The influence of astaxanthin supplemented dry pellets on spawning of striped jack

Transcription

1 FISHERIES SCIENCE 2001; 67: Original Article The influence of astaxanthin supplemented dry pellets on spawning of striped jack ROBERT VASSALLO-AGIUS, 1 HITOSHI IMAIZUMI, 2 TAKESHI WATANABE, 1, * TETSUO YAMAZAKI, 2 SHUICHI SATOH 1 AND VISWANATH KIRON 1 1 Laboratory of Fish Nutrition, Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato, Tokyo and 2 Komame Station of Japan Sea Farming Association, Ohtsuki, Kochi , Japan SUMMARY: This experiment was conducted to investigate the effect of astaxanthin supplemented steam-dry pellets on the spawning of striped jack Pseudocaranx dentex. Five months before spawning, 7-year-old fish were divided into three groups, each consisting of five females and five males. The control group was fed a raw fish mix (RF) and the test diets consisted of steam-dry pellets (DP) and steam-dry pellets supplemented with 10 p.p.m. astaxanthin (a-dp). Feeding was carried out once per day, three times a week in floating net cages. The RF and the a-dp groups produced more than three times the amount of eggs produced by the DP group. The buoyancy, fertilization, and hatching rates were higher and similar for the RF and the DP groups. Total lipid contents of the diets, eggs, and larvae were similar among the groups. Highly unsaturated fatty acids (n-3 HUFA) and polar and non-polar lipids in eggs and larvae were also related to their dietary availability. These results indicate that astaxanthin, due to its various physiological functions, improved the condition of the broodstock and enhanced total egg production but not egg quality as striped jack do not incorporate carotenoids into their eggs. Therefore we confirm that the nutritional quality of diets given to striped jack prior to spawning plays a fundamental role in egg production, quality and egg composition. KEY WORDS: striped jack. astaxanthin, broodstock, Pseudocaranx dentex, spawning, steam-dry pellets, INTRODUCTION As the optimization of marine fish seed production is one of the major bottlenecks of aquaculture development, it is important to understand the nutritional requirement of parent fish as nutrient availability influences various aspects of reproductive physiology. 1 With an increase in fish culture production, farmers tend to shift from the more traditional raw fish diets to the more advantageous dry pellets. 2 Thus, for a sufficient production of goodquality eggs, specific broodstock dry pellets for the various high-value marine species are required. The time-consuming process of selecting and preparing the spawners for their annual spawning coupled with the need for large-scale facilities are *Corresponding author: Tel: Fax: watana@tokyo-u-fish.ac.jp Received 18 July Accepted 10 October the primary reasons for the slow pace at which broodstock nutrition research has been progressing. Work on yellowtail Seriola quinqueradiata has already indicated that carotenoids, specifically astaxanthin, improve spawning and egg quality and can be transferred from diets to eggs via the broodstock. 3 5 Our previous work has shown that striped jack Pseudocaranx dentex is capable of spawning after being fed on the so-called soft-dry pellets, prepared by using a large-sized twin-screw extruder, but eggs obtained from the control group fed on a raw fish mix were colorless and did not contain any carotenoids even though the diet contained up to 7.7 p.p.m. astaxanthin. 6 8 Further work indicated that Spirulina, a blue-green alga abundant in carotenoids and already shown to improve pigmentation and flesh quality in striped jack, did not enhance the spawning performance of this species Moreover, the number of eggs produced

2 Astaxanthin and spawning in striped jack FISHERIES SCIENCE 261 and the percentages of normal larvae obtained from total eggs were lower for the fish fed soft-dry pellets when compared to the fish fed raw fish diets. 6,12 For the present study we used steam-dry pellets (low total lipid content) rather than soft-dry pellets as other work indicated that after being fed on soft-dry pellets, striped jack suffered from fat accumulation in their abdomen (Imaizumi H, Japan Sea Farming Association, unpubl. data, 1998). This work was performed to investigate whether the supplementation of 10 p.p.m. pure astaxanthin could improve spawning in striped jack, and to examine the effects of these comparatively lower-lipid pellets on the lipid and fatty acid components of eggs and yolk-sac larvae produced. MATERIALS AND METHODS Feeding of broodstock All the fish used in this experiment were of a cultured origin and had been reared on commercial dry pellets in floating net cages. The average weight of the fish at the start of the experiment was 3.7 ± 0.6 kg. Five months before the spawning manipulation, the fish were split into three groups of 10 fish each (male : female ratio, 5 : 5) and test feeding commenced immediately. The control group was fed a raw fish mix (RF) and the two trial groups were fed either steam-dry pellets similar to those used for red sea bream Pagrus major culture (DP) or the same type of pellets supplemented with 10 p.p.m. astaxanthin (a-dp). Each group was stocked in separate m floating net cages belonging to the Komame Station of the Japan Sea Farming Association (JASFA) in Kochi Prefecture, Japan. The fish were fed once a day, three times a week to near satiation at approximate rates of 5% and 2% of the average bodyweight for the RF and the dry pellet groups, respectively. Experimental diets Table 1 shows the ingredients used for the diets under study. The control diet (RF) was the standard striped jack broodstock diet used at JASFA, and consisted of a mixture of jack mackerel Trachurus japonicus, squid Todarodes pacificus, and shrimps (various species) mixed at a ratio of 2:2:1, respectively. All these were stored at -40 C and defrosted and chopped to a suitable size before feeding. This diet was supplemented with 1% (wet weight) feed oil, vitamin C, and vitamin E. The dry pellets used for the other two groups were produced by Table 1 Ingredients of the experimental diets Ingredients (%) DP a-dp RF Fish meal Mackerel, Soybean meal squid Corn gluten meal and Wheat flour shrimp (2:2:1) Vitamins and minerals Fish oil Supplementation (%) Feed oil 1.0 Vitamin mix 1.0 Astaxanthin (p.p.m.) 10.0 DP, steam-dry pellets; a-dp, steam-dry pellets supplemented with 10 p.p.m. astaxanthin; RF, raw fish mix. 1 Jack mackerel, produced in Chile. 2 Defatted (crude protein: 45.0%, crude lipid: 1.0%). 3 Crude protein 60%. 4 Watanabe T. et al. 7 Table 2 Proximate composition, n-3 HUFA, and carotenoid contents of the experimental diets DP a-dp RF Proximate composition (%) Crude protein Crude lipid Crude ash Moisture Dry matter basis weight (g/100 g) Crude protein Crude lipid Crude ash n-3 HUFA Carotenoid contents (p.p.m.) Astaxanthin Zeaxanthin Others Total carotenoids DP, steam-dry pellets; a-dp, steam-dry pellets supplemented with 10 p.p.m. astaxanthin; RF, raw fish mix. Sakamoto Feed Co. Ltd. (Chiba, Japan), and were similar except that the a-dp diet was supplemented with 10 p.p.m. pure astaxanthin. This was supplied as a synthetic carotenoid, Carophyll Pink (Roche Vitamin, Tokyo, Japan) which contained 8% astaxanthin. The main protein and lipid sources were fish meal and fish oil, respectively. The proximate compositions of the three diets are shown in Table 2. Although on a dry matter basis the crude lipid contents were similar for all the diets, the protein and n-3 highly unsaturated fatty acid (HUFA) contents were higher for the

3 262 FISHERIES SCIENCE R Vassallo-Aguis et al. RF diet. The DP and a-dp contained a high amount of xanthophylls other than astaxanthin or zeaxanthin, probably derived from the corn gluten meal and soybean meal. The quantity of astaxanthin was highest in the RF diet, whereas the a- DP contained 7.7 p.p.m., a quantity similar to the RF diet in our previous experiments. 6,12 Compared to the astaxanthin content of the RF diet in our previous work, 6,12 the content in this study was quite high, highlighting the inconsistency of RF diets which are too dependent on the quality of the raw materials available at the time of purchase. Table 3 shows the main lipid classes and fatty acids of the experimental diets. The total lipid contents and the non-polar and polar lipid proportions were similar among the diets. As expected, the main non-polar lipids in the diets were triglycerides and the main polar lipid was phosphatidylcholine. The major differences in fatty acid contents were that the RF diet contained a markedly lower amount of 18 : 2n-6 (linoleic acid) and a markedly higher amount of 22 : 6n-3 Table 3 Main lipid classes and fatty acid compositions of the experimental diets DP a-dp RF Total lipid (% dry basis) Non-polar lipids Sterol esters Triglycerides Free fatty acids Free sterols Others Polar lipids Phosphatidylethanolamine Phosphatidylcholine Others Main fatty acids (% area) 16: :1n : :1n-(9 + 7) :2n : :4n :5n : :5n :6n S Saturates S Monoenes S n-3 HUFA DHA/EPA DP, steam-dry pellets; a-dp, steam-dry pellets supplemented with 10 p.p.m. astaxanthin; RF, raw fish mix. (docosahexaenoic acid, DHA). This higher DHA content was reflected in a higher n-3 HUFA content as well as a higher DHA/EPA ratio. The higher levels of 18 : 2n-6 in the DP and a-dp were due to the soybean and the corn gluten meals incorporated in these diets, however, the 18 : 2n-6 content of RF in our previous study 8 was also markedly higher than our current value. Spawning manipulation After 5 months of feeding, the fish were transferred to 65 m 3 concrete indoor spawning tanks equipped with a sand-filtered, heated, flow-through system, through which seawater was directly supplied from the nearby bay. Fish transfer and husbandry procedures were carried out according to our previous work 6 but apart from raising the water temperature to 22 C, hormone injection (HCG, 600 IU/kg bodyweight) was used because the fish were virgin spawners. 13 The fish were allowed to spawn at the optimum 22 C for 1 month, during which the water flow through the spawning tanks was sufficient to maintain a total water exchange rate of five times per day. Investigation of spawning and the evaluation of egg quality Egg collection was performed as described in our previous work 6 and buoyant eggs were separated from deposited eggs. Aliquots of buoyant eggs ( eggs) were examined for fertilization and abnormality rate estimations. Egg and oilglobule diameter measurements were carried out on 25 buoyant eggs using a Profile Projector V-12 equipped with a Data Processor DP-301 (Nikon, Tokyo, Japan). Normal, fertilized eggs were recorded as eggs viable at the Morula stage and were characteristically buoyant, spherical, colourless, transparent, and possessed only one oil-globule. 14 Eggs with more than one oil-globule were considered to be abnormal, even though recent work on Japanese sea bass Lateolabrax japonicus has shown that these eggs do not hatch into abnormal larvae as fusion of the multiple oil-globules occurs before hatching. 15 For hatching rate estimations, ml of buoyant eggs were stocked into hatching nets with optimum rearing conditions for striped jack eggs. 6 Before and after hatching, egg or larval counts were performed and hatching rates were estimated. Samples for counting were taken from 10 cm below the surface in a 100 ml container, from five differ-

4 Astaxanthin and spawning in striped jack FISHERIES SCIENCE 263 ent areas of the hatching net after 30 s of strong aeration. Survival activity index Survival activity index tests (SAI) 16 were carried out on hatched larvae as an indication of larval vitality under starvation at 22 C. Sample collection Eggs and yolk-sac larvae were sampled from each diet group during the first week of spawning. Eggs or larvae were scooped from the hatching nets with a hand net and excess water was removed by placing the hand net on soft tissue paper for less than 1 min. Eggs were sampled at the morula stage and larvae were sampled at 0-d, 2-d, 4-d, and 6-d (d = days after hatching). All samples were immediately stored at -20 C until analysis. All egg samples were ground in a glass tube homogenizer prior to analysis. Analytical methods Eggs and larvae were analyzed for total lipid contents, lipid classes, and fatty acids. All analytical procedures were carried out according to our previous study. 8 Statistical analysis Significance of difference (P < 0.05) of fish and egg quality parameters between dietary treatments was determined by Scheffé s test. RESULTS Broodstock status The average weight of all the fish at the start of the feeding experiment was 3.7 ± 0.6 kg and the average sizes of the fish at the start of spawning are shown in Table 4. After 5 months of feeding, the group average weights of all the fish of each group were around 3.4 and 3.5 kg. Although the weight of the fish decreased from the start of the experiment, this decrease was not significant (P < 0.05). The females and males of the RF and DP groups were of a similar size whereas the females of the a-dp group were significantly larger (P < 0.05) than the males of the same group. Despite this, there were no significant differences (P < 0.05) between male averages or female averages among the diet groups. The condition factors of the fish at the start of the experiment were significantly larger (P < 0.05) than at the start of spawning for all the diet groups. At spawning there were no significant differences (P < 0.05) among the groups. Spawning performance Fish of all groups started spawning approximately 36 h after the hormone injection when the temperature was 22 C. The total spawning results of the three diet groups are shown in Table 5. The total results are shown as eggs or larvae produced per female, as well as eggs or larvae produced per female per day due to the different number of spawning days among the groups. Egg and larval production results of the RF group were calculated per four females as one of the females of this group died soon after transfer to the spawning tanks. Post-mortem examination revealed that the fish had not yet spawned and the cause of death was Table 4 Average size parameters of striped jack at the start of spawning Parameters 1 RF DP a-dp Male Female Male Female Male Female Bodyweight (kg) 3.4 ± ± ± ± ± ±0.7 2 Fork length (cm) 52.4 ± ± ± ± ± ±3.3 Condition factor ± ± ± ± ± ±1.3 Group average Bodyweight (kg) 3.4 ± ± ± 0.7 Fork length (cm) 53.0± ± ±3.5 Condition factor 22.9 ± ± ± 1.7 DP, steam-dry pellets; a-dp, steam-dry pellets supplemented with 10 p.p.m. astaxanthin; RF, raw fish mix. 1 Mean ± SD, n = 5 for male or female values, n = 10 for group averages. 2 Values are significantly different within the diet group (P < 0.05). 3 [Bodyweight (g) 1000]/[Fork length (cm)] 3.

5 264 FISHERIES SCIENCE R Vassallo-Aguis et al. Table 5 Spawning results of striped jack fed the RF, DP, and a-dp diets Diet RF DP a-dp Sex ratio (M : F) 5 : 4 1 5:5 5:5 Spawning days (Days) Spawning temperature ( C) Egg production Total eggs ( 10 3 /female) Av. total eggs ± SD ( 10 3 /female per day) 203±104 a 68±10 c 139 ± 15 b Buoyant eggs ( 10 3 /female) Av. buoyant eggs ± SD ( 10 3 /female per day) 172 ± 98 a 58±39 b 109 ± 79 ab Deposited eggs ( 10 3 /female) Fertilized eggs 2 ( 10 3 /female) Av. fertilized eggs±sd ( 10 3 /female per day) 160 ± 85 a 51±35 b 90±67 b Egg quality Buoyancy rate among total eggs (%) Fertilization rate among buoyant eggs (%) among total eggs (%) Abnormality rate ± SD 3 (%) 5.6±8.2 a 14.0±11.5 a 7.5±8.2 a Egg diameter±sd (mm) 984 ± 23 a 990 ± 26 a 980 ± 22 a Oil-glob. diameter ± SD (mm) 247 ± 11 a 250 ± 10 a 253 ± 11 a Larval production Hatched larvae ( 10 3 /female) Av. hatched larvae ± SD ( 10 3 /female per day) 129 ± 84 a 43±37 b 77±61 b Larval quality Average SAI ± SD ±10.0 a 13.3±11.2 a 14.2±11.3 a Rate of hatched larvae among fertilized eggs (%) among buoyant eggs (%) among total eggs (%) DP, steam-dry pellets; a-dp, steam-dry pellets supplemented with 10 p.p.m. astaxanthin; RF, raw fish mix. 1 One female died during the experiment. 2 Morphologically normal, fertilized eggs at the morula stage. 3 Eggs with more than one oil-globule. 4 Starvation tolerance of larvae. Values in the same line with a different superscript are significantly different (P < 0.05). For SD, n = number of spawning days. not nutritional as it died because of atresia in the genital pore. Egg production and quality Figure 1 shows the daily total egg production per female for each diet group throughout the spawning experiment. During the first 2 days of spawning the fish produced a larger number of eggs due to the effect of the hormone injection. Thereafter, the egg production decreased and started increasing again on the 4th day of spawning for the RF group and on the 5th day of spawning for the DP and a-dp groups. In conjunction with our previous work, 6,12 egg production gradually increased to a peak which was reached on the 14th, 12th, and 15th spawning days for the RF, DP, and a-dp groups, respectively. These groups spawned a total of 18, 15, and 24 times, respectively. It can be seen in Table 5 that the total eggs produced by the RF and a-dp groups per female over the experimental period were more than thrice that of the DP group. Moreover, the differences in the average daily egg production between the three groups were significant (P < 0.05), and were in the following order: RF > a-dp > DP. Figure 2 shows the daily fertilized egg production per female throughout the spawning period and displays a pattern similar to Fig. 1, apart from the first 4 days where the numbers of fertilized eggs produced by the DP and a-dp groups were markedly lower than the numbers of total eggs produced. Both the DP and a-dp groups required more time to start producing good fertilized eggs after the initial hormone effect.

6 Astaxanthin and spawning in striped jack FISHERIES SCIENCE 265 Fig. 1 Total eggs produced per female per day by striped jack broodstock during the experimental period. ( ) RF; ( ) DP; ( ) a-dp. Fig. 2 Fertilized eggs produced per female per day by striped jack broodstock during the experimental period. ( ) RF; ( ) DP; ( ) a-dp. Referring to Table 5, it can be seen that throughout the spawning experiment, the RF group produced the highest number of fertilized eggs, 1.2 times the amount produced by the a-dp group. However, because of the higher number of spawning days for the a-dp group, the difference between the two groups was greater for the average number of fertilized eggs produced per day. This was significantly higher (P < 0.05) for the RF group. Between the two dry-pellet groups, the average fertilized eggs produced were higher for the a-dp group, but the difference was not significant (P > 0.05). The average daily total number of eggs produced by the a-dp group was significantly higher (P < 0.05) than that of the DP group, but significantly lower (P < 0.05) than the production of the RF group. Egg quality was better for the RF and DP groups (the a-dp group showed the lowest buoyancy and fertilization rates) but the average number of total and fertilized eggs produced per day by the DP group was significantly lower (P < 0.05). Average egg diameters, oil-globule diameters and abnormality rates of all the diet groups were within the same ranges throughout the spawning experiment and showed no significant differences (P > 0.05) among the groups. Larval production and quality Figure 3 shows the daily larval production per female and follows the same pattern as that of Figs

7 266 FISHERIES SCIENCE R Vassallo-Aguis et al. Fig. 3 Larvae produced per female per day by striped jack broodstock during the experimental period. ( ) RF; ( ) DP; ( ) a-dp. 1 and 2. The spawning results in Table 5 show that there were no marked differences in the hatching rates among fertilized eggs among the groups. As the number of viable larvae produced is a more reliable indicator of egg quality, the hatching rates from total eggs produced are of great importance when examining broodstock diets. 17 The numbers of larvae produced by the RF and a-dp groups were, respectively, 3.6 and 2.8 times more than those produced by the DP group. Although the average number of larvae produced per day by the a-dp group was higher than that of the DP group, the number of larvae produced by the RF group was significantly higher (P < 0.05) than both the pellet groups. The SAI values obtained for all the diet groups were similar and the averages showed no significant differences (P < 0.05) between the groups. The SAI has been used as an indication of larval activity for striped jack 16 and yellowtail 18 and it is carried out using newly hatched, unfed larvae. In this test, larvae only survive on nutrients inherited from the broodstock diets via the parent fish. Total lipids and lipid classes of eggs and unfed larvae Table 6 presents total lipids and main lipid classes of eggs and yolk-sac larvae produced by the broodstock fed the experimental diets. Eggs of all the three diet groups show similar total lipid contents, reflecting the similar total lipid content in the diets. The egg total lipids ranged between 22.34% and 23.74% on a dry matter basis and decreased to between 11.73% and 12.42% by the time the larvae reached the 6-d stage. Non-polar lipids in eggs made up % of the total lipids. These values decreased to % of the total lipids in 6-d larvae. This decrease in non-polar lipids was mainly due to the decrease in triglycerides and sterol esters as the eggs hatched and unfed larvae developed to 6-d. On the other hand, polar lipids increased in proportion to total lipids as phosphatidylcholine showed a decreasing trend and phosphatidylethanolamine increased. Main fatty acids of eggs and unfed larvae The main fatty acids of eggs and yolk-sac larvae produced in this study are shown in Table 7. Corresponding to the trends of dietary fatty acids shown in Table 3, the eggs and larvae of the RF group showed lower amounts of 18 : 2n-6 and markedly higher quantities of DHA. Also, 18 : 2n-6, 20 : 5n-3 (eicosapentaenoic acid, EPA), and total monoenes decreased with larval development whereas 20 : 4n-6 (arachidonic acid), DHA, and total n-3 HUFA as percentage area increased but decreased on a dry matter basis (data not shown). This decrease in DHA content (dry basis) with development (eggs to larvae) is in accordance with other work on striped jack, cod Gadus morhua, red sea bream, and Japanese flounder Paralichthys olivaceus as reported by Watanabe. 19 Due to these opposing trends of DHA and EPA, the DHA/EPA ratios, obtained from percentage area (wet basis) data, increased until the 6-d larval stage.

8 Astaxanthin and spawning in striped jack FISHERIES SCIENCE 267 Table 6 Total lipids and main lipid classes of eggs and larvae produced by striped jack fed the experimental diets DP a-dp RF Egg 0-d 2-d 4-d 6-d Egg 0-d 2-d 4-d 6-d Egg 0-d 2-d 4-d 6-d Total lipid (% dry basis) Non-polar lipids Sterol esters Triglycerides Free fatty acids Free sterols Others nd nd nd nd nd nd Polar lipids Phosphatidylethanolamine Phosphatidylcholine Others DP, steam-dry pellets; a-dp, steam-dry pellets supplemented with 10 p.p.m. astaxanthin; RF, raw fish mix. nd = Not detected. d = Age of larvae in days. Table 7 Main fatty acids of eggs and larvae produced by striped jack fed the experimental diets Fatty acid (% area) DP a-dp RF Egg 0-d 2-d 4-d 6-d Egg 0-d 2-d 4-d 6-d Egg 0-d 2-d 4-d 6-d 16: :1n : :1n-(9 + 7) :2n : :4n :5n : :5n :6n S Saturates S Monoenes S n-3 HUFA DHA/EPA DP, steam-dry pellets; a-dp, steam-dry pellets supplemented with 10 p.p.m. astaxanthin; RF, raw fish mix. d = Age of larvae in days.

9 268 FISHERIES SCIENCE R Vassallo-Aguis et al. DISCUSSION Broodstock status and spawning performance From the beginning of the 5-month feeding period until the start of the spawning manipulation, the bodyweight of the fish decreased, probably because the greater part of the feeding experiment was carried out during the winter months (before the spawning season) when the feeding activity was lower due to the lower metabolic rate. Apart from the lower feeding activity, the low lipid steam-dry pellets used in this study may have contributed to a reduction in abdominal lipid accumulation, a phenomenon frequently known to disturb captive spawning in this species. This decrease in bodyweight was not significant (P > 0.05) and the different diet groups showed a similar decrease, so it was not due to any dietary shortcomings. As all the fish used in this study were only 7 years old and it was their first spawning in captivity, spawning was induced by injecting the fish with HCG (600 IU/kg) and raising the water temperature to 22 C. Even though this inducement method is more stressful (involves individual fish handling for HCG injection) than only raising the water temperature, it is more reliable for egg production when using virgin spawners. In our previous studies, 6,12 the fish were not virgin spawners and they were at least 10 years old and capable of reliably producing eggs without hormone inducement. All three groups in the present study started spawning only 36 h after hormone injection. This is less than the required 48 h and shows that at least some females in all groups were at an advanced maturation stage even before the hormone injection. Egg and larval production and quality The markedly higher number of eggs produced by the RF group during the first spawning day (Fig. 1) and the higher number of fertilized eggs produced during the first 2 days (Fig. 2) indicates that the fish of the RF group were more mature at the time of hormone injection and were immediately able to produce good fertilized eggs. This is also indicated by the lower egg quality of the pellet groups during these first few days. Table 5 shows the total egg production over the spawning period. When compared to the DP diet, the a-dp produced a significantly higher number of eggs per day (P < 0.05), indicating that astaxanthin supplementation in the steam-dry pellets had a positive effect on egg production. Eggs from all groups were colorless and contained no carotenoids. Therefore, despite the fact that striped jack do not incorporate carotenoids into their eggs, astaxanthin was beneficial to the broodstock and enhanced egg production. 8 Carotenoids are known to have a diversity of biological functions in animals, namely their beneficial effect on the endocrine system in respect to gonadal development and maturation in fish and crustacea. 20 When comparing the hatching rates produced by the diets as ratios of the RF values, we can see that despite being slightly inferior, the hatching ratio of the a-dp group is 0.87 that of the values produced by DP and RF diet groups in this experiment. This value is higher than the values of our previous studies, 6,12 where the ratios were 0.77 and 0.74 indicating that the steam-dry pellets used in this study were superior to soft-dry pellets. This improvement may be attributed to the lower total lipid content (dry basis) in the steam-dry pellets used in this experiment ( %). They are similar to the total lipid content (dry basis) of the RF diet (17.2%) and are much lower than the values of the soft-dry pellets (24.1% and 22.3%). 6,12 Although the DP group produced eggs of better quality than the a-dp group, its significantly lower (P < 0.05) egg production renders it impractical for the large number of eggs required for large-scale production. Total lipids and lipid classes of eggs and unfed larvae The similarity in the total lipids of the eggs produced by the RF and steam-dry pellet groups (Table 6) shows that the eggs produced in this study had lipid contents lower than those obtained from the soft-dry pellet groups in our previous work. 8 This confirms that dietary lipids directly affect egg lipids in striped jack and the lower lipid steam-dry pellets used were instrumental in decreasing the lipid contents of the eggs. Despite this, further work needs to be done to investigate the optimal lipid levels for hatching and survival of striped jack eggs. Due to lipid metabolism during development, the lipid contents decreased as the larvae developed. The decrease in total lipid contents was also similar between the diet groups and followed a similar trend to that of larvae produced after being fed the RF diet in our previous work. 8 Triglycerides were lower in the RF diet (Table 3) as well as in eggs produced by the fish of this group. On the other hand, sterol esters were higher in eggs produced by the RF diet as in our previous work. 8 Despite these small differences between eggs pro-

10 Astaxanthin and spawning in striped jack FISHERIES SCIENCE 269 duced by the RF and steam-dry pellet groups, the main non-polar lipids were triglycerides and sterol esters in all fertilized eggs. Phosphatidylcholine, the main polar lipid in the diets, was also the main polar lipid in the eggs of all groups and decreased with larval development. All the main lipid class composition trends agreed with those of our previous work 8 and were in accordance with those reported in other work on red sea bream 21,22 and yellowtail. 4 Main fatty acids of eggs and unfed larvae The main fatty acids in fertilized eggs were also dependent on those of the diets and complied with our previous work 8 and other observations on red sea bream 21 and yellowtail. 4 The higher DHA and the lower 18 : 2n-6 contents in eggs of the RF group reflected the levels in the diet and may have contributed to the better egg production or quality of this group. The n-3 HUFA levels and DHA/EPA ratios were lower in eggs obtained from the pellet diets. Although DHA has improved larval vitality in red sea bream, 23 the percentage of n-3 HUFA of eggs of gilthead sea bream cannot be used as the sole criterion to determine egg quality, even though the fertilization rate of middle and late spawning season eggs of this species was negatively affected by an n-3 HUFA-deficient broodstock diet. 24,25 Other authors have also suggested the importance of optimum DHA/EPA or (n-3)/ (n-6) polyunsaturated fatty acid ratios, rather than their absolute levels. 26 Furthermore, the relative quantity of arachidonic acid (20 : 4n-6) with respect to DHA and EPA, as well as the overall balance of polyunsaturated, monounsaturated, and saturated fatty acids, and their relative proportions in dietary phospholipids and triacylglycerols should also be considered for marine diet formulations. 27 The results of this experiment show that 10 p.p.m. astaxanthin supplementation in dry pellets improved egg production of striped jack. In this experiment the spawning performances of all groups were observed over 1 month and not until the end of spawning because of two reasons. First, the data of our previous studies on striped jack 6,12 indicated that the highest quantity and quality of eggs were produced during the first month of spawning and, second, to prevent late repeated spawnings that are reported to cause Striped Jack Nervous Necrosis Virus (SJNNV). 28 When compared directly with the DP, the a-dp produced a significantly higher number of eggs that, in turn, had a lower quality with lower buoy- ancy and fertilization rates. Our previous experiment 8 showed that carotenoids are not incorporated into striped jack eggs, so, although it improved egg production significantly, some other nutritional factor was lacking and egg quality degraded. Moreover, the 10 p.p.m. supplementation in this experiment may not have been the optimum level required for striped jack broodstock and may have diminished egg quality as previously observed for yellowtail. 3 The egg quality of the a- DP group could have also been influenced by other factors such as the release of over-mature eggs, poorer sperm quality, or bad synchronization of egg and sperm release. This work indicates that astaxanthin, which has been beneficial to various species of broodstock such as yellowtail 3 and red sea bream, 29 is also beneficial for egg production in striped jack. However, unlike yellowtail and red sea bream, astaxanthin did not improve egg quality, which is probably dependent on other nutritional factors. The dietary and egg fatty acid profiles indicate that the higher DHA and the lower 18 : 2n-6 levels in the RF diet may have, among other factors, influenced egg production and quality. Apart from astaxanthin supplementation, future work should focus on n-3 HUFA levels or squid meal as a protein source as these have already been shown to affect egg quality in gilthead sea bream, which is, like striped jack, a continuous spawner with short vitellogenic periods. 24,25,30,31 Finally, although RF diets have better palatability and digestibility than the pellet diets, they have to be supplemented with vitamins and oils and are more inconvenient to use, transport and prepare. For these reasons, we aim to develop a broodstock dry pellet that enables striped jack to produce a sufficient number of eggs with an egg quality comparable or superior to the eggs produced by broodstock fed a RF diet. REFERENCES 1. Bromage N. Broodstock management and the optimization of seed supplies. Suisanzoshoku 1998; 46: Watanabe T. Status quo: Fish feed development in Japan. Suisanzoshoku 1996; 44: Verakunpiriya V, Mushiake K, Kawano K, Watanabe T. Supplemental effect of astaxanthin in broodstock diets on the quality of yellowtail eggs. Fisheries Sci. 1997; 63: Verakunpiriya V, Watanabe T, Mushiake K, Kiron V, Satoh S, Takeuchi T. Effect of broodstock diets on the chemical components of milt and eggs produced by yellowtail. Fisheries Sci. 1996; 62: Verakunpiriya V. Trials on the development of dry pellets for broodstock yellowtail. PhD Thesis, Tokyo University of Fisheries, Tokyo, 1996.

11 270 FISHERIES SCIENCE R Vassallo-Aguis et al. 6. Watanabe T, Vassallo-Agius R, Mushiake K, Kawano K, Kiron V, Satoh S. The first spawn-taking from striped jack broodstock fed soft-dry pellets. Fisheries Sci. 1998; 64: Watanabe T, Sakamoto H, Abiru M, Yamashita J. Development of a new type of dry pellet for yellowtail. Nippon Suisan Gakkaishi 1991; 57: Vassallo-Agius R, Watanabe T, Mushiake K, Kawano K, Satoh S. Chemical components of eggs and yolksac larvae obtained from striped jack broodstock fed on a raw fish mix and dry pellets. Fisheries Sci. 1998; 64: Miki W, Yamaguchi K, Konosu S. Carotenoid composition of Spirulina maxima. Nippon Suisan Gakkaishi 1986; 52: Okada S, Liao WL, Mori T, Yamaguchi K, Watanabe T. Pigmentation of cultured striped jack reared on diets supplemented with the blue-green alga Spirulina maxima. Nippon Suisan Gakkaishi 1991; 57: Liao WL, Takeuchi T, Watanabe T, Yamaguchi K. Effect of dietary Spirulina supplementation on extractive nitrogenous constituents and sensory test of cultured striped jack flesh. J. Tokyo Univ. Fish. 1990; 77: Vassallo-Agius R, Mushiake K, Imaizumi H, Yamazaki T, Watanabe T. Spawning and quality of eggs of striped jack fed raw fish or dry pellets with 2% Spirulina. Suisanzoshoku 1999; 47: Mushiake K. Studies on the technical development of broodstock management in striped jack and yellowtail. PhD Thesis, Hiroshima University, Hiroshima, Murai M, Kato K, Nakano T, Takashima F. Egg development and morphological changes of larvae of striped jack Caranx delicatissimus. Suisanzoshoku 1987; 34: Makino N, Uchiyama M, Iwanami S, Tohyama T, Tanaka M. Developmental changes in multiple oil-globules of Japanese sea bass eggs. Nippon Suisan Gakkaishi 1999; 65: Mushiake K, Sekiya S. A trial evaluation of activity in striped jack, Pseudocaranx dentex larvae. Suisanzoshoku 1993; 41: Kjørsvik E, Mangor-Jensen A, Holmefjord I. Egg quality in fishes. In: Blaxter JHS, Southward AJ (eds). Advances in Marine Biology. Academic Press, London. 1990; Mushiake K, Fujimoto H, Shimma H. A trial of evaluation of activity in yellowtail, Seriola quinqueradiata larvae. Suisanzoshoku 1993; 41: Watanabe T. Importance of docosahexaenoic acid in marine larval fish. J. World Aquacult. Soc. 1993; 24: Latscha T. Carotenoids Their Nature and Significance in Animal Feeds. F. Hoffmann La Roche Ltd, Basel Watanabe T, Ohhashi S, Itoh A, Kitajima C, Fujita S. Effect of nutritional composition of diets on chemical components of red sea bream broodstock and eggs produced. Nippon Suisan Gakkaishi 1984; 50: Watanabe T, Itoh A, Satoh S, Kitajima C, Fujita S. Effect of dietary protein levels and feeding period before spawning on chemical components of eggs produced by red sea bream broodstock. Nippon Suisan Gakkaishi 1985; 51: Watanabe T, Izquierdo MS, Takeuchi T, Satoh S, Kitajima C. Comparison between eicosapentaenoic acid and docosahexaenoic acid in terms of essential fatty acid efficiency in larval red sea bream. Nippon Suisan Gakkaishi 1989; 55: Fernández-Palacios H, Izquierdo MS, Robaina L, Valencia A, Salhi M, Vergara JM. Effect on n-3 HUFA level in broodstock diets on egg quality of gilthead sea bream (Sparus aurata L.). Aquaculture 1995; 132: Rodríguez C, Cejas JR, Martín MV, Badía P, Samper M, Lorenzo A. Influence of n-3 highly saturated fatty acid deficiency on the lipid composition of broodstock gilthead sea bream (Sparus aurata L.) and on egg quality. Fish Physiol. Biochem. 1998; 18: Bromage N. Broodstock management and seed quality general considerations. In: Bromage NR, Roberts RJ (eds). Broodstock Management and Egg and Larval Quality. Blackwell Science, Oxford. 1995; Sargent J, McEvoy L, Estevez A, Bell G, Bell M, Henderson J, Tocher D. Lipid nutrition of marine fish during early development: Current status and future directions. Aquaculture 1999; 179: Mushiake K, Nishizawa T, Nakai T, Furusawa I, Muroga K. Control of VNN in striped jack: selection of spawners based on the detection of SJNNV gene by Polymerase Chain Reaction (PCR). Fish Pathol. 1994; 29: Watanabe T, Kiron V. Red sea bream (Pagrus major). In: Bromage NR, Roberts RJ (eds). Broodstock Management and Egg and Larval Quality. Blackwell Science, Oxford. 1995; Fernández-Palacios H, Izquierdo M, Robaina L, Valencia A, Salhi M, Montero D. The effect of dietary protein and lipid from squid and fish meals on egg quality of broodstock for gilthead sea bream (Sparus aurata). Aquaculture 1997; 148: Almansa E, Pérez MJ, Cejas JR, Badía P, Villamandos JE, Lorenzo A. Influence of broodstock gilthead sea bream (Sparus aurata L.) dietary fatty acids on egg quality and egg fatty acid composition throughout the spawning season. Aquaculture 1999; 170: