growth production due to their lower feed requirement for a fixed weight increase. If enhanced weight in the transgenic males was not the result 0f increasing lean deposition, the question remains as to which body parts accounted for the general body weight increase? The data on organ weights indicate that viscera likely accounted for a significant part ofthis increase. All organs measured were larger in transgenic males than in controls. This was especially true for liver and spleen, which were approximately twice as heavy in transgenics. In terms of organ weight relative to body weight, liver and spleen were the only organs to display disproportionately increased growth. Indeed, both heart and testis were smaller in uansgenic animals when expressed as a percentage of body weight. However, after adjustment to a common body weight, fiver and spleen remained larger in transgenics while all other organs were of similar weight among genotypes. The present results agree well with those of Shea et al. ( 1987 ) using MTrat GH transgenic mice, in that liver and spleen appear to exhibit special enlargement in transgenic mice, while other organs may grow in proportion to overall body weight increases. Data from Brem et al. (1989) with MT-hGH also show disproportionate growth of liver, but not for spleen. Brem et al. (1989) discuss possible causes for different responses of various transgenic lines in terms of organ development. In transgenic pigs (Pursel et al., 1990) there is also significant change m the growth of certain organs, while transgenic sheep with the oMTIa-oGH construct had larger livers and kidneys than controls, even though body weight had not been significantly altered (Nancarrow et al., 1991 ). The growth and related production-oriented traits of these transgenic mice, as well as those reported in other studies, clearly indicate a great potential for transgenic technology in improving livestock production systems. The preliminary evidence from transgenic pigs supports this notion, although several critical problems concerning reproduction and health need to be addressed. The overall value of a transgene not only depends on how it affects the target trait, but also on its effect on correlated traits which contribute to economic merit (Smith et al., 1987 ). Currently, steady and effective genetic progress is achieved in many livestock breeding systems using sophisticated computeraided selection programs. In addition, marker-assisted selection may soon in-

EFFICIENCY AND COMPOSITION OF TRANSGENIC MICE

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crease the efficiency of these breed tng programs. Clearly, this is the standard against which transgenic techniques must be judged. The oMT I a-oGH fusion gene is unique when used in transgenic mice, as expression can be controlled by provision or withdrawal of zinc (Shanahan et al., 1989). Further studies are required to define time periods oftransgene activation to optimize growth, efficiency and production of lean product while minimizing adverse side effects of chronically high levels of circulating growth hormone. ACKNOWLEDGEMENTS

The authors wish to thank J.S. Conrad-Brink and T. Martin for technical assistance. We are also grateful to G.E. Bradford, E.J. Eisen, S.N. Newman and two anonymous referees for very helpful reviews. REFERENCES Bartke, A., Steger, R.W., Hodges, S.L., Parkening, T.A., Collins, T.J., Yon, J.S. and Wagner, T.E., 1988. Infertility in transgenic female mice with human growth hormone expression: evidence for luteai failure. J. Expt. Zool., 248:121- ! 24. Bereskin, B. and Steele, N.C., 1985. Efficiencyof feed utilization in swine: A review of research and current applications. USDA-ARS Production Research Report, Number 184. Bhuvanakumar, C.K., Lynch, C.B., Roberts, R.C. and Hill, W.G., 1985. Heterosis among lines of mice selected for body weight. 1. Growth. Theor. Appl. Genet., 71: 44-51. Brem, G., Wanke, R., Wolf, E., Buchmilller,T., Mfiller, M., Brenig, B. and Hermann, W., 1989. Multiple consequences of human growth hormone expression in transgenic mice. Mol. Biol. Med., 6: 531-547. Bremner, I. and Beattie, J.H., 1990. Me~,ailothioneinand the trace minerals. Annu. Rev. Nutr., 10" 63-83. Brody, S., i 945. Bioenergeneticsand Growth. Chapter !". Reinhold Publishing, New York. Calvert, C.C., Famula, T.R., Bern;er, J.F. and Bradford, G.E., 1985. Serial composition dunng growth in mice with a major gene for rapid postweantag growth. Growth, 49: 246-257. Campbell, R.G., Steele, N.C., Capema, T.J., McMurtry, .~P., Soloman, M.B. and Mitchell, A.D., 1988. Interrelationships between enertry intake ant, exogenous porcine growth hormone administration on the performance, body composition, and protein and energy metabolism ,rowing pigs weighing 25 tu 55 kilograms live weight. J. Anim. Sci., 66:1643-1655. C . Jell, R.G., Steele, N.C., Capema, T.J., McMunry, J.P., Solomon, M.B. and Mitchell, A.D., t 989. Interrelationships between sex and exogenousgrowth hormone administration on performance, body composition and protein and fat accretion of growing pigs. J. Anita. Sci., 67: 177-186. Eisen, E.J., 1987. Selection for components related to body composition in mice: direct responses. Theor. Appl. Genct., 74: 793-801. Eisen, EJ. and Leatherwood, J.M., 1981. Predicting percent fat in mice. Growth, 45: 100-107. Evock, C.M., Etherton, T.D., Chung, C.S. and Ivy, R.E., 1988. Pituitary porcine growth hormone (pGH) and a recombinant pGH analog stimulate pig growth performance in a similar manner. J. Anita. Sci., 66: 1928-1941. Fowler, V.R., Bichard, M. and Pease, A., 1976. Objectives in pig breeding. Anita. Prod., 23: 365-387.

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Gould, S.J., 1966. AIIome~ryand size in ontogeny and phylogeny. Biol. Rev., 41: 587-640. Hammer, R.E., Brinster, R.L. and Palmlter, R.D., 1983a. U~ ofgene transfer to increase animal growth. Cold Spring Harbor Syrup. Quant. Biol., 50: .'r79--387. Hammer, R.E., Brinster, R,L., Rosenfeld, M.G., Evans, R.M. and Mayo, K.E., 1985b. Expression of human growth hormone releasing factor in trans/~enic mice results in increased somatic growth. Nature (London), 315:413-416. Huxley, J.S., i 932. Problems of Relative Growth. M,thuen, London. Mathews, L.S., Hammera, R.E., Behrin~r, R.R., D'Ercole, A.,I., Bell, G.I., Brinster, R,L. and Palmiter, R.D., 1988. Growth enhancement of transgenic mice expressing human insulinlike growth factor I. Endocrinology, 123: 2827-2833. MeCusker, R.H. and Campion, D.R., 1986. Effect ofgrowth hormone-secreting tumors on Ix,dy composition and feed intake in young female Wistar-Furth rats. J. Anita. Sci., 63:1126!133. Medrano, J.F., Pomp, D., Sharrow, L, Bradford, G.E., Downs, T.R. and Frohman, L.A., 1991. Growth hormone and insulin-like growth factor I measurements in high growth (hg) mice. Genet. Res., Camb. 58: 67-74. M~rello, D., Moore, G., Salmon, A.M., Yaniv, M. and Babinet, C., 1986. Studies on the expression of an l-l-2K/human growth hormone fusion gene in giant transgenic mice. EMBO J., 5: 1877-1883. Nancarrow, C.D., Marshall, J.T.A., Clarkson. J.L.~ Murray, J.D, Millard, R.M., Shanahan, C.M., Wyn,1, P.C. and Ward, K.A, 1991. Expression and physiology of performance regulating genes in transgenic sheep. J. Reprod. Fert., Suppl., 43: 277-291. Orian, J.M., Scong Lee, C., Weiss, L.M. and Brandon, M.R., 1989. The expression of a metalIozhionein--ovine growth hormone fusion gene in transgenic mice does not impair fertility but results in pathologlcalqesions in the liver. Endocrinology, ! 24: 455-453. Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer, M.G., Rosenfeld, M.G., Birnberg, N.C. and Evans, R.M., i 982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature ( London ), 300:611-615. Palmiter, R.D., Norstedt, G., Gelinas, R.E., Hammer, R.E. and Brinster~ R.L., 1983. Metallothionein-human growth hormone fusion genes stimulate growth of mice. Science, 222: 809814. Pomp, D., Murray, J.D. and Medrano, J.F., 1991. Single day detection of transgenic mice by PCR of toe-clips. Mouse Genome, 89: 279. Pursel, V.G., Pinkert, C.A., Miller, K.F., Bolt, D.J., Campbell, R.G., Palmiter, R.D., Brinster, R.L. and Hammer, R.E., 1989. Genetic engineering of livestock Science. 244:1281-1288. Pursel, V.G., Bolt, D.J., Miller, K.F., Pinkert, C.A., Hammer, R.E., Palmiter, R.D. and Brinster, R.L., 1990. Expression and performance in transgenic pigs. J. Reprod. Fert., Suppl. 40: 235245. Rexroad, C.E., Jr, Hammer, R.E., Bolt, D.J., Mayo, K.E., Frohman, L.A., Palmiter, R.D. and Brinster, R.L., 1989. Production oftransgenic sheep with growth-regulating genes. Mol. Reprod. Dev., !: 164- i 69. Rogers, P. and Webh, G.P., 1980. Estimation of body fat in normal and obese mice. Br. J. Nutr., 43: 83. SAS Institute, 1988. SAS/STAT User's Guide, Release 6.03. Editor, SAS Institute, Inc., Cary, NC. Searle, T.W., Murray, J.D. and Baker, P.J., 1992. the effect of increased growth hormone production or body composition in mice- transgenic versus control. J. Endo. in press. Shanahan, C.M,, Rigby, N.B., Murray, J.D., Marshall, J.T., Townrow, C.A., Nancarrow, C.D. and Ward, K.A., 1989. Regulation of expression of a sheep metallothionein l a-shecp growth hormone fusion gene in transgenic mice. Mol. Cell. Biol., 1989: 5473-5479. Shea, B.T., Hammer, R.E. and Brinster, R.L, 1987. Growth allometry of the organs in giant transgenic mice. Endocrinology, 121: 1924-1930.

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Smith, C., Meuwisse, T.T.E. and Gibson J.P., 1987. On the use of transgenes in livestock imi~rovement. Anita. Breed. Abstr., 55: I-I0. Steele, N.C. and Pursel, V.G., 1990. Nutrient partitioning by ,ansgenic animals. Annu. Rev. Nutr., I0: 213-232. Wieghar;, M., Hoover, J.L.,McOrane, M.M., Hanso ~, R.W. and Rottman, F.M., 1990. Production oftransgenic swine harboring a rat phosphoenolpyruvate carboxykinase-bovine grog~h hormone fusion gene. J. Reprod. Ferlil.,Suppl. 4 !: 89-96. RESUME Pomp, D., Nancarrow, C.D., Ward, K.A. et Murray, J.D., 1992. Croissance, efficacit~alimentaire et composition corporelle de souris transg~niques experimant un g~ne de fusion m~talIothion~ine ovine la-Hormone de croissance ovine. Livcst. Prod. Sci., 31:335-350 (en anglais). On a ~tudi~ la croissance, I'efficacit~ alimentaire et la composition corporelle de souris males possc~dant un transg~ne M~tallothion~ine ovine la-Hormone de croissance ovine (oMTla= OHG). Le trans~ne ~tait activ~ au sevrage ( 21 jours) par 25 mM de sulfate de zinc additionn~s reau de boisson. Le poids viler la consommation alimentaire ont ~t~ mesur~ de fagon hebdomadairejusqu'i 70 jours. Les males transg~niques ont eu une croissance sup~rieure de 53,3% celle des t~rnoins pendant la p~riode de contr61e (P< 0.001 ) alors que leur consommation n',~ augment~ que de 10,9% (P<0.001). La consommat~on corrig~e pour le poids m~tabolique a ~t~ idemique pour les deux g~otypes. Les transg~niques ont eu plus de gras souscutan~ et ~ i didymaire a 70 jours (P<0.001) mais moins de gras aux memes endmits (P<0.05) apr~s ajustement au m~bmepoids. Le tissu maigre, estim~ par le poids de carcasse arrive d~,raiss~, a et~ plus important chez les transg~niques au m~me age (P< 0.01 ) mais inf~rieur au meme poids vif (P<0.0001). En raisonnant ~t gain de poids identique, les transg~niques manifestent une efficacit~ sup~rieure pour la production de tissu maigre, en raison d'une consommation inf~rieure. Les viscc~resont ~t~ plus importams chez les transg~niques, mais seuls le foie et la rate ont augment~ davantage que ie poids vff (P<0.001). Ces r~sultats montrent que les souris poss~dant un trans~ne oMTla-oOH montrent une efficacit~ am¢lion~e pour la croissance et la production de tissu maigre, particuli~rement quand on consid~re une d u r ~ standard de croissance. KURZFASSUNG Pomp, D., Nancarrow, C.D., Ward, K.A. und Murray, J.D., 1992. Wachstum. Futterverwenung und K0rperzusammensetzung tra~sgener Mlluse welche ein Wachstumshormon Fusionsgen Metallothionein la vom Schafexprimieren. Lirest. Prod. Sci., 3 !: 335-350 (aufenglisch). Wachstum, Futterverwertung und K0rperzusammensetzung wurden bei mlinnlichen M~usen untersucht, die ein ovines metailothionein la-Schaf Wachstumshormon Transgen (oMTIaoGH) besitzen. Das Transgen wurde beim Absetzen (21 Tage~. durch die Verabreichung yon mM Zinksulfat halti~m Trinkwasser aktiviert. K~rpergewicht und Futteraufnahme wurden bis zum 70 Alterstas w0chentilich erhoben. Transgene Mluse nahmen in der Testperiode um 53% mehr zu gegenilber Kontrolltieren (P<0.001) und fressen nur 10.9% mehr (P<0.001). Nach Korrektur auf das metabolische Gewicht war die Futteraufnahme bei beiden Genotypen tthniich. Transgene Tiere hatten bei 70 Tagen mehr epididymes und subcutanes Fett (P<0.001), jedoch weniger in beiden Depots (P< 0.05) nach Korrektur aufein gemeinsames Gewicht. Mageres Fleischgewebe, gescMtzt anhand des Hinterbeingewichtes, war bei u'ansgenen Tieren h0her verglichen bei gleichem Alter (P<0.01) und tiefer verglichen bei gleichem K0rpergewicht

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(P< 0.0C I ). In der Auswertung bei gleichem Gewicht, wic~en Transgene eine viel bessere Et'fizienz in der Magerfleischprt~ctuktion auf, well viei wenigcs Fatter zur Produktion yon Fleisch ben6tigt wurde. Innere Organc waren bei Transscncn schwcrer als bei der Kontrolle, abet nur Leber und Milz waren Uberproportional grOsser verglichen mit tier Zunahme am ganzen K~,rper (P<0.00!). Diese Resultate zeigen, dass Mtiuse im Besitz yon oMTla--oGH Transgenen ein h6hercs Wachslumsvcrm0gen und bessere Magerfleichprodukt~on aufweisen, vorallem wena eine standardisiene Wachstumsperiode berUcksichtigt wird.