Effect of two maturation diet combinations on reproductive performance of domesticated Penaeus monodon

Aquaculture 263 (2007) 75 – 83 www.elsevier.com/locate/aqua-online

Effect of two maturation diet combinations on reproductive performance of domesticated Penaeus monodon G.J. Coman ⁎, S.J. Arnold, T.R. Callaghan, N.P. Preston CSIRO Food Futures National Research Flagship, CSIRO Marine and Atmospheric Research, Cleveland, Qld 4163, Australia Received 29 August 2006; received in revised form 11 October 2006; accepted 12 October 2006

Abstract The reproductive performance of tank-reared, 2nd generation domesticated Penaeus monodon was compared when fed on two experimental maturation diet combinations; a control diet (CD) containing a treatment portion of 32.5% squid (Photololigo sp.) and 32.5% bivalves (Plebidonax sp.); and a shrimp-supplemented diet (SSD) containing a treatment portion of 21.6% squid (Photololigo sp.), 21.6% bivalves (Plebidonax sp.) and 21.6% shrimp (sexually mature Penaeus sp. and Metapenaeus sp.). The remaining portion of both diets consisted of 5% polychaetes and 30% commercial shrimp pellets. Broodstock were fed on the diets from approximately 10 months of age until commencement of reproductive assessment at 11 months, and until completion of the assessment when females had completed two moult periods post-ablation. No significant difference in growth, survival, ovarian maturation, spawning and egg production was found between diet treatments (P N 0.05). However, the percentage of spawnings that hatched (mean ± standard error) (CD 77.5 ± 6.7%; SSD 41.2 ± 8.6%) (P b 0.01), egg fertility rates per spawning (CD 60.2 ± 6.1%; SSD 34.4 ± 8.4%) (P b 0.05), hatch rates per spawning (including both unhatched and hatched spawnings) (CD 23.3 ± 4.2%; SSD 5.2 ± 1.7%) (P b 0.01) and nauplii per spawning (× 103) (CD 41 ± 9; SSD 6 ± 2) (P b 0.01) were significantly lower for broodstock fed the SSD than the CD. No difference in spermatophore weight or sperm quantity was found between diet treatments (P N 0.05). These results indicated that partial replacement of squid and bivalves with sexually mature shrimp in the maturation diet of tankdomesticated P. monodon had a negative effect on egg fertility and hatching. Furthermore, these results highlight the large effect that the final maturation diet can have on reproductive output of domesticated P. monodon broodstock. © 2006 Elsevier B.V. All rights reserved. Keywords: Prawn; Broodstock; Domestication; Tank-rearing; Tank-reared; Fresh-frozen diets; Giant tiger prawn

1. Introduction Despite the long farming history of the giant tiger shrimp, Penaeus monodon, virtually all commercial stocks are still produced from wild-caught broodstock (Clifford and Preston, 2001; Moss and Crocos, 2001).

⁎ Corresponding author. Tel.: +61 7 3826 7103; fax: +61 7 3826 7222. E-mail address: [email protected] (G.J. Coman). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.10.016

Progressing from using wild broodstock to domesticated stocks could alleviate problems of broodstock shortages, enable selection of stocks with genetically superior attributes, and allow more control over pathogens in the farming system (Gjedrem and Fimland, 1995; Primavera, 1997; Browdy, 1998; Crocos et al., 1999). Considerable efforts have been made towards domesticating P. monodon over the past two decades (e.g. Millamena et al., 1986; Menasveta et al., 1993; Pratoomchat et al., 1993; Makinouchi and Hirata, 1995; Hall et al., 2003; Coman et al., 2005, 2006). However, difficulties in

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controlling disease and providing appropriate nutrition and environmental conditions for rearing broodstock have contributed to the absence of commercial-scale breeding programs for P. monodon. The development of suitable broodstock maturation diets and feeding regimes is critical for domestication of any species. In penaeids, the nutritional requirements for reproduction are far less understood than the requirements for the grow-out phase of production, largely due to the greater complexity of physiological processes involved in reproduction, and the demanding requirements and expense of conducting reproductive trials to evaluate broodstock diets (Wouters et al., 2001a). Considerable efforts have been made to understand nutrient requirements and develop artificial (semi-purified) diets for maturation of several penaeid species (e.g. Millamena et al., 1986; Bray and Lawrence, 1990a,b; Cahu et al., 1994; Xu et al., 1994; Marsden et al., 1997; Wouters et al., 2001b). However, despite these efforts, successful broodstock maturation still typically relies on nutrition derived from the provision of a variety of natural food organisms, such as squid, annelid worms (polychaetes), bivalves (mussels, clams, and oysters), crustaceans (shrimp, crab, krill, enriched Artemia) and fish, as a large portion of the diet (Primavera, 1983; Menasveta et al., 1993; Cavalli et al., 1997; Browdy, 1998; Wouters et al., 2001a). Maturation diets consisting of different combinations of natural marine organisms generally produce high reproductive performance from wild P. monodon broodstock (e.g. Primavera and Caballero, 1992; Menasveta et al., 1993; Hansford and Marsden, 1995). However, the reproductive outputs of domesticated P. monodon stocks have commonly been inferior to the wild stocks when fed on similar diets based wholly or largely on combinations of natural marine organisms (Millamena et al., 1986; Menasveta et al., 1993, 1994; Makinouchi and Hirata, 1995; Coman et al., 2005, 2006). In many instances, the higher performances of the wild stocks may be due to a higher uptake of key nutrients required for reproduction while in the natural environment prior to being brought into captivity. In contrast, the domesticated stocks must obtain all nutrients required for reproduction from the diet provided. Any deficiencies or inappropriate balances of these nutrients in the diet would be apparent in lower reproductive performances. For this reason, it is important to know the contribution that all components of the diet have towards the observed reproductive performance when developing maturation diets for domesticated broodstock. Crustacean tissues have been a key component of maturation diets for several penaeid species (e.g. Crocos

and Kerr, 1986; Menasveta et al., 1993; Cavalli et al., 1997; Crocos and Coman, 1997; Peixoto et al., 2004; Preston et al., 2004). The inclusion of fresh-frozen shrimp within these maturation diets has provided a nutritional source to replace a variety of crustacean prey items occurring in the natural diet of the penaeid broodstock (Rothlisberg, 1998). Furthermore, the inclusion of sexually mature shrimp within these maturation diets may have non-nutritional benefits by providing a source of reproductive hormones that contribute to the endocrinological cycle (Wouters et al., 2001a). However, due to the risks of disease transmission, feeding of crustacean tissues to penaeid broodstock is seen as a risk to biosecure broodstock production and considered unsuitable for commercial application (Wouters et al., 2001a). For this reason, shrimp and other crustacean tissues have been removed from the diet of tank-domesticated P. monodon stocks at CSIRO Marine and Atmospheric Research (CMAR) since 1999. Importantly, it is not known whether the removal of this crustacean component from the maturation diet has contributed to the moderate to low hatching rates found in these tankdomesticated P. monodon stocks in recent years (Coman et al., 2005, 2006). The current study evaluated the reproductive performance of tank-reared, 2nd generation domesticated P. monodon when fed on two experimental maturation diets; a control diet (CD) containing a treatment portion of 32.5% squid (Photololigo sp.) and 32.5% bivalves (Plebidonax sp.); and a shrimp-supplemented diet (SSD) containing a treatment portion of 21.6% squid (Photololigo sp.), 21.6% bivalves (Plebidonax sp.) and 21.6% shrimp (sexually mature Penaeus sp. and Metapenaeus sp.). The effect of partial replacement of squid and bivalve with shrimp on reproductive performance of the broodstock was evaluated in order to identify whether the shrimp ingredient contains key nutrients that influence reproductive output. 2. Methods 2.1. Source and rearing of stocks The experimental stocks were 2nd generation P. monodon reared in tanks at CMAR, which originated from founder stocks collected from a population off the coast of Weipa (12°48′, 141°32′), Gulf of Carpentaria, Queensland, Australia. The stocks were comprised of seven families, which were each reared to PL15 (15 d post-metamorphosis from mysis to postlarval stage 1) in three replicate 100 L tanks (0.6 m dia.). From PL16 to PL30, the families were reared

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in two replicate 200 L cages suspended within a communal 10 000 L tank (3.6 m dia.). At PL30, individuals from all families were stocked into 10 000 L sandsubstrate tanks (Crocos and Coman, 1997) at a density of 10 individuals m − 2 . At 3 months, densities were reduced to 6 individuals m− 2 in all tanks. At 5.5 months, the stocks were weighed and individuals from the smallest 50% of each family were allocated among six 10 000 L sand-substrate tanks. All tanks had equivalent numbers of individuals from all families. The stocks were reared in these tanks at a density of 3.5 individuals m− 2 and at a sex ratio of 1.3 females:1 male for a further 5.5 months until reaching an age of 10 months. From PL30 until 10 months of age, stocks were fed on a diet consisting of approx. 20% squid (Photololigo sp.) (% dry weight), 10% bivalves (Plebidonax sp.) and 70% commercial pellets (Lucky Star, Taiwan Hung Kuo Industrial Pty Ltd). Proximate analysis of the batch of commercial diet used found the pellets contained 10.1% moisture, and on an as fed basis, approx. 12% ash, 61% crude protein and 13% total lipid. Food was provided three times daily (0900, 1300 and 1700 h) until 5.5 months of age, and twice daily beyond this age (0900 and 1700 h). At 10 months, all broodstock were captured from tanks and weighed, and from this age fed on their respective maturation treatment diet. Three of the six tanks were fed on a control diet (CD) consisting of 32.5% squid (% dry weight), 32.5% bivalves, 5% polychaetes (Marphysa sp.) and 30% pellets. The other three tanks were fed on a shrimp-supplemented diet (SSD) consisting of 21.6% squid, 21.6% bivalves, 21.6% shrimp (Penaeus sp. and Metapenaeus sp.), 5% polychaetes and 30% pellets. Squid, bivalve and shrimp were fresh-frozen and chopped prior to feeding. Polychaetes were fed live. All natural food organisms fed throughout the duration of the trial were obtained from the same source. Shrimp included in the SSD were collected from the same location as the experimental broodstock (Gulf of Carpentaria, Queensland, Australia). Based on both their size, and external observations of ovarian development in the females, the shrimp fed in the SSD contained a high proportion (approx. 80%) of sexually mature individuals. The six 10 000 L sand-substrate tanks used for the experiment were fitted with a sub-sand water circulation system to maintain the sand substrate under aerobic conditions (Crocos and Coman, 1997). Seawater flowed through the tanks at 3.5 L min− 1 maintaining water temperatures and salinities at (mean ± S.D.) 29.0 ± 0.2 °C and 35 ± 1‰. Photoperiod was maintained at 14 h light:10 h dark.

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2.2. Reproductive performance trial At 11 months of age, broodstock were captured from the tanks. Females at moult stages C, D1, D2 and D3 (Smith and Dall, 1985) were weighed, ovary staged (Tan-Fermin and Pudadera, 1989), eye-tagged for individual recognition, moult-tagged (water-proof labels glued to the carapace) to identify when each female had moulted, and restocked back into the tanks. Males were also weighed and restocked back into the tanks. Stocking densities and sex ratios were similar in both treatments (CD: 2.4 individuals m− 2, 0.9 females:1 male; SSD: 2.3 individuals m− 2, 0.9 females:1 male). Moulting of each female was monitored daily throughout the trial. Females were unilaterally eye-stalk ablated using hot forceps 3 d after their first moult and their subsequent reproductive performance was assessed. Reproductive performance of each female was assessed for two moult periods (approx. 6 wk) after ablation. All broodstock were maintained throughout the reproductive performance assessments under the same conditions as described for the 10 months to 11 months diet conditioning period. Females were examined daily for ovarian maturation. Ripe females (Tan-Fermin and Pudadera, 1989) were transferred to circular spawning tanks (0.66 m dia., water flow 0.7 L min− 1, water temperature 29 °C) filled to 80 L and allowed to spawn. Spawning tanks were fitted with an automated spawning detection and alarm system (Coman et al., 2003) to enable early detection of spawnings and assessment of fertilization rates of the eggs at the first or second mitotic division. Immediately after spawning detection, a 50 mL sample of water was removed from the spawning tanks to determine the time since spawning and whether a valid fertilization assessment could be performed. Microscopic observation of the earliest stages of egg development, including cortical rod protrusion and initial formation of the transparent hatching envelope, indicated that the eggs had been detected within 8 min of spawning (PongtippateeTaweepreda et al., 2004), and were used as criteria determining the possibility of performing valid fertilization assessment. After spawning, or if the ovaries had regressed, females were weighed and returned to the maturation tanks. For spawnings detected early enough to allow a valid fertility assessment (within 8 min post-spawning), fertilization rates were estimated from the percentage of eggs developing between 45 min and 75 min postspawning (undergoing mitotic division) from a 250 mL sample of the spawning tank water. For all spawnings, eggs per spawning was estimated from the total number

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of eggs collected in four 250 mL samples taken from the spawning tank water between 4 h and 8 h post-spawning. Eggs were then allowed to hatch in the spawning tanks and nauplii numbers per spawning were estimated from the total number of nauplii collected from four 250 mL samples taken 2 to 5 h after observation of first hatching (after thorough mixing to ensure eggs and nauplii were homogeneous within the water). Spermatophore and sperm quality of males matured on the two treatment diets were assessed at the end of the reproductive performance trial (13 months of age). Two spermatophores were collected from each one of three males randomly selected from each tank. Spermatophores were extruded from the males by gently pressing the coxas of the fifth pair of pereiopods (Lin and Ting, 1986). Spermatophores were placed in calcium-free saline (Leung-Trujillo and Lawrence, 1987) and the external condition was analyzed according to PerezVelazquez et al. (2001). The spermatophores were then blotted dry to remove excess saline and weighed. One of the two spermatophores from each male was randomly selected to estimate sperm quantity. These spermatophores were ground in 2 mL of calcium-free saline using a glass rod to release sperm from the sperm mass. The resulting sperm-saline suspension was mixed several times to ensure homogeneity. Estimates of sperm number within each spermatophore were determined by counting cells within the suspension using a haemocytometer and light microscopy (Leung-Trujillo and Lawrence, 1987). 2.3. Measures of broodstock performance Size of broodstock during the maturation period was expressed for each diet treatment as female and male weights at 10 months and 11 months of age. Female survival during the reproductive assessment was expressed for each diet treatment as the percentage of ablated females surviving the first and second moult periods after ablation in each tank. Survival of males during the reproductive assessment was expressed as the percentage of males stocked in each tank at the start of the trial (11 months) surviving until 13 months. Moult period durations were recorded for individual females. Reproductive performance was expressed in terms of the % of females that matured prior to ablation (ovary stages III or IV) and after ablation (ovary stage IV) (TanFermin and Pudadera, 1989), % of females that spawned, number of spawnings per ablated female (i.e. includes all females that were ablated, one spawning), duration from ablation to first spawning,

female weight at first spawning, eggs per spawning, percentage of spawnings that hatched, hatch rate for all spawnings and for spawnings that hatched only, nauplii per spawning, spermatophore weight, and sperm count. Reproductive performance of the subset of spawnings detected early enough for fertility assessment was expressed in terms of eggs per spawning, percentage of spawnings that were fertile (i.e. where eggs were observed undergoing first and second mitotic division), percentage of fertile spawnings that hatched, percentage of eggs fertilized within the fertile spawnings (i.e. percentage of eggs undergoing mitotic division within the fertile spawnings only), percentage hatch rate of the fertile spawnings, and percentage of fertilized eggs hatching per spawning (i.e. the percentage of fertilized eggs in each spawning that developed into nauplii; this measure excludes non-fertilized eggs; calculated as 100 × hatch rate of fertile spawnings / fertilization rate of fertile spawnings). 2.4. Statistical analyses Weight of broodstock from each sex was analyzed by ANOVA at each age using Generalized Linear Model I (PROC GLM; SAS Institute Software, 1999). Survival of the broodstock in each tank was analyzed by ANOVA for each sex using Model II. Duration of the female moult period was analyzed using Model I. Ovarian maturation prior to ablation of females (at 11 months) in each tank was analyzed using Model II. All other reproductive measures were analyzed using Model I. Both Models I and II included diet as a fixed effect. Model I included a tank nested within diet effect. Where the nested tank term was found significant, indicating significant differences between replicate tanks within either or both diet treatments, the analyses were repeated by Model II using mean values from each tank. Significant differences between treatments were determined from the ANOVA F statistic with the significance level set at α = 0.05. Yij ¼ l þ Dieti þ Tank ðDietÞj þ e Yi ¼ l þ Dieti þ e

Model I Model II

For Model I, Yij is the performance of an individual broodstock (e.g. percentage of females spawning), or spawning (e.g. eggs per spawning) within the ith diet and the jth tank. For Model II, Yi is the total performance of all broodstock within a tank (e.g. % of females surviving one moult period post-ablation) within the ith diet. μ is the overall mean; Dieti is the fixed effect of the

G.J. Coman et al. / Aquaculture 263 (2007) 75–83 Table 1 Mean (± S.E.) weights of Penaeus monodon in control (CD) and shrimp-supplemented diet (SSD) treatments, prior to (10 months), and after one month of feeding on the treatment diet (11 months) Weight measure

CD

SSD

Female weight at 10 months (g) Male weight at 10 months (g) Female weight at 11 months (g) Male weight at 11 months (g)

97.1 (2.2) n = 43

99.0 (2.1) n = 41

74.4 (1.6) n = 39

76.7 (1.5) n = 41

106.3 (2.5) n = 36

108.5 (2.2) n = 35

80.5 (1.4) n = 37

83.1 (1.5) n = 37

The CD contained 32.5% squid and 32.5% bivalves; the SSD contained 21.6% squid, 21.6% bivalves and 21.6% shrimp. No significant differences in broodstock weights were found between diet treatments for either sex (within rows) (P N 0.05). n = numbers of individual broodstock from the three replicate tanks.

ith diet; Tankj is the effect of the jth tank nested within diet (Model I); e is the random error.

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throughout the first and second moult periods, male survival from 11 months to 13 months and female moult period duration did not differ between diet treatments (P N 0.05) (Table 2). 3.2. Ovarian maturation, spawning and weight at first spawning Only a single female in the CD treatment had mature ovaries (stage IV; Tan-Fermin and Pudadera, 1989) at stocking of the reproductive trial (i.e. prior to ablation of the females). After ablation, no significant differences were found in the percentages of females maturing and spawning between the diet treatments (P N 0.05) (Table 3). The numbers of spawnings per ablated female and per spawning female did not differ between treatments (P N 0.05). The durations from ablation to first spawning and weights at first spawning were also similar for females fed on both diets (P N 0.05) (Table 3). 3.3. Eggs, hatching and nauplii production per spawning

3. Results 3.1. Broodstock size, survival and female moult period duration Weights of broodstock did not differ between diet treatments at the start of the maturation period (10 months) or the start of the reproductive trial (11 months) (P N 0.05) (Table 1). Female survival Table 2 Mean (±S.E.) % survival, and female moult period duration of Penaeus monodon reared on a control (CD) and a shrimp-supplemented diet (SSD) Measure

CD

SSD

% of females surviving one moult period post-ablation a % of females surviving second moult period post-ablation a % of males surviving from 11 to 13 months b Mean female moult period duration (d) for 1st and 2nd periods c, d

67.7 (8.1) n = 34 41.2 (8.6) n = 34 84.4 (3.3) n=3 23.4 (0.6) n = 23

81.3 (7.0) n = 32 50.0 (9.0) n = 32 80.7 (11.2) n=3 23.9 (0.7) n = 26

Refer to Table 1 for details of the two diets. No significant differences were found between diet treatments for any survival measure (within rows) (P N 0.05). a n = numbers of individual broodstock. b n = numbers of tanks. c n = numbers of females completing at least one moult period. d Mean value for completed moult periods per individual female (i.e. where two moult periods were completed, value is a mean for both 1st and 2nd moult period) post-ablation throughout reproductive assessment.

The Model I ANOVA of the eggs (×103) per spawning found a significant nested tank term (P b 0.05), indicating differences in eggs per spawning between tank replicates within the diet treatments. Consequently, the analysis was repeated by Model II using mean values of eggs per spawning from each tank. No differences in eggs per spawning were found between diet treatments when reanalyzed (P N 0.05) (Table 4). Table 3 Mean (±S.E.) maturation and spawning performance of Penaeus monodon broodstock reared on a control (CD) and a shrimp-supplemented diet (SSD) Reproductive measure

CD

SSD

% of females maturing to ovary stage IV a, b % of females spawning b Number of spawnings per ablated female b Number of spawnings per spawning female c Duration from ablation to first spawning (d) c Female weight at first spawning c

55.9 (8.8) n = 34

65.6 (8.5) n = 32

44.1 (8.6) n = 34 1.2 (0.4) n = 34

53.1 (9.0) n = 32 1.1 (0.2) n = 32

2.7 (0.7) n = 15

2.0 (0.3) n = 17

19.2 (3.6) n = 15

21.8 (2.9) n = 17

109.6 (4.6) n = 15

110.5 (3.6) n = 17

Refer to Table 1 for details of the two diets. No significant differences were found between diet treatments for any reproductive measure (within rows) (P N 0.05). a Ovary stages outlined in Tan-Fermin and Pudadera (1989). b n = numbers of ablated females. c n = numbers of spawning females.

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The percentage of spawnings that hatched was significantly higher for broodstock fed the CD (77.5 ± 6.7%) than the SSD (41.2 ± 8.6%) (P b 0.01). Hatch rates of spawnings that hatched were higher for spawnings from broodstock fed the CD (30.0 ± 4.7%) than the SSD (12.8 ± 3.2%), however this difference was not statistically significant (P N 0.05). When all spawnings were analyzed (unhatched and hatched), hatch rates were significantly higher from spawnings of broodstock fed the CD (23.3 ± 4.2%) than the SSD (5.2 ± 1.7%) (P b 0.01). The higher hatch rates of broodstock fed the CD also resulted in significantly higher numbers of nauplii (× 103) per spawning (41 ± 9) than broodstock fed the SSD (6 ± 2) (P b 0.01) (Table 4).

The external condition of all spermatophores obtained from males fed either the CD or the SSD appeared healthy. No significant difference in spermatophore weight or sperm count was found between diet treatments (P N 0.05) (Table 4). 3.5. Fertility assessments For the subset of spawnings for which fertility was assessed, the number of eggs (× 103) per spawning did not differ between diet treatments (P N 0.05) (Table 5).

Table 4 Mean (±S.E.) performance of spawnings, spermatophore weights and sperm counts of Penaeus monodon broodstock reared on a control (CD) and a shrimp-supplemented diet (SSD) Reproductive measure

CD

SSD

Eggs per spawning (× 103)1

144 (12) n = 40 77.5 (6.7)a n = 40 30.0 (4.7) n = 31 23.3 (4.2)a n = 40 41 (9)a n = 40 0.18 (0.02) n = 18 9.38 (2.84) n=9

132 (14) n = 34 41.2 (8.6)b n = 34 12.8 (3.2) n = 14 5.2 (1.7)b n = 34 6 (2)b n = 34 0.19 (0.04) n = 18 7.10 (1.58) n=9

% hatch rate per spawning for spawnings that hatched2 % hatch rate per spawning for all spawnings (unhatched and hatched)1 Nauplii per spawning (unhatched and hatched) (×103)1 Male-Spermatophore weight (g)3 Male-Sperm count (×106)3

Reproductive measure 3 1

Eggs per spawning (× 10 )

% of spawnings that were fertile (i.e. undergoing mitotic division)1,2 % of fertile spawnings that hatched2,3 % of eggs fertilized within the fertile spawnings2,3 % hatch rate of the fertile spawnings2,3 % of fertilized eggs hatching within each spawning2,3,4

3.4. Spermatophore and sperm assessments

% spawnings that hatched1

Table 5 Mean (±S.E.) performance of a subset of spawnings detected early enough to allow a valid fertility assessment (within 8 min postspawning) from Penaeus monodon broodstock reared on a control (CD) and a shrimp-supplemented diet (SSD)

Refer to Table 1 for details of the two diets. Diet treatments with different superscripts are significantly different for each reproductive measure (within rows) (P b 0.01). 1 n = numbers of spawnings. 2 n = numbers of spawnings that hatched. 3 n = numbers of spermatophores.

CD

SSD

144 (13) n = 33 75.8 (7.6) n = 33 96.0 (4.0) n = 25 60.2 (6.1)a n = 25 34.6 (5.4)a n = 25 50.2 (6.5)a n = 25

114 (15) n = 19 57.9 (11.6) n = 19 90.0 (9.1) n = 11 34.4 (8.4)b n = 11 10.7 (4.0)b n = 11 24.7 (5.6)b n = 11

Refer to Table 1 for details of the two diets. Values in table from spawnings assessed between 45 min and 75 min post-spawning. Diet treatments with different superscripts are significantly different for each reproductive measure (within rows) (P b 0.05). 1 n = numbers of spawnings (detected within 8 min post-spawning). 2 Fertility estimated from the percentage of eggs undergoing mitotic division. 3 n = numbers of fertile spawnings (detected within 8 min post-spawning). 4 % of the fertilized eggs within each spawning which developed into nauplii; this measure excludes non-fertilized eggs; calculated as 100 × hatch rate of fertile spawnings / fertilization rate of fertile spawnings.

The percentage of spawnings that were fertile was higher on average in the CD (75.8 ± 7.6%) than the SSD treatment (57.9 ± 11.6%), however this difference was not statistically significant (P N 0.05). The percentage of fertile spawnings that hatched was high for both diet treatments (N 90%). The percentage of eggs fertilized within the fertile spawnings, and the hatching rates from these fertile spawnings, were significantly higher for the CD (60.2 ± 6.1%, 34.6 ± 5.4%) than the SSD treatment (34.4 ± 8.4%, 10.7 ± 4.0%) (P b 0.05). Furthermore, the percentage of fertilized eggs hatching was also significantly higher in the CD (50.2 ± 6.5%) than the SSD treatment (24.7 ± 5.6%) (P b 0.05) (Table 5). 4. Discussion The present study found that P. monodon broodstock fed on a maturation diet containing a treatment portion of equal amounts of squid, bivalve and sexually mature shrimp (shrimp-supplemented diet: SSD) produced fewer spawnings that hatched and had lower fertility and hatch rates per spawning than broodstock fed on a diet containing a treatment portion of squid and bivalve

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only (control diet: CD). The lower percentage of fertilized eggs hatching into nauplii from spawnings of females fed the SSD than the CD indicated that diet had a large affect on embryo development. In addition, the lower fertilization rates of spawnings from broodstock fed the SSD suggested that diet also affected egg fertilization. Notably, diet had no effect on broodstock growth, survival, maturation, spawning and egg production. These results demonstrate the large effect that different maturation diets can have on egg fertility and hatching of domesticated P. monodon broodstock, even when fed for a relatively short period prior to spawning. Furthermore, these findings suggest that the shrimp ingredient does not contain a suite of additional nutrients that will augment the existing maturation diet and improve the reproductive output of tank-domesticated stocks. Fertilization and hatching rates of the eggs are two key parameters which have limited the reproductive output of domesticated penaeid stocks (e.g. AQUACOP, 1979; Menasveta et al., 1994; Makinouchi and Hirata, 1995; Coman et al., 2005, 2006). The maturation diet fed to the broodstock immediately prior to spawning has been shown to greatly affect fertilization and hatching of the eggs (e.g. Millamena et al., 1986; Cahu et al., 1995; Xu et al., 1994; Wouters et al., 1999; Mengqing et al., 2004). Similarly, the present study demonstrated the large effect that the maturation diet can have on egg fertilization and hatching in domesticated penaeid stocks. The difference in embryo development between diet treatments suggested that egg quality was lower for broodstock fed on the SSD than the CD. While several factors affect fertilization and hatching, including mating success, sperm quantity, sperm quality and the mechanism of fertilization at the time of egg release, the quality of the eggs largely determines embryo development beyond fertilization. Results of the present study suggest that nutritional differences between the experimental diets produced eggs of differing quality, which resulted in poorer embryo development and hatching of spawnings from females fed the SSD than the CD. Previous studies of pond- and tank-domesticated P. monodon have also suggested that egg quality largely influences egg hatching (Millamena et al., 1986; Millamena, 1989; Peixoto et al., 2005; Coman et al., 2006) and maturation diet has been shown to have a large influence on egg quality (e.g. Millamena et al., 1986; Millamena, 1989; Bray and Lawrence, 1990a,b; Xu et al., 1994; Cahu et al., 1994, 1995; Wouters et al., 1999). Consequently, further development of maturation diets tailored at improving egg quality will be a critical step for improving reproductive output of the tank-domesticated broodstock.

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The lower fertilization rates of spawnings from broodstock fed the SSD suggested that diet also affected egg fertilization. Sperm quantity was consistent for males fed on both diets, and consequently, differences in sperm quality or egg quality were likely responsible for the different fertilization rates found between broodstock fed on the two diets. While it was not possible to separate the effects of sperm and egg quality on fertilization in this study, it does seem plausible that egg quality may have also contributed to the lower fertilization rates of spawnings from females fed the SSD. Future studies incorporating independent evaluations of sperm quality and egg quality will be invaluable for understanding the contribution that these two factors have toward egg fertilization. Importantly, improvements in egg quality obtained through further diet development could serve to increase both the percentage of fertilized embryos hatching into nauplii and percentage of spawned eggs that are fertilized. Squid, bivalves (mussels, oysters and clams), and polychaetes are the most common ingredients used in maturation diet of penaeid broodstock (Moss and Crocos, 2001). Due to the high cost of polychaetes, squid and bivalves are typically fed at the highest daily ratios, and polychaetes at a supplemental level (Wouters et al., 2001a). The importance of these three fresh ingredients to successful reproduction is believed attributable to their nutritional profiles, particularly their content and ratios of certain amino acids, lipid fractions and critical fatty acids, such as arachidonic acid, eicosapentaenoic acid and docosahexanoic acid, which are known to have significant metabolic and physiological roles in penaeid reproduction (for review Harrison, 1990; Wouters et al., 2001a). Crustacean tissues have formed significant components of successful penaeid maturation diets. Notably, an earlier study of wild-caught P. monodon found that the reproductive performance of broodstock fed on a diet comprising squid, bivalves and shrimp was not inferior to a diet comprising squid and bivalves only (Crocos et al., 1992). The contrasting results found in the present study may reflect differences in the nutritional condition of wild-caught and domesticated broodstock due to their prior rearing history in the natural and captive environments. From the present study, it was not possible to determine whether the presence of the shrimp component in the SSD treatment had a negative influence on reproductive performance, or whether the greater proportion of either squid or bivalve in the CD had a positive effect on performance. However, the results of this study clearly demonstrated that the substitution of part of the squid and bivalve components of the diet with a shrimp component

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had a negative effect on egg fertility and hatching. Moreover, the inferred reduction in egg quality found when broodstock were fed on the SSD suggests that the nutritional balance of fatty acids and other key nutrients influencing embryo development was compromised by the inclusion of shrimp component at the expense of squid and bivalve components. To date, maturation diets for both wild-caught and domesticated penaeid broodstock rely predominantly on provision of natural food organisms, either fed live or fresh-frozen (Browdy, 1998; Wouters et al., 2001a). The relatively poor understanding of the different compounds required for reproduction in penaeids (Wouters et al., 2001a) has contributed to this reliance on natural food organisms in broodstock diets. Large variations in the biochemical composition of natural food organisms, due to location, season, stage of sexual maturity, quality of storage and various other factors, mean that the nutritional value of maturation diets based on freshfrozen ingredients is not always consistent (Bray and Lawrence, 1992). As a result, suitable maturation diets for penaeid broodstock are often developed based on empirical trialing of different combinations and proportions of artificial diets and available natural food organisms. Future studies evaluating diets of known biochemical composition, likely incorporating both natural food and artificial components, will be essential for improving our understanding of the dietary requirements for improved egg quality in the tank-domesticated P. monodon stocks. With this knowledge, artificial diets can be developed to progressively replace larger proportions of the fresh-frozen ingredients, thereby increasing the consistency and reliability of maturation diets for domesticated P. monodon. The present study demonstrated that maturation diet affected egg fertilization and hatching in our tankdomesticated stocks. While other factors, such as mating success, sperm quantity or sperm quality may still be limiting fertilization and hatching to some degree, improvements in egg quality through the development of a more suitable maturation diet will play a key role in increasing the reproductive output of these stocks. At a practical level, these results indicate that the removal of the shrimp component from the diet of our tank-domesticated P. monodon stocks over recent years would not have compromised the reproductive performance of these stocks. Acknowledgements We thank Melony Sellars and Frank Coman for their assistance in running the trial. This research

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