Growth performance studies in transgenic Cyprinus carpio

Aquaculture 173 Ž1999. 285–296

Growth performance studies in transgenic Cyprinus carpio Yaniv Hinits ) , Boaz Moav Department of Zoology, Faculty of Life Sciences, Tel AÕiÕ UniÕersity, Ramat AÕiÕ 69978, Israel Accepted 14 October 1998

Abstract Two growth experiments with transgenic common carp Ž Cyprinus carpio . containing growth hormone ŽGH. gene constructs were conducted in earthen ponds using the communal testing method. The first experiment was performed in summer under optimal growth conditions Žhigh ambient temperatures and optimal feeding.. It tested three groups of F2 transgenic carp containing the FV-1rcsGH gene construct Žthe common carp b-actin proximal promoter fused to the chinook salmon growth hormone cDNA. from different integration events, and a non-transgenic control group. No significant difference in growth rates was found between the transgenic groups and the control. The second experiment was performed in winter under low ambient temperatures and minimal feeding. It compared three groups of fish: one group of F1 transgenic carp had the FV-1rcsGH gene construct; another the FV-2rcGH gene construct—a complete common carp b-actin promoter ŽLiu et al., 1990. fused to the common carp growth hormone cDNA; and the third was a non-transgenic control group of the commercial carp in Israel. In this experiment, the two transgenic groups showed higher growth rates as compared to the control Ž P - 0.001.. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Transgenic fish; Cyprinus carpio; Growth performance; Growth hormone

1. Introduction A major goal in introducing new genes into the fish genome is to establish new, improved commercial strains for use in aquaculture. Since the mid 1980s many studies have introduced growth hormone genes and growth-related genes from mammalian or piscine sources fused to a variety of promoters, into fish Žsee reviews by Hackett, 1993; )

Corresponding author

0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 4 5 2 - 9

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Gong and Hew, 1995; Iyengar et al., 1996.. Expression of GH transgenes has been documented ŽZhang et al., 1990; Du et al., 1992; Devlin et al., 1995., but very few studies have tested transgenic fish in reliable growth trials. Growth rate of fish depends on temperature, food and density; in addition, size heterogeneity is very common in fish culture ŽWohlfarth and Moav, 1972; Chen et al., 1993.. Iyengar et al. Ž1996. noted that firm evidence for enhanced growth as a direct result of the circulating novel GH can only be obtained by carrying out effective growth trials. Upon examining studies claiming the establishment of fast growing transgenic fish, we found that some of them had used the F0 generation Že.g., Gross et al., 1992.. This generation is very heterogeneous and includes non-transgenic fish and mosaic fish that differ from one another. Because establishing a family of transgenic fish requires the breeding of one of the transgenic individuals, the performance test must be at the F1 or F2 generation to have significance regarding future use of a particular genotype of transgenic fish. Other studies used aquaria rather than aquaculture systems Že.g, Du et al., 1992.. Using separate aquaculture ponds requires many repetitions because ponds have been found to vary greatly even if built adjacent to each other on a uniform soil substrate ŽWohlfarth and Moav, 1985.. In testing growth performance by the communal testing method, however, only genetic differences between groups of fish are measured while environmental conditions are common for all groups. The number of ponds needed for this method is thus reduced significantly ŽWohlfarth and Moav, 1985.. In communal testing, a comparison must be made between the weight gains of the tested genotypes, and it is sometimes necessary to correct the observed weight gains in order to diminish the effects of differences in initial weights ŽWohlfarth and Moav, 1985.. Testing final weights Že.g., Martinez et al., 1996. that are clearly affected by the initial weights may dramatically change the experiment conclusions. Reliable quantitative growth tests are needed to assay differences between different genetically engineered genotypes. In the present work we study the growth performance of a second and a third generation of transgenic common carp Ž Cyprinus carpio . carrying different ‘all-fish’ growth hormone transgenes under conditions common in aquaculture. 2. Materials and methods 2.1. Microinjection, fish maintenance and breeding of F0 transgenic carp Common carp ŽYugoslavian or Dor 70 strains. eggs and sperm were provided by Dr. S. Rothbard ŽGan Shmuel Fish Breeding Center.. After fertilization the carp zygotes were microinjected with plasmids carrying the transgenes as described previously ŽMoav et al., 1992.. After hatching, larvae were transferred to glass aquaria and kept for 3–4 weeks and then stocked in ponds at the Aquaculture Center in Dor until they reached a size Žabove 20 g. that allowed individual tagging with PIT tags ŽDestronrID, Biosonics. and sampling. For breeding, selected sexually mature transgenic and non-transgenic carp were transferred to indoor plastic tanks at Yafit laboratory, the Gan Shmuel Fish Breeding Center. The fish were induced to spawn by GnRH injection ŽDrori et al., 1994., and artificial insemination was performed according to Rothbard Ž1981..

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2.2. Preparation of the transgenes FV-1rcsGH ŽFig. 1a. was prepared as described in Moav et al. Ž1995.. FV-2rcGH ŽFig. 1b. contains the complete common carp b-actin promoter fused to the carp growth hormone ŽcGH. cDNA. The source of the common carp b-actin promoter was construct a6 of Liu et al. Ž1990.. The construct was sequentially cut with KpnI Žthe site is located between the b-actin promoter and the CAT gene., the protruding 3X termini were filled in with T4 DNA polymerase, and the construct was digested with EcoRI to remove the CAT gene. The construct was then dephosphorylated and ligated to the 1.2 kb piece cut with EcoRI from the pBRcGH-carp growth hormone cDNA ŽKoren et al., 1989. obtained from Dr. V. Daniel, Department of Biochemistry, Weizmman Institute, Re-

Fig. 1. Ža. The FV-1rcsGH plasmid. Diagram of the plasmid FV-1rcsGH containing the proximal promoter of the carp b-actin gene fused to the chinook salmon growth hormone cDNA. Žb. The FV-2rcGH plasmid. Diagram of the plasmid FV-2rcGH containing the maximal promoter of the carp b-actin gene Žincluding the X 5 flanking sequence, the first axon and the first intron. fused to the carp growth hormone cDNA.

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hovot, Israel. The clones carrying the correct orientation were chosen by restriction analysis with HindIII and EcoRV. 2.3. DNA screening Screening the F0 , F1 and F2 transgenic generations was performed by polymerase chain reaction ŽPCR.. DNA samples were extracted from caudal fin, blood and sperm samples of adult carp as described previously ŽMoav et al., 1995.. The PCR reaction mixture Žfinal volume of 50 ml. contained 50–100 ng DNA, 1 unit of Dynazyme II DNA polymerase ŽFinnzymes Oy., Dynazyme buffer, 0.1 mM of the four dNTPs and 50 pmole of each primer. The PCR protocol used for both transgenes was as follows: 5 min denaturation at 958C followed by 30 cycles of 30 s at 928C, 30 s at 558C and 40 s at 728C and then a final elongation step for 7 min at 728C Ža similar PCR program was used for the 1994 summer experiment detailed in Moav et al., 1995.. To detect the transgene FV-1rcsGH we used the primers: 5X-CACTCGGTCAGCTGTTCGTGC-3X and 5X-GCGTCTCAGCCTCACTTTGAG-3X with a 331 bp expected PCR product. To detect FV-2rcGH we used the primers: 5X TAATGAGAATGCAGAGGG3X and 5X CAGAAAGACAGAGGGAAG3X with an expected 373 bp product. 2.4. Radioimmunoassay (RIA) RIA for cGH was performed according to Fine et al. Ž1996.. The procedure was modified by using as second antibody Kodak-Amerlex-M donkey anti-rabbit magnetic separation reagent for immunoassay ŽAmersham.. Rabbit anti-carp growth hormone antibodies were prepared in our lab as described previously ŽFine et al., 1993.. The plasma samples were separated from blood cells by centrifuging the fish blood in an eppendorf centrifuge for 10 min at 4000 rpm. 2.5. Communal testing experiments We used the communal testing method ŽWohlfarth and Moav, 1985. to compare the growth rates of different genetic groups of transgenic and non-transgenic carp stocked together in a series of communal ponds. The different genetic groups were transferred initially as larvae to separate earthen ponds of 400 m2 at the Aquaculture Research Station in Dor, Israel, for 1 month of nursing in the summer experiment and 7 weeks in the winter experiment. When the fish reached 50–100 g average weight Žin summer. or 20 g Žin winter., they were marked by hot branding with a low voltage electrically heated marking tool ŽMoav et al., 1960a,b.. The genotypes that were co-stocked in ponds were specifically marked to differentiate them clearly. The marked fish were then transferred to communal ponds Žfour ponds in the summer experiment and one pond in the winter experiment.. During the summer experiment ŽJuly–October 1994., the weather conditions were fair and water temperature was 25–298C. The fish were fed with a high-protein pelleted

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diet Ž40%., 5% of body weightrday. In the winter ŽDecember 1996–March 1997. water temperature ranged from 78C to 188C and the fish were fed a 20% protein pelleted diet, 0.5% of body weightrday. Each test group was weighed at the beginning of the experiment and then every 2 weeks for a period of 3 months in the summer test and every 4 weeks in the winter. The winter experiment had to be stopped after 3 months due to heavy predation by birds. The weighing was done by collecting a sample from each pond by net and then sorting the families by their different marks and weighing the fish. At both the onset of the experiment and its conclusion all of the fish were weighed. In the summer experiment the fish were weighed in groups and in the winter experiment they were weighed individually. The results of communal testing require correction of the observed weight gains due to differences in initial weights between the tested progenies. This must be performed if the results show a clear correlation between initial weight and weight gain of the tested groups ŽWohlfarth and Moav, 1985.. We calculated the correction factor Ž b . by using the prediction equation: b s 4.543 y 0.113 X.q 0.00671Y. generated from many empirical results of growth experiments in common carp using the multiple nursing method ŽWohlfarth and Milstein, 1987. Ž b s predicted correction factor, X.s mean initial weight, and Y.s mean weight gain.. Statistic analyses were performed by the ‘Statistica’ software. Specific growth rate Ž% SGRrday. was calculated as follows: % SGRrdays Žln W2 y ln W1 . = 100rt, where W1 Žg. was the initial weight, W2 Žg. was the final weight and t was the length Žin days. of the growing period.

3. Results 3.1. Summer 1994 experiment In a previous study ŽMoav et al., 1995., two ‘all-fish’ gene constructs FV-1rcsGH and FV-2rcsGH, were injected into carp zygotes obtained from a cross between Dor 70 male common carp and female Koi carp. Eight of the transgenic F0 carp that carried the transgene in their germ line Žas demonstrated by PCR screening. were used to establish the F1 generation by crossing with a Yugoslavian common carp. These F1 families had different percentages of transgenic offspring, due to different degrees of mosaicism in the germ cells of their F0 transgenic parents. The sexually mature F1 carp were screened by PCR to find non-mosaic transgenic individuals suitable to establish the F2 generation for the growth performance experiments. At the onset of the experiment we obtained transgenic F1 male carp that carried the FV-1rcsGH construct. Three of these, a90522, a77E22 and a30066, originating from different integration events were crossed with a Dor 70 female carp. For comparison, a Yugoslavian male carp was crossed with the same Dor 70 female carp; the resulting crossbred progeny are the commercial common carp variant in Israel and have been found to have better growth and improved qualities as compared to other carps ŽWohlfarth, 1993.. The four sets of progeny were each stocked in separate earthen ponds of 400 m2 at the Aquaculture Research Center in Dor, Israel. After 1 month of nursing in separate ponds, the fish were marked and co-stocked in four identical ponds for communal testing. Each pond was stocked as follows: 29 fish

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from crossing transgenic male a90522 with Dor 70 female, 41 fish from crossing transgenic male a77E22 with Dor 70 female, 60 fish from crossing transgenic male a30066 with Dor 70 female and 60 fish from crossing Yugoslavian male with Dor 70 female. The average weights of each transgenic family during the experiment and average weight gain from all four communal ponds are detailed in Table 1a. The correction of observed weight gains for the differences in initial weights Žincluding the calculation of the correction factor, b . is detailed in Table 1b. Comparison of the observed weight gains of the four groups in the test Žby t-test. shows that only the 90522= Dor 70 group weight gain Ž454.6 g. was significantly higher Ž P s 0.026. than the control group weight gain Ž378.2 g.. The other groups; Ž77E22= Dor 70 and 30066 = Dor 70. weight gains Ž416.6 g and 410.5 g. were not significantly different from the control Ž P s 0.128 and P s 0.125.. However, after the corrections were made, the differences in weight gains between 90522= Dor 70 Ž434.7 g., 30066= Dor 70 Ž426.1 g., 77E22= Dor 70 Ž375.9 g. group, and the control group Ž423.5 g. were insignificant by t-test Ž P s 0.686, P s 0.900 and P s 0.064, respectively.. Another way of comparing growth rates of the different groups is to calculate the % SGRrday. The % SGRrday of the control group exceeded the % SGRrday of all the transgenic genotypes ŽTable 1a.. 3.2. Winter 1997 experiment 3.2.1. DNA screening of the F0 generation The construct FV-2rcGH ŽFig. 1b. was microinjected into Yugoslavian = Yugoslavian common carp zygotes. Ninety-four of this F0 generation attained a size that allowed individual tagging with PIT tags. They were weighed and sampled Žfin clips and blood were collected from all fish, and sperm collected from the males.. Only 59 of these had survived by the time that PCR screening was performed. Of these fish, only eight were found to contain the transgene ŽPCR positive. in one of the tissues tested Ž13.5%., and only one transgenic male was found to contain the transgene in its sperm and was, therefore, fit to establish the next transgenic generation Žsee Table 2.. Notes to Table 1: a Corrected weight gains were compared by t-test. 90522=Dor 70 Ž P s 0.686., 77E22=Dor 70 Ž P s 0.900. and 30066=Dor70 Ž P s 0.064. were not significant compared to the control group ŽYugoslavian=Dor 70.. X Y sY y bŽ X y X... X Y sCorrected weight gain. Y sObserved weight gain Žfinal weightyinitial weight.. bsCorrection factor. X s Initial weight of each genetic group. X.s Mean initial weight of all groups. Y.s Mean weight gain of all groups. Yˆ s Mean observed weight gain of all groups. Prediction equation Žaccording to Wohlfarth and Milstein, 1987.: bs 4.543y0.113 X.q0.00671)Y. X.s 79.4. Yˆ s 494.3. Y.s 494.3y79.4 s 414.9. bsy1.645.

Table 1 Ža. Weights of transgenic carp during summer experiment Day of experiment

Group 77E22=Dor70

30066=Dor70

Yug=Dor70 Žcontrol.

91.5Ž"1.8. 165.9Ž"7.8. 259.4Ž"22.5. 334.6Ž"28.1. 392.5Ž"25.2. 459.1Ž"32.9. 546.1Ž"40.9. 454.6Ž"41.2.) a 2.2

104.2Ž"2.6. 181.1Ž"10.4. 255.3Ž"18.7. 323.8Ž"30.0. 395.4Ž"33.4. 481.0Ž"36.0. 520.8Ž"34.1. 416.6Ž"33.0. a 2.0

69.9Ž"2.4. 153.9Ž"19.2. 229.7Ž"14.3. 284.1Ž"6.9. 359.5Ž"17.6. 441.1Ž"30.0. 480.4Ž"23.4. 410.5Ž"25.5. a 2.4

51.9Ž"2.5. 123.6Ž"12.7. 169.9Ž"6.9. 235.6Ž"4.6. 299.2Ž"16.8. 349.3Ž"26.8. 430.1Ž"18.7. 378.2Ž"18.3. a 2.6

Average weight Žg. of each group in the summer growth experiment as a function of days from co-stocking in communal ponds, average weight gain Žg. and % SGRrday. The data are a mean of four replicate ponds. a Observed weight gains were compared by t-test. 90522=Dor 70 was found significantly higher than the control group Ž P s 0.026.. 77E22=Dor 70 Ž P s 0.128. and 30066=Dor70 Ž P s 0.125. were not significant. Žb. Correction of the weight gain due to differences in the initial weight Genetic group

Initial weight Ž X .

Deviation from average initial weight Ž ds X y X..

Correction for the weight gain Žy bd .

Weight gain Ž Y .

Corrected weight gain Ž Y X sY y bd .

90522 77E22 30066 control

91.5 104.2 69.9 51.9

12.1 24.8 y9.5 y27.5

y19.9 y40.7 15.6 45.3

454.6 416.6 410.5 378.2

434.7 a 375.9 a 426.1a 423.5a

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0 13 27 39 53 67 80 Average weight gain % SGRrday

90522=Dor70

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Table 2 PCR screening for F0 carp injected with FV-2rcGH Fish code

Sex

PCR result for fin DNA

PCR result for blood DNA

27260 27270 30577 3092F 76874 77D2E 83C5E 8486B

Female Female Female Male Male Male Male Female

q q q q y q q q

q y q q y y y Not tested

PCR result for sperm DNA

y q y y

Results of PCR screening in different tissue samples of F0 carp injected with FV-2rcGH. Data shown refer only to the fish found positive.

3.2.2. Radioimmunoassay for GH in plasma The levels of carp growth hormone in the plasma of the F0 carps injected with FV-2rcGH were determined by RIA. The fish were sampled and tagged on three additional occasions during the year in order to eliminate any seasonal fluctuations. The levels of GH in most of the fish were 0–2 ngrml, with very few exceptions of higher levels. Similar levels of GH were found in plasma of control common carps. 3.2.3. Communal testing experiments The winter experiment tested three progenies. The first transgenic group was the progeny of the F0 Yugoslavian male carp carrying the FV-2rcGH transgene in its sperm Ža76874. crossed with a non-transgenic Dor 70 female carp. The second transgenic group was the progeny of an F1 carp carrying the FV-1rcsGH construct Ža26E1A., as in the summer experiment, crossed with the same non-transgenic Dor 70 female carp. The comparison group was the progeny of a non-transgenic Yugoslavian male carp

Table 3 Weights of transgenic carp during winter experiment Days of experiment

0 29 63 96 Average weight gain % SGRrday Žall 96 days.

Group 26E1A=Dor70

76874=Dor70

Yug=Dor70 Žcontrol.

16.7"14.8 24.3"20.7 35.4"40.2 44.5"45.5 27.8))) a 1.0

18.6"11.8 24.2"23.4 32.2"34.1 53.4"58.6 34.8))) a 0.9

21.8"17.4 21.9"21.1 25.4"16.2 31.3"22.3 9.5 0.4

Average weight Žg. of each group in the winter growth experiment as a function of days from co-stocking in communal pond, average weight gain Žg. and % SGRrday. a Growth rates were compared by F-test for difference between regression coeficients. Groups 76874=Dor 70 and 26E1A=Dor70 were each found significant Ž P - 0.001. when compared to the control group ŽYugoslavian=Dor 70..

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crossed with the same non-transgenic Dor 70 female carp. The three groups of progeny were transferred to earthen ponds of 400 m2 in the Aquaculture research center in Dor on mid October. After 6 weeks of nursing in separate ponds, the fish were marked and co-stocked in one communal pond for communal testing Žthe number of fish in each group was too small for pond repetitions.. The pond was stocked as follows: 51 fish from crossing transgenic male a76874 with Dor 70 female, 80 fish from crossing transgenic male a26E1A with Dor 70 female and 80 fish from crossing Yugoslavian male with Dor 70 female. The average weights of each transgenic group during the experiment and the average weight gain are detailed in Table 3. Testing the growth rates by F-test for difference between regression coefficients revealed that both the transgenic groups 26E1A= Dor 70 and 76874= Dor 70 had a significantly higher growth rate than the comparison group of Yugoslavian= Dor 70 Ž P - 0.001.. Examining the growth rate by the % SGRrday ŽTable 3. shows a similarly higher % SGRrday for the transgenic groups than for the control group. 4. Discussion Wohlfarth and Moav Ž1985. noted that although many interesting fish variants and stocks had been produced in various fish. In most cases, their growth performance had not been adequately tested. Although many publications have appeared on transgenic fish Žreviewed by Hackett, 1993 and Iyengar et al., 1996., there is little available information on their growth characteristics. Testing each group of fish separately requires many ponds, due to the necessity for repetitions for each group. The communal testing method, in which many groups are co-stocked into the same pond, is more economical, and it was shown that groups with faster growth in communal ponds Žcompetition conditions. also grew faster in separate ponds Žno competition. ŽWohlfarth and Moav, 1985.. To properly compare the weight gains of different genotypes, weight gains must be corrected when they clearly correlate with the initial weights of the groups. Correction and standardization is done by calculating the correction factor from the prediction equation based on empirical data from growth experiments using the multiple nursing method ŽWohlfarth and Milstein, 1987.. Corrected weight gains have been shown to be non-correlated with initial weights and are, therefore, considered reliable estimates of relative growth rates of the stocks tested in communal ponds ŽWohlfarth and Moav, 1985.. Comparison of the corrected weight gains of the four groups in the summer test shows no significant differences between the transgenic families and the non-transgenic comparison group. The summer experiment posed no technical problems, and conditions of temperature, feeding and density were optimal for growth. Growth was rapid; on average the fish grew from 80 to 500 g in 80 days, similar to the rate shown in previous experiments in the same season ŽWohlfarth and Moav, 1972, 1985.. Survival rate was quite high; of 190 fish stocked in each pond, 170, 170, 173 and 139 individuals were counted at the end of the experiment in the replicate ponds, with no significant differences between the different genotypes.

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Fine et al. Ž1996. found that carp growth hormone administration increased the % SGRrday in fish fed a low protein diet Ž21%. while there was no effect on % SGRrday in fish fed a high protein diet Ž40%.. In addition, they found that the effect of cGH administration on % SGRrday was higher when fish were kept at 178C, as compared with fish kept at 268C Ž26% vs. 10%, respectively.. Our summer experiment was conducted in conditions Žtemperature and feeding. similar to those in which Fine et al. Ž1996. found little effect of exogenous carp growth hormone administration. It is therefore suggested that, the results of comparing GH transgenic phenotypes performance in sub-optimal conditions may be more definite and significant. Concerning the transgene’s presence in the genome of the nominal transgenic fish groups, it should be noted that all the progenies are from crosses between an F1 transgenic fish and a non-transgenic one, which should yield 50% of the offspring carrying the GH transgene, if the transgene is transmitted in a simple Mendelian fashion. No clear positive evidence as yet exists to show transgene expression in either of the transgenic groups. It is possible that transgenes are suppressed and not expressed, or that they are expressed at a very low efficiency. One reason for low level of expression in the summer experiment may be the promoter strength, which is itself weak because it contains only the proximal promoter of the common carp b-actin gene. The same promoter when fused to the CAT reporter gene has shown minimal levels of expression when compared to the expression of FV-2rCAT that contains the full promoter element of the carp b-actin gene Žincluding the first intron with the regulatory elements found in it. in zebrafish ŽMoav et al., 1993. and common carp ŽMoav et al., 1992.. Similar differences were found when these promoters were tested on tissue cultured carp epithelial cells ŽMoav et al., 1992.. We therefore had reason to expect that F2 transgenic carp containing the FV-2 transgene would achieve a higher growth rate than carp with the FV-1 transgene. The winter growth test included F1 progeny of a male carp that carried the FV-2rcGH, in addition to a group of F2 carp with the FV-1rcsGH similar to the previous summer experiment, but not of the same genotype. The third group was the comparison group, Yugoslavian= Dor 70, as in the summer test. It should be noted that we employed an ‘all-carp’ transgene by using the carp growth hormone cDNA instead of the chinook salmon growth hormone cDNA. In order to select the FV-2rcGH transgenic F0 that would be the parent for the F1 generation, we screened the adult F0 fish. The amount of transgene integration was 13.5%, which is similar to our findings in a previous study ŽMoav et al., 1995.. The mosaic nature of these fish ŽTable 2. is also typical for transgenic fish of the first generation and was demonstrated previously in carp ŽMoav et al., 1995. as well as in other fish ŽPenman et al., 1990; Culp et al., 1991.. Only one transgenic male contained the transgene in its sperm ŽTable 2. and we used it to establish the F1 generation for the growth experiment. F0 carp plasma samples were also tested for carp GH by RIA. Carp growth hormone ŽcGH. in plasma was found at normal levels Ž0–2 ngrml. in fish from all four genotypes. The result of the RIA for cGH in the F0 FV-2rcGH carp showed no correlation between levels of cGH and the transgenic status of the fish. It is important to note that only 13.5% of the fish were found to be transgenic and only a part of them were transgenic in their blood samples. Thus it seems that RIA for GH will be of significance only for full transgenic fish of F1 or later generations.

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Very few growth experiments have been performed in the past during the winter season. Wohlfarth et al. Ž1964. compared fall and winter growth of seven groups of carp. In the fall they found no significant differences between the growth rates of any of the groups. In winter, in contrast, the same groups showed differences in growth rate, and growth rate had no correlation with differences in their initial body weight. They concluded that it is worthwhile to select for specific winter growth capacity. This could also be done with transgenic carp, which might be found to be superior to the commercial strains in their growth at low temperatures while having the same growth rate in summer. The findings of Fine et al. Ž1996. reinforce this notion. The winter experiment contained fewer fish than the summer one and was, therefore, conducted in only one communal pond. Unfortunately, the winter experimental was stopped before its conclusion, but the two transgenic groups Ž26E1A= Dor 70 and 76874= Dor 70. that were initially smaller than the comparison group, exceeded it at the time the experiment was stopped and their growth rates were found to be significantly Ž P - 0.001. higher than the control group. It should be noted that the effects of differences in initial weight on weight gains are almost negligible in winter, eliminating the need for correction of weight gains ŽWohlfarth et al., 1964; Wohlfarth and Moav, 1985.. These winter weight gains of the transgenic genotypes are impressive considering the low temperatures at which they are achieved. Acknowledgements This work is supported by the ISRAEL-USA Binational Agricultural Research and Development Fund-BARD grant No. US-2305-93RC and the Basic Research Fund of Tel Aviv University. References Chen, T.T., Kight, K.K., Lin, C.M., Powers, D.A., Hayat, M., Chatakondi, N., Ramboux, A.C., Duncan, P.L., Dunham, R.A., 1993. Expression and inheritance of RSVLTR-rtGH complementary DNA in transgenic common carp, Cyprinus carpio. Mol. Mar. Biol. Biotechnol. 2, 88–95. Culp, P., Nusslein-Volhard, C., Hopkins, N., 1991. High-frequency germ-line transmission of plasmid DNA ¨ sequences injected into fertilized zebrafish eggs. Proc. Natl. Acad. Sci. USA 88, 7953–7957. Devlin, R.H., Yesaki, T.Y., Donaldson, E.M., Hew, C.-L., 1995. Transmission and phenotypic effects of an antifreezer6H gene construct in coho salmon Ž Oncorhynchus kisutch.. Aquaculture 137, 161–169. Drori, S., Ofir, M., Levavi-Sivan, B., Yaron, Z., 1994. Spawning induction in common carp Ž Cyprinus carpio . using pituitary extract or GnRH superactive analogue combined with metoclopramide: analysis of hormone profile, progress of oocytes maturation and dependence on temperature. Aquaculture 19, 393–407. Du, S.J., Gong, Z., Fletcher, G.L., Shears, M.A., King, M.J., Idler, D.R., Hew, C.L., 1992. Growth enhancement in transgenic salmon by the use of an ‘all-fish’ chimeric growth hormone gene construct. BiorTechnology 10, 176–181. Fine, M., Sakal, E., Vashdi, D., Daniel, V., Lipshitz, O., Gertler, A., 1993. Preparation and comparison of biological properties of recombinant carp Ž Cyprinus carpio . growth hormone and its Cys-123-Ala mutant. Fish Biochem. Physiol. 11, 353–361. Fine, M., Zilberg, D., Cohen, Z., Degani, G., Moav, B., Gertler, A., 1996. The effect of dietary protein level, water temperature and growth hormone administration on growth and metabolism in the common carp Ž Cyprinus carpio .. Comp. Biochem. Physiol. 114, 35–42.

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