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Assessment of egg quality in atlantic salmon, Salmo salar, treated with testosterone—II. Amino acids
Camp. Biochem. Physiol.Vol. 103A, No. 2, pp. 397402, 1992
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ASSESSMENT OF EGG QUALITY IN ATLANTIC SALMON, SALMO SALAR, TREATED WITH TESTOSTERONE-II. AMINO ACIDS RAKESH
K.
SRWASTAVA*
and
JOSEPHA. BROWN
Ocean Sciences Centre/Department of Biology, Memorial University of Newfoundland, St John’s, NF AlC 5S7, Canada (Received 6 February 1992; accepted 6 March 1992) Atlantic salmon (Sabno salar) were treated with Silastic pellet implants containing testosterone (200 pg/g body weight) four times in a year. Eggs stripped from control (sham implantation) and testosterone-treated fish were fertilized and comparisons of free and total amino acid compositions made until first feeding. 2. Despite having eggs which were smaller in diameter, lighter in weight and lower in total amino acid contents, alevins from testosterone-treated fish were heavier in wet weight and larger in body length, and exhibited enhanced free amino acid contents at first feeding. 3. The qualitative composition of total amino acids in eggs from treated and control fish did not differ. 4. Total amino acid pool of eggs and alevins declined during development, but an increase in the free amino acid pool was noticed through development. The increase in free amino acid pool was higher in eggs and alevins from treated fish than controls, perhaps due to enhanced mobilization of the free amino acid pool.
Abstract-l.
INTRODUCTION Various
androgens
have
been
used
to
enhance
in teleosts (Crim et al., 1989; Crim, 1991), but their consequences on amino acid compositions of eggs have never been examined. Gonadal recrudescence can also be accelerated by long term hormonal therapy consisting of analogs of gonadotropic hormone releasing hormone (GnRH) and/or steroids in various species of salmonids (Crim and Evans, 1983; Crim et al., 1983, 1989; Crim, 1991). Part I of this paper deals with the effects of testosterone treatment during gonadal recrudescence on subsequent protein, lipid, carbohydrate, polyamine, dry matter and ash contents of eggs and alevins, and early growth, development and survival of Atlantic salmon (Srivastava and Brown, 1992). Recently, Korsgaard (1990) reported significant increases in free amino acids, total RNA, RNA:DNA and RNA:protein in the liver and plasma of estradioltreated eel pout, Zoarces viuiparus L. males over controls. Therefore, in this paper we examine the consequences of testosterone treatment during gonadal recrudescence on subsequent amino acid compositions (free and total) of eggs, and their changes during early development of Atlantic salmon (Salmo salar). Amino acids are both precursors for proteins and substrates for energy production. A deficiency or an excess of one or more of these amino acids can limit protein synthesis (Tews et al., 1979), growth (Harper et al., 1970) or both (Tews et al., 1980). Following gonadal
recrudescence
*Present address: Department of Zoology, College of Biological Science, University of Guelph, Guelph, Ontario NlG 2W1, Canada.
fertilization and during development of the embryo, the yolk proteins are degraded and resorbed, and amino acids are utilized for the synthesis of somatic proteins (Love, 1980). MATERIALS AND METHODS
Animals
The broodstock originated as fry (wild population) from the Exploits river and-were held at-St Ma&-Bay (46”48’N; 53”39’W). Fish were maintained in a circular tank (1500 1) and fed an artificial diet (Corey Feed Mills, Fredericton, NB): Water source was brackish and varied from 12.0 to 16.7 ppt over the study. Photoperiod was ambient, varied from 8 hr light: 16 hr dark (December) to 16 hr light : 8 hr dark (June). There were no significant differences in body weight (control: 2.52 + 0.11 kg; treatment: 2.6 + 0.09 kg, P > 0.05) and body length (control: 57.8 + 0.42 cm; treatment: 57.6 + 0.47 cm, P z 0.05) among control and treated fish at the time of egg collection. Fecundity varied by less than 100 eggs out of 1800 among the groups. We followed the protocol of Crim et a/. (1989) for testosterone administration. Fish received either sham implantation treatment (control fish) or testosterone hormone implantation (treated fish) bimonthly. More specifically, treatment fish were administered with Silastic pellet (1 x 0.2 cm in diameter) implants containing testosterone (200 pg/g body weight) four times (February, April, June and August) in a year. Testosterone implantation results in measurable increases in testosterone levels in plasma of the same broodstock of Atlantic salmon (Dr L. W. Crim, personal communication). Control and treated fish were tagged for identification. Mating design and incubation
Eggs and sperm were collected in November and brought to the Marine Sciences Research Laboratory. Two replicates of egg batches (two from control and two from treated fish) were made. In each replicate, 2000 eggs from four females were fertilized with a pool of milt from three males. After 397
398
RAKESHK. SRIVASTAVAand JOSEPHA. BROWN
water hardening, the eggs were transported to an incubator where the temperature varied from 6 to 8°C.
with a Beckman 121 MB amino acid analyser (Srivastava et al., 1992).
Sampling
Statistical analyses
Samples were collected at five different developmental stages, i.e. before fertilization, after fertilization, eyed-stage, hatching and first feeding. From each replicate, 20 eggs and alevins were collected to determine egg weight/diameter, and alevin weight/length, respectively.
The data were not normally distributed (Shapiro-Wilk statistic), therefore, non-parametric statistics (Mann-Whitney test) were used. There were no significant differences between replicates (P > 0.05), so replicates were pooled. Absolute values of free and total amino acid were calculated from nmol/mg of tissues to nmol/egg or alevin. All 40 eggs or alevins (pooled from two replicates of 20 each) were analysed individually for each amino acid. These eggs or alevins represented variability within the population from which the samples were drawn. Procedures for these analyses are described in Sokal and Rohlf (1981). A probability level of P -C0.05 was considered statistically significant. Computations were performed using the SAS (Statistical Analysis System, release 6.06) package.
Biochemical analyses
For biochemical analyses, samples of 50 eggs or alevins were randomly collected from each replicate to determine qualitative composition (nmol/mg of tissues). All 50 eggs or alevins were homogenized in a ground glass homogenizer with a motor driven pestle. Egg or alevin homogenates were sonicated (85 W, 20 kHz, 30 set) to ensure complete disruption of the shells. Subsequent aliquots were prepared from homogenized tissues of each replicate to determine free and total amino acids (nmol/mg of tissues). These values were later converted into absolute values (nmol/mg of tissues to nmol/egg or alevin) on the basis of egg or alevin weights (20 eggs or alevins randomly on the basis of egg or alevin weights (20 eggs or alevins randomly collected from each replicate for weight/diameter or length measurements). All the assays were carried out in triplicate. Total amino acid
Amino acid levels were determined as described by Shahidi et al. (1990). Samples were freeze-dried and then hydrolysed for 24 hr at 110°C with 6 N HCI (Blackburn, 1968). The hydrolysed amino acids were then separated and identified (Shahidi et al., 1990). Tryptophan was determined separately, according to the method of Penke et al. (1974). Cysteine and methionine were converted to cysteic acid and methionine sulphone, respectively, during the HCI hydrolysis (Blackbum, 1968). Free amino acid
For determination of free amino acid levels, samples were deproteinized with 10% sulfosalicylic acid (four parts sample, one part sulfosalicylic acid) and diluted 1:2 with lithium citrate buffer, pH 2.2,0.3 N Li (Mondino er al., 1972; Ohara and Ariyoshi, 1979). Deproteinized samples were analysed Table
RESULTS
Eggs collected from the testosterone-treated fish were significantly lighter in wet weight (control: 95.01 + 5.34 mg; treatment: 89.24 f 8.30 mg, N = 40, P < 0.05) and smaller in diameter (control: 6.00 + 0.21 mm; treatment: 5.78 + 0.24 mm, N = 40, P < 0.05)than those from controls. Alevins from the testosterone-treated fish were significantly heavier in wet weight (control: 108.55 + 12.35 mg; treatment: 118.34 + 16.49 mg, N = 40, P < 0.05) and larger in fork length (control: 2.29 + 0.13 cm; treatment: 2.47 + 0.12 cm, N = 40, P < 0.05) than those from controls at first feeding. The total amino acid content per egg from control groups (178, 104 nmol/egg) was higher than eggs from treatment groups (169,801 nmol/egg) (Tables 1 and 2). The total amino acid content per egg or alevin declined continuously through development, with a minimum at first feeding (control = 113,295 nmol/alevin; treatment = 103,397 nmol/alevin). Eggs collected from treated fish had higher amounts of cysteine, hydroxy-
I. Levelsof amino acids in eaas or alevins at different developmental stages from control group -
Amino acid Alanine Argininet Aspartic acid Cysteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine* Leucine* Lysine’ Methionine eq.’ Phenylalanine’ Proline Serine Threonine* Tryptophan Tyrosine* Valine* Essential Non-essential Total
Before fertilization
Developmentalstages After fertilization Eyed stage
20,929t 8330’1 14,916t 3017t 16,062t 7749t 3907t 286t 10,606.t 16,470t
2l,o8lt 8390t 14,882t 2914t 16,235t 7673t 3900t 234% 10,796t 16,313t
13,262t
13,270t
3392t 6814t 10,128t 11,358t 978lt 1344t 516lt 14,594t 92,316 85,788 178,104
3260t 6809t
9832t
11,411t 9754t 1195t 5099t 14,675t 92,267 85,457 177,724
18,880t 79101 13,716: 1641: 15,703t SlSS$ 3717% 215% 9316f 14,978% 12,349f 1926% 6341% 9607% 10,669t 9031% 1821% 4834% 13,075f 83,476 80,438 163,914
_____ Hatching 17.6696 71985 12,2105 2619 13,700$ 78427 349@ 276t 888of, I3,805§ I I ,845g 30855 57838 752% 9641% 80475 I0865 43835 12,454$ 78,968 72,559 151,527
First feeding
I I,35611 5493 (1 998411 170611 12,078$ IO.0765 2929 11 I245 5860 I/ 9873/j 871511 197911 4375/l 526311 64OW 571711 406 II 309411 78679 55,901 57,394 113,295
Means (nmol/egg, alevin) followed by diflerent symbols in a horizontal row were significantly different (P -z 0.05, N = 40). Standard errors have been omitted for clarity. *Essential amino acids
Egg quality in Atlantic salmon Table 2. Levels of amino acids in eggs or alevins at different developmental treated WOUD Developmental
Amino acid Alanine Argininet Asoartic acid Cykteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine’ Leucine’ Lysine* Methionine eq.* Phenylalanine” Proline Serine Threonine* Tryptophan Tyrosine’ Vahne’ Essential Non-essential Total
Before fertilization 19,927t 7950t 14,188t 3248t 15,242t 7376t 3674t 288T 10,139t 15,589t 12,540t 34537 6436t 9554t 10,866t 93497 1255t 48427 13,887t 87,859 81,944 169.803
After fertilization 19,781t 7806t 14,037t 3166% 15,563t 7248t 3627t 169% 9790t 15,267t 12,431t 3782% 6358t 9326t 10,892t 9245t Il98t 4783t 13,613t 86,702 81,380 168.082
399 stages from testosterone
stages
Eyed stage 18,622f 7702t 13.332% 27355 15,368t 7967% 3618t l5l$ 92735 14,449f 12,114.t 3Ow 6178t 9006% 10,329t 8797% 1286t 4495% 12,929t 82,559 78,796 161.355
Hatching 17.2135 7058? 12.0378 225011 13,784$ 8010% 3491% 2045 867911 13,3775 ll,517% 28455 5613% 74065 9305% 77719 1027% 41185 I I ,992f 76,461 71,236 147.697
First feeding ~0,544/1 47985 10,948 Ij 203611 I I ,635$ 91845 26355 22011 4 I6971 815411 7662s; 251611 36525 471211 48Ol$ 4721 II 376§ 2261 I/ 8373$ 48,941 54,456 103.397
Means (nmol/egg, alevin) followed by different symbols in a horizontal row were significantly different (P < 0.05, N = 40). Standard errors have been omitted for clarity. *Essential amino acids.
lysine and methionine than eggs collected from controls (Tables 1 and 2). Alanine content was the highest among all protein amino acids present in eggs of both groups (control = 11.75%, treatment = 11.74%). When expressed in quantitative terms (nmol/egg), the first five amino acids in descending order were alanine, leucine, glutamic acid, aspartic acid and valine in both groups. In control groups, there were no significant changes in glutamic acid and serine until the eyed-stage (Table 1). On the other hand, in treatment groups, arginine, glutamic acid, histidine, lysine, phenylalanine, serine, tryptophan and valine did not decline significantly until the eyed-stage (Table 2). Glycine was the only amino acid whose levels were higher at eyed-stage, hatching and first feeding over initial levels in both groups (Tables 1 and 2). In control groups, alanine, aspartic acid, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine were found to decrease continuously during development (Table 1). In contrast, alanine, arginine, aspartic acid, cysteine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine declined continuously during development in treatment groups (Table 2). Over the development period, maximum decline in total amino acids was noticed from hatching to first feeding in both groups (control = 25.23%; treatment = 30%). Total essential amino acids (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryosine and valine; Walton, 1985) were lower in eggs from treated fish (87,859 nmol/egg) than eggs from controls (92,316 nmol/egg), and declined through development. Total non-essential amino acids in eggs collected from treated fish (81,944 nmol/egg) were also lower than in eggs from controls (85,788 nmol/egg), and declined through development. In eggs of both groups, essential amino acids were higher than non-essential amino acids
(control = 51.83% essential; treatment = 5 1.74% essential). During development, the decline in essential amino acids was higher than non-essential amino acids (control: essential = 39.45%; nonessential = 33.1%; treatment: essential = 44.3%; non-essential = 33.54%). When expressed in nmol/mg dry weight of egg tissues (qualitative composition), there were no significant differences in protein-bound amino acids between control and treatment groups (P > 0.05, Table 3). Essential and non-essential amino acids in
Table 3. Qualitative composition of amino acids in eggs (unfertilized) collected from control and testosterone-treated fish Amino acid Alanine Arginine’ Aspartic acid Cysteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine* Leucine* Lysinet Methionine eq.* Phenylalanine’ Proline Serine Threonine* Tryptophan Tyrosine* Valine’ Essential Non-essential Total
Control
Treatment
622t 264t 472t 95t 5087 245t 124t 9t 336t 521t 420t 107t 216t 320t 359t 3097 43t I63t 462t 2920 2714 5634
670t 267t 477t 109t 513t 248t 124t lot 3417 524t 422t ll6t 216t 3217 365t 314t 42t I63t 467t 2955 2756 5711
Means (nmol/mg dry weight of egg tissues) followed by same symbols in a horizontal row were not significantly different (P > 0.05, N = 6). Standard errors have been omitted for clarity. *Essential amino acids.
400
RAKFSH
K.
SRIVASTAVAand JOSEPHA. BROWN
Table 4. Levels of free amino acids in eggs or alcvins at different developmental stages from control PTOUD Developmental stages Amino acid Alanine Arginine’ Asparagine Aspartic acid Cysteine eq. Glutamic acid Glutamine Glycine Histidine* Isoleucine* Leucine’ Lysine’ Methionine eq.* Phenylalanine’ Proline Serine Threonine’ Tryptophan Tyrosine* Valine’ Essential Non-essential Total
Before fertilization I96t 49t
14t 528t 318t 360t 94t 84t 37t 71t 107t 83t
31t
37t 73t 192t 64t 2:: 132t 638 1868 2506
After fertilization
Eyed stage
182t 40t 29%
468% 438% BLD
377% 436% lolt 73t 36t
:$ 6005 218% 196% 178% 222% 2395 6555 138% 181% 1w 664% 233% 905 38% 3355 2657 2847 5504
137f
W 137% 96%
w 39t 52% 177t
73t 13% 35% l58t 136 2177 2913
Hatching 3945 3750 BLD 16711 15711 439% 1415 224 184% 1835 19411 50211
128% 1427 101II 623% 1824 66 II llO§ 26011 2260 2314 4574
First feeding 140911 602/I 1st 549t 17411 856/l 499 II 1124(/ :; 9w 916T
4175 3716
52411
12035 445 II 9911 342 II 713!1 5603 6461 12,064
Means (nmol/egg, alevin) followed by different symbols in a horizontal row were significantly different (P < 0.05, N = 40). Standard errors have been omitted for clarity. BLD, Below the limits of detection: *essential amino acids.
dry egg tissues (nmol/mg dry weight) from treated fish were not significantly different from controls (control: essential = 2920 nmol/mg; non-essential = 2714 nmol/mg; treatment: essential = 2955 nmol/mg; non-essential = 2756 nmol/mg; Tables 1 and 2). Free amino acids in eggs from treated fish were significantly higher than in eggs from controls (P c 0.05, Tables 4 and 5). Over the developmental period, the highest amount of free amino acids (total)
was noticed at first feeding in both groups (control = 12,064 nmol/alevin; treatment = 23,950 nmol/alevin). During development (before fertilization to first feeding), the increase in the free amino acid pool was higher in treatment groups than controls (treatment = 500.70%; control = 381.40%). Total essential amino acids (free) in eggs were significantly lower than non-essential amino acids in both groups (control = 25.45%; treatment = 24.08%).
Table 5. Levels of free amino acids in eggs or alevins at different developmental testosterone-treated wxm
stages from
Developmental stages Amino acid Alanine Arginine’ Asparagine Aspartic acid Cysteine eq. Glutamic acid Glutamine Glycine Histidine* Isoleucine’ Leucine’ Lysine* Methionine eq.’ Phenylalanine’ Proline Swine Threonine’ Tryptophan Tyrosine’ Valine* Essential Non-essential Total
Before fertilization 298t 64t 27t 972t 48lt 548t 1487 I34T
45t 114t 167t 132t 49t 56t 116t 290t 97) 13t 4Ot 196t 960 3027 3987
After fertilization 370% 79t 52% 8667 564t 763% 162t lost 63% 150% 238% 180% 66% 78% 107t 334% 132% 27% 69% 255% 1310 3354 4664
Eyed stage 2245 169% BLD 83% 62% 23% 63% 89% 73% ;; 2535 61% 73% 69% 2526 93t 418 155 1375 1041 1120 2161
Hatching 748 II 5838 BLD 3115 174 7467 218t 42% 280§ 271 II 34511 78011 2294 2315 2285 ill@ 3121 9911 16711 423 II 362 I 4065 7686
First feeding 278271 1167/l 1505 121211
11811 176111 7675 210711 57911 85311 2614’11 209271 77011 73911 1112/l 189311 99811 14211 64311 145111 11,906 12,044 23,950
Means (nmol/egg, alevin) followed by different symbols in a horizontal TOWwere significantly different (P < 0.05, N = 40). Standard errors have been omitted for clarity. BLD, Below the limits of detection; *essential amino acids.
Egg quality in Atlantic salmon DISCUSSION
The data indicated that amino acids were found in high amounts in Atlantic salmon eggs at spawning, and progressively decreased until the time of final yolk absorption. Quantitatively, the first five amino acids (nmoliegg) in descending order were alanine, leucine, glutamic acid, aspartic acid and valine in both groups. After fertilization, a period of increasing metabolic activity starts in the eggs, which has been reported to correlate with embryonic cell divisions, cellular differentiation and organogenesis. These processes are energy demanding, with the energy coming from the catabolism of body stores i.e., protein, iipid and carbohydrate. For protein utilization, it must be broken down into amino acids which are then either catabolized for energy production or utilized in the building of new body tissues. Total amino acid content per egg from treated fish was lower than eggs from controls due, in part, to their smaller size, but the qualitative composition (nmol/mg dry weight) of amino acids did not differ. These data are in agreement with those of Timoshina et al. (1982) who reported higher quantitites of protein-bound amino acids in larger eggs, but the qualitative composition of the amino acids in eggs was the same. Moreover, in another study on Atlantic salmon, we found that the eggs collected from a wild (anadromous) stock were larger and heavier, and had a significantly higher amino acid content due to their larger size, compared with eggs from a cultured stock (Srivastava er al., 1992). The amino acid pool in eggs and alevins declined continuously through development in both groups. The maximum decline in the amino acid content was noticed after hatching. The decline in total amino acid content during development was much higher for the treatment groups than the controls. For successful embryonic development, adequate amounts of amino acids and nucleic acids are necessary, otherwise development will halt or slow down at a later developmental stage (Monroy and Maggio, 1964; Harper et al., 1970; Tews et al., 1979, 1980; Metcoff, 1986). Proteins obtained from the maternal circulation later serve as amino acid and energy sources for the growing embryo (Tyler er al., 1988). In control groups, alanine, aspartic acid, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and vaiine were found to decrease during development. However, alanine, arginine, aspartic acid, cysteine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine declined continuously during development in treatment groups. The differences in the types of amino acids utilized during development may be due to the differential growth and developmental rates of embryos and alevins. Despite having eggs which were smaller in diameter, lighter in weight and lower in total amino acid content, alevins from treated fish were heavier in wet weight and larger in body Iength, and exhibited enhanced free amino acid contents at first feeding. The free amino acid pool increased during early development in both groups. This may be due to enhanced synthesis and mobilization of the free amino acid pool to meet an increasing and immediate energy requirement. Korsgaard (1990) reported significant increases
401
in free amino acids, total RNA, RNA:DNA and RNA:protein in the liver and plasma of estradioltreated Zoarces viviparus L. males over controls. Therefore, it can be hypothesized that the protein turnover of embryos and alevins from treated fish would have been higher than controls. Changes in the free amino acid pool during embryonic development of fresh water fishes have never been studied before. However, contrary to this work, a decrease in the free amino acid pool of Atlantic cod (Gadus morrhua) embryos has been reported (Fyhn and Serigstad, 1987; Fyhn et al., 1987; Fyhn, 1989). Reasons for the decline of the free amino acid pool in Atlantic cod embryos may be due to less energy reserves being present in cod eggs to support the metabolic requirements of embryos, or the yolk protein breakdown rate is slower than metabolic requirement of embryos, Total essential amino acids were higher than nonessential amino acids in the eggs of both groups. Moreover, the decline in essential amino acids was higher than non-essential amino acids during development. Fish cannot synthesize essential amino acids which means they have to come from external sources i.e., diet (Walton, 1985). Therefore, at the time of formulating salmon diets it should be made sure that the essential amino acids are present in adequate amounts. Although we have not measured if testosterone injections or implantations elevate the endogenous testosterone levels in the plasma of broodstock, Dr L. W. Crim (personal communication) found that testosterone administration resulted in a significant increase in testosterone levels in the plasma of Atlantic salmon broodstock the next month. Testosterone implantations increase the rate of rematuration of female Atlantic salmon kelt, in other words the number of fish which spawned is increased (Crim et al., 1989). In conclusion, although eggs collected from testosterone-treated fish were smaller in diameter and lighter in weight, their qualitative compositions of all protein amino acids did not differ. Embryos and alevins from treated fish mobilized more free amino acids, and, as a result, alevins were larger and heavier at first feeding compared to controls. This may be due to the anabolic effect of testosterone being transferred from mother to eggs during the period of oocyte growth. The higher synthesis of free amino acids by alevins from testosterone-treated fish would mean more protein available for future use, which would enhance growth and survival. Ack~owledgemenfs-We
thank Dr L. W. Crim and Connie Short for allowing us access to their broodstock. We also thank Dr Jozef Synowiecki and Ronald B. Pegg for their valuable suggestions and help in conducting the experiments. Technical support of D. Hall and S. Banfield for amino acid analyses is greatly appreciated. Financial support for this project was provided by the Newfoundland and Labrador Department of Fisheries, St John’s, NF, Canada. REFERENCES
Blackburn S. (1968) Amino Acid Determination. Methods and Techniques, 1st Edn. Marcel Dekker, New York. Crim L. W. (1991) Hormonal manipulation of fish seasonal reproductive cycles, In Proc. 4th Int. Symp. Reprod.
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Physiol. Fish (Edited by Scott A. P., Sumpter J. P., Kime D. E. and Rolfe M.). vv. 43-47. Universitv of East Analia Press, U.K. ” _Crim L. W. and Evans D. M. (1983) Influence of testosterone and/or luteinizing hormone releasing hormone analogue on precocious sexual development in the juvenile rainbow trout. Biol. Reprod. 29, 137-142. Crim L. W., Evans D. M. and Vickery B. H. (1983) Manipulation of the seasonal reproductive cycle of the landlocked Atlantic salmon (Salmo s&r) by LHRH analoaues administered at various stages of aonadal development. Can. J. Fish. Agric. Sci. &, 61-67: Crim L. W.. Wilson C. E.. So Y. P. and Idler D. R. (1989) Influence ‘of testosterone treatment on the rematurationj spawning performance of female Atlantic salmon kelt reared in captivity. In Abs. 6th amt. Meeting Aquaculture Assoc. Can. 31, 89-3.
Fyhn H. J. (1989) First feeding of marine fish larvae: are free amino acids the source of energy? Aquaculture 80, 11l-120. Fyhn H. J. and Serigstad B. (1987) Free amino acids as energy substrate in developing eggs and larvae of the cod, Gadus morhua. Mar. Biol. 96, 335-341. Fyhn H. J., Serigstad B. and Mangor-Jensen A. (1987) -Free amino acids in developing eggs and yolk-sac larvae of the cod. Gadus morhua L. Sarsia 72. 363-365. Harper A. E., Benevenga N. J. and Wolhueter R. M. (1970) Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50, 428-557. Korsgaard B. (1990) Estrogen treatment and its influence on protein synthesis and amino acid metabolism in Zoarces viviparus (L) males. Fish Physiol. Biochem. 8, 121-127. Love R. M. (1980) The Chemical Biology of Fishes, Advances 1968-1977, Vol. 2. Academic Press, London. Metcoff J. (1986) Intracellular amino acid levels as predictors of protein synthesis. J. Am. College Nutr. 5, 107-120. Mondino A., Bongiovanni G., Fumero S. and Rossi L. (1972) An improved method of plasma deproteination with sulphosalicylic acid for determining amino acids and related compounds. J. Chromatogr. 74, 255-263. Monroy A. and Maggio R. (1964) Biochemical studies
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0300-9629/92 $5.00 + 0.00 0
Printedin Great Britain
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ASSESSMENT OF EGG QUALITY IN ATLANTIC SALMON, SALMO SALAR, TREATED WITH TESTOSTERONE-II. AMINO ACIDS RAKESH
K.
SRWASTAVA*
and
JOSEPHA. BROWN
Ocean Sciences Centre/Department of Biology, Memorial University of Newfoundland, St John’s, NF AlC 5S7, Canada (Received 6 February 1992; accepted 6 March 1992) Atlantic salmon (Sabno salar) were treated with Silastic pellet implants containing testosterone (200 pg/g body weight) four times in a year. Eggs stripped from control (sham implantation) and testosterone-treated fish were fertilized and comparisons of free and total amino acid compositions made until first feeding. 2. Despite having eggs which were smaller in diameter, lighter in weight and lower in total amino acid contents, alevins from testosterone-treated fish were heavier in wet weight and larger in body length, and exhibited enhanced free amino acid contents at first feeding. 3. The qualitative composition of total amino acids in eggs from treated and control fish did not differ. 4. Total amino acid pool of eggs and alevins declined during development, but an increase in the free amino acid pool was noticed through development. The increase in free amino acid pool was higher in eggs and alevins from treated fish than controls, perhaps due to enhanced mobilization of the free amino acid pool.
Abstract-l.
INTRODUCTION Various
androgens
have
been
used
to
enhance
in teleosts (Crim et al., 1989; Crim, 1991), but their consequences on amino acid compositions of eggs have never been examined. Gonadal recrudescence can also be accelerated by long term hormonal therapy consisting of analogs of gonadotropic hormone releasing hormone (GnRH) and/or steroids in various species of salmonids (Crim and Evans, 1983; Crim et al., 1983, 1989; Crim, 1991). Part I of this paper deals with the effects of testosterone treatment during gonadal recrudescence on subsequent protein, lipid, carbohydrate, polyamine, dry matter and ash contents of eggs and alevins, and early growth, development and survival of Atlantic salmon (Srivastava and Brown, 1992). Recently, Korsgaard (1990) reported significant increases in free amino acids, total RNA, RNA:DNA and RNA:protein in the liver and plasma of estradioltreated eel pout, Zoarces viuiparus L. males over controls. Therefore, in this paper we examine the consequences of testosterone treatment during gonadal recrudescence on subsequent amino acid compositions (free and total) of eggs, and their changes during early development of Atlantic salmon (Salmo salar). Amino acids are both precursors for proteins and substrates for energy production. A deficiency or an excess of one or more of these amino acids can limit protein synthesis (Tews et al., 1979), growth (Harper et al., 1970) or both (Tews et al., 1980). Following gonadal
recrudescence
*Present address: Department of Zoology, College of Biological Science, University of Guelph, Guelph, Ontario NlG 2W1, Canada.
fertilization and during development of the embryo, the yolk proteins are degraded and resorbed, and amino acids are utilized for the synthesis of somatic proteins (Love, 1980). MATERIALS AND METHODS
Animals
The broodstock originated as fry (wild population) from the Exploits river and-were held at-St Ma&-Bay (46”48’N; 53”39’W). Fish were maintained in a circular tank (1500 1) and fed an artificial diet (Corey Feed Mills, Fredericton, NB): Water source was brackish and varied from 12.0 to 16.7 ppt over the study. Photoperiod was ambient, varied from 8 hr light: 16 hr dark (December) to 16 hr light : 8 hr dark (June). There were no significant differences in body weight (control: 2.52 + 0.11 kg; treatment: 2.6 + 0.09 kg, P > 0.05) and body length (control: 57.8 + 0.42 cm; treatment: 57.6 + 0.47 cm, P z 0.05) among control and treated fish at the time of egg collection. Fecundity varied by less than 100 eggs out of 1800 among the groups. We followed the protocol of Crim et a/. (1989) for testosterone administration. Fish received either sham implantation treatment (control fish) or testosterone hormone implantation (treated fish) bimonthly. More specifically, treatment fish were administered with Silastic pellet (1 x 0.2 cm in diameter) implants containing testosterone (200 pg/g body weight) four times (February, April, June and August) in a year. Testosterone implantation results in measurable increases in testosterone levels in plasma of the same broodstock of Atlantic salmon (Dr L. W. Crim, personal communication). Control and treated fish were tagged for identification. Mating design and incubation
Eggs and sperm were collected in November and brought to the Marine Sciences Research Laboratory. Two replicates of egg batches (two from control and two from treated fish) were made. In each replicate, 2000 eggs from four females were fertilized with a pool of milt from three males. After 397
398
RAKESHK. SRIVASTAVAand JOSEPHA. BROWN
water hardening, the eggs were transported to an incubator where the temperature varied from 6 to 8°C.
with a Beckman 121 MB amino acid analyser (Srivastava et al., 1992).
Sampling
Statistical analyses
Samples were collected at five different developmental stages, i.e. before fertilization, after fertilization, eyed-stage, hatching and first feeding. From each replicate, 20 eggs and alevins were collected to determine egg weight/diameter, and alevin weight/length, respectively.
The data were not normally distributed (Shapiro-Wilk statistic), therefore, non-parametric statistics (Mann-Whitney test) were used. There were no significant differences between replicates (P > 0.05), so replicates were pooled. Absolute values of free and total amino acid were calculated from nmol/mg of tissues to nmol/egg or alevin. All 40 eggs or alevins (pooled from two replicates of 20 each) were analysed individually for each amino acid. These eggs or alevins represented variability within the population from which the samples were drawn. Procedures for these analyses are described in Sokal and Rohlf (1981). A probability level of P -C0.05 was considered statistically significant. Computations were performed using the SAS (Statistical Analysis System, release 6.06) package.
Biochemical analyses
For biochemical analyses, samples of 50 eggs or alevins were randomly collected from each replicate to determine qualitative composition (nmol/mg of tissues). All 50 eggs or alevins were homogenized in a ground glass homogenizer with a motor driven pestle. Egg or alevin homogenates were sonicated (85 W, 20 kHz, 30 set) to ensure complete disruption of the shells. Subsequent aliquots were prepared from homogenized tissues of each replicate to determine free and total amino acids (nmol/mg of tissues). These values were later converted into absolute values (nmol/mg of tissues to nmol/egg or alevin) on the basis of egg or alevin weights (20 eggs or alevins randomly on the basis of egg or alevin weights (20 eggs or alevins randomly collected from each replicate for weight/diameter or length measurements). All the assays were carried out in triplicate. Total amino acid
Amino acid levels were determined as described by Shahidi et al. (1990). Samples were freeze-dried and then hydrolysed for 24 hr at 110°C with 6 N HCI (Blackburn, 1968). The hydrolysed amino acids were then separated and identified (Shahidi et al., 1990). Tryptophan was determined separately, according to the method of Penke et al. (1974). Cysteine and methionine were converted to cysteic acid and methionine sulphone, respectively, during the HCI hydrolysis (Blackbum, 1968). Free amino acid
For determination of free amino acid levels, samples were deproteinized with 10% sulfosalicylic acid (four parts sample, one part sulfosalicylic acid) and diluted 1:2 with lithium citrate buffer, pH 2.2,0.3 N Li (Mondino er al., 1972; Ohara and Ariyoshi, 1979). Deproteinized samples were analysed Table
RESULTS
Eggs collected from the testosterone-treated fish were significantly lighter in wet weight (control: 95.01 + 5.34 mg; treatment: 89.24 f 8.30 mg, N = 40, P < 0.05) and smaller in diameter (control: 6.00 + 0.21 mm; treatment: 5.78 + 0.24 mm, N = 40, P < 0.05)than those from controls. Alevins from the testosterone-treated fish were significantly heavier in wet weight (control: 108.55 + 12.35 mg; treatment: 118.34 + 16.49 mg, N = 40, P < 0.05) and larger in fork length (control: 2.29 + 0.13 cm; treatment: 2.47 + 0.12 cm, N = 40, P < 0.05) than those from controls at first feeding. The total amino acid content per egg from control groups (178, 104 nmol/egg) was higher than eggs from treatment groups (169,801 nmol/egg) (Tables 1 and 2). The total amino acid content per egg or alevin declined continuously through development, with a minimum at first feeding (control = 113,295 nmol/alevin; treatment = 103,397 nmol/alevin). Eggs collected from treated fish had higher amounts of cysteine, hydroxy-
I. Levelsof amino acids in eaas or alevins at different developmental stages from control group -
Amino acid Alanine Argininet Aspartic acid Cysteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine* Leucine* Lysine’ Methionine eq.’ Phenylalanine’ Proline Serine Threonine* Tryptophan Tyrosine* Valine* Essential Non-essential Total
Before fertilization
Developmentalstages After fertilization Eyed stage
20,929t 8330’1 14,916t 3017t 16,062t 7749t 3907t 286t 10,606.t 16,470t
2l,o8lt 8390t 14,882t 2914t 16,235t 7673t 3900t 234% 10,796t 16,313t
13,262t
13,270t
3392t 6814t 10,128t 11,358t 978lt 1344t 516lt 14,594t 92,316 85,788 178,104
3260t 6809t
9832t
11,411t 9754t 1195t 5099t 14,675t 92,267 85,457 177,724
18,880t 79101 13,716: 1641: 15,703t SlSS$ 3717% 215% 9316f 14,978% 12,349f 1926% 6341% 9607% 10,669t 9031% 1821% 4834% 13,075f 83,476 80,438 163,914
_____ Hatching 17.6696 71985 12,2105 2619 13,700$ 78427 349@ 276t 888of, I3,805§ I I ,845g 30855 57838 752% 9641% 80475 I0865 43835 12,454$ 78,968 72,559 151,527
First feeding
I I,35611 5493 (1 998411 170611 12,078$ IO.0765 2929 11 I245 5860 I/ 9873/j 871511 197911 4375/l 526311 64OW 571711 406 II 309411 78679 55,901 57,394 113,295
Means (nmol/egg, alevin) followed by diflerent symbols in a horizontal row were significantly different (P -z 0.05, N = 40). Standard errors have been omitted for clarity. *Essential amino acids
Egg quality in Atlantic salmon Table 2. Levels of amino acids in eggs or alevins at different developmental treated WOUD Developmental
Amino acid Alanine Argininet Asoartic acid Cykteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine’ Leucine’ Lysine* Methionine eq.* Phenylalanine” Proline Serine Threonine* Tryptophan Tyrosine’ Vahne’ Essential Non-essential Total
Before fertilization 19,927t 7950t 14,188t 3248t 15,242t 7376t 3674t 288T 10,139t 15,589t 12,540t 34537 6436t 9554t 10,866t 93497 1255t 48427 13,887t 87,859 81,944 169.803
After fertilization 19,781t 7806t 14,037t 3166% 15,563t 7248t 3627t 169% 9790t 15,267t 12,431t 3782% 6358t 9326t 10,892t 9245t Il98t 4783t 13,613t 86,702 81,380 168.082
399 stages from testosterone
stages
Eyed stage 18,622f 7702t 13.332% 27355 15,368t 7967% 3618t l5l$ 92735 14,449f 12,114.t 3Ow 6178t 9006% 10,329t 8797% 1286t 4495% 12,929t 82,559 78,796 161.355
Hatching 17.2135 7058? 12.0378 225011 13,784$ 8010% 3491% 2045 867911 13,3775 ll,517% 28455 5613% 74065 9305% 77719 1027% 41185 I I ,992f 76,461 71,236 147.697
First feeding ~0,544/1 47985 10,948 Ij 203611 I I ,635$ 91845 26355 22011 4 I6971 815411 7662s; 251611 36525 471211 48Ol$ 4721 II 376§ 2261 I/ 8373$ 48,941 54,456 103.397
Means (nmol/egg, alevin) followed by different symbols in a horizontal row were significantly different (P < 0.05, N = 40). Standard errors have been omitted for clarity. *Essential amino acids.
lysine and methionine than eggs collected from controls (Tables 1 and 2). Alanine content was the highest among all protein amino acids present in eggs of both groups (control = 11.75%, treatment = 11.74%). When expressed in quantitative terms (nmol/egg), the first five amino acids in descending order were alanine, leucine, glutamic acid, aspartic acid and valine in both groups. In control groups, there were no significant changes in glutamic acid and serine until the eyed-stage (Table 1). On the other hand, in treatment groups, arginine, glutamic acid, histidine, lysine, phenylalanine, serine, tryptophan and valine did not decline significantly until the eyed-stage (Table 2). Glycine was the only amino acid whose levels were higher at eyed-stage, hatching and first feeding over initial levels in both groups (Tables 1 and 2). In control groups, alanine, aspartic acid, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine were found to decrease continuously during development (Table 1). In contrast, alanine, arginine, aspartic acid, cysteine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine declined continuously during development in treatment groups (Table 2). Over the development period, maximum decline in total amino acids was noticed from hatching to first feeding in both groups (control = 25.23%; treatment = 30%). Total essential amino acids (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryosine and valine; Walton, 1985) were lower in eggs from treated fish (87,859 nmol/egg) than eggs from controls (92,316 nmol/egg), and declined through development. Total non-essential amino acids in eggs collected from treated fish (81,944 nmol/egg) were also lower than in eggs from controls (85,788 nmol/egg), and declined through development. In eggs of both groups, essential amino acids were higher than non-essential amino acids
(control = 51.83% essential; treatment = 5 1.74% essential). During development, the decline in essential amino acids was higher than non-essential amino acids (control: essential = 39.45%; nonessential = 33.1%; treatment: essential = 44.3%; non-essential = 33.54%). When expressed in nmol/mg dry weight of egg tissues (qualitative composition), there were no significant differences in protein-bound amino acids between control and treatment groups (P > 0.05, Table 3). Essential and non-essential amino acids in
Table 3. Qualitative composition of amino acids in eggs (unfertilized) collected from control and testosterone-treated fish Amino acid Alanine Arginine’ Aspartic acid Cysteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine* Leucine* Lysinet Methionine eq.* Phenylalanine’ Proline Serine Threonine* Tryptophan Tyrosine* Valine’ Essential Non-essential Total
Control
Treatment
622t 264t 472t 95t 5087 245t 124t 9t 336t 521t 420t 107t 216t 320t 359t 3097 43t I63t 462t 2920 2714 5634
670t 267t 477t 109t 513t 248t 124t lot 3417 524t 422t ll6t 216t 3217 365t 314t 42t I63t 467t 2955 2756 5711
Means (nmol/mg dry weight of egg tissues) followed by same symbols in a horizontal row were not significantly different (P > 0.05, N = 6). Standard errors have been omitted for clarity. *Essential amino acids.
400
RAKFSH
K.
SRIVASTAVAand JOSEPHA. BROWN
Table 4. Levels of free amino acids in eggs or alcvins at different developmental stages from control PTOUD Developmental stages Amino acid Alanine Arginine’ Asparagine Aspartic acid Cysteine eq. Glutamic acid Glutamine Glycine Histidine* Isoleucine* Leucine’ Lysine’ Methionine eq.* Phenylalanine’ Proline Serine Threonine’ Tryptophan Tyrosine* Valine’ Essential Non-essential Total
Before fertilization I96t 49t
14t 528t 318t 360t 94t 84t 37t 71t 107t 83t
31t
37t 73t 192t 64t 2:: 132t 638 1868 2506
After fertilization
Eyed stage
182t 40t 29%
468% 438% BLD
377% 436% lolt 73t 36t
:$ 6005 218% 196% 178% 222% 2395 6555 138% 181% 1w 664% 233% 905 38% 3355 2657 2847 5504
137f
W 137% 96%
w 39t 52% 177t
73t 13% 35% l58t 136 2177 2913
Hatching 3945 3750 BLD 16711 15711 439% 1415 224 184% 1835 19411 50211
128% 1427 101II 623% 1824 66 II llO§ 26011 2260 2314 4574
First feeding 140911 602/I 1st 549t 17411 856/l 499 II 1124(/ :; 9w 916T
4175 3716
52411
12035 445 II 9911 342 II 713!1 5603 6461 12,064
Means (nmol/egg, alevin) followed by different symbols in a horizontal row were significantly different (P < 0.05, N = 40). Standard errors have been omitted for clarity. BLD, Below the limits of detection: *essential amino acids.
dry egg tissues (nmol/mg dry weight) from treated fish were not significantly different from controls (control: essential = 2920 nmol/mg; non-essential = 2714 nmol/mg; treatment: essential = 2955 nmol/mg; non-essential = 2756 nmol/mg; Tables 1 and 2). Free amino acids in eggs from treated fish were significantly higher than in eggs from controls (P c 0.05, Tables 4 and 5). Over the developmental period, the highest amount of free amino acids (total)
was noticed at first feeding in both groups (control = 12,064 nmol/alevin; treatment = 23,950 nmol/alevin). During development (before fertilization to first feeding), the increase in the free amino acid pool was higher in treatment groups than controls (treatment = 500.70%; control = 381.40%). Total essential amino acids (free) in eggs were significantly lower than non-essential amino acids in both groups (control = 25.45%; treatment = 24.08%).
Table 5. Levels of free amino acids in eggs or alevins at different developmental testosterone-treated wxm
stages from
Developmental stages Amino acid Alanine Arginine’ Asparagine Aspartic acid Cysteine eq. Glutamic acid Glutamine Glycine Histidine* Isoleucine’ Leucine’ Lysine* Methionine eq.’ Phenylalanine’ Proline Swine Threonine’ Tryptophan Tyrosine’ Valine* Essential Non-essential Total
Before fertilization 298t 64t 27t 972t 48lt 548t 1487 I34T
45t 114t 167t 132t 49t 56t 116t 290t 97) 13t 4Ot 196t 960 3027 3987
After fertilization 370% 79t 52% 8667 564t 763% 162t lost 63% 150% 238% 180% 66% 78% 107t 334% 132% 27% 69% 255% 1310 3354 4664
Eyed stage 2245 169% BLD 83% 62% 23% 63% 89% 73% ;; 2535 61% 73% 69% 2526 93t 418 155 1375 1041 1120 2161
Hatching 748 II 5838 BLD 3115 174 7467 218t 42% 280§ 271 II 34511 78011 2294 2315 2285 ill@ 3121 9911 16711 423 II 362 I 4065 7686
First feeding 278271 1167/l 1505 121211
11811 176111 7675 210711 57911 85311 2614’11 209271 77011 73911 1112/l 189311 99811 14211 64311 145111 11,906 12,044 23,950
Means (nmol/egg, alevin) followed by different symbols in a horizontal TOWwere significantly different (P < 0.05, N = 40). Standard errors have been omitted for clarity. BLD, Below the limits of detection; *essential amino acids.
Egg quality in Atlantic salmon DISCUSSION
The data indicated that amino acids were found in high amounts in Atlantic salmon eggs at spawning, and progressively decreased until the time of final yolk absorption. Quantitatively, the first five amino acids (nmoliegg) in descending order were alanine, leucine, glutamic acid, aspartic acid and valine in both groups. After fertilization, a period of increasing metabolic activity starts in the eggs, which has been reported to correlate with embryonic cell divisions, cellular differentiation and organogenesis. These processes are energy demanding, with the energy coming from the catabolism of body stores i.e., protein, iipid and carbohydrate. For protein utilization, it must be broken down into amino acids which are then either catabolized for energy production or utilized in the building of new body tissues. Total amino acid content per egg from treated fish was lower than eggs from controls due, in part, to their smaller size, but the qualitative composition (nmol/mg dry weight) of amino acids did not differ. These data are in agreement with those of Timoshina et al. (1982) who reported higher quantitites of protein-bound amino acids in larger eggs, but the qualitative composition of the amino acids in eggs was the same. Moreover, in another study on Atlantic salmon, we found that the eggs collected from a wild (anadromous) stock were larger and heavier, and had a significantly higher amino acid content due to their larger size, compared with eggs from a cultured stock (Srivastava er al., 1992). The amino acid pool in eggs and alevins declined continuously through development in both groups. The maximum decline in the amino acid content was noticed after hatching. The decline in total amino acid content during development was much higher for the treatment groups than the controls. For successful embryonic development, adequate amounts of amino acids and nucleic acids are necessary, otherwise development will halt or slow down at a later developmental stage (Monroy and Maggio, 1964; Harper et al., 1970; Tews et al., 1979, 1980; Metcoff, 1986). Proteins obtained from the maternal circulation later serve as amino acid and energy sources for the growing embryo (Tyler er al., 1988). In control groups, alanine, aspartic acid, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and vaiine were found to decrease during development. However, alanine, arginine, aspartic acid, cysteine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine declined continuously during development in treatment groups. The differences in the types of amino acids utilized during development may be due to the differential growth and developmental rates of embryos and alevins. Despite having eggs which were smaller in diameter, lighter in weight and lower in total amino acid content, alevins from treated fish were heavier in wet weight and larger in body Iength, and exhibited enhanced free amino acid contents at first feeding. The free amino acid pool increased during early development in both groups. This may be due to enhanced synthesis and mobilization of the free amino acid pool to meet an increasing and immediate energy requirement. Korsgaard (1990) reported significant increases
401
in free amino acids, total RNA, RNA:DNA and RNA:protein in the liver and plasma of estradioltreated Zoarces viviparus L. males over controls. Therefore, it can be hypothesized that the protein turnover of embryos and alevins from treated fish would have been higher than controls. Changes in the free amino acid pool during embryonic development of fresh water fishes have never been studied before. However, contrary to this work, a decrease in the free amino acid pool of Atlantic cod (Gadus morrhua) embryos has been reported (Fyhn and Serigstad, 1987; Fyhn et al., 1987; Fyhn, 1989). Reasons for the decline of the free amino acid pool in Atlantic cod embryos may be due to less energy reserves being present in cod eggs to support the metabolic requirements of embryos, or the yolk protein breakdown rate is slower than metabolic requirement of embryos, Total essential amino acids were higher than nonessential amino acids in the eggs of both groups. Moreover, the decline in essential amino acids was higher than non-essential amino acids during development. Fish cannot synthesize essential amino acids which means they have to come from external sources i.e., diet (Walton, 1985). Therefore, at the time of formulating salmon diets it should be made sure that the essential amino acids are present in adequate amounts. Although we have not measured if testosterone injections or implantations elevate the endogenous testosterone levels in the plasma of broodstock, Dr L. W. Crim (personal communication) found that testosterone administration resulted in a significant increase in testosterone levels in the plasma of Atlantic salmon broodstock the next month. Testosterone implantations increase the rate of rematuration of female Atlantic salmon kelt, in other words the number of fish which spawned is increased (Crim et al., 1989). In conclusion, although eggs collected from testosterone-treated fish were smaller in diameter and lighter in weight, their qualitative compositions of all protein amino acids did not differ. Embryos and alevins from treated fish mobilized more free amino acids, and, as a result, alevins were larger and heavier at first feeding compared to controls. This may be due to the anabolic effect of testosterone being transferred from mother to eggs during the period of oocyte growth. The higher synthesis of free amino acids by alevins from testosterone-treated fish would mean more protein available for future use, which would enhance growth and survival. Ack~owledgemenfs-We
thank Dr L. W. Crim and Connie Short for allowing us access to their broodstock. We also thank Dr Jozef Synowiecki and Ronald B. Pegg for their valuable suggestions and help in conducting the experiments. Technical support of D. Hall and S. Banfield for amino acid analyses is greatly appreciated. Financial support for this project was provided by the Newfoundland and Labrador Department of Fisheries, St John’s, NF, Canada. REFERENCES
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