Direct feminization of lumpfish (Cyclopterus lumpus L.) using 17β-oestradiol-enriched Artemia as food

Aquaculture Aquaculture 123 (1994) 137-151

Direct feminization of lumpfish (Cyclopterus lumpus L. ) using 17P-oestradiol-enriched Artemia as food D. J. Martin-Robichaud”,b7*,

R.H. Petersona, T.J. Benfeyb, L.W. Grim”

“Department ofFisheries and Oceans, St. Andrews, N.B., EOG 2X0, Canada bDepartment ofBiologv, University ofNew Brunswick, Fredericton, N.B., E3B 6E1, Canada ‘Ocean Sciences Centre, Memorial University of Newfoundland, St. JohnS, NJd., AIC 5S7, Canada (Accepted 8 November 1993 )

Abstract Artemia nauplii cultured in lipid-enriched media containing 0, 5, 10 or 20 mg 17P-oestradiol/l for 24 h contained 0, 140,90 and 23 1 ng 1‘Ipoestradiol/mg dry wt., respectively. Feminization of lumpfish larvae was essentially 100% when the larvae were fed Artemia containing 231-407 ng l’lb-oestradiol/mg dry wt. at first feeding for 20 days. Immersing eggs or newly hatched larvae at 22,29 and 36 days post-fertilization in 200 ,ug 17jLoestradial/l for 2 h resulted in 68% and 46% females in duplicate treatments. The ovaries of some fish which consumed Artemia containing 17j$oestradiol were smaller, filiform and less developed than those from similar size control females although no spermatogenic tissue was detected in these filiform ovaries. This may indicate that females with filiform ovaries were feminized, genotypic males. It appears that direct sex-reversal techniques, based upon steroid-supplemented Artemia nauplii, has promise of producing monosex populations in fish larvae requiring a diet of live prey during the period of sex determination.

1. Introduction Characteristics conditions have

which influence the success of teleost species under aquaculture led to the development of sex control techniques to produce

monosex stocks (Johnstone et al., 1978; Hunter and Donaldson, 1983; Yamazaki, 1983; Shelton, 1989; Dunham, 1990). Production of monosex rainbow trout (Oncorhynchus mykiss) (Solar et al., 1984; Bye and Lincoln, 1986), coho (0. kisutch) and chinook salmon (0. tshawytscha) (Hunter et al., 1986; Piferrer and Donaldson, 1993 ), tilapia (Oreochromis mossambicus) (Pandian and Varada*Corresponding author. 0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ0044-8486(93)E0266-C

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raj, 1990), grass carp (Ctenopharyngodon idella) (Shelton, 1986) and channel catfish (Ictaluruspunctatus) (Goudie et al., 1983) has eliminated early male maturation and/or optimized sex-specific preferential growth rates, egg production, flesh quality and marketability. Investigations into the culture potential of other teleost species may find gender-specific characteristics which affect the protitability of an aquaculture operation. Direct and indirect sex-reversal procedures are routinely used to produce monosex stocks (Hunter and Donaldson, 1983). Both practices require treatment of early developmental stages with sex steroids during the sexually labile period before the onset of sexual differentiation (Yamamoto, 1969; Piferrer and Donaldson, 1993). The period of gonadal ontogenesis is species-specific and, depending on the timing of sexual differentiation, either immersion treatments or oral application of the steroid are suitable (Goetz et al., 1979). Sexual differentiation in gonochoristic species frequently occurs around the time of first feeding (Piferrer and Donaldson, 1993 ). Feeding fish commercial diets treated with steroids is suitable for species that will accept an artificial diet at the onset of exogenous feeding. However, the culture of many marine fish, which have relatively small undeveloped larvae with small mouth gapes, usually requires an initial diet of live prey (Watanabe et al., 1983a; Tilseth, 1990). Under these circumstances, feeding steroid-treated pelleted food is inappropriate and alternative techniques are required. Rotifers and Artemia are common zooplankton cultured as food for larval fish (Watanabe et al., 1983a; LCger et al., 1986; Tilseth, 1990). Nutritional inadequacies of Artemia have resulted in the development of enrichment techniques using fatty acid emulsions to increase the concentration of highly unsaturated fatty acids (HUFA) in the predator diet (Watanabe et al., 1983b; LR;ger et al., 1986; Walford and Lam, 1987; Gatesoupe, 1991). In addition to nutritional enrichment, various prophylactics, such as antibiotics, have been incorporated into the tissues of brine shrimp (Mohney et al., 1990; Touraki et al., 199 1; Verpraet et al., 1992). Garrett ( 1989) seemed to successfully enrich Artemia with steroids, since sex reversal of largemouth bass was accomplished when the larvae were fed Artemia hatched in steroid-treated water. Lumpfish, Cyclopterus lumpus, is a gonochoristic, semi-pelagic teleost inhabiting the North Atlantic (Scott and Scott, 1988 ). A commercial fishery exists for lumpfish roe in Newfoundland, Iceland and some European countries (Anonymous, 1989). Considering that roe is the marketable product, any commercial aquaculture production of lumplish would necessitate the culture of female fish to be most economical (Martin-Robichaud, 199 1). Feminization of lumpfish larvae would ensure the availability of more roe-producing fish for caviar production, thereby enhancing the profitability of aquaculture operations. Although lumpfish larvae are well-developed at hatch and will accept pelleted food at first feeding, better survival and growth are achieved with live prey (Benfey and Methven, 1986; Martin-Robichaud, 199 1) . Sexual differentiation in lumpfish occurs by the time fish are 5.8-6.0 mm standard length (SL) which is within the first month post-hatch (Martin-Robichaud, 1993 ) . Therefore, for successful feminization, 17P-oestradiol should be introduced to first feeding larvae during

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this interval. This investigation tested the feasibility of enriching Artemia with 17poestradio1, a natural vertebrate Oestrogen, to enable its introduction to firstfeeding lumpfish larvae. The accumulation rate of 17j$oestradiol into the Artemia was determined. In addition, the feasibility of directly feminizing genotypic male lumptish in order to increase the proportion of phenotypic females was investigated by feeding lumpfish larvae live Artemia nauplii enriched with 17/3-oestradiol.

2. Materials and methods

17B-Oestradiol enrichment ofArtemia nauplii San Francisco Brand@ Great Salt Lake strain Artemia cysts ( 18 g) were decapsulated by immersion in 750 ml of 0.25 M NaOH in 33% sodium hypochlorite with aeration for lo- 12 min. The decapsulated cysts were rinsed thoroughly with water on 120 pm nitex screening. Hypochlorite deactivation was achieved by aerating the cysts in 500 ml of 0.06 M sodium thiosulphate for 10 min, then filtering and rinsing again. The decapsulated cysts were weighed and equal quantities placed into six 1-litre graduated Imhoff cones containing 950 ml of seawater (3 l33 ppt ) at 18-20” C (3 g of dry cysts or approximately 750 000 embryos per litre). Each cone was sealed at the bottom with a rubber stopper, through which a 22gauge needle was inserted to supply aeration. The cysts were incubated for 42 h, allowing approximately 30 h for hatching, and 12 h to reach the first feeding instar II stage (Sorgeloos et al., 1986). Continuous light intensity ranged from 2 15 to 538 lux. After 42 h nauplii survival and hatching success were confirmed microscopically before adding the HUFA-enriched diet. The HUFA-enriched diet (Provesta@ Microfeast Plus L-10,0.8 g), was blended in 50 ml of water and added to the cones containing the nauplii. Four 17/3-oestradiol treatments (0, 5, 10, and 20 mg/l) were prepared by adding 17poestradiol dissolved in 70% ethyl alcohol to the HUFA-enriched solution before blending. The control treatment received the same concentration of alcohol. Each treatment consisted of 6 cones containing brine shrimp cultured in identical concentrations of 17/3-oestradiol and diet for 24 h. Different treatments were run sequentially to minimize cross-contamination. Two brine shrimp samples were collected from different cones at 0, 2, 4, 8, 16, and 24 h to determine the tissue 17fioestradiol concentration. Additional control samples of nauplii were taken at 42 h before food or 17goestradiol was added. Artemia samples were collected by shutting off aeration for 3-5 min to allow nauplii to settle and concentrate. From the dense concentration of nauplii, 10 ml was removed by pipet and filtered through a 120 ,um nitex screen. The supematant was collected and frozen, and the nauplii rinsed thoroughly with distilled water before transfer to preweighed test tubes. The nauplii samples were weighed and stored at - 80°C. After lyophilization samples were reweighed and frozen at - 80’ C until radioimmunoassay (RIA) analysis.

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Radioimmunoassay 17BOestradiol concentration in brine shrimp tissue and in the enriched brine shrimp rearing water was determined by RIA. Subsamples of dry brine shrimp tissue were sonicated in 1 ml of 0.6% NaCl, kept at room temperature for 0.5 h and then centrifuged (4°C 2000 xg, 15 min). Replicate tissue samples (28% of total samples) were spiked with radiolabelled ( 1251) 17P-oestradiol for determination and correction of the percent recovery after digestion and ether extraction of the samples. Ether extraction efficiency for each water sample was calculated by determining the losses of tritiated 17fi-oestradiol. Brine shrimp tissue extracts and water samples were diluted 1: 10 000 and 1: 20 000, respectively to obtain readings within the range of the 17poestradiol RIA standard curve using the methodology described by Crim et al. ( 1992). In brief, the ether-extracted tissue and water samples were evaporated under nitrogen and the samples were redissolved with 1 ml absolute ethanol. The 17/I-oestradiol concentration in duplicate 100 ~1 ( 10%) ethanol aliquots was determined within the linear range of the 17&oestradiol standard curve from 1 to 1000 pg. Serial dilutions of samples were investigated ensuring a linear response parallel to the 17/3-oestradiol standard curve. Curvilinear regressions were calculated for each treatment using logarithmic transformation of both variables to equalize variances among treatment means.

LumpJish feminization One pair of adult lumpfish caught in Passamaquoddy Bay, New Brunswick in April 199 1, spawned on a rock substrate in a 400-litre tank at 5.5 “C on 2 May 199 1. After the development of eye pigmentation (eyeing), at 115 degree days, the egg mass was broken up into small clumps of 2-l 0 eggs each and placed into two 5-litre McDonald hatching jars at a density of about 2800 eggs/l. Mean salinity, temperature and flow rate were 32 ppt, 8.9 “C and 1.7 l/min, respectively. The feminization experiment consisted of four different 17P-oestradiol treatments: control, 17Boestradiol immersion only, 17poestradiol diet only, and 17/3oestradiol immersion plus 17Boestradiol diet, with duplicates of each. The immersion treatment entailed three immersions in the hatching jars at 173 and 2 17 degree days ( ‘C d) (22 and 29 days post-fertilization (dpf ) ) for eyed-eggs, and at 250’ C d ( 36 dpf ) when hatching was 90- 100% complete. For each immersion treatment, 1 ml of 0.1% 17Boestradiol in absolute EtOH was mixed with 1 litre of seawater and added to the hatching jar, resulting in a concentration of 200 ,ug 17poestradiol/l in a 5-litre volume. Controls received the same concentration of ethyl alcohol. During the 2 h immersion treatment, water flow was turned off, solutions were aerated and temperature maintained at 9 ‘C by immersing the jars in a cold water bath. Every 1O-l 5 min the eggs were gently agitated to ensure adequate circulation and exposure. After 2 h the water flow was resumed to the jars at 1 l/min, resulting in total water exchange within 5 min. After the final immersion treatment, batches of 500 newly hatched larvae were transferred to each of 8 tanks, 4 tanks receiving 17poestradiol treated larvae and 4 receiving control larvae.

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The plexiglass tanks (26 1) had a surface inflow ( 1.1 l/min) at one end and two screened, bottom outflow tubes connected to exterior standpipes, opposite. Four gray PVC plates supported vertically from the tank bottom provided additional substrate and shelter for the larvae which had functional ventral suction discs. A 14 h light/ 10 h dark photoperiod was maintained for 102 days post-hatch (dph). Light intensity, measured with a Research Radiometer IL 1700 with photopic filter Y no. 10156 on SUD038 silicon detector, ranged from 5.84 to 18.3 1LW. Feeding was initiated at 5 dph. Lumptish larvae were fed either the 17/3-oestradiol or control Artemia diet for 20 days. Artemia were decapsulated as described previously and transferred to 10-litre containers ( 3 g cysts/l). Vigorous aeration maintained embryos and nauplii in suspension. The containers were covered with lids and a plastic sheet to prevent aerosal contamination, Embryos were incubated for 42 h at 19-22 ‘C before adding a blended emulsion containing the Provesta Microfeast Plus L- 10@HUFA-enriched diet. 17/?-oestradiol-treated brine shrimp had 40 ml of 0.5% 17/3-oestradiol in 70% EtOH added to the enriched emulsion before blending. The final concentration in the lo-litre Artemia tanks was 20 mg 17P-oestradiol/l. Control brine shrimp received an identical amount of EtOH in their diet. The duration of the enrichment interval was 24 h. Brine shrimp nauplii were collected, rinsed thoroughly with tap water and transferred to two 6-litre Erlenmyer flasks containing aerated 4’ C sea water ( 30 ppt). Variable speed, peristaltic pumps with 1.O mm ID tubing, continuously pumped nauplii to individual lumpfish rearing tanks at 4 ml/min at 09 : 00-20 :00 h, daily. Additional seawater was added to the flasks throughout the day to extend the feeding period. Assuming 100% hatch and survival, lumpfish larvae in each tank received about 1.9 x 1O6Stage II or III nauplii daily. Prey densities appeared more than adequate. To determine if steroid levels decreased during the feeding interval, samples of brine shrimp were collected and analyzed for 17poestradiol as described above, except that control samples were not diluted and treated samples were diluted 1: 1000. Larvae were fed the control Artemia diet for an additional 5 days before weaning to pelleted food commenced at 30 dph. To facilitate weaning, assorted larval feeds such as Provesta Microfeast@ diets and macerated salmon starter were slowly incorporated into the feeding regime. Automatic larval feeders (Aquaresearch Ltd. ) fed the pelleted diet for 1 min intervals 5 times daily during daylight hours through holes in the tank lids. Supplemental feeding of live brine shrimp nauplii occurred until 55 dph. Tanks were siphoned as necessary. All larvae received periodic prophylactic treatments with formalin ( 1: 8000, 1 h) and chloramine-T (5 mg/l, 0.5 h) to combat infections resulting from tail biting and other aggressive behaviour. To measure the concentration of 17P-oestradiol in lumptish tissue after 20 days of feeding, 10 lumpfish that were only exposed to dietary 17poestradiol and 10 control fish were sampled. The larvae were rinsed in distilled water and placed in preweighed microcentrifuge tubes, then frozen, lyophilized and coarsely ground.

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1‘I&oestradiol concentration was determined by RIA using the methodology described above. No dilution of sample aliquots was required. The temperature ranged from 9.5 to 16.5”C through the subsequent 6.5-month rearing period. At 100 dph, increased aggression resulting in mortalities necessitated that the lumpfish be transferred to larger aquaria (52 litres). Survival to date was determined for each treatment. The lumpfish received an assorted pelleted diet until they attained a size adequate to allow sexing by gross gonadal morphology and observation of oocytes and ovarian lamellae under magnification. Fish were initially sampled for sexing at 40-55 mm standard length (SL). Experience in sexing these relatively large juveniles subsequently allowed sexing of smaller, moribund fish ( 2 18 mm SL) previously sampled. Periodic sampling of larger fish inadvertently graded the population, and a reduction in aggressive encounters ensued resulting in reduced mortalities. All fish were sampled by 196 dph. Juvenile lumpfish were preserved in 5% formalin after standard lengths (SL) were measured to 0.1 cm. Gonads were excised and fixed in 5% formalin. A subsample of 45 gonads was prepared for cytological confirmation of sex. These gonads were specifically selected on the basis of treatment, size and morphological features to clarify developmental differences noticed between treatments. Standard histological techniques of paraffin infiltration and embedding were done before staining the 6-7 pm sections with Gill-2 haematoxylin (Shandon) and eosin. Deviations from an expected 1: 1 sex ratio were calculated using an adjusted chi-square formula incorporating the Yates conservative correction of continuity (Sokal and Rohlf, 198 1).

3. Results

17@Oestradiol enrichment ofArtemia nauplii Recovery of 17koestradiol from brine shrimp tissue spiked with iodinated 17/3oestradiol indicated a digestion efficiency of 69% (before ether extraction). The efficiency of ether extraction only was 77%, resulting in a total extraction efflciency of 53%. Recovery of tritiated oestradiol from water samples after ether extraction was 85%. In incubation water containing 0,5, 10, and 20 mg 17P-oestradiol/l , the initial concentrations detected by RIA analysis were 0, 4.04, 8.27 and 12.56 mg 17poestradiol/l, respectively. Oestrogen levels in the incubation water decreased during the initial 8 h at the two highest treatment concentrations (Fig. 1) . After 24 h, brine shrimp had accumulated 140,90, and 231 ng 17/3-oestradiollmg dry wt. of tissue at 17/3-oestradiol treatment concentrations of 5, 10 and 20 mg/l, respectively (Fig. 2 ) .

D.J. Martin-Robichaud et al. / Aquaculture 123 (I 994) 13 7-151

0

4

8

12

16

20

143

24

Time (h)

Fig. 1. 17&Oestradiol concentration in Artemia rearing water during the 17poestradiol loading experiment. Data points represent the mean of duplicate RIA aliquots from each water sample. Lines fitted by eye.

0

4

8

12

16

20

24

Time(h)

Fig. 2. Uptake of 17/.?-oestradiol by Artemia nauplii over 24 h. Data points represent the mean of duplicate RIA aliquots from each Artemia sample. Lines fitted using logarithmic regressions: 5 mg/l, r’z0.745; 10 mg/l, log[ 17P-oestradiol] = log[ l’l&oestradiol] = 1.5179+0.4558(logtime), 1.3948+0.4039(logtime), r’=0.703; 20 mg/l, log[ 17B-oestradiol] = 1.8234+0.3916(logtime), r2=0.617.

3.2. Lumpfish feminization 17Poestradiol levels in brine shrimp maintained alive at 4’ C during the daily feeding interval declined from an initial concentration of 407 2 100 to 355 t 9 and 195 +- 11 ng 17&oestradiol/mg dry wt. after 4 and 7 h, respectively. Control brine shrimp contained 0.58 4 0.359 ng 17poestradiol/mg dry wt. One replicate tank of lump&h larvae treated with 17&oestradiol immersion plus 17/3-oestradiol diet was lost due to a water failure. No treatment-related mortality was evident at 100 dph (Table 1) . In one replicate of the 17j?-oestradiol immersion-only and 17jSoestradiol diet-only treatments some larvae were lost due to screen dislodgement, and therefore survival observations on these tanks were invalid. The 17fi-oestradiol concentration in lumpfish larvae was 2.29 and 100.3 1 ng/ mg dry wt. for larvae receiving the control diet and the 17/I-oestradiol treated

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Table 1 Gonadal sex and survival of lumpfish larvae for various treatments Treatment

n

Percent Female

Control 17/%Oestradiol immersion 17/3-Oestradiol diet 17POestradiol immersion plus 17/?-oestradiol diet

Male

Survival

62 51 53 28

45 49 68 46

55 51 32” 53

19 17 17

49 57 56

98 100 100

;:

16 21

Ob

Significant differences of sex ratios from expected 1: 1 ratio indicated by a= Pc 0.025 and b = PC 0.00 1 (adjusted chi-square) .

brine shrimp, respectively (t-test, P-c0.00 1). The sex ratio of the control groups did not differ significantly from an expected 1: 1 sex ratio (Table 1). The 178 oestradiol diet resulted in almost 100% females. One fish (39 mm SL) receiving the l’lgoestradiol diet had testes. This was confirmed histologically and no cytological abnormalities were detected (Fig. 3E and F). All female lumpfish were so classified on the basis of overall gross gonadal morphology as exemplified in Fig. 3A. Oocytes and/or lamellae were noticeable, and ovaries were larger and thicker than filiform testes (Fig. 3C) from lish of similar size. Some ovaries from females fed 17/I-oestradiol contained oocytes which were tiliform (Fig. 3G). The presence of oocytes in a subsample of liliform ovaries was confirmed histologically (Fig. 3H). Histological examination of a portion of ovaries from female lump&h fed the 17poestradiol diet ( 15% of treatment total) or subjected to 17/?-oestradiol immersion (4% of treatment total) revealed no spermatogenic tissue. A gradation of this frliform ovarian appearance was apparent in some ovaries excised from fish fed the 17/3-oestradiol diet (Fig. 4). Immersing eggs and newly hatched lump&h larvae in 200 ,ug 17poestradiol/l significantly increased the percentage of female fish in one replicate only (Table 1). The sex ratios were not significantly different (P < 0.05 ) from a normal 1: 1 ratio for the immersion treatment when duplicates were combined. Gonad weight increases more rapidly in females than in males up to 60 mm SL (standard length) (Fig. 4A). The ratio of gonad weight to SL effectively separates the sexes in the control and 17/?-oestradiol immersion treatments (Fig. 4A and B ) _ Ratios from female fish fed the 17P-oestradiol diet are more heterogeneous than control and 17Boestradiol immersion females (Fig. 4C and D), with some females having lower gonad ratios than other females of a similar length. This heterogeneity was also noticeable while dissecting ovaries from lumpfish fed 17goestradiol as described previously.

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Fig. 3. Morphology and cytology of gonads from juvenile lumpfish. Typical morphology (A) and cytology (B) of ovary from control fish (SL= 34 mm). Note ovarian lamellae (01) containing oocytes (oc). Typical morphology (C) and cytology (D) of testis from control fish (SL= 35 mm) showing spermatogonia (sg) in spermatogenic cysts. Morphology (E) and cytology (F) of testis from sole male fish (SL=39 mm) subjected to 17&oestradiol-diet. Note spermatogenic cyst (c). Morphology (G) and cytology (H) of tilifonn ovary from lumpfish fed 17/7-oestradiol (SL= 35 mm). Scale bar = 1 mminA,C,E,Gand50,nminB,D,FandH.

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10

20

30

40

50

60

10

20

30

. .

40

50

60

D IMMERSION

AND

DIET

.

.

.

10

20

30

Length

40

50

(mm)

60

10

20

30

Length

40

50

60

(mm)

Fig. 4. Changes in ratios of gonadal weight to standard length (SL) with fish length for various treatments. Females V; males 0.

4. Discussion

Significant concentrations of 17/3-oestradiol were incorporated into Artemia tissue; concentrations were reduced by 50% over the cold storage and feeding period, but remained sufficiently high to effect sex-reversal. Successful sex-reversal of largemouth bass also occurred when larvae were fed Artemia cultured in steroid-treated water without an enrichment diet, although control sex ratios indicate that cross-contamination may have occurred (Garrett, 1989). These results indicate that enrichment of Artemia with steroids is a feasible procedure for introducing steroids to first feeding fish larvae. Low 17Soestradiol levels (0.58 ng/mg dry wt.) were detected in control Artemia prepared for lumplish ingestion whereas the concentration in control prey in the 17/3-oestradiol loading experiment was below detectable levels. This may be due to the different dilutions used in the assays or cross-contamination. For the 17j?-oestradiol loading experiment, all samples were diluted 1: 10 000 whereas digested samples of dietary Artemia prepared for lumpfish ingestion were not diluted before RIA. It is unknown whether the detection of l’ira-oestradiol in con-

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trol samples is caused by cross-reactivity with interfering substances specific to brine shrimp or by cross-contamination. The concentration of endogenous 17/S oestradiol in mature Artemia is very low compared to the levels measured in 17/S oestradiol-enriched Artemia (Van Beek and De Loof, 1988). Treated brine shrimp prepared for larval ingestion contained higher oestrogen concentrations than detected during the initial loading experiment (407.2 vs. 23 1 ng 17/?-oestradiollmg dry wt.). This difference may be due to the different assay dilutions and/or to brine shrimp culture variability. The decrease in 17P-oestradiol levels in the brine shrimp-rearing water during the initial 8 h could be due to its accumulation by the nauplii. Enrichment of 17p oestradiol into Artemia nauplii was possible when the zooplankton were cultured in a dietary enrichment medium containing 5-20 mg 17@-oestradiolll. The higher 17poestradiol concentrations accumulated in Artemia reared in 5 than in 10 mg/ 1 may result from variability in quality and viability of brine shrimp batches under identical regimes. Feeding larval lumpfish Artemia, which were previously incubated in 20 mg 17Boestradiol/l, produced 100% gonadal females based on the occurrence of ovarian tissue in juvenile fish. It has yet to be determined if all females have fully functional ovaries at maturity. Garrett ( 1989) also achieved 100% sex-reversal of largemouth bass larvae by adding oestrogenic and androgenic steroids at a concentration of 5 mg/l to brine shrimp-rearing water before feeding as prey to bass larvae for 10 weeks. No lipid-enriched diet was used in Garrett’s ( 1989) experiment, the incubation interval was not stated and, although the loading concentration was 25% less than the level used in this investigation, 100% sex-reversal was accomplished. Garrett’s ( 1989 ) enrichment technique was more effective at sexreversing largemouth bass than was spraying hormones on inert diets. This may be due to loss of steroids from the diets into water during feeding or higher ingestion rates of live feed. Maintaining a low metabolic rate of Artemia sustained nutritional quality (Urger et al., 1986) and minimized excretion of 17Boestradiol. High larval mortalities resulting from 17/Soestradiol treatments have been reported (Bye and Lincoln, 1986; Hunter et al., 1986). Although no treatmentrelated mortalities were detected at the concentrations used in these experiments, further tests to reline treatment conditions and minimize steroid use may find that lower 17poestradiol concentrations are adequate for successful sex-reversal if applied at the optimum time. The process of sex differentiation consists of two phases: anatomical (gonadogenesis) and cytological (gametogenesis ) differentiation. The labile period (when gonadal cells are most susceptible to the influence of exogenous steroids) of coho salmon is approximately 3-4 weeks before sexual differentiation and immersion treatments are required to optimize treatment success (Piferrer and Donaldson, 1989). Gonadal tissues are still capable of responding to gonadal hormone treatments after sexual differentiation, but successful sex-reversal requires higher doses and durations (Piferrer and Donaldson, 1993 ) . Immersion of lumpfish eggs and pre-feeding larvae in 17poestradiol did not consistently result in sex-reversal of genotypic males. In one duplicate, this immersion treat-

14%

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ment produced 68% females. Considering that 100% sex-reversal occurred when larvae were fed the 17/$oestradiol diet from 5 to 25 dph, the immersion treatments may not have subjected primordial gonadal tissue to steroid levels at their most susceptible period or at an adequate concentration. To optimize treatment procedures, further tests could be conducted to accurately identify the labile interval by administering steroids for short periods at several doses (Hunter and Donaldson, 1983; Hunter et al., 1986; Piferrer and Donaldson, 1993). The lumpfish, which were fed the 17fl-oestradiol diet and had smaller ovaries containing fewer oocytes than those of other juveniles of the same size and treatment, may be sex-reversed genotypic males. In grass carp cytological differentiation begins at 120 mm and is complete by 180-200 mm TL (total length) but two gonad types can be distinguished anatomically at 50-60 mm (Shelton, 1986). Boney et al. (1984) found that if androgenic treatment occurs after gross anatomical gonadal features develop in grass carp, then spermatogenic tissue may develop in an ovarian-shaped gonad. Piferrer and Donaldson ( 1992) also found ovaries in 17P-oestradiol-treated chinook salmon which they classified as “affected” ovaries. These “affected” ovaries contained a reduced number of oocytes, the cross-sectional area of the ovary was reduced, and a vascular system typical of testes was present. No evidence of a male vascular system was observed in ovaries sampled in this experiment, but the other characteristics correspond to the morphological differences detected in some of the 17/I-oestradiol-fed lumpfish. Piferrer and Donaldson ( 1992) consider the chinook salmon having “affected” ovaries as genotypic males. If the lumptish with smaller, less-dense ovaries were genotypic males, then this may confirm that the 17poestradiol diet was responsible for the higher percentage of females, and that this increase was not due to higher male mortalities. This technique of comparing anatomical gonadal differences of one sex was used by Boney et al. ( 1984) to distinguish between normal and sex-reversed male grass carp. Morphological differences detected in this experiment may indicate that the steroid treatments triggered sex-reversal before cytological differentiation, but in some cases not before anatomical differentiation had been initiated. A subsample of sex-reversed fish has been retained to compare the egg mass of mature females to determine if this reduction in oocyte density is permanent. The 17/?-oestradiol concentration administered to lumpfish larvae via Artemia was about 200-400 ng/mg dry wt., which is lo-fold higher than concentrations frequently used to feminize teleosts by application to diets (Hunter and Donaldson, 1983; Yamazaki, 1983). This dosage was selected to compensate for any steroid loss due to excretion by Artemia during the feeding interval. Sathyanarayana Rao and Satyanarayana Rao ( 1983) fed high concentrations of dietary oestradiol (200 mg/kg) to common carp fry for 13 1 days, resulting in a significant retardation of gonadal development and high sterility. Oestradiol treatments also resulted in diminished growth and gonadal size of coho and chum salmon (Goetz et al., 1979; Redding et al., 1987). These abnormalities detected by other researchers seemed to affect all fish, not a certain portion as evident in our investigation.

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Direct feminization techniques, such as those employed in the present study, which directly expose marketable fish to steroids is a practice disdained by consumers. Indirect feminization would reduce the steroid quantities required since only a small percentage of females would be masculinized for broodstock. This would also eliminate direct exposure of the marketable product to steroids. Determination of the heterogametic sex is required to develop indirect feminization techniques (Solar et al., 1984). In either case, utilizing enrichment techniques to introduce steroids to fish larvae should allow production of monosex populations of teleosts which require live prey at first feeding. Acknowledgements

Connie Wilson provided RIA training and assistance. The HMSC Atlantic Reference Centre kindly provided the photomicrograph system. F. Cunningham prepared the figures and Drs. K. Haya and J. Bailey reviewed the manuscript. The primary author was supported by a Marquerite and Murray Vaughan Graduate Fellowship. Addendum

Since this experiment was conducted, three female lumpfish from the 17poestradiol immersion plus diet treatment were grown to maturity. Two of these females successfully spawned with wild males in laboratory tanks. The sex ratio of progeny from one female was close to the expected 1: I ratio (0.025
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