Metabolic Effects of Bovine Growth Hormone and ...

Metabolic Effects of Bovine Growth Hormone and Genetica Engineered Rainbow Trout Growth Hormone in Rainbow Tr Oncorhynchus mykiss Reared at a High Temperature Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by 199.201.121.12 on 06/04/13 For personal use only.

Roy G. Danzrnann' john Hopkins University, Chesapeake Bay Institute, Shady Side, MD 26764 USA

Glen I. Van Der Kraak, University of Guelph, Beparfment of Zoology, Guelph, Ont. N 7 C 2 W 1 Canada

Thomas T. Chen, and Dennis A. Powers2 john Hopkires University, Chesapeake Bay Institute, Shady Side, M B 20744. USA, and john Hopkins University, Department of Biology, Baltimore, MD 2 12 18 USA and University of Maryland, Center sf Marine Bisteehnslogy, Baltimore, MD 2 1282 USA

Danzmann, R. G.,6.j. Van Der Keaak, S: T* CChen, and D. A. Powers. 1990. Metabolic effects of bovine growth hormone and genetically engineered rainbow trout growth hormone in rainbow trout (Oncsahynchus mykiss) reared at a high temperature. Can. j. Fish. Aquat. %cia47: 1292-1 301.

The growth promoting ability of bovine growth hormone purified from natural sources (bGH) and genetically engineered rainbow trout growth hormone (rtGH) were compared in rainbow trout (Oncsrhynchus mykiss) reared at 17.O0C. Fish were fed high (on-demand feeding) and low (2.8-4.676 of body weighvd) rations. On both rations, length specific growth rates were significantly higher in bGH treated fish than in all other treatments. No significant differences in weight specific growth rates were detected between treatments. Testosterone levels in fish fed the high ration and treated with 1 .Q eglg rtGH or bGH were significantly elevated compared with controls as were 19-p-estradiol levels in females. bGH significantly reduced condition factors of low ration fish, and bGH and rtGH significantly depressed hepatosomatic indicies of high ration fish compared with controls. Bn a second experiment, weight and length specific growth rates were higher in rtGH and bGH injected fish than in saline controls but these differences were not significant. DNA standardized RNA levels were higher in rtGH and bGH treated fish than in saline control fish and these differences were significant in GGH treated fish. Results indicate that at high rearing temperatures GH administration does not have a major influence on growth but does stimulate steroidsgenic and metabolic activity. La capacit6 de favoriser la croissance de I'hsrmone de croissance d'origine bovine purifike de sources naturelies (bGH) et de I'hsrmone de croissance provenant de la truite arc-en-ciel (rtGH) obtenue par genie g6nktique a 6te comparee chez la truite arc-en-ciel (Oncorhynchur mykiss) elevee A 19,O0C. Les poiss~nssnt r e p des rations alimentaires elevees (3 la demande) et faibles (2,8 3 4,6% du psids par jour). Dans le cas des deux types d'alimentation, fe taux de croissance specifique en fonctisn de la longueur etait significativernent plus elevke chez les poissons auxquels on a administre B'horrnone de croissance d'origine bovine que chez les poissons auxquels on a administre tous Bes autres traitements. Aucune difference significative n'a 6t6 dkcelke au niveau du taux de crsissance specifique en fonction du poids entre les diffkrents traitements adrninistr4s aux poissons. Le taux de testosterone chem les poissons ayant recu une ration elevee et auxquels on a administr6 une dose de 1,0 pg/g de rtGH ou de bGH etait significativernent plus eleve par rapport aux poissons t6rnoins tout comrne le taux de 17pestradiol chez les femelles. L'administration de bGH a abaisse significativement Be coefficient de condition des poissons auxquels on a adrninistr6 une ration faible, et I'administration de bGH et de rtGH a abaiss6 sigwificativement les indices h6patosornatiques des poissons ayant r e p une portion klevee par rapport aux poissons tkmoins. Dans une deuxierne experience, le taux de croissance specifique en fonction dks poids et de la longueur etait plus dev6 chez les pissons auxquels on a inject6 de la rtGH et de la bGH que chen les t h o i n s agsxquels on a administre une solution physiolsgique, mais ces differences n'etaient pas significatives. ke taux d'ARN normalls6 par rapport a I'ADN etait plus eleve chez les poissons ayant r e p de la rtGH et de Ja bGH que chez les poissons temoins auxquels on a administre une solution physiologique et ces differences etaierat significatives chez les poissons ayant resu de Ba bGH. D'apres les rksultats obtenus, 3 ternp6rature elevee, I'adrninistratisn d'hsrmone de croissance n'a pas d'effet important sur la croissance mais elle stimule la steroidogenese et le rnetabolisrne.

Received August 23, 1989 Accepted February 27, 7 990

(JA290)

"esent address: University sf Guelph, Department of Zoology, Guelph, Ont. NlG 2W1 Canada. 2Pmse~taddress: Stanford University, Hopkins Marine Station, Department of BioHogica% Sciences, Pacific Grove, GA. 93950-3094 USA.

Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by 199.201.121.12 on 06/04/13 For personal use only.

n recent years there have been several studies examining the growth promoting abilities of growth homone (GH) in salmsnid fishes (Higgs et d. 1975, 1976,1977, 1978; Clarke et al. 1977; Markert et al. 1977; Domldson et al. 1979; Gill et al. 1985; Sekine et al. 1985; Kawauchi et al. 1986; Agellon et al. 1988; Down et al. 1988, 1989). The GH used in these studies was either purified from mammalian or piscine sources, or synthetically produced from cloned mammalian or piscine growth homone genes. Generally, all of these studies have documented an increased growth rate of salmonid fishes in response to GH administration. Agellon et al . (I 988) reported that recombinant rainbow trout GH (rtGH) at a dosage of 1.0 ~ g l was g the most effective in stimulating growth in rainbow trout (Bncorhywhus mykiss) reared at 13-15°C. In other studies, bovine GH (bGH) at dosages of 1-0 pg/g body weight or higher were necessary to elicite significant growth enhancement in coho salmon reared at 10°C (Higgs et al. 1977, 1978). Hn contrast, bGH administered at a dose of 0.25 kg/$ body weight significantly enhanced growth rates in sockeye salmon (0. n e r h ) reared at 17.5"C (Clarke et al. 1977). These results indicate that the dosage of K H necessary to affect growth rates may be reduced in salmonids reared at w m e r temperatures. Most studies investigating the effects of GH on growth have been conducted at lower temperatures than the thermal optimum for growth. In this study we examine how GH administration effects growth rates in rainbow trout reared at the thermal optimum for growth (17-18°C). Two types of GH were used: (1) a recombinant rainbow trout GH (rtGH) (Agellon md Chen 1986) and; (2) natural bGH. When food is not limiting, salmonid growth rates are directly proportional to environmental temperature (up to the thermal optimum for the species) (Brett et al. 1969; Weatherley 1972). It was of interest, therefore, to determine if rtGH also has enhanced growth promoting ability at wanner temperatures and to compare the growth promoting ability of rtGH and bGH at temperatures where growth is maximally stimulated. In addition, the interaction between ration size (high and low) and GH administration on growth rate was examined.

Materials and Methods Source of the Fish Experiment 1: the Reynoldsdale strain of rainbow trout was used in this work. Gametes were obtained from the Reynoldsdale State Fish Hatchery, Fishertown, Pennsylvania, om June 3, 1987. Approximately equal proportions of gametes from 16 females and 27 males were mixed separately and then pooled together to make a Barge pooled cross. Embryos were raised at the Chesapeake Bay Institute in vertical FAL incubating trays at 12-24°C until hatching. The fish were then acclimated to summer well water temperatures at the Institute (16-18°C) prior to experimentation. The experiment was initiated Sanuargr 25, 1988, when the fish were 246 d old, postfertilization. Experiment 2: juvenile Reynoldsdale rainbow trout obtained on July 28, 1987, were acclimated at 1618°C at the Chesapeake Bay Institute for approximately 2 mo prior to the initiation of the experiment on September 21. Experimental Protocols Experiment 1 was conducted to measure the influence of GH on weight and length specific growth rates, physiological Can. J . Fish. Aqesat. Sci., Vol. 47, 1990

changes, and steroidogenic activity in individual fish. Experiment 2 was conducted to measure the influence of GH on length and weight specific growth rates and nucleic acid ievels in individual fish. Experiment 1 : 2 wk prior to the initiation of the experiment, lots of 20 fish each were weighed and measured. Each lot of 20 fish was housed in a circular 30 gallon tank on a 12:12 1ight:da-k cycle. Twenty experimental lots (400 fish) were used for the experiment. The fish were divided into high and low feeding rations ( 2 0 fish in each). Fish were either: (1) fed using on-demand feeding hoppers or; (2) fed a hatchery ration calculated from tables of feeding rations for salmonid fishes (Bardach et al, 1972). According to the tables, fish were fed a ration which was approximately 4,6% of body weight at the beginning of week 1. This ration was gradually decreased to 2.8% of body weight after week 6. Rations were recalculated at each weighing and adjusted for any mortalities. Low ration fish were fed once daily. The diet fed was Matins 3/32" 84G pellets (40% protein; 10% fat; 3% fibre) from Martins Feed Mills, Elmira, Ont. Five treatments were used for each feeding ration: (1) an uninjected control; (2) an injected control group receiving an extract of bacterial protein from strain HE3101 (the bacterial strain used to clone rtGH); (3) a bGH treatment receiving (USDA-bGH-BZ) from the National Hormone and Pituitary Program, at a concentration sf 1.6 ~ g / body g weight; (4) a rtGH treatment receiving a dose of 1.O pg/g body weight; and (5) a rtGH treatment receiving a dose of 0.I pg/g body weight. RGH was prepared according to the protocols outlined in AgelIon and Chen (1986) and Agellon et al. (1988). All fish were measured (fork length) and weighed, and injected (if needed according to treatment) intraperitoneally weekly at the beginning of week 1, 2, 3 , 4 , and 6 . Doses were recalculated according to the weights of each individual fish at these times. Fish were not fed on the day of these measurements. The fish were anaesthetiaed with 2-phenoxyethanol (Sigma Chemical Co.) at a 1:2800 dilution to facilitate handling. The experiment was terminated at the end of week 8. At this time, a blood sample was obtained from 15 fish in the uninjected control, bGH, and rtGH (1.0 pg/g) treatments, for measurements sf circulating testosterone and 17-f3-estradiol levels. The average weight and length of the fish at the beginning of the experiment was 11.79 g (range: 4.79-30.03 g) m d 9.25 cm (range: 7.2-12.9 cm), respectively. Mortalities throughout the course of the experiment were low, ranging from 0 to 3.75% of the fish in a treatment, averaged over both feeding rations. Experiment 2: 36 fish were randomly assigned to one of three treatment groups consisting o t (I) a saline control; (2) bGH; or (3) ffiH. Fish were measured (fork length), weighed, and injected 4 x at 8-d intervals, and the experiment was terminated on day 32. At this time, a muscle sample was excised from each fish and immediately frozen in liquid nitrogen for nucleic acid determinations. Flsh were fed ad libitum twice daily w i ~ Martins 84G pellets. Fish in the saline control received 280 pb, of 0.8% saline at each injection, while bGH treated fish received I5 pg of hormone in 150 pE of saline at each in~ection.PtGH treated fish received 200 pL of recombinant protein extract which was initially estimated (using an immunoblot analysis) to be an equivalent dosage to the bGH treatment, but was subsequently shown to contain 10 p,g of rtGH. Therefore, PtGH treated fish received a lower dose of GH than bGH treated fish. The initial starting

TABLE1 . Final mean weights (g) and percent increases in weight based upon the initial mean starting weight of fish in the different treatments in experiment 1.

TABLE2. Mean weight specific growth rates" (Day 1-56) of replicates (N = 4) in the different experimental treatments used in expeHimeHnt 1.

Treatment

Treatment Feeding ration

High k SE


Sig. Low Q SE

Sig.

NomaI HE3101 control control 49.68 3.26

48.29 2.54

4-

+

44.50 3.01

39.85 2.35

+

+

bGH 1.0 g g 55.68 3.81

rig;H rtGH 1.0 yiglg 0. I pgl$ 47.82 3.22

-I-

+

45.17 2.53

41.70 3.00

-I-

+

47-01 3.14

Feeding ration High k SE

+

Sig." Low

41.08 2.93

Sig .

+

2 SE

Normal HBlOl control control

316 288

309 263

368 266

297 26 B

297 24 1

"Results from a GT-2 pairwise muBtipHe comparison test. Treatments are not significantly different from one another (across rows) if underscored by the same symbol.

weight of the fish in each treatment were: (saline csnb-sl; mean = 40.00 g; range = 27.07-54.87 g), (bGH; mean = 39.2 1 g; range = 3 1.89-60.27 g), (HfGH; mean = 39.27; range = 20.09-8 1.08 g). Corrected for mortalities, the initid starting weights were: (saline control; N = 8; mean = 38.02 g; range = 27.0747.62 g), (bCH; N = 8; mean = 41.78 g; range = 33.9240.27 g), (fiGH; N = 6; mean = 44.32 g; range = 33.03-81.00 g). Biochemical Analyses For nucleic acid measurements, 0. B -g samples (approximate weight) were homogenized by ssnlcation in 4 rnL of cold phssphate buffered saline (PBS). After centrifugation, an 800 pL aliquot was removed md further diluted to 4 mL in cold PBS. AH samples were analyzed in duplicate for both RNA and DNA content, using the methods of Karsten and Wollenkrger ( 1972; 11977). Total nucleic acids (RNA +DNA) were measured, and DNA levels were detemined by treating replicate samples with RNase using the protocol described by Ferguson md DmhusBBch& (1990). Protein determinationswere made using the Lowry method (Lowry et al. 1951). For the analysis of steroid levels, plasma samples were extracted with ether and the testosterone and 17-@-estradiol content determined by RIA as previously described (Van Der &a& et a%.11984). Statistical Analyses Experiment 1: differences in length and weight specific growth rates between treatments over the course sf the experiment were ccampaed using a two-factorial ANOVA based upon replicate means (Sskal and Wohlf 1981). The five treatments were considered the fist factor and feeding ration the second factor. Within each feeding level 280 fish were divided between five treatments, such that each treatment consisted of four replicates divided between two tanks with 1O fish in each replicate. Ten fish within each tank were caudal fin branded and 10 fish were unmarked. Replicates were moved to a new experimental tank after each weighing to minimize environmental and social influences upon the growth dynamics of the fish. However, only fish sf one treatment class were housed together.

rtGH

1.O pglg 0.1 pglg

2.708 0.167

2.713 0.037

2.932 0.137

2.653 0.016

2.659 8.120

2.511 0.072

2.399 8.047

2.347 0.039

2.339 0.052

2.228 0.054

+ +

+

+

+

+

+

-

"Specific growth rates calculated as:

Inwt,

Percentage increase in weight since the beginning of the experiment

High 6%) Low (96)

rtGH

bGH 1 .0 yglg

- Inwt,

f2 -

4

+

+

x 108,

where wt, is the mean weight of fish in the group at time t , and wt, is the mean weight of fish in the same group at time 8,. bResultsfrom a GT-2 painvise multiple comparison test. Treatments are not significantly different from one another (across rows) if underscored by the same symboI.

TABLE3. Final mean lengths (mm)and percent increases in length based upon the initial mean starting length of fish in the different treatments in experiment 2 . Treatment Feeding ration

High

Normal HI3181 control control

5 SE

151.2 3.38

158.3 2.84

Sig." kasw

144.8

2 SE

Sig .

+

3.30

+

bGH

B .B pglg

rtGH d2-I I .0 ~ g / g 0.1 pglg

157.3 3.43

147.5 3.26

9

+

848.9 3.43

140.3 2.91

149.5 2.47

142.2 3.38

142.5 3.31

Q

t

-I-

-I-

+

+

Percentage increase in length since the beginning of the experiment High (96) Low (8)

61.5 55.3

60.1 52.1

72.3 59.3

62.4 55.9

61.3 53.2

"Results from a GT-2 pairwise multiple compa~sontest. Treatments are not significantly different from one mother (across rows) if underscored by the same symbol.

For the analysis of final lengths, weights, hepato-somatic indices (HSHs), initial condition factors (CF), and semm steroid levels, individual fish were considered replicates within each experimental treatment to meet the assumptions of homogeneity of variance. Values for serum steroid levels were squareroot transformed. Differences in find CF between treatments were tested using non-puametric procedures as variances between treatments were refractory to nomalization using several transformations. A Kmskal-Wallis test was used ts test for significant differences between treatments. bf significance was detected, gair-wise comp&sons between treatments were made using a Wilcoxon two-sample test (Sokal and Rohlf 1198%). An alpha-level of 0.05/2c9where c is the number of pair-wise comparisons, was considered significant. This correction was adopted to minimize type I error. Experiment 2: length and weight specific growth rates and CF were calculated for the fish as described in experiment 1. Differences in the means between treatments were compared using a one-way ANOVA. RNAIDNA ratios were reciprocally transformed (B/value) to meet the assumptions of homogeneity of variances between treatments. Protein values were analyzed Can. J . Fish. Aquar. Sci., Vol. 47, 1990

TABLE4. Mean kngth specific growth rates (Day 1-56] sf replicates (N= 4) in the different experimental treatments ~ s e in d experiment 1.

Treatment Nsmal HBBOI control control

1.0 pglg

8.917 0.050

1.836 0.043

0.934 0.011

0.921 8.807

Sig."

+

8.985 0.010

Low

0.814 0.019

8.779

8.$48

0.815

0.006

0.808 0.017

0.776 0.816

Feeding ration High 2 SE


k SE

Sig .

+

bGH

+ +

*

+

G gbGH 1.O pg/g 0. I &g/g

+ ,*

+ ,*

+

+

"Results from a GT-2 painwise multiple comparison test. Treatments not significantly different from one another (across rows) if underscored by the same symbol.

using a non-parametric Kmskal-Wallis test, since it was not possible to satisfactorily transform these data.

Experiment I Weight While no significant differences in final weights (P = 0.166), or weight specific growth rates (P-0.240) were detected between treatments, highly significant differences in final weights (P