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Changes in the amino acid pool during embryonic development of cultured and wild Atlantic salmon (Salmo salar)
Aquaculture ELSEVIER
Aquaculture
131 (1995)
115-124
Changes in the amino acid pool during embryonic development of cultured and wild Atlantic salmon (Salmo salar) Rakesh Kumar Srivastava”.” “Ocean Sciences Centre/Department
, Joseph A. Browna, Fereidoon Shahidib of Biology, Memorial University of Newfoundland St. John’s,
NF, AI C 5S7, Canada hDepartment of Biochemistry, Memorial University of Newfoundland St. John’s, NF, AIC 5S7, Canada Accepted 25 July 1994
Abstract Changes in amino acid content (free and protein-bound) of cultured and wild (anadromous) Atlantic salmon eggs and alevins were compared during development. Eggs collected from wild Atlantic salmon had higher amounts of free and protein-bound amino acids due to their larger size than those collected from cultured stock. The qualitative composition of amino acids (nmol/mg) in egg tissues from cultured and wild stocks did not differ. Total amino acid pool of eggs and alevins declined during development, but an increase in the free amino acid pool was noted through development. At the same temperature, the development of embryos and alevins of the wild stock was faster than that of the cultured stock. Protein-bound amino acids, alanine, arginine, aspartic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine were decreased in both cultured and wild salmon, suggesting their importance in embryonic and alevin development. Keywords: Protein and amino acids; Salmo salar; Embryology;
Alevins; Growth-fish
1. Introduction
Amino acids are precursors of many biological compounds, notably protein, and can act as substrates for energy production (Walton, 1985). Deficiencies or excess of one or more of the amino acids are known to limit protein synthesis, growth or both (Ogino, 1980; Wilson and Halver, 1986; Cho and Kaushik, 1990; Cowey, 1992; Murai, 1992). Therefore, * Corresponding 2W1, Canada.
author, present address: Department
SSDIOO44-8486(94)00202-9
of Zoology,
University of Guelph, Guelph, Ontario, NlG
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amino acids should be present in proper balance in the body tissues in order to promote optimum growth and development. It is well known in teleosts that oocytes accumulate several energy reserves such as glycogen, lipids (triglycerides, neutral lipids, fatty acids) and proteins (phosphoproteins, glycoproteins, lipoproteins) during oogenesis (Heming and Buddington, 1988; Mommsen and Walsh, 1988). This accumulation of nutrients in the oocyte provides a reservoir of materials used during various biosynthetic activities essential for the early stages of embryogenesis (Heming and Buddington, 1988; Mommsen and Walsh, 1988). As the embryo develops, the absolute and relative composition of the yolk changes (Nakagawa and Tsuchiya, 1976; Boulekbabache, 1981). After fertilization and during development of the embryo, the yolk proteins are degraded and resorbed, and the amino acids are utilized either for the synthesis of somatic proteins (Love, 1980) or as an energy source for the developing embryos (Tyler et al., 1988). For salmonids held in captivity, it has been found that diet influences reproduction by affecting egg size, fecundity, egg hatchability, fry viability and chemical composition of eggs (Hardy, 1985; Springate et al., 1985). The situation in wild salmonids is less clear. Several studies have been conducted on egg quality of Atlantic salmon (e.g., Hamor and Garside, 1977)) but no attempt has been made to determine the utilization of amino acids during embryogenesis. The purpose of this study was therefore to compare the changes in amino acid content (free and protein-bound) in cultured and wild (anadromous) Atlantic salmon eggs and alevins during development.
2. Materials and methods Animals Wild broodstock of Salmo salar (weight = 2.54 f0.09 kg, length =57.2 f0.62 cm, n = 15) were collected from the Northeast Placentia River (47”,48’N, 53”,52’W), Newfoundland, during their upstream migration in November, and were held at the Marine Sciences Research Laboratory (MSRL) until stripping. Cultured broodstock (weight = 2.52 f 0.11 kg, length = 57.8 + 0.42 cm, n = 40) were raised at St. Mary’s Bay (46*,48’N, 53”,39’W), Newfoundland. Mating design and incubation The cultured fish were stripped at St. Mary’s Bay, and eggs and sperm were brought to the MSRL while the wild broodstock was stripped at the MSRL. Both broodstocks were stripped in November. Two replicates of egg batches were made in both cultured and wild stocks. There were no significant differences in body weight or body length of the broodstocks either between replicates or between cultured and wild stocks (P > 0.05). Eggs were mixed and counted prior to fertilization, and no attempt was made to maintain individual families of eggs. In each replicate, 2000 eggs from 4 females were fertilized with a pool of milt from 3 males, and water-hardened. After water-hardening, the eggs were transferred to an incubator where water temperature varied from 6 to 8°C. The water temperature was measured every day. There were no differences in incubation temperature between the two groups.
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In order to check the success of fertilization, eggs were collected 22 h after fertilization and placed in a solution of glacial acetic acid, methanol and water ( 1: 1: 1, v/v) until they became completely clear. After clearing, the eggs were examined under a dissecting microscope. Eggs were fertilized if cleavage of the germinal disc was seen. When the majority of eggs had developed eye pigment, the trays containing the eggs were given a mild physical shock. Eggs that became opaque were removed. Sampling From each replicate, 20 eggs or alevin were randomly collected for measurements (egg weight and diameter or alevin fork length and weight with yolk-sac) before fertilization, after fertilization, eyed stage, hatching, and first feeding. The criteria for the various stages were when at least 75% eggs were eyed, hatched or when at least 75% of alevins began feeding. Dead eggs were removed and recorded regularly during incubation. Biochemical analysis For biochemical analyses, 3 batches of 50 eggs or alevins were randomly collected from each replicate at the above-mentioned stages. Eggs or alevins (with yolk-sac) were thoroughly homogenized, and subsequent sub-samples, for biochemical analyses, were taken Table 1 Amino acid composition Amino acid
Alanine Arginine* Aspartic acid Cysteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine* Leucine* Lysine* Methionine eq.* PhenylaIanine* Proline Serine Threonine* Tryptophan Tyrosine* VaIine* Essential Non-essential Total
of eggs or alevins from cultured Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
20.7 + 0.3” 8.3+0.1” 14.4 f 0.2” 2.7+0.1” 15.7 f 0.2” 7.7f0.1” 3.9fO.l” o.3*o.oa 10.6 f 0.2a 16.4 k 0.2” 13.2+0.1” 3.4 + 0.0” 6.8f0.1a 10.1 *O.la 11.2*o.2a 9.7f0.1a 1.4 f O.Ob 5.1*0.1a 14.5 f 0.2” 91.7 84.0 175.7
20.9 f0.3” 8.4*0.2= 14.1 f 0.2” 2.5 kO.1” 15.8 fO.la 7.6f0.1b 3.9 fO.0” 0.2*0.0b 10.8*0.1” 16.2 f 0.2” 13.2akO.l” 3.2 fO.Oa 6.8 fO.la 9.8fO.l” 11.2*0.1a 9.7*0.1= 1.2 f O.Ob 5.1 ltO.1” 14.5 f0.1” 91.5 83.4 174.9
18.4f0.1b 7.5kO.lb 13.5 f 0.2a 1.4 f O.Ob 15.1 f 0.3” 8.0f0.1b 3.5 f O.Ob 0.2 f O.Ob 9.1*0.1b 14.7fO.lb 11.7*0.1b 1.8rtO.O” 6.2kO.l” 9.5+0.1= 10.0*0.1” 8.8fO.lb 1.7*0.1” 4.8 f O.Ob 12.7f0.1b 80.8 77.8 158.6
17.3f0.1C 6.8kO.l’ 12.0*0.1b 2.5fO.l” 13.3 fO.lb 7.6f0.1b 3.3fO.O’ 0.3 f0.0” 8.7f0.1c 13.6fO.l’ 11.3k0.1b 2.0fO.Ob 5.6&0.0b 7.4fO.lb 9.0fO.lb 7.9 *o.o= l.O*o.Ob 4.3 f 0.0’ 12.2fO.lb 75.7 70.4 146.1
9.9 f o.3d 4.9kO.2“ 9.4 f 0.2’ l.5f0.1b 11.2*0.1’ 9.0+~0.2” 2.5 i-0.1” 0.1 f 0.0” 5.4*o.ld 9.0f0.1d 7.s*o.1c 1.6f0.0C 4.0 f 0. I’ 4.7*o.oc 5.2kO.l” 5.3f0.1d 0.3 f 0.0’ 2.8f0.1d 7.2Yc0.1c 50.3 51.5 101.8
Means f s.e.m. (Fmol/egg, alevin) with different superscript letters in each row are significantly different (P < 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; * = essential amino acid.
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from the homogenized tissues. All the assays were carried out in triplicate. Means of these values were used in later calculation. Amino acid levels were determined as described by Shahidi et al. ( 1990). Samples were freeze-dried and then hydrolysed for 24 h at 110°C with 6N HCl (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 oxidized to cysteic acid and methionine sulphone respectively during HCI hydrolysis (Blackburn, 1968). They are therefore reported as cysteine equivalents equivalents (methionine (cysteine eq. = 2 * cystine + cysteic acid), and methionine eq. = methionine + methionine sulphone). For determination of free amino acid levels, samples were deproteinized with 10% sulphosalicylic acid (4 parts sample, 1 part sulphosalicylic acid) and diluted 1: 2 with lithium citrate buffer (pH 2.2,0.3N Li) (Mondino et al., 1972; Ohara and Ariyoshi, 1979). Deproteinized samples were analyzed with a Beckman 121 MB amino acid analyzer using Benson D-X8.25 resin and a single column, 3-buffer lithium method as per Beckman 121 MB-TB-017 application notes.
Table 2 Amino acid composition Amino acid
Alanine Arginine* 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
of eggs or alevins from wild Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
25.2+0.8” 9.9 * 0.2” 16.7 f 0.5” 2.9 f O.Oa 18.010.4a 9.2 f 0.3a 4.6fO.l” 0.4 f o.oa 12.7 f 0.7” 19.5 f 0.7” 15.9kO.6” 4.5*0.1a 8.1 f0.5” 11.2+0.8a 13.8 f 0.9 11.5+0.8” l.2+o.la 6.2 f 0.7a 17.6 f 0.6” 110.5 98.6 209.1
24.7k0.6a 9.9fO.l” 16.9 f 0.6” 3.5+0.1” 18.8~kO.7’ 9.3 f 0.3” 4.6k0.1a 0.3 f O.Ob 12.2f0.4” 19.2 f 0.6” 15.5 f0.7” 4.6*0.1a 8.0*0.4” 12.0 f 0.8” 13.5 *0.7” 11.4*0.8” 1.3+0.1” 5.8 f 0.3” 16.7fO.S’ 107.9 100.3 208.2
21.5f0.3b 8.1 kO.lb 16.9 f 0.5” 3.8kO.l” 16.6 f 0.3b 8.0f0.2b 4.2rtO.l” 0.3fO.Ob 8.9 f 0.4b 16.8 f 0.5b 12.9 f 0.3b 3.8rtO.lb 6.5 f 0.4b 9.5 f 0.6” 10.0 f 0.6b 8.4 f 0.7b 0.7fO.Ob 4.6 f 0.3b 15.3 f0.2b 89.5 87.3 176.8
18.7 + 0.2” 7.2kO.l’ 14.1 f0.2b 2.3 f 0.0” 13.2 + 0.2’ 7.7*0.1’ 3.9fO.lb 0.2 f0.0’ 7.2 f 0.3” 13.2*0.4’ 11.3 i-0.2’ 2.8kO.l’ 5.7*0.3’ 7.7 * 0.5’ 7.9 f 0.5’ 7.0*0.4” 0.4 f 0.0” 3.8fO.l” 13.2 f 0.2’ 75.3 72.2 147.5
12.9 f0.2d 5.7fO.ld 8.6 f0.2’ 2.0fO.Ob 11.3+0.1d 9.1 jZO.3” 3.1 f0.0’ 0.2 f 0.0’ 4.8f0.2d 7.2 f 0.3“ 8.2kO.4’ 2.2fO.ld 4.5fO.l” 5.6f0.4d 5.3*0.46 5.6f0.5d 0.4 f o.oc 2.8 f O.od 9.3 f o.5d 53.4 55.4 1108.8
Means f s.e.m. (pmollegg, alevin) with different superscript letters in each row are significantly different (P < 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; * = essential amino acid.
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Statistical analysis Since the data were not normally distributed as tested by Shapiro-Wilk statistics, nonparametric statistics (the Mann-Whitney test) were used. The procedures for these statistical analyses were as described by Sokal and Rohlf ( 1981). A probability level of P < 0.05 was considered statistically significant. Absolute values of amino acids were calculated for each egg and alevin (from nmol/mg of tissue to nmol/egg or alevin) based on the weights of 20 eggs/alevins collected from each replicate. In this paper, qualitative and quantitative compositions of amino acids refer to nmol/mg tissue and nmol/egg or alevin, respectively. Since there were no significant differences between replicates of all the variables studied (P > 0.05)) the replicates were pooled. Computations were performed using the Statistical Analysis System, release 6.06 package (SAS Institute Inc., 1990). 3. Results Eggs collected from the wild stock were significantly heavier and larger than those from cultured stock (mean weight, wild = 112.90 f 5.13 mg, cultured = 95.01 f 5.34 mg, n = 40, Table 3 Free amino acid composition Amino acid
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
of eggs or alevins from cultured Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
196k4.6’ 49*1.1= 14kO.9” 528 f 8.9’ 31853.6’ 360 f 5.2” 94 + 2.4” 84k2.1” 31 f 0.6” 71 f3.2” 107 f 2.5a 83 f 2.3a 31 zhO.7” 37 kO.8” 131h3.2~ 192k5.2” 64rt2.5a 9 f0.2a 26 f 0.8” 132k4.3” 638 1868 2506
182 f 3.9= 40f2.1” 29 f 0.4b 731 f 9.gd 317 f 5.3d 436 f 6.1b 101 f 3.3” 13 f 2.5a 36 + 0.5” 89+4.1a 137 f4.5b 96 f 3.9b 33 f 0.8” 39 f 0.8a 52 f 2.8” 177f7.8” 73 f 2.8” 13f0.3b 35 rt 1.9b 158 * 4.2b 136 2177 2913
468k5.6’ 438k5.8’ BLD 264 *4.5b 202 f 2.8b 600 f 8.6’ 218f5.6b 196 f4.4b 178f5.2b 222 f 6.8b 239 + 5.4’ 655 *9.8’ 13855.4” 181 f 15b 144f5.44 664rt 18b 233f7.6” 90f2.5’ 38f 1.7” 335 f 8.6d 2651 2847 5504
394 f 5.4b 315 f4.9b BLD 167 f 2.5” 157*4.5a 439 * 5.9b 141 rt4.8’ 226 f 5.9’ 184f7.8b 183*5.8’ 194 k 4.6d 502 k 6.8d 128 f 6.5b 142f11b 101 + 3.6” 623 f 16b 182 j~4.6~ 66 f 3.5c 110*5.9= 260 f 5.4’ 2260 2314 4514
1409 f 38.6d 602 f 7.9d 18*0.6” 549 * 9.7’ 114 f 7.4” 856 f 12.5’ 499 f l.gd 1124 f 25.6d 449*9.9c 445k 11.8’ 904 f 10.9e 916 f 14.7e 417 f 7.9’ 371 f 8.8c 524 f 8.9’ 1203 f 36.5’ 445 rt 8.gd 99 f 3.4e 342 k 4.5’ 713f9.8e 5603 6461 12 064
Means f s.e.m. (pmol/egg, alevin) with different superscript letters in each row are significantly different (P < 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; BLD = below the limits of detection; * =essentiaf amino acid.
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Table 4 Free amino acid composition 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
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of eggs or alevins from wild Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
299f 11.8” 83 k5.2a 32i0.Sb 1113 f 24.5e s70*9.se 390 f 19.4a 274+3.3b 57 + 2.3a 54*2.1a 84i5.2a 128*9.1a 157*7.t? 48 f 3.3” 61 f3.5” 51 k2.6” 242 f4.4b 77 + 4.7= 19k0.2a 44k3.2” 142 f 9.4a 879 3347 4226
242 f 7.7’ 66*4.5” 34 + 0.4b 845 f 7.5d 598 f 8.6d 310*9.5a 195 f 4.4a 64*2.1= 48 f 2.2” 61 f 3.6” 971t6.1” 133 + 9.2” 36k 1.8” 51 k2.3” 661k3.8” 192k3.4” 67 + 6.2” 16kO.2” 41 f 2.8” 107 f 8.7” 707 2565 3272
459 f 3.4b 437f5.7b BLD 243 f 3.7b 471 *4.3c 612* 15’ 157*8.7a 203 f 5.2” 185 ~k5.2~ 191 i6.2b 220 f 9.9b 600 f 6.7b 164k8.5b 184f9.8b 99 f 4.2b 76 1 + 6.4” 197 f 7.3b 93f2.3b 51*4.2* 300f6.3b 2528 3099 5627
642 f 7.2’ 441 f4.Sb BLD 194 f 2.6” 330 f 4.3b 570* lib 176k7.8” 414*7.3c 276 f 4.3” 220 f 8.9b 316f8.3’ 6lOk 8.9b 199*9.@ 226 f 6.3’ 151 f 7.2” 847 f 9.46 223 + 12b 94*2.1b 167 f4.5b 349 f 6.9’ 3028 3418 6446
1384 f 22.4’ 545 f 4.8’ 15kO.2’ 573 f 7.3” 151*4.5a 831 f9.6’ 473 f 8.3” 606 f lo.5d 405 f 8.46 437 * 12= 884 f 9.9d 857 f 7.9” 397 f 7.6” 387f9.5d 546 f 8.4d 1139*44.5’ 460 k 16.5’ 93 f 4.3b 337 *4.5= 700 f 19.4d 5409 5811 11220
Means f s.e.m. (pmol/egg, alevin) with different superscript letters in each row are significantly different (P< 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; BLD = below the limits of detection; * = essential amino acid.
P < 0.05; mean diameter, wild = 6.17 + 0.35 mm, cultured = 6.00 f 0.21 mm, 12= 40, P < 0.05). The development (degree-days = dd) of embryos and alevins of the wild stock was faster compared to the cultured stock (cultured, eyed-stage = 457 dd, hatching = 575 dd, first feeding = 841 dd; wild, eyed-stage = 424 dd, hatching = 520 dd, first feeding = 753 dd). When expressed in nmol/mg dry weight of unfertilized egg tissues (qualitative composition), there were no significant differences in amino acid content between cultured and wild stocks (P > 0.05; data not shown). The amounts of essential and non-essential amino acids in unfertilized egg tissues were similar in both cultured and wild stocks (data not shown). The amino acid (protein-bound) content per egg from the wild stock (209.1 pmol/egg) was higher than eggs from the cultured stock ( 175.7 pmol/egg) (Tables 1 and 2). Eggs from wild stock had higher amounts of amino acids than eggs from cultured stock except for tryptophan. Alanine content was highest among all amino acids present in eggs of both stocks (cultured= 12.05% alanine, wild = 11.78% alanine). The amino acid content per egg or alevin in both the cultured and wild stocks declined continuously during development.
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Essential amino acid content was higher in eggs or alevins from wild stock than in eggs or alevins from cultured stock, and declined during development (Tables 1 and 2). Nonessential amino acid content of eggs collected from wild stock (98.6 pmol/egg) was also higher than that of eggs collected from cultured stock ( 84 pmol/egg) , and declined during development up to first feeding. In eggs of both stocks, the amount of essential amino acids was higher than that of the non-essential amino acids (cultured eggs = 52.2% essential, wild eggs = 52.8% essential). The free amino acid pool was higher in eggs of wild stock (4226 nmol/egg) than of cultured stock (2506 nmol/egg) , and increased in both stocks until first feeding (cultured, 12 064 nmol/alevin; wild, 11 226 nmol/alevin; Tables 3 and 4). Essential free amino acids in eggs were lower than non-essential free amino acids in both stocks (cultured = 25.5% essential, wild = 20.8% essential; Tables 3 and 4).
4. Discussion This is the first study demonstrating that amino acids (protein-bound) are found in large amounts in Atlantic salmon eggs at spawning, and progressively decrease to low levels at first feeding. After fertilization, there is a period of increasing metabolic activity accompanied by high energy demand. This energy comes from the catabolism of body stores ie. protein, lipid and carbohydrate (Srivastava and Brown, 1991). Protein must be broken down into amino acids which are then either catabolized for energy production or utilised for building up new body tissues. Quantitatively (nmol/egg), the amino acid (protein-bound) content of eggs from wild stock was higher than that of eggs from cultured stock but the qualitative composition (nmol/mg dry weight) of amino acids in egg tissues from cultured and wild stocks did not differ. Our data are in agreement with those of Timoshina et al. ( 1982) who reported higher quantities of amino acids in larger eggs, but the qualitative composition of the amino acids in eggs was the same. The decline in amino acid content during development was greater in wild stock than cultured stock perhaps because the embryos from wild stock developed faster, i.e. took less time to reach the eyed stage, and hatched earlier. For successful embryonic development, an adequate amount of amino acids is necessary, otherwise development will be halted or slowed down at a later stage (Harper et al., 1970; Ogino, 1980; Metcoff, 1986; Wilson and Halver, 1986; Cho and Kaushik, 1990; Cowey, 1992; Murai, 1992). Proteins obtained from the maternal circulation later serve as amino acid and energy sources for the growing embryo (Tyler et al., 1988). In the present study, the amino acids (protein-bound) alanine, arginine, aspartic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine were found to decrease in both stocks because of their utilization by embryos and alevins. In Arctic charr, it has been noted that isoleucine, lysine, serine, threonine, tryptophan and valine were important for embryonic and fry development (Srivastava, 1991; Srivastava and Brown, 1994). The difference in the types of amino acids may be due to their species-specific roles (Mertz, 1977; Metcoff, 1986). The levels of protein-bound essential amino acids were always higher than those of nonessential amino acids in both stocks, and declined during development. By comparison, the
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levels of free essential amino acids were lower than those of non-essential amino acids, and increased through development in both stocks. It seems that preferential retention of essential amino acids occurs during embryonic and alevin development in both stocks. At present it is not clear how specific amino acids might be retained. It is possible that the processes associated with their catabolism are suppressed or not developed until later in early development, thereby preventing losses. In this study, the free amino acid pool was higher in eggs of the wild stock than of the cultured stock, and increased during development. This suggests that the protein breakdown rate would have been higher than the anabolic and catabolic losses of developing embryos. Timoshina et al. (1982) have also reported higher quantities of free amino acids in the larger eggs of rainbow trout due to their larger size, but there were no qualitative (nmol/ mg of tissues) differences in amino acids. In another study with rainbow trout eggs, total free amino acid concentrations declined just after fertilization, increased to near initial values in the blastula and were nearly doubled at hatching (Zeitoun et al., 1977). However, in contrast to these findings, a continuous decrease in the free amino acid pool of Atlantic cod embryos has been reported (Fyhn and Serigstad, 1987; Fyhn et al., 1987; Fyhn, 1989). The reason for the differences observed in egg quality of the wild and cultured Atlantic salmon is not clear. The two stocks were from different areas, and thus genetic differences cannot be dismissed. Moreover, other factors such as age of parents and diets could also account for the differences observed in the present study (Srivastava and Brown, 1991). The amino acid content of carp eggs was shown to be related to the quality of food eaten by the broodstocks (Lavrovskaya, 1980). Apart from amino acids, there may be some other nutrients (vitamins, trace elements, carotenoids and essential fatty acids etc.) which could contribute to the better performance of embryos and alevins (Luquet and Watanabe, 1986). The cultured stock was fed a commercial diet while the wild stock fed on naturally occurring organisms. It is possible that the diet of the wild population was better and that this contributed to their higher amino acid levels and larger eggs, which means more amino acid would be available for the development of the embryos and alevins. In conclusion, it can be suggested that amino acids in eggs may play a role in the successful development of embryos and alevins. On the basis of our findings, it is reasonable to suggest that the amino acids alanine, arginine, aspartic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine should be present in the diets of broodstock, so that they can be transported to developing oocytes during vitellogenesis.
Acknowledgements We express our thanks to Dr. Garth Fletcher and David Methven for their comments on an earlier version of the manuscript. We also thank Dr. Jozef Synowiecki and Ronald B. Pegg for their valuable suggestions and help in conducting the experiments. Technical support by D. Hall and S. Banfield with 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.
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References Blackbum, S., 1968. Amino Acid Determination. Methods and Techniques, 1st edition. Marcel Dekker, Inc., New York, NY, 21 pp. Boulekbabache, H., 198 1. Energy metabolism in fish development. Am. Zool., 2 1: 377-389. Cho, C.Y. and Kaushii, S.J., 1990. Nutrition energetics in fish: energy and protein utilization in rainbow trout (Salmo gairdneri). In: G.H. Boume (Editor), Aspects of Food Production, Consumption and Energy Values, World Rev. Nutr. Diet. Vol. 61, Karger, Basel, pp. 132-172. Cowey, C.B., 1992. Nutrition: estimating requirements of rainbow trout. Aquaculture, 100: 177-189. Fyhn, H.J., 1989. First feeding of marine fish larvae: are free amino acids the source of energy? Aquaculture, 80: 111-120. Fyhn, H.J. and Serigstad, B., 1987. Free amino acids as energy substrate in developing eggs and larvae of the cod, Gadus marhua. 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. Hamor, T. and Garside, E.T., 1977. Quantitative composition of the fertilized ovum and constituent parts in the Atlantic salmon Salmo salar L. Can. J. Zool., 55: 1650-1655. Hardy, R.W., 1985. Salmonid broodstock nutrition. In: R.N. Iwamoto and S. Sower (Editors), International Symposium on Salmonid Reproduction, Bellevue, WA, 1983. Washington Sea Grant Program. Univ. Washington, Seattle, WA, pp. 98-108. 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. Heming, T.A. and Buddington, R.K., 1988. Yolk absorption in embryonic and larval fishes. In: W.S. Hoar and D.J. Randall, (Editors), Fish Physiology, Vol. XIA. Academic Press, Inc., New York, NY, pp. 407-446. Konurbayev, A.O., 1978. Effect of egg size on the growth rate and variability of larvae of Issyk-Kul’ trout, Kirghiz Academy of Sciences Press, 3: 73-75. Lavrovskaya, N.R., 1980. Sovremennyye issledovaniya po biokhimii ryb (Recent Studies on the Biochemistry of Fish). Tsniieirkh Press, Moscow. Love, R.M., 1980. The Chemical Biology of Fishes, Vol. 2. Advances 1968-1977. Academic Press, London, p. 943. Luquet, P. and Watanabe, T., 1986. Interaction “nutrition-reproduction” in fish. Fish Physiol Biochem., 2: 121129. Mertz, E.T., 1977. The protein and amino acid needs. In: J.E. Halver (Editor), Fish Nutrition. Academic Press, New York, NY, pp. 105-143. Metcoff, J., 1986. Intracellular amino acid levels as predictors of protein synthesis. J. Am. Coll. Nutr., 5: 107120. Mommsen, T.P. and Walsh, P.J., 1988. Vitellogenesis and oocyte assembly. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. XIA. Academic Press, Inc., New York, NY, pp. 347-406. 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. Murai, T., 1992. Protein nutrition of rainbow trout. Aquaculture, 100: 191-207. Nakagawa, H. and Tsuchiya, Y. 1976. Studies on rainbow trout eggs (Salmo gairdnerii irideus). VI. Changes of lipid composition in yolk during development. J. Fat. Fish. Anim. Husb. Hiroshima Univ., 15: 3546. Ogino, C., 1980. Requirements of carp and rainbow trout for essential amino acids. Bull, Jpn. Sot. Sci. Fish., 46: 171-174. Ohara, I. and Ariyoshi, S., 1979. Comparison of protein precipitants for the determination of free amino acids in plasma. Agric. Biol. Chem., 43: 1473-1478. Penke, B., Ferenczi, R. and Kovacs, K. 1974. A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal. Biochem., 60: 45-50. SAS Institute, Inc., 1990. Statistical Analysis System, release 6.06. SAS Institute, Inc., Cary, NC. Shahidi, F., Synowiecki, J. and Naczk, M., 1990. Seal meat - a potential source of muscle food: chemical composition, essential amino acids and colour characteristics. Can. Inst. Food Sci. Tecbnol. J., 23: 137-139. Sokal, R.R. and Rohlf, F.J., 1991. Biometry: The Principles and Practice of Statistics in Biological Research. Freeman and company, San Francisco, p. 467.
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Springate, J.R.C., Bromage, N.R. and Cumaranatunga, P.R.T., 1985. The effects of different ration on fecundity and egg quality in the rainbow trout (Saho gairdneri). In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 371-393. Srivastava, R.K., 1991. Measures of egg quality and hatching performance of Arctic charr (Saluelinus alpinus L.) and Atlantic salmon (Salmo salar L.) eggs. M.Sc. thesis, Memorial University of Newfoundland, St. John’s, NF, Canada, pp. 166. Srivastava, R.K. and Brown, J.A., 1991. The biochemical characteristics and hatching performance of cultured and wild Atlantic salmon (Salmo salar) eggs. Can. J. Zool., 69: 2436-2441. Srivastava, R.K. and Brown, J.A., 1994. Developmental changes in egg composition over the incubation period in Arctic charr. Can. J. Zool., (in press). Timoshina, L.A., Shabalina, A.A. and Mel’nikov, A.S., 1982. Amino acid composition of the sperm and spawn of trout. Hydrobiol. J., 17: 66-72. Tyler, C.R., Sumpter, J.P. and Bromage, N.R., 1988. Selectivity of protein sequestration by vitellogenic oocytes of the rainbow trout, Salmo gairdneri. J. Exp. Zool., 248: 199-206. Wallace, J.C., Kolbeinshavn, A. and Aasjord, D., 1988. Observation on the effect of light intensity on the growth of Arctic charr fingerlings (Saloelinus alpinus) and salmon fry (Salmo salar) Aquaculture, 72: 8 l-84. Walton, M.J., 1985. Aspects of amino acid metabolismin teleost fish. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London, pp. 47-67. Wilson, R.P. and Halver, J.E. 1986. Protein and amino acid requirements of fishes. Annu. Rev. Nutr., 6: 225-244. Zeitoun, I.H., Ullrey, D.E., Bergen, W.G. and Magee, W.T., 1977. DNA, RNA, protein, and free amino acid during ontogenesis of rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can., 34: 83-88.
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Changes in the amino acid pool during embryonic development of cultured and wild Atlantic salmon (Salmo salar) Rakesh Kumar Srivastava”.” “Ocean Sciences Centre/Department
, Joseph A. Browna, Fereidoon Shahidib of Biology, Memorial University of Newfoundland St. John’s,
NF, AI C 5S7, Canada hDepartment of Biochemistry, Memorial University of Newfoundland St. John’s, NF, AIC 5S7, Canada Accepted 25 July 1994
Abstract Changes in amino acid content (free and protein-bound) of cultured and wild (anadromous) Atlantic salmon eggs and alevins were compared during development. Eggs collected from wild Atlantic salmon had higher amounts of free and protein-bound amino acids due to their larger size than those collected from cultured stock. The qualitative composition of amino acids (nmol/mg) in egg tissues from cultured and wild stocks did not differ. Total amino acid pool of eggs and alevins declined during development, but an increase in the free amino acid pool was noted through development. At the same temperature, the development of embryos and alevins of the wild stock was faster than that of the cultured stock. Protein-bound amino acids, alanine, arginine, aspartic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine were decreased in both cultured and wild salmon, suggesting their importance in embryonic and alevin development. Keywords: Protein and amino acids; Salmo salar; Embryology;
Alevins; Growth-fish
1. Introduction
Amino acids are precursors of many biological compounds, notably protein, and can act as substrates for energy production (Walton, 1985). Deficiencies or excess of one or more of the amino acids are known to limit protein synthesis, growth or both (Ogino, 1980; Wilson and Halver, 1986; Cho and Kaushik, 1990; Cowey, 1992; Murai, 1992). Therefore, * Corresponding 2W1, Canada.
author, present address: Department
SSDIOO44-8486(94)00202-9
of Zoology,
University of Guelph, Guelph, Ontario, NlG
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amino acids should be present in proper balance in the body tissues in order to promote optimum growth and development. It is well known in teleosts that oocytes accumulate several energy reserves such as glycogen, lipids (triglycerides, neutral lipids, fatty acids) and proteins (phosphoproteins, glycoproteins, lipoproteins) during oogenesis (Heming and Buddington, 1988; Mommsen and Walsh, 1988). This accumulation of nutrients in the oocyte provides a reservoir of materials used during various biosynthetic activities essential for the early stages of embryogenesis (Heming and Buddington, 1988; Mommsen and Walsh, 1988). As the embryo develops, the absolute and relative composition of the yolk changes (Nakagawa and Tsuchiya, 1976; Boulekbabache, 1981). After fertilization and during development of the embryo, the yolk proteins are degraded and resorbed, and the amino acids are utilized either for the synthesis of somatic proteins (Love, 1980) or as an energy source for the developing embryos (Tyler et al., 1988). For salmonids held in captivity, it has been found that diet influences reproduction by affecting egg size, fecundity, egg hatchability, fry viability and chemical composition of eggs (Hardy, 1985; Springate et al., 1985). The situation in wild salmonids is less clear. Several studies have been conducted on egg quality of Atlantic salmon (e.g., Hamor and Garside, 1977)) but no attempt has been made to determine the utilization of amino acids during embryogenesis. The purpose of this study was therefore to compare the changes in amino acid content (free and protein-bound) in cultured and wild (anadromous) Atlantic salmon eggs and alevins during development.
2. Materials and methods Animals Wild broodstock of Salmo salar (weight = 2.54 f0.09 kg, length =57.2 f0.62 cm, n = 15) were collected from the Northeast Placentia River (47”,48’N, 53”,52’W), Newfoundland, during their upstream migration in November, and were held at the Marine Sciences Research Laboratory (MSRL) until stripping. Cultured broodstock (weight = 2.52 f 0.11 kg, length = 57.8 + 0.42 cm, n = 40) were raised at St. Mary’s Bay (46*,48’N, 53”,39’W), Newfoundland. Mating design and incubation The cultured fish were stripped at St. Mary’s Bay, and eggs and sperm were brought to the MSRL while the wild broodstock was stripped at the MSRL. Both broodstocks were stripped in November. Two replicates of egg batches were made in both cultured and wild stocks. There were no significant differences in body weight or body length of the broodstocks either between replicates or between cultured and wild stocks (P > 0.05). Eggs were mixed and counted prior to fertilization, and no attempt was made to maintain individual families of eggs. In each replicate, 2000 eggs from 4 females were fertilized with a pool of milt from 3 males, and water-hardened. After water-hardening, the eggs were transferred to an incubator where water temperature varied from 6 to 8°C. The water temperature was measured every day. There were no differences in incubation temperature between the two groups.
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In order to check the success of fertilization, eggs were collected 22 h after fertilization and placed in a solution of glacial acetic acid, methanol and water ( 1: 1: 1, v/v) until they became completely clear. After clearing, the eggs were examined under a dissecting microscope. Eggs were fertilized if cleavage of the germinal disc was seen. When the majority of eggs had developed eye pigment, the trays containing the eggs were given a mild physical shock. Eggs that became opaque were removed. Sampling From each replicate, 20 eggs or alevin were randomly collected for measurements (egg weight and diameter or alevin fork length and weight with yolk-sac) before fertilization, after fertilization, eyed stage, hatching, and first feeding. The criteria for the various stages were when at least 75% eggs were eyed, hatched or when at least 75% of alevins began feeding. Dead eggs were removed and recorded regularly during incubation. Biochemical analysis For biochemical analyses, 3 batches of 50 eggs or alevins were randomly collected from each replicate at the above-mentioned stages. Eggs or alevins (with yolk-sac) were thoroughly homogenized, and subsequent sub-samples, for biochemical analyses, were taken Table 1 Amino acid composition Amino acid
Alanine Arginine* Aspartic acid Cysteine eq. Glutamic acid Glycine Histidine* Hydroxylysine Isoleucine* Leucine* Lysine* Methionine eq.* PhenylaIanine* Proline Serine Threonine* Tryptophan Tyrosine* VaIine* Essential Non-essential Total
of eggs or alevins from cultured Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
20.7 + 0.3” 8.3+0.1” 14.4 f 0.2” 2.7+0.1” 15.7 f 0.2” 7.7f0.1” 3.9fO.l” o.3*o.oa 10.6 f 0.2a 16.4 k 0.2” 13.2+0.1” 3.4 + 0.0” 6.8f0.1a 10.1 *O.la 11.2*o.2a 9.7f0.1a 1.4 f O.Ob 5.1*0.1a 14.5 f 0.2” 91.7 84.0 175.7
20.9 f0.3” 8.4*0.2= 14.1 f 0.2” 2.5 kO.1” 15.8 fO.la 7.6f0.1b 3.9 fO.0” 0.2*0.0b 10.8*0.1” 16.2 f 0.2” 13.2akO.l” 3.2 fO.Oa 6.8 fO.la 9.8fO.l” 11.2*0.1a 9.7*0.1= 1.2 f O.Ob 5.1 ltO.1” 14.5 f0.1” 91.5 83.4 174.9
18.4f0.1b 7.5kO.lb 13.5 f 0.2a 1.4 f O.Ob 15.1 f 0.3” 8.0f0.1b 3.5 f O.Ob 0.2 f O.Ob 9.1*0.1b 14.7fO.lb 11.7*0.1b 1.8rtO.O” 6.2kO.l” 9.5+0.1= 10.0*0.1” 8.8fO.lb 1.7*0.1” 4.8 f O.Ob 12.7f0.1b 80.8 77.8 158.6
17.3f0.1C 6.8kO.l’ 12.0*0.1b 2.5fO.l” 13.3 fO.lb 7.6f0.1b 3.3fO.O’ 0.3 f0.0” 8.7f0.1c 13.6fO.l’ 11.3k0.1b 2.0fO.Ob 5.6&0.0b 7.4fO.lb 9.0fO.lb 7.9 *o.o= l.O*o.Ob 4.3 f 0.0’ 12.2fO.lb 75.7 70.4 146.1
9.9 f o.3d 4.9kO.2“ 9.4 f 0.2’ l.5f0.1b 11.2*0.1’ 9.0+~0.2” 2.5 i-0.1” 0.1 f 0.0” 5.4*o.ld 9.0f0.1d 7.s*o.1c 1.6f0.0C 4.0 f 0. I’ 4.7*o.oc 5.2kO.l” 5.3f0.1d 0.3 f 0.0’ 2.8f0.1d 7.2Yc0.1c 50.3 51.5 101.8
Means f s.e.m. (Fmol/egg, alevin) with different superscript letters in each row are significantly different (P < 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; * = essential amino acid.
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from the homogenized tissues. All the assays were carried out in triplicate. Means of these values were used in later calculation. Amino acid levels were determined as described by Shahidi et al. ( 1990). Samples were freeze-dried and then hydrolysed for 24 h at 110°C with 6N HCl (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 oxidized to cysteic acid and methionine sulphone respectively during HCI hydrolysis (Blackburn, 1968). They are therefore reported as cysteine equivalents equivalents (methionine (cysteine eq. = 2 * cystine + cysteic acid), and methionine eq. = methionine + methionine sulphone). For determination of free amino acid levels, samples were deproteinized with 10% sulphosalicylic acid (4 parts sample, 1 part sulphosalicylic acid) and diluted 1: 2 with lithium citrate buffer (pH 2.2,0.3N Li) (Mondino et al., 1972; Ohara and Ariyoshi, 1979). Deproteinized samples were analyzed with a Beckman 121 MB amino acid analyzer using Benson D-X8.25 resin and a single column, 3-buffer lithium method as per Beckman 121 MB-TB-017 application notes.
Table 2 Amino acid composition Amino acid
Alanine Arginine* 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
of eggs or alevins from wild Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
25.2+0.8” 9.9 * 0.2” 16.7 f 0.5” 2.9 f O.Oa 18.010.4a 9.2 f 0.3a 4.6fO.l” 0.4 f o.oa 12.7 f 0.7” 19.5 f 0.7” 15.9kO.6” 4.5*0.1a 8.1 f0.5” 11.2+0.8a 13.8 f 0.9 11.5+0.8” l.2+o.la 6.2 f 0.7a 17.6 f 0.6” 110.5 98.6 209.1
24.7k0.6a 9.9fO.l” 16.9 f 0.6” 3.5+0.1” 18.8~kO.7’ 9.3 f 0.3” 4.6k0.1a 0.3 f O.Ob 12.2f0.4” 19.2 f 0.6” 15.5 f0.7” 4.6*0.1a 8.0*0.4” 12.0 f 0.8” 13.5 *0.7” 11.4*0.8” 1.3+0.1” 5.8 f 0.3” 16.7fO.S’ 107.9 100.3 208.2
21.5f0.3b 8.1 kO.lb 16.9 f 0.5” 3.8kO.l” 16.6 f 0.3b 8.0f0.2b 4.2rtO.l” 0.3fO.Ob 8.9 f 0.4b 16.8 f 0.5b 12.9 f 0.3b 3.8rtO.lb 6.5 f 0.4b 9.5 f 0.6” 10.0 f 0.6b 8.4 f 0.7b 0.7fO.Ob 4.6 f 0.3b 15.3 f0.2b 89.5 87.3 176.8
18.7 + 0.2” 7.2kO.l’ 14.1 f0.2b 2.3 f 0.0” 13.2 + 0.2’ 7.7*0.1’ 3.9fO.lb 0.2 f0.0’ 7.2 f 0.3” 13.2*0.4’ 11.3 i-0.2’ 2.8kO.l’ 5.7*0.3’ 7.7 * 0.5’ 7.9 f 0.5’ 7.0*0.4” 0.4 f 0.0” 3.8fO.l” 13.2 f 0.2’ 75.3 72.2 147.5
12.9 f0.2d 5.7fO.ld 8.6 f0.2’ 2.0fO.Ob 11.3+0.1d 9.1 jZO.3” 3.1 f0.0’ 0.2 f 0.0’ 4.8f0.2d 7.2 f 0.3“ 8.2kO.4’ 2.2fO.ld 4.5fO.l” 5.6f0.4d 5.3*0.46 5.6f0.5d 0.4 f o.oc 2.8 f O.od 9.3 f o.5d 53.4 55.4 1108.8
Means f s.e.m. (pmollegg, alevin) with different superscript letters in each row are significantly different (P < 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; * = essential amino acid.
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Statistical analysis Since the data were not normally distributed as tested by Shapiro-Wilk statistics, nonparametric statistics (the Mann-Whitney test) were used. The procedures for these statistical analyses were as described by Sokal and Rohlf ( 1981). A probability level of P < 0.05 was considered statistically significant. Absolute values of amino acids were calculated for each egg and alevin (from nmol/mg of tissue to nmol/egg or alevin) based on the weights of 20 eggs/alevins collected from each replicate. In this paper, qualitative and quantitative compositions of amino acids refer to nmol/mg tissue and nmol/egg or alevin, respectively. Since there were no significant differences between replicates of all the variables studied (P > 0.05)) the replicates were pooled. Computations were performed using the Statistical Analysis System, release 6.06 package (SAS Institute Inc., 1990). 3. Results Eggs collected from the wild stock were significantly heavier and larger than those from cultured stock (mean weight, wild = 112.90 f 5.13 mg, cultured = 95.01 f 5.34 mg, n = 40, Table 3 Free amino acid composition Amino acid
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
of eggs or alevins from cultured Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
196k4.6’ 49*1.1= 14kO.9” 528 f 8.9’ 31853.6’ 360 f 5.2” 94 + 2.4” 84k2.1” 31 f 0.6” 71 f3.2” 107 f 2.5a 83 f 2.3a 31 zhO.7” 37 kO.8” 131h3.2~ 192k5.2” 64rt2.5a 9 f0.2a 26 f 0.8” 132k4.3” 638 1868 2506
182 f 3.9= 40f2.1” 29 f 0.4b 731 f 9.gd 317 f 5.3d 436 f 6.1b 101 f 3.3” 13 f 2.5a 36 + 0.5” 89+4.1a 137 f4.5b 96 f 3.9b 33 f 0.8” 39 f 0.8a 52 f 2.8” 177f7.8” 73 f 2.8” 13f0.3b 35 rt 1.9b 158 * 4.2b 136 2177 2913
468k5.6’ 438k5.8’ BLD 264 *4.5b 202 f 2.8b 600 f 8.6’ 218f5.6b 196 f4.4b 178f5.2b 222 f 6.8b 239 + 5.4’ 655 *9.8’ 13855.4” 181 f 15b 144f5.44 664rt 18b 233f7.6” 90f2.5’ 38f 1.7” 335 f 8.6d 2651 2847 5504
394 f 5.4b 315 f4.9b BLD 167 f 2.5” 157*4.5a 439 * 5.9b 141 rt4.8’ 226 f 5.9’ 184f7.8b 183*5.8’ 194 k 4.6d 502 k 6.8d 128 f 6.5b 142f11b 101 + 3.6” 623 f 16b 182 j~4.6~ 66 f 3.5c 110*5.9= 260 f 5.4’ 2260 2314 4514
1409 f 38.6d 602 f 7.9d 18*0.6” 549 * 9.7’ 114 f 7.4” 856 f 12.5’ 499 f l.gd 1124 f 25.6d 449*9.9c 445k 11.8’ 904 f 10.9e 916 f 14.7e 417 f 7.9’ 371 f 8.8c 524 f 8.9’ 1203 f 36.5’ 445 rt 8.gd 99 f 3.4e 342 k 4.5’ 713f9.8e 5603 6461 12 064
Means f s.e.m. (pmol/egg, alevin) with different superscript letters in each row are significantly different (P < 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; BLD = below the limits of detection; * =essentiaf amino acid.
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Table 4 Free amino acid composition 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
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of eggs or alevins from wild Atlantic salmon at different developmental
Developmental
stages
stage
Before fert.
After fert.
Eyed stage
Hatching
First feeding
299f 11.8” 83 k5.2a 32i0.Sb 1113 f 24.5e s70*9.se 390 f 19.4a 274+3.3b 57 + 2.3a 54*2.1a 84i5.2a 128*9.1a 157*7.t? 48 f 3.3” 61 f3.5” 51 k2.6” 242 f4.4b 77 + 4.7= 19k0.2a 44k3.2” 142 f 9.4a 879 3347 4226
242 f 7.7’ 66*4.5” 34 + 0.4b 845 f 7.5d 598 f 8.6d 310*9.5a 195 f 4.4a 64*2.1= 48 f 2.2” 61 f 3.6” 971t6.1” 133 + 9.2” 36k 1.8” 51 k2.3” 661k3.8” 192k3.4” 67 + 6.2” 16kO.2” 41 f 2.8” 107 f 8.7” 707 2565 3272
459 f 3.4b 437f5.7b BLD 243 f 3.7b 471 *4.3c 612* 15’ 157*8.7a 203 f 5.2” 185 ~k5.2~ 191 i6.2b 220 f 9.9b 600 f 6.7b 164k8.5b 184f9.8b 99 f 4.2b 76 1 + 6.4” 197 f 7.3b 93f2.3b 51*4.2* 300f6.3b 2528 3099 5627
642 f 7.2’ 441 f4.Sb BLD 194 f 2.6” 330 f 4.3b 570* lib 176k7.8” 414*7.3c 276 f 4.3” 220 f 8.9b 316f8.3’ 6lOk 8.9b 199*9.@ 226 f 6.3’ 151 f 7.2” 847 f 9.46 223 + 12b 94*2.1b 167 f4.5b 349 f 6.9’ 3028 3418 6446
1384 f 22.4’ 545 f 4.8’ 15kO.2’ 573 f 7.3” 151*4.5a 831 f9.6’ 473 f 8.3” 606 f lo.5d 405 f 8.46 437 * 12= 884 f 9.9d 857 f 7.9” 397 f 7.6” 387f9.5d 546 f 8.4d 1139*44.5’ 460 k 16.5’ 93 f 4.3b 337 *4.5= 700 f 19.4d 5409 5811 11220
Means f s.e.m. (pmol/egg, alevin) with different superscript letters in each row are significantly different (P< 0.05, n = 40). Before fert. = before fertilization; After fert. = after fertilization; BLD = below the limits of detection; * = essential amino acid.
P < 0.05; mean diameter, wild = 6.17 + 0.35 mm, cultured = 6.00 f 0.21 mm, 12= 40, P < 0.05). The development (degree-days = dd) of embryos and alevins of the wild stock was faster compared to the cultured stock (cultured, eyed-stage = 457 dd, hatching = 575 dd, first feeding = 841 dd; wild, eyed-stage = 424 dd, hatching = 520 dd, first feeding = 753 dd). When expressed in nmol/mg dry weight of unfertilized egg tissues (qualitative composition), there were no significant differences in amino acid content between cultured and wild stocks (P > 0.05; data not shown). The amounts of essential and non-essential amino acids in unfertilized egg tissues were similar in both cultured and wild stocks (data not shown). The amino acid (protein-bound) content per egg from the wild stock (209.1 pmol/egg) was higher than eggs from the cultured stock ( 175.7 pmol/egg) (Tables 1 and 2). Eggs from wild stock had higher amounts of amino acids than eggs from cultured stock except for tryptophan. Alanine content was highest among all amino acids present in eggs of both stocks (cultured= 12.05% alanine, wild = 11.78% alanine). The amino acid content per egg or alevin in both the cultured and wild stocks declined continuously during development.
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Essential amino acid content was higher in eggs or alevins from wild stock than in eggs or alevins from cultured stock, and declined during development (Tables 1 and 2). Nonessential amino acid content of eggs collected from wild stock (98.6 pmol/egg) was also higher than that of eggs collected from cultured stock ( 84 pmol/egg) , and declined during development up to first feeding. In eggs of both stocks, the amount of essential amino acids was higher than that of the non-essential amino acids (cultured eggs = 52.2% essential, wild eggs = 52.8% essential). The free amino acid pool was higher in eggs of wild stock (4226 nmol/egg) than of cultured stock (2506 nmol/egg) , and increased in both stocks until first feeding (cultured, 12 064 nmol/alevin; wild, 11 226 nmol/alevin; Tables 3 and 4). Essential free amino acids in eggs were lower than non-essential free amino acids in both stocks (cultured = 25.5% essential, wild = 20.8% essential; Tables 3 and 4).
4. Discussion This is the first study demonstrating that amino acids (protein-bound) are found in large amounts in Atlantic salmon eggs at spawning, and progressively decrease to low levels at first feeding. After fertilization, there is a period of increasing metabolic activity accompanied by high energy demand. This energy comes from the catabolism of body stores ie. protein, lipid and carbohydrate (Srivastava and Brown, 1991). Protein must be broken down into amino acids which are then either catabolized for energy production or utilised for building up new body tissues. Quantitatively (nmol/egg), the amino acid (protein-bound) content of eggs from wild stock was higher than that of eggs from cultured stock but the qualitative composition (nmol/mg dry weight) of amino acids in egg tissues from cultured and wild stocks did not differ. Our data are in agreement with those of Timoshina et al. ( 1982) who reported higher quantities of amino acids in larger eggs, but the qualitative composition of the amino acids in eggs was the same. The decline in amino acid content during development was greater in wild stock than cultured stock perhaps because the embryos from wild stock developed faster, i.e. took less time to reach the eyed stage, and hatched earlier. For successful embryonic development, an adequate amount of amino acids is necessary, otherwise development will be halted or slowed down at a later stage (Harper et al., 1970; Ogino, 1980; Metcoff, 1986; Wilson and Halver, 1986; Cho and Kaushik, 1990; Cowey, 1992; Murai, 1992). Proteins obtained from the maternal circulation later serve as amino acid and energy sources for the growing embryo (Tyler et al., 1988). In the present study, the amino acids (protein-bound) alanine, arginine, aspartic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine were found to decrease in both stocks because of their utilization by embryos and alevins. In Arctic charr, it has been noted that isoleucine, lysine, serine, threonine, tryptophan and valine were important for embryonic and fry development (Srivastava, 1991; Srivastava and Brown, 1994). The difference in the types of amino acids may be due to their species-specific roles (Mertz, 1977; Metcoff, 1986). The levels of protein-bound essential amino acids were always higher than those of nonessential amino acids in both stocks, and declined during development. By comparison, the
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levels of free essential amino acids were lower than those of non-essential amino acids, and increased through development in both stocks. It seems that preferential retention of essential amino acids occurs during embryonic and alevin development in both stocks. At present it is not clear how specific amino acids might be retained. It is possible that the processes associated with their catabolism are suppressed or not developed until later in early development, thereby preventing losses. In this study, the free amino acid pool was higher in eggs of the wild stock than of the cultured stock, and increased during development. This suggests that the protein breakdown rate would have been higher than the anabolic and catabolic losses of developing embryos. Timoshina et al. (1982) have also reported higher quantities of free amino acids in the larger eggs of rainbow trout due to their larger size, but there were no qualitative (nmol/ mg of tissues) differences in amino acids. In another study with rainbow trout eggs, total free amino acid concentrations declined just after fertilization, increased to near initial values in the blastula and were nearly doubled at hatching (Zeitoun et al., 1977). However, in contrast to these findings, a continuous decrease in the free amino acid pool of Atlantic cod embryos has been reported (Fyhn and Serigstad, 1987; Fyhn et al., 1987; Fyhn, 1989). The reason for the differences observed in egg quality of the wild and cultured Atlantic salmon is not clear. The two stocks were from different areas, and thus genetic differences cannot be dismissed. Moreover, other factors such as age of parents and diets could also account for the differences observed in the present study (Srivastava and Brown, 1991). The amino acid content of carp eggs was shown to be related to the quality of food eaten by the broodstocks (Lavrovskaya, 1980). Apart from amino acids, there may be some other nutrients (vitamins, trace elements, carotenoids and essential fatty acids etc.) which could contribute to the better performance of embryos and alevins (Luquet and Watanabe, 1986). The cultured stock was fed a commercial diet while the wild stock fed on naturally occurring organisms. It is possible that the diet of the wild population was better and that this contributed to their higher amino acid levels and larger eggs, which means more amino acid would be available for the development of the embryos and alevins. In conclusion, it can be suggested that amino acids in eggs may play a role in the successful development of embryos and alevins. On the basis of our findings, it is reasonable to suggest that the amino acids alanine, arginine, aspartic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine should be present in the diets of broodstock, so that they can be transported to developing oocytes during vitellogenesis.
Acknowledgements We express our thanks to Dr. Garth Fletcher and David Methven for their comments on an earlier version of the manuscript. We also thank Dr. Jozef Synowiecki and Ronald B. Pegg for their valuable suggestions and help in conducting the experiments. Technical support by D. Hall and S. Banfield with 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.
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