a mammalian growth hormone-releasing hormone increases serum growth hormone levels and somatic...

JOURNALOFTHE WORLD AQUACULTURE SOCIETY

Vol. 27, No. 4 December, 1996

A Mammalian Growth Hormone-Releasing Hormone Increases Serum Growth Hormone Levels and Somatic

Growth at Suboptimal Temperatures in Tilapia

ANITA M. KELLY', AND CHRISTOPHER c. KOHLER Fisheries Research Laboratory and Department of Zoology,

Southern Illinois University at Carbondale. Carbondale, Illinois 62901 -651 1 USA

E. GORDON GRAU Hawaii Institute of Marine Biology, P.O. Box 1346, Coconut Island,

Kaneohe, Hawaii 96744-1346 USA

Abstract The effects of a mammalian growth hormone-releasing hormone (GHRH), PrebCRF(1-

78)OH (bCHRH), on growth and serum growth hormone (GH) levels were investigated in tilapias Oreoehromis rnossambicus and 0. niloticus X 0. aureus. Fish were injected intramuscularly or implanted intraperitoneally (Silastic or cholesterol implants) with distilled water, 0.1 pgkg bCHRH, 1.0 pgkg bGHRH, 10.0 pgkg bGHRH, or 100.0 pgkg bCHRH and compared to untreated controls, Bsh implanted with 60 mgkg 17a-methyltestosterone (MT), or a combination of bGHRH concentrations plus either MT, 0.01 p g k g of a thyroid hormone (T3), or 0.01 pgkg of a glucocorticoid (DEX). The bGHRH increased serum GH levels in tilapia maintained at suboptimal temperatures (18 C). Serum GH levels were highest (5.3 f 0.45 ng/mL) for fish injected with 10.0 pgkg bCHRH. Fish implanted with a Silastic implant containing 10.0 pgkg bGHRH had significantly higher (4.35 2 0.35 ng/mL) serum GH levels than those with an equivalent dosage in a cholesterol implant. The addition of MT, thyroid hormones, and glucocorticoids did not increase serum GH levels above those obtained for fish receiving bGHRH alone. Tilapia reared at suboptimal temperatures and implanted with 10.0 pgkg and 100.0 pgkg hGHRH had significantly greater increases in weight and length than control Bsh. Fish implanted with bGHRH, MT or hGHRH plus MT had significantly higher moisture and protein content, while fat and ash contents were significantly lower than controls or sham-implanted fish. Fish implanted with bCHRH or bGHRH plus MT had significantly higher gonadosomatic indices than fish implanted with MT alone, shams or non- treated controls. This study demonstrates that a mammalian GHRH stimulates release of GH, promotes somatic and gonadal growth and may affect reproductive performance in tilapia.

Grace Pickford demonstrated in 1948 that mammalian growth hormone (GH) was active in teleost fish. Until the 1980s, the GH utilized in experiments with fish were all of bovine origin (Donaldson et al. 1979). Subsequently, researchers have utilized purified fish GH to estimate the biological activity of this preparation. These experiments have demonstrated the pleiotropic effects of purified fish GH, which plays an important role in osmoregulation, reproduction and in protein and lipid metabolism.

In many vertebrates, GH secretion is reg-

' Corresponding author.

ulated by two hypothalamic factors: somatostatin, which exerts an inhibitory effect, and growth hormone-releasing hormone (GHRH), which exerts a stimulatory effect. The regulation of GH release in teleosts has not been as extensively studied as in mammalian systems. Marchant and Peter (1989) were unable to elicit an increase in GH from goldfish pituitary cells utilizing a human GHRH, but reported increases in GH concentrations for fish treated with GnRH. Subsequent studies have added cre- dence to the hypothesis that GH secretion is regulated by GnRH (Marchant et al. 1989; Chang et al. 1993; Melamed et al. 1995). However, mammalian GHRH uti-

Q Copyright by the World Aquaculture Society 1996

38'4

MAMMALIAN GHRH AND GROWTH OF TILAPIA 385

lized in previous studies was not very ho- mologous to fish GHRH which has been sequenced and, therefore, much less suc- cessful in stimulating GH release in fish than the fish GHRH (Marchant et al. 1989; Vaughan et al. 1992; Le Gac et al. 1993; Lin et al. 1993; Melamed et al. 1995). The low degree of homology between the mammalian GHRH and the fish GHRH may ex- plain the lack of success in stimulating GH release in many teleost species (Vaughan et al. 1992).

Several studies have shown that cells which produce GH in teleosts are capable of synthesizing and secreting GH in vitro (Ingleton et al. 1973; Baker and Ingleton 1975; Luo et al. 1990). Fish somatostatins identical in amino acid sequence and with a high homology to mammalian SST-14 have been identified in anglerfish Lophius americanus (Noe et al. 1979), channel catfish Ictalurus punctatus (Andrews and Dix- on 1981) and coho salmon Oncorhyncus kisurch (Plisetskaya et al. 1986). Synthetic mammalian somatostatin has been shown to inhibit GH secretion in the Mossambique tilapia Oreochromis mossambicus, in vitro (Fryer et al. 1979) and goldfish Carassius auratus (Cook and Peter 1984; Marchant et al. 1987). Marchant and Peter (1989) also demonstrated an inverse relationship between the circulating concentrations of GH and somatostatin in the goldfish. However, little research has been done on the role of either exogenous or endogenous growth hormone releasing factors (GHRH) in fish.

Immunological evidence shows that GHRH-like material is present in teleosts (Pan et al. 1985; Luo and McKeown 1989). Olivereau et al. (1990) reported localizing GHRH-like immunoreactivity in nine teleost species. GHRH-like neuronal systems have been demonstrated in a number of teleost species including codfish Gadus mor- hua (Pan et al. 1985), sea bass Dicentrar- chus lubrax (Marivoet et al. 1988), eel An- guilla anguilla, common carp Cyprinus carpio, goldfish and several salmonid species (Olivereau et al. 1990). Peter et al.

(1984) reported that synthetic hGHRH stimulated GH release in vivo in a teleost fish. To date, only common carp GHRH has been sequenced (Vaughan et al. 1992). Vaughan et al. (1992) demonstrated that carp GHRH (cGHRH) is closely related to the N-terminal portion of mammalian GHRH. Stimulation of GH secretion by cGHRH has been demonstrated in vitro utilizing pituitary cells of goldfish (Vaughan et al. 1992) and rainbow trout (Luo and McKeown 1989).

Parker and Sherwood (1990) concluded that salmon GHRH is immunologically more closely related to hGHRH than to rat GHRH (rGHRH). Conversely, Ackland et al. (1989) characterized the GHRH from the codfish and found it to be similar to rGHRH (1-43). Vaughan et al. (1992) demonstrated that the primary structure of isolated c- GHRH was as similar to hGHRH as to rGHRH. Robberecht et al. (1985) showed that the biological activity of GHRH is dependent on the N-terminus of the molecule. Since codfish GHRH is bioactive on rat pituitary cells (Ackland et al. 1989) and human pancreatic GHRH stimulated release of GH in goldfish (Peter et al. 1984), Ackland et al. (1989) suggested that the evolutionary changes in the structure of GHRH between teleosts and mammals occurred within the C-terminal half of the molecule. They concluded that phylogenetically, GHRH is an ancient molecule with its biological activity and certain immunoreactive domains con- served, at least, from teleost to mammal.

Several studies have provided strong evidence suggesting the importance of thyroid hormone andor glucocorticoids in the regulation of GH secretion in mammals (Mich- el et al. 1984; Root et al. 1985; Frohman and Jansson 1986). Thyroid hormone promotes fish growth (Gross et al. 1964; Don- aldson et al. 1979; Weatherley and Gill 1987). protein synthesis (Higgs et al. 1982) and improves food conversion (Higgs and Eales 1978), all of which are similar to the effects of GH.

Higgs et al. (1979) demonstrated that fish

386 KELLY ET AL

fed thyroid hormone (T,) supplemented diets grew faster in length and weight than controls. The condition factors for fish receiving T, were lower, but T, stimulated food consumption and improved food conversion efficiency. T, is more potent than T4 in promoting cartilage and bone growth (Qureshi 1976).

Glucocorticoids have been reported to stimulate GH gene transcription in rat pituitary cell cultures (Nyborg et al. 1984), and consistently enhance T, stimulated GH gene transcription in rat pituitary cell cultures (Yaffe and Samuels 1984) and in por- tions of the human growth hormone gene (Rousseau et al. 1987). Nishioka et al. ( I 985) reported that glucocorticoids may effect in vitro secretion of GH in tilapia while T, does not.

The positive effect of 17a-methyltestosterone (MT) on growth responses have been demonstrated in several species of fish including tilapias (Oreochromis spp.) (Guer- rero 1975). Many androgens have growth promoting effects on sex-related tissue and under some conditions on the total organ- ism (Donaldson et al. 1979). In fish MT has been demonstrated to stimulate the thyroid (Sage and Bromage 1970; Van Overbeeke and McBride 1971; Higgs et al. 1977) and insulin secretion from the pancreas (Higgs et al. 1977).

Seasonal variations in growth occur in a wide range of teleost species including the suckers Catostomus catostomus and C. commersoni (Basset 1957), barbel Barbus barbus (Hunt and Jones 1975), black crappie Pomoxis nigromaculatus (Haines 1980), bluegill Lepomis macrochirus (Gerking 1966), the perches Perca fluviatilis and P.

Jlavescens (LeCren 1951; Kearns and At- chinson 1979). northern pike Esox lucius (Diana and Mackay 1979), brown trout Sal- mo trutta (Swift 1961) and various coregonid species (Hogman 1968; Berg 1970; Hagen 1970). Generally, somatic growth is highest in the summer and very reduced in the winter. Originally, researchers theorized that seasonal variations in GH contributed

to the growth cycle observed (Pickford 1957; Gross et al. 1965; Gerking 1966; Adelrnan 1977; Kayes 1977; Brett 1979). Due to the lack of radioimmunoassays, several studies utilized indirect assessments of the pituitary GH content concluding that pituitary GH content varied on a seasonal ba- sis (Scruggs 1951; Swift and Pickford 1965; Bhargava and Raizada 1973; Kaul and Vollrath 1974). Subsequent development of radioimmunoassays has provided evidence that seasonal variations in blood GH levels do occur in fish (Marchant and Peter 1986).

Significant diel variations in cortisol (Pe- ter et al. 1978), gonadotropin (Hontela and Peter 1978) and thyroid hormones (Spieler and Noeske 1979) in goldfish have been demonstrated. Cook (1981) and Marchant and Peter (1986) found no diel pattern to GH secretion in goldfish, while Le Bail et al. (1991) reported diel variations in GH in chinook salmon. Whether diel GH patterns exist in Oreochromis species remains to be elucidated.

The purpose of this study was to examine the effects of an exogenous GHRH, and combinations of GHRH, 17a-methyltestosterone, a thyroid hormone and a glucocorticoid on growth and serum GH concentrations in tilapia.

Materials and Methods Growth Hormone-Releasing Hormone A non-proprietary supply of DHP’D

AO-PrebGRF( 1 -78)OH (bGHRH) was provided by Dr. Mark Heiman, Lilly Research Laboratories, as a lyophilized powder. The bGHRH was hydrolyzed in 0.1 N acetic acid, diluted to a concentration of 1.0 mg/mL and stored in 1 mL aliquots in liq- uid nitrogen. Aliquots were thawed when needed and diluted in distilled water.

Fish The euryhaline Mossambique tilapia Or-

eochromis mossambicus and a hybrid tilapia 0. niloticus X 0. aureus (white tilapia) served as the experimental organisms. Mos-


sambique tilapia were utilized in the studies to determine the effect of bGHRH on serum GH levels. Fish were maintained in circular 900-L tanks on a freshwater flow-through system. Temperature was maintained at 24 2 1 C. Fish were fed a floating commercial 36% crude protein diet (Co-op Complete Catfish Diet, Farmland Industries, Law- rence, Kansas, USA) once daily.

White tilapia were utilized in the growth studies. Fish were maintained in either 110-L aquaria or 1,500-L concrete tanks, both of which were recirculating systems equipped with biofilters. Temperatures were maintained at either 18 5 0.5 C, 24 -+ 0.5 C, or 30 2 0.5 C, depending upon the study objectives.

Fish were anesthetized prior to tagging or implantation by immersion in 0.05% tricane methanesulfonate (Finquil) solution buf- fered 2:l with sodium bicarbonate. In the hormone studies, each group of fish was tagged with a different color Floy tag. In- dividual fish within each experimental group were identified by the number on the Floy tag. In growth studies, fish were in- dividually tagged with a passive integrated transponder (PIT) tag with different treatment groups being kept in separate aquaria. The PIT tag system consisted of encapsu- lated PIT tags (12 mm length X 2 mm di- ameter) and an electronic tag detector, scale and digitizing pad which were connected to a computer. Each tag was uniquely number coded and could be read by the detector in situ when a tagged fish was passed through the detector loop. Following individual identification, fish length was measured on a digitizing pad and weight on the scale, and both measurements were automatically recorded into the computer.

Hormone Treatments

Fish were injected intramuscularly (IM) or implanted intraperitoneally with either a cholesterol or Silastic implant containing either bGHRH, 17a-methyltestosterone (MT), a thyroid hormone (3,5,3’-triiodo-L- thyronine; T,) (Sigma, St. Louis, Missouri,

USA), the glucocorticoid dexamethasone (DEX) (Sigma), or distilled water (sham). A dose of 0.01 p g k g was utilized for T, and DEX, 60 mgkg for MT, and either 0.1, 1.0, 10.0 or 100.0 p g k g for bGHRH. The IP implants were inserted based on a modification of the method described by Woods et al. (1995). The implants were inserted into the abdominal cavity just anterior to the pelvic fins. The incision was sealed utilizing cyanoacrylate adhesive (Nemetz and MacMillan 1988).

Injections

Injections of bGHRH were given IM 3- 4 rows down from the middle of the dorsal fin, by sliding the needle under the skin a few millimeters, puncturing the muscula- ture and evacuating the fluid from a 1-cc syringe equipped with a 22-gauge needle. This method resulted in little to no back- wash of fluid from the injection site.

Implants

Cholesterol pellets were prepared following the procedure of Sherwood et al. (1988). The pellets utilized in this study contained only cholesterol (and the hormone or sham concentration) which provided slow release of the hormone over several weeks (Sherwood et al. 1988). Silastic implants were made following the techniques outlined by Lee et al. (1986). Sham implants for both the cholesterol and Silastic implants were produced in the same manner as the hormone implants with distilled water replacing the hormone. All implants were stored at -20 C until needed.

Blood Sampling

Blood samples were taken from the cau- dal vasculature utilizing a 22-gauge hepa- rinized 5-cc syringe. The heparin (ammonia heparin from porcine intestinal mucosa, Sigma) was diluted to a concentration of 11.6 mg/mL. The blood was placed on ice immediately after sampling, and centri- fuged at 10,OOO rpm at 4 C for 15 min. The

388 KELLY ET AL.

serum was removed and stored at -80 C until analyzed.

Radioimmunoassay (RIA) for Growth Hormone

The RIA for tilapia growth hormone (tGH) utilized the method outlined by Ay- son et al. (1993). The RIA procedure utilized the double antibody method, in which the anti-tGH, G-4-4, was precipitated by goat anti-rabbit IgG. Nonspecific binding was less than 1% of the total radioactivity and specific binding was 17%. Sensitivity of the assay was between 0.17 and 0.40 pg/mL. Samples were run in duplicate. Re- peated measures within the same assay and in subsequent assays of a pool of 0. mossambicus plasma gave an intra- and inter- assay coefficients of variation of 7.6 and 10.3% for tGH, respectively.

White Tilapia Growth Study

Three separate thermal regimes, 30 2 0.5 C (hyperoptimal), 24 2 0.5 C (warmwater) and 18 2 0.5 C (coolwater), were utilized to determine if bGHRH increases growth rates in the white tilapia. In the hyperopti- ma1 and warmwater thermal regimes, 10 fish weighing 24 -f 1.5 g and 22.0 2 1.3 g, respectively, were placed into each of three groups: non-implanted control, sham implant, and 100 p g k g bGHRH implant. The studies ran for 84 d with fish being weighed and measured at 14, 28, 42, 56, and 84 d from the start of the experiment.

In the coolwater thermal regime two separate size groups were utilized but kept separate. One 113-L aquarium was utilized for each experimental group which consisted of ten white tilapia weighing 18.5 ? 2.5 g in the small fish group and 47.0 2 3.0 g in the large fish group. The experimental groups consisted of non-implanted control, sham implant, MT implant, 10 F g k g b- GHRH implant, 100 p g k g bGHRH implant, 10 p g k g bGHRH plus MT implant, and 100 p g k g bGHRH plus MT implant. The studies ran for 56 d with fish being

weighed and measured at 28, 42, and 56 d after start of the experiment.

At the completion of the coolwater growth study, the fish were euthanized and placed in a sealed plastic bag. The fish were frozen at -25 C and analyzed the following day. Since white tilapia become sexually mature at small sizes, the sex of each fish was determined by external examination of the genital papilla; males have one genital pore while females have two. The gonadosomatic index (GSI) was calculated as fol- lows:

Ovary weight X 100 Weight of fish '

GSI =

The visceral somatic index (VSI) was determined by dissection of each fish. The VSI was determined by removing and weighing the entire visceral mass in the abdominal cavity to the nearest 0.01 g. The following equation was utilized:

Weight of empty carcass X 100 Total weight of fish

VSI =

All fish in the study were examined for external side effects which might be caused by exposure to the bGHRH andor other hormone treatments. Skin color, fin color, fin development, and liver color were qual- itatively compared between each group to untreated and sham treated controls.

Proximate Analyses

Each experimental group was analyzed for tissue composition using standard AOAC ( I 984) methods except the percent crude protein, which was determined utilizing the Hach modification of the AOAC methods (Watkins et al. 1987). Ten fish in each group were dried at 65 C for approx- imately 7 d to determine moisture content, and then separated into two equal groups for analyses as either whole body or empty carcass. The two groups were ground in a W h y @ mill in preparation for protein, lipid and ash determinations. All analyses were run in triplicate.

MAMMALIAN GHRH AND GROWTH OF TILAPlA 389

TABLE I . Effect of IP injection for three dosages of bGHRH versus sham and non-injected controls on serum GH levels (ng/mL) at 0, I , 3, and 24 h post-injection in female Oreochromis mossambicus. Values are means _t SEM. Values in the same column not sharing the same letter are significantly different (P < 0.05).

Time (h)

Treatment” 0 1 3 24

Control 0.10 2 0.05 A 0.45 % 0.1 1 A 0.10 ? 0.06 A 0.10 2 0.04 A Sham 0.10 % 0.07 A 0.20 t 0.09 A 0.30 % 0.1 1 A 0.10 t 0.05 A bGHRHO.1 0.10 t 0.05 A 0.55 2 0.15 A 0.75 t 0.25 A 1.12 ? 0.06 B bGHRH1.O 0.10 t 0.02 A 0.55 % 0.15 A 0.10 -+ 0.02 A 3.75 % 0.50 C bGHRH 10.0 0.10 2 0.07 A 0.40 2 0.30 A 0.95 2 0.85 A 5.30 % 1.10 C

8 Values accompanying treatment abbreviations are concentrations of the hormone in pgkg. N size for all groups at each time period was 5.

Statistical Analyses

Results from all experiments were analyzed using the computer-based statistical analysis system, version 6.08 (SAS Institute Inc. 1989). All data were analyzed for the assumption of normality and heterogeneity. The general linear model was used to fit a regression model to % moisture; % crude protein; % crude fat; % ash; VSI; GSI; weight gain; and length gain, all of which were log transformed. The treatment and control groups were compared on the dependent variable previously listed using Duncan’s post hoc multiple F-test. All de- cisions on significance were made at the P < 0.05 level.

Log-logit transformations were applied to the standard curves of all GH assays (Rodbard 1974), and were analyzed using ANOVA.

Results

Serum Growth Hormone

Serum GH levels obtained 24 h post-injection for Mossambique tilapia receiving a single injection of bGHRH at 0.1 p,g/kg, 1 .O pg/kg, and 10.0 p,g/kg, were significantly higher than control and sham serum growth hormone levels (Table 1). There was a dose-response relationship between b- GHRH and serum GH levels 24 h post-injection.

Mossambique tilapia given 10 p g k g bGHRH via cholesterol implants had serum GH levels significantly higher 24 h and 2 wk post-implantation when compared with all other treatments (Table 2). Serum GH levels in fish given doses of 0.1 and 1.0 p,g/ kg bGHRH were not significantly different from controls throughout the sampling pe-

TABLE 2. Mean _f SEM of serum GH levels for female Oreochromis mossambicus implanted with cholesterol implants containing one of three dosages of bGHRH versus sham and non-injected controls at 0, I , 3, and 24 h, and I . 2, and 3 wk post-implantation. Values in the same row not sharing the same letter are signiJcantly different (P < 0.05).

Treatments

Time Control bGHFUIO. 1 bGHRHl .O bGHRH 10.0 Sham

Oh 0 . 1 0 2 0 . 0 2 A 0 . 1 0 % 0 . 0 1 A 0 . 1 0 2 0 . 0 4 A 0 . 4 5 t 0 . 3 1 A 0 . 3 5 2 0 . 2 7 A 3 h 1.65 t 0.65 A 0.65 t 0.25 A 0.75 ? 0.43 A 0.75 ? 0.27 A 1.15 % 0.21 A

24 h 1.45 % 0.80 A 0.50 % 0.14 A 0.75 2 0.31 A 3.00 t 2.10 B 1.15 -t 0.36 A 1 wk 1.60 -t 0.80 A 1.25 ? 0.25 A 0.95 % 0.24 A 2.25 % 1.20 A 1.75 t 0.90 A 2 w k 2 . 1 5 ? 0 . 4 0 A 1 . 7 5 t 0 . 6 0 A 1 . 4 5 t 0 . 5 0 A 3 . 2 0 2 0 . 6 0 B 2 . 5 0 ? 0 . 1 3 A 3 w k 1 .00%0.09A 0 . 8 5 % 0 . 1 0 A 1.60%0.95A 2 . 0 5 5 1.10A 2.20% 1.30A

a Values accompanying treatment abbreviations are concentrations of the hormone in pgkg. N size for all treatment groups at each time period was 5.

390 KELLY ET AL.

TABLE 3. Serum G H levels for female Oreochromis mossambicus implanted with silastic implants containing one of three dosages of b G H R H alone, or one of three dosages of b G H R H plus MT. or one of three dosages of b C H R H plus T3 and DEX versus sham, MT, Tj, DEX and non-implanted controls at 0, I , 3, and 24 h, and I , 2, and 3 wk post-implantation. Values are means _f SEM. Values in the same column not sharing the same letter are signijicuntly different (P < 0.05).

Time

Treatment” O h 3 h 24 h I wk 2 wk 3 wk ~ ~ _ _ _ _ ~~

Control Sham bGHRHO.1 bGHRH1.O hGHRHIO.0 MT bGHRHO. 1 MT bGHRH 1 .OMT bGHRHIO.OMT T, DEX bGHRHO. lTDh bGHRHI.OTD bGHRH1O.OTD

~ ~~~~

0.15 f 0.50 A 1.15 f 0.95 A 1.40 f 0.30 A 0.30 2 0.20 A 0.80 f 0.05 A 1.50 f 0.05 A 1.15 f 0.75 A 1.25 2 0.20 A 0.80 f 0.50 A 0.55 f 0.35 A 1.00 2 0.20 A 0.80 f 0.50 A 0.95 2 0.45 A 1.95 f 0.56 A 1.25 f 0.15 A 1.20 f 0.50 A 0.75 2 0.09 A 0.80 f 0.10 A 0.50 f 0.20 A 0.10 f 0.30 A 0.65 f 0.25 A

0.90 2 0.10 A 0.75 f 0.60 A 7.00 f 3.90 C 0.90 ? 0.20 A 1.95 f 0.45 A 0.15 f 0.05 A 0.90 f 0.45 A 0.60 f 0.15 A 0.90 ? 0.35 A 1.05 f 0.35 A 0.85 f 0.25 A 0.75 f 0.25 A 0.70 f 0.20 A 0.60 f 0.15 A 1.90 f 0.55 A 0.90 f 0.55 A 1.25 f 0.60 A 1.65 2 0.35 A

0.70 2 0.10 A 0.10 f 0.04 A 0.65 f 0.07 A

1.60 f 0.80 A 2.15 f 0.40 A 1.00 f 0.09 A 0.95 2 0.10 A 1.10 f 0.20 A 1.50 ? 0.50 A 0.40 f 0.05 A 1.15 2 0.15 A 1.75 2 0.45 A 1.25 2 0.75 A 1.55 f 0.25 A 0.85 f 0.45 A 1.00 2 0.05 A 3.15 2 0.75 B 4.35 2 0.35 C 1.35 ? 0.45 A NIA NIA 1.70 f 0.25 A NIA NIA 1.70 f 0.05 A NIA NIA 1.35 f 0.45 A NIA NIA 1.35 f 0.60 A NIA NIA 1.10 f 0.45 A 0.90 f 0.25 A NIA 1.35 f 0.45 A 1.70 f 0.30 A NIA 1.20 f 0.35 A 0.90 f 0.25 A NIA 1.20 f 0.45 A 2.10 f 0.65 A NIA

* Values accompanying treatment abbreviations are concentrations of the hormone in p&g for bGHRH. MT

h T = T,; D = DEX. concentrations utilized were 60 mglkg. N size for all treatment groups at all time periods is 5.

riod, indicating that a threshold level must be reached prior to increases in GH levels.

Female Mossambique tilapia serum GH levels were significantly higher 2 and 3 wk post-implantation for 10 p g k g bGHRH administered via Silastic implants when compared with controls and other treatments of bGHRH alone and combinations of b- GHRH with thyroid hormone and glucocorticoid (Table 3). Female Mossambique tilapia administered 10 pg/kg bGHRH via Silastic implants revealed a less random pattern of serum GH concentrations than did females given 10 p g k g bGHRH via cholesterol implants. Two weeks post-implantation serum GH levels for Silastic implanted fish increased to 3.15 2 0.75 ng/mL and increased further to 4.35 2 0.35 ng/mL 3 wk post-implantation. However, fish implanted with cholesterol implants had significantly higher serum GH concentrations at 24 h and 2 wk post-implantation. This indicates that the Silastic implant released the hormone in a relatively uniform manner,

whereas the cholesterol implants may release significant quantities at various times.

Female Mossambique tilapia having Si- lastic implants of 10 pgkg bGHRH plus MT had significantly higher serum GH concentrations 24 h post-implantation when compared with other treatments (Table 3). GH concentrations 24 h post-implantation were 7.0 2 3.9 ng/mL for fish receiving 10 p g k g bGHRH plus MT, which is 3.7 to 10 times higher than the serum GH concentrations obtained for the other treatment groups. Fish receiving the combination of 10 p g k g bGHRH plus MT had serum GH concentrations 5.6 times higher than fish receiving 10 p g k g bGHRH alone.

The use of thyroid hormone T, or the glucocorticoid DEX in combination with bGHRH did not increase serum GH levels significantly, but both appeared to suppress GH perhaps by depressed stimulation by bGHRH. Female Mossambique tilapia implanted with 10 p g k g bGHRH had significantly higher serum GH concentrations

MAMMALIAN GHRH AND GROWTH OF TILAPIA 39 1

TABLE 4. Weight ( g ) and length (mm) increases f o r small Oreochromis aureus X 0. niloticus (IR.5 * 2.5 g) implanted with silastic implants containing either I0 p g k g bCHRH, 100 pgkg bCHRH, MT. I0 pgkg bCHRH plus MT. or 100 pgkg bCHRH plus MT versus sham and non-implanted controls. Fish were maintained at 18 C. Values are means _f SEM. Values in the same column not sharing the same letter are significantly diflerent (P < 0.05).

Time (wk)

4 6 8

Treatment.' Weight Length Weight Length Weight Length

Control 1.7 I 0.2 A 1.4 2 0.8 A 1.7 I 0.4 A 3.0 2 0.6 A 4.4 5 0.5 A 4.0 2 0.7 A Sham 1.4 I 0.5 A 2.4 2 0.2 A 2.2 2 0.4 A 3.0 2 0.5 A 4.0 2 0.5 A 4.6 I 0.9 A bGHRH 10 2.5 2 0.4 B 1.3 I 0.9 A 3.5 2 0.2 B 6.0 2 0.6 B 4.4 2 0.4 A 11.0 2 1.9 B bGHRH 100 2.9 2 0.7 B 3.6 2 0.5 A 3.5 I 0.6 B 6.0 2 0.8 B 4.2 5 1.8 A 5.0 2 1.3 A MT 1.7 I 0.4 A 2.8 2 0.7 A 3.0 2 0.8 B 3.3 2 1.3 A 4.3 2 0.8 A 6.8 2 1.4 A bGHRHlOMT 2.9 2 0.7 B 2.0 I 1.0 A 3.3 2 1.0 B 2.5 I 1.5 A 4.2 2 0.5 A 9.0 2 1.0 B bGHRH100MT 3.2 2 0.3 B 1.9 2 0.9 A 3.9 2 0.7 B 5.4 2 1.0 B 5.0 2 1.2 A 7.0 I 0.9 A

a Values accompanying treatment abbreviations are concentrations of the hormone in pglkg for bGHRH. MT concentrations utilized were 60 m@g. N sizes for all treatment groups at all time periods was 10.

when compared with fish implanted with 10 p g k g bGHRH plus T, and DEX at 2 and 3 wk post-injection (Table 3).

White Tilapia Growth Studies

White tilapia, ranging from 16-21 g initial weight and maintained at 18 C had significantly greater weight increases when treated with either 10 p g k g bGHRH or 100 p g k g bGHRH when compared with controls or sham implanted fish at 4 and 6 wk post-implantation (Table 4). At 4 wk post- implantation, weight increases ranged from 1.8 to 2.0 times higher for 10 p g k g b- GHRH and 100 p g k g bGHRH when compared with controls and sham implanted fish. At 6 wk post-implantation, weight increases of 1.3 to 1.4 times higher were obtained for 10 Kgkg bGHRH and 100 pg/ kg bGHRH, respectively, when compared to all other treatment groups. By week eight no significant differences existed between any of the treatment groups.

Fish implanted with any dosage of b- GHRH plus MT had significantly greater weight increases (2.1 to 2.3 times) when compared with controls and fish implanted with MT alone up to week 6 of the growth study, at which time fish treated with MT alone were also significantly different from the controls (Table 4). At week 8, no sig-

nificant differences in weight increases existed between any of the treatment groups.

Length increases were not significantly different until 6 wk post-implantation (Ta- ble 4). Fish implanted with 10 p g k g b- GHRH increased 2.0 times the lengths obtained for controls and sham implanted fish. Fish implanted with 100 p g k g bGHRH plus MT had increased 1.7 to 2.2 times more than all the controls or other MT treatment groups. However, 8 wk post-implantation, fish implanted with 10 pg/kg bGHRH and 10 p g k g bGHRH plus MT had significantly greater length increases when compared with other treatment groups. Length increases were 1.5 to 2.8 times longer than the other treatment groups.

White tilapia averaging 30-35 g and 25- 30 g initial weight and maintained at 24 C and 30 C, respectively, did not significantly differ in weight or length increases throughout a 12 and 8-week study comparing non- implanted controls, 10 p g k g bGHRH implanted fish and sham implanted fish.

White tilapia, ranging from 44-50 g initial weight, implanted with either 10 p g k g bGHRH, 100 p g k g bGHRH, MT, 10 pg/ kg bGHRH plus MT or 100 p g k g bGHRH plus MT and maintained at 18 C, also had significantly greater increases in weight

392 KELLY ET AL.

TABLE 5. Weighr ( g ) and length (mm) increases for large Oreochromis aureus X 0. niloticus (47.0 f 3.0) implanred wirh silasric implants containing either I0 pgkg bGHRH, 100 pgAg bGHRH, MT, I0 pgkg bGHRH plus MT, or 100 pgkg bGHRH plus MT versus sham and non-implanted controls. Fish were maintained at 18 C. Values are means _f SEM. Values in the same column not sharing rhe same letter are significantly differenr (P < 0.05).

Time (wk)

4 6 8

Treatment" Weight Length Weight Length Weight Length

Control 3 . 0 2 0 . 1 A 0 . 7 2 0 . 8 A 5 . 4 2 1 .4A 3 . 7 2 0 . 3 A 9 . 6 2 1 . 8 A 4 . 7 2 0 . 5 A Sham 3 . 0 % 0 . 5 A 1 . 3 2 0 . 6 A 5 . 4 ? 1 . 2 A 2 . 7 2 0 . 1 A 8 . 5 ? 2 . 6 A 4 . 3 r t 0 . 4 A bGHRHlO 4.5 ? 0.5 B 2.3 2 0.1 B 7.5 2 1.2 B 3.0 2 0.3 B 8.4 2 3.7 A 6.3 2 1.3 B bGHRHlOO 4 . 7 2 1 .0B 4 . 7 % 1.OC 6 . 6 2 0 . 9 B 6 . 6 2 0 . 9 C 1 2 . 2 2 2 . 6 A 1 1 . 2 2 1 . 6 C MT 5.8 ? 0.8 B 1.0 2 0.7 A 7.1 L 1.4 B 2.6 2 0.9 A 9.6 rt 2.1 A 5.0 2 0.2 A bGHRHlOMT 5.8 2 1.0 B 2.3 2 0.3 B 7.0 2 1.2 B 4.5 rt 0.8 B 10.7 rt 1.3 A 8.5 rt 2.1 B bGHRHlOOMT 5.2 t 0.6 B 4.3 2 0.5 C 6.6 rt 1.0 B 5.8 2 0.5 C 11.5 2 2.4 A 10.7 2 0.9 C

a Values accompanying treatment abbreviations are concentrations of the hormone in F a g . MT concentration was 60 m a g . N size for all treatment groups at all time periods is 10.

through week 6 of the study when compared with controls (Table 5 ) . Fish implanted with either 10 p g k g bGHRH or 100 pg/ kg bGHRH had weight increases of 1.2 to 1.5 times the weight increases for non-implanted and sham implanted controls through week 6.

No significant differences in weight increases existed throughout the study between white tilapia implanted with MT, 10 p g k g bGHRH, 1 0 0 pgkg bGHRH, 10 pg/

GHRH plus MT However, white tilapia implanted with 100 p g k g bGHRH and 100 p g k g bGHRH plus MT had significantly greater increases (2.0 to 6.1 times, respectively) in length when compared with control fish or fish implanted with 10 pg/kg bGHRH, 10 pgkg bGHRH plus MT and MT alone 4 wk post-implantation.

Significant differences in length increases for fish implanted with 100 p g k g b- GHRH or 100 pgkg bGHRH plus MT existed when compared with all other treatments throughout the study (Table 5) . The greatest differences in lengths occurred at 4 wk post-implantation; length increases were 1.9 to 6.7 times higher for 1 0 0 pg/kg b- GHRH fish compared with all other treatment groups. At week six of the study, length increases for 100 p g k g bGHRH

kg bGHRH PIUS MT and 100 p g k g b-

were 1.8 to 2.4 times greater than all other treatment groups, which is significantly less than the increases seen at week 4. Values for 1 0 0 p g k g bGHRH 8 wk post-implantation were 1.8 to 2.6 times higher than all other treatment groups, which is similar to the difference in weight increases seen at week 4.

Proximate Analyses

Carcass compositions for white tilapia maintained at 18 C were significantly different between the controls and all other treatment groups with the exception of the % ash content for empty carcass composition (Table 6). In whole body composition analyses, fish implanted with 10 p g k g bGHRH, 1 0 0 pg/kg bGHRH, MT, 10 pg/ kg bGHRH plus MT or 100 p g k g bGHRH plus MT had significantly higher % moisture (1.90-3.33%) and % protein (1.45- 2.83%), but significantly lower % fat (1.42- 2.25%) and % ash (2.08-3.14%) content.

Empty carcass composition analyses demonstrated that fish implanted with 10 pgkg bGHRH, 100 pg/kg bGHRH, MT, 10 p g k g bGHRH plus MT and 100 p g k g bGHRH plus MT had significantly higher % moisture (2.33-2.91%) and % protein (1.64-4.28%) when compared to controls. The % fat in the controls (range 5.81-5.93)

MAMMALIAN GHRH AND GROWTH OF TlLAPlA 393

TABLE 6. Proximate analyses of Oreochromis niloticus X 0. aureus reared at 18 C and treated with either a mammalian releasing factor (bGHRH) andor I7 a-methyliesiosierone (MT). Percen rages ore based on dry weights. Values not sharing rhe same letters within a column are significantly different (P < 0.05).

Treatment’ % Moisture % Protein % Fat % Ash

Whole body composition Control 70.55 A 15.15 A 7.27 A 7.15 A Sham 70.73 A 15.64 A 6.67 A 7.21 A bGHRH 10 72.45 B 17.08 B 5.85 B 4.69 B bGHRH100 72.76 B 16.60 B 5.43 B 5.13 B MT60 72.84 B 17.98 B 5.21 B 5.02 B bGHRH IOMT60 73.23 B 17.74 B 5.29 B 4.07 B bGHRH100MT60 73.88 B 17.29 B 5.02 B 4.41 B

Empty carcass Control 71.55 A 15.62 A 5.93 A 4.19 A Sham 71.03 A 15.23 A 5.81 A 4.24 A bGHRHlO 74.46 B 17.59 B 4.41 B 4.05 A bGHRH 100 74.18 B 17.26 B 4.73 B 3.90 A MT60 73.44 B 17.81 B 4.01 B 3.42 A bGHRH 1 OMT60 73.81 B 19.07 B 3.89 B 3.89 A bGHRH 100MT60 73.36 B 19.90 B 3.82 B 3.74 A

a Values accompanying treatment abbreviations indicate p g k g of bGHRH and mgkg of MT utilized. N size for each treatment group is 5.

were significantly higher than all other treatments (range 3.82-4.73), while no significant differences in the % ash content were detected.

Effects of bGHRH on Visceral Somatic and Gonadosomatic Indices

There were no significant differences in the VSI for males or females in either group of white tilapia utilized in the 18 C growth study.

The GSI values were significantly higher for females treated with bGHRH or b- GHRH plus MT in both the small and large treatment groups (Table 7). In the small group, female GSI for fish treated with bGHRH or bGHRH plus MT values were 4.6 to 11.2 times the values obtained for all other treatment groups. Female GSI values in the large group for fish treated with bGHRH or bGHRH plus MT were 1.6 to 2.5 times higher than the other treatment groups. The GSI values for males did not differ significantly between any treatment in either the small or large group (Table 7).

Discussion

The bGHRH, DHP’D AO-PrebGHRH( 1 - 78)OH, stimulated GH releases in vivo as well as increased somatic growth at below optimum temperatures. Peter et al. (1984) demonstrated previously that serum GH increases in goldfish injected with a hGHRH. The similarities between fish and mammalian GH and GHRH are well documented. The releasing factor utilized in this study, bGHRH, was a hybrid between an analog of GHRH (44 aa) and a human GHRH C-peptide (1 -44 aa) which is similar to carp GHRH. This hybrid was originally produced to prevent rapid degradation of GHRH by E. coli proteases (Smith et al. 1992). The resulting hybrid GHRH, is not as degradable by blood enzymes as compared to other unprotected mammalian GHRHs (Mark Heiman, Lilly Research Labs, personal communication). Therefore, the increases in GH observed in serum of Mossambique tilapia were not unexpected. Another observation is that bGHRH is capable of stimulating GH release for several

394 KELLY ET AL

Table 7. Gonadosomatic indices for Oreochromis niloticus X 0. aureus mainrained at 18 C and treated with either a mammalian releasing facror (bCHRH) andor I7 a-methyltestosterone (MT). Values are means 2 SEM. Values not sharing the same letiers within a column are significantly different (P < 0.05).

Treatmenta

Small group Large group (18 t 2.5 g) (47 2 3.0 g)

Female Male Female Male

Control MT60 bGHRHlO bGHRHlOO bGHRH 1 OOMT bGHRH 1 OMT Sham

0.45 ? 0.27 A 0.61 2 0.34 A 1.81 2 0.81 A 0.99 2 0.93 A 0.25 2 0.20 A 0.47 2 0.43 A 1.52 t 0.10 A 0.51 t 0.29 A 2.81 2 0.24 B 0.31 2 0.20 A 2.98 2 0.34 B 0.44 -t 0.36 A 2.06 2 0.40 B 0.15 ? 0.08 A 3.46 2 0.51 B 0.56 2 0.53 A 2.48 ? 0.35 B 0.25 ? 0.14 A 3.81 2 0.53 B 0.28 t 0.25 A 2.76 ? 0.57 B 0.48 -t 0.29 A 0.43 2 0.31 A 0.36 2 0.28 A 1.71 5 0.56 A 0.43 2 0.33 A

0.57 2 0.55 A 3.12 2 0.32 B

~

a Values accompanying treatment abbreviations are concentrations of the hormones in figkg body weight for bCHRH and mgkg body weight for MT. For treatment where MT is utilized in conjunction with bGHRH, the dosage utilized was 60 mgkg body weight. N size for all treatment groups by sex is 5.

weeks in fish with Silastic implants containing bGHRH at 1.0 and 10 p g k g body weight.

Differences in growth, weight, and length increases between fish reared at 18 C, 24 C, and 30 C and implanted with bGHRH suggest that pituitary hormonal synthesis and secretion, and tissue responsiveness may be temperature sensitive. Studies in which bovine GH (bGH) was administered to black bullhead catfish (Kayes 1977) and common carp (Adelman 1977, 1978) at different temperatures revealed significant increases in length and weight of fish receiving bGH and maintained at temperatures below and above optimum. Adelman (1 977, 1978) concluded that there was a decrease or inhibition of endogenous GH production at higher temperatures enabling the effects of the exogenous GH to be measured. An attempt to repeat Adelman’s (1978) study was unsuccessful which may be attributable to study length, fish stock, hormone preparation, or hormone type utilized.

While this study utilized bGHRH rather than bGH, significant increases in weight and length for treated fish were obtained at 18 C. This would suggest that GH secretion, although it may be diminished, can be stimulated at below optimal temperatures. However, at temperatures above optimum, 30 C, endogenous GH secretion is either

inhibited, reduced to such minute amounts that it is not detectable above background levels, or is metabolized at a high rate making detection difficult.

Studies by Pickford (1957, 1959) and Swift and Pickford (1965) also suggested that temperature influences endocrine-mediated growth. These studies demonstrated that the growth-promoting activity of the pituitary in the perch Perca Juviatifis began to increase above winter levels in early spring, with activity peaking in June, a month prior to the period of maximum growth. Swift and Pickford (1965) suggested that rising water temperatures and/or increased day length stimulated perch GH synthesis.

Failure of hypophysectomized bullheads (Kayes 1977) and Fundulus (Swift and Pickford 1965) treated with bGH to grow in length at suboptimal temperatures suggests reduced levels of tissue responsiveness to low levels of bGH.

Serum concentrations of GH in Mossam- bique tilapia were not significantly different from controls when fish were treated with T, or DEX (a glucocorticoid) alone or in combination. Serum GH concentrations were not enhanced by T3 or DEX when utilized in combination with bGHRH, but ac- tually decreased serum GH concentrations. This is partially consistent with results re-


ported by Luo and McKeown (1991). They demonstrated that lower dosages of DEX alone had no effect on GH release; lower dosages of T, alone had marginal effect on GH release; but higher dosages of DEX and T, potentially reduce GH release in rainbow trout. They also demonstrated that T, and DEX when used in conjunction with carp GHRH increased GH secretion in cultured carp pituitary cells. However, in the present study, serum GH secretion was not increased. The reason for the difference between these studies may be due to differences in species, differences in naturally occurring iodogens in the diet, fish size, time of year, or the fact that this was an in vivo versus an in vitro study (Hopper 1952; Bar- rington et al. 1961; LaRoche et al. 1966; Narayansingh and Eales 1975). The utilization of T, and DEX in conjunction with bGHRH is likely a dose-dependent phenomenon in Oreochromis, or there may be some other hormonal or enzyme interaction occurring with T, or DEX in the white tilapia. While the dosage in this study failed to elicit an increase in serum GH levels, further studies are necessary to determine their potential as a GH stimulant.

While injections of bGHRH allowed for immediate measurement of increments of GH in fish serum, it did not allow for ob- servations over an extended period of time. Prolonged hormone release was accom- plished through the utilization of cholesterol and Silastic implants. Weil and Crim (1983) compared various ways of admin- istering LHRH-a to salmon and concluded that a single hormone implant made from either cholesterol or Silastic tubing was equally effective as frequent injections of LHRH-a.

The Silastic implant is utilized mainly for the administration of steroid hormones (Crim 1985). Therefore, to examine the effects of bGHRH on GH release over an extended period of time while minimizing stress in fish, cholesterol and Silastic implants were utilized.

The release of bGHRH from cholesterol

and Silastic implants appeared to differ. The cholesterol implant containing 10 kg/kg bGHRH caused peaks in GH at 24 h and 2 wk post-implantation. While serum GH measured at these times were significantly higher than those in the controls and sham implants, the overall levels (3.0 ng/mL and 3.2 ng/mL) were not much above background levels. The Silastic implants caused a peak in GH at 2 wk and continued to rise 3 wk post-implantation. While the response in elevated serum GH levels was delayed compared to the cholesterol implants, the concentrations of GH were about 1.5 times greater than those obtained for cholesterol implants. Sherwood et al. (1988) examined the rate of GnRH release from cholesterol pellets and demonstrated that only 36-38% of the hormone utilized (LHRH in her study) was released by day 28. Tamaru et al. ( 1 990) examined the release rate of three steroids from Silastic tubes and demonstrated that the steroids were released for at least 21 d, which was the duration of the study, and that the release rate from the Silastic tubes was constant.

The advantage of implants is that they offer protection of the hormone from enzyme degradation over long periods of time. The animal receiving the implant is not continually stressed from the handling associated with the injection protocols, and the animal does not receive a bolus dose but a slow continuous dose of the hormone over time. Thyrotropin-releasing hormone and LHRH release have been demonstrated from silicon implants in rats (Lotz and Syll- wasschy 1979). In fish, Shelton (1982) demonstrated that testosterone was released from Silastic implants for a period of a year when he utilized this technology to produce monosex populations of grass carp.

The release of bGHRH from cholesterol pellets may be too slow to see greater changes in serum GH concentrations. It appeared that the Silastic implant allowed for diffusion of bGHRH into the blood, thus allowing elevated levels of GHRH and resulting in GH release. It may well be that

396 KELLY ET AL

GH could have continued to increase or fall back to background levels if the study had been continued.

The % CP was increased significantly in white tilapia receiving either bGHRH, MT or a combination of the two hormones when compared to controls. Since in white tilapia bGHRH had increased serum GH concentrations, the increase in carcass protein levels were not totally unexpected. Higgs et al. (1975, 1976) demonstrated that the protein content of bGH-treated coho salmon was substantially higher than observed in the controls. Enomoto (1964) demonstrated that the ratios of CP to crude ash in whole body composition of rainbow trout treated with bGH were larger than controls. Total plasma protein was reduced in the sculpin Cottus scorpius after being treated with mammalian GH (Matty 1962). These studies indicate that GH treatment creates a positive nitrogen balance in fish due to the stimulatory effects that GH has on protein synthesis (Donaldson et al. 1979). While this study did not utilize GH directly, the increase in protein could be an indirect effect of the bGHRH. Since b- GHRH resulted in increased serum GH levels, this should have resulted in stimulation of protein synthesis.

Fish treated with bGHRH also had significantly lower levels of lipids in whole body and empty carcass composition suggesting reduced fat deposition. Higgs et al. (1975, 1976, 1977) and Markert et al. ( 1977) demonstrated that the percentage of lipid in the muscle of coho salmon declines following bGH treatment. Clarke (1976) also found that bGH treatment decreased whole body lipid of underyearling sockeye salmon. Frye ( 1 97 1) and Snyder and Frye (1972) suggested that more of the amino acids would be available for growth since lipid would be preferentially utilized as an energy source. This would result in higher protein level and lower lipid level in the treated fish. We believe that the increased protein and decreased lipid levels are a re-

sult of increased endogenous GH levels in bGHRH-treated fish.

The fish treated with MT and bGHRH plus MT exhibited patterns of protein and lipid deposition that are similar to those observed in fish treated only with bGHRH. Killan and Kohler (1991) found increased protein and decreased lipid percentages in the hybrid red tilapia Oreochromis mossambicus X 0. niloticus which were treated with MT. In salmonids, feeding of MT generally reduced body fat while increasing weight gain (Fagerlund et al. 1979). How- ever, treating tilapia with both MT and bGHRH did not significantly decrease the amount of body fat and increase the amount of body protein over fish treated with either MT or bGHRH alone, suggesting the two hormones do not work synergistically. This could also be a matter of dose, if effects of either hormone alone were at or near maximum responses.

The VSI of bGHRH-treated fish did not significantly differ from the control group, although lower values were obtained for all hormone-treated fish. This suggests that the weight gain was not due to increases in weight of the viscera. These results differ from those obtained by Killan and Kohler (1991) and Lone and Matty (1980). both of whom examined the effects of MT alone. The difference could be associated with the effect of bGHRH on GH release.

The GSI for females in both the small and large group were significantly higher when treated with bGHRH or bGHFU-I plus MT This suggests that bGHRH may indirectly cause some gonadotropic activity. The pos- sible involvement of GH in the regulation of ovarian follicular growth and development in mammals has been suggested by several researchers (Jia et al. 1986; Hutchinson et al. 1988; Manson et al. 1990). The growth promoting effects of GH are mediated by insulin growth factor-I (IGF-I) as well as other poly- peptides that are released from several isolated cells and tissues (Davoren and Hsueh 1986; Hsu and Hammond 1987; Baxter 1988). Recent studies indicate that GH aug-


ments the response of the human ovary to stimulation by gonadotropins (Homburg et al. 1988; Blumenfeld and Lunenfeld 1989; Owen et al. 1991). The same phenomenon may be occurring in fish. It is established that bGHRH increases serum GH levels in fish and this increase may augment the response of the fish ovary to stimulation by endogenous gonadotropins. The tilapia treated with MT had depressed GSI values, suggesting MT may inhibit gonad development. How- ever, tilapia treated with bGHRH plus MT had higher GSI values than those treated with MT alone, indicating that bGHRH may over- ride the effect of MT in white tilapia.

This study demonstrated that a mammalian GHRH increased serum GH levels in a teleost fish, and that it increased somatic growth at suboptimum temperatures. GSI values in female tilapia were significantly higher in bGHRH-treated fish than in non- treated fish, indicating that bGHRH may affect the reproductive process.

Acknowledgments This article i s based, in part, upon re-

search conducted by the senior author for the Doctor of Philosophy degree in the De- partment of Zoology, Southern Illinois Uni- versity at Carbondale, Carbondale, Illinois. We thank Dr. T. Sakamoto of the University of Hawaii Institute of Marine Biology for conducting the growth hormone radi- oimmnoassays. This research was funded by Eli Lilly Research Laboratories. We thank the Edwin J. Pauley Summer Pro- gram at the Hawaii Institute of Marine Bi- ology and the Indiana-Illinois Sea Grant Program for providing travel support for this research. We thank W.L. Muhlach and A. Bartke for providing review comments.

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