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Nitrogen budgets for juvenile big-bellied seahorse Hippocampus abdominalis fed Artemia, mysids or pelleted feeds
Aquaculture 255 (2006) 233 – 241 www.elsevier.com/locate/aqua-online
Nitrogen budgets for juvenile big-bellied seahorse Hippocampus abdominalis fed Artemia, mysids or pelleted feeds Z. Wilson, C.G. Carter ⁎, G.J. Purser School of Aquaculture, Tasmania Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia Received 2 September 2005; received in revised form 5 December 2005; accepted 6 December 2005
Abstract This study aimed to compare nitrogen budgets for juvenile (1.17 ± 0.07 g, mean ± S.D.) big-bellied seahorses fed live, frozen and pelleted feeds and to examine the potential of non-destructive measurements of excretion to predict feed performance. Three feeds were tested against live Artemia: frozen mysid shrimp species (Paramesopodopsis rufa, Tasmanomysis oculata,Tenagomysis sp.), a pellet prepared from the mysid shrimps and a commercial crumbled feed. Nitrogen budgets were constructed from nitrogen retention measured over a 30 day growth experiment, ammonia and urea excretion measured over 24 h on days 15 and 30, and nitrogen digestibility. After 30 days seahorses fed Artemia and frozen mysid had significantly (P b 0.001) higher final weights and growth than the other two feeds. Although seahorses successfully weaned onto the commercial feed, the final weight was not significantly different from their initial weight. Seahorses fed the mysid pellet were not successfully weaned and lost weight. Feed conversion ratio was significantly (P b 0.001) affected by feed and seahorses fed Artemia and frozen mysid had the most efficient conversion of feed to growth. Despite the poor growth performance of seahorses fed the mysid and commercial pellets survival over 30 days was above 95% and there was no significant difference between treatments. Ammonia excretion was significantly affected by feed on day 15 (P b 0.05) and day 30 (P b 0.001), excretion was higher for Artemia than for mysid pellets on both days. Urea excretion was significantly (P b 0.05) affected by feed only on day 30 and was higher for Artemia and frozen mysids. Nitrogen retention was above 45% on Artemia and frozen mysid treatments. An indirect method of assessing nitrogen retention was an accurate technique where seahorses were growing but not where growth was near or below maintenance. © 2005 Elsevier B.V. All rights reserved. Keywords: Excretion; Larval pelleted feed; Live feed; Nitrogen balance; Nitrogen budget; Seahorse
1. Introduction The demand for seahorses has resulted in many animals being taken from the wild, for example approximately 20 million seahorses were taken in 1995 for a variety of markets such as the aquarium and medicine ⁎ Corresponding author. Tel.: +61 3 63243823; fax: +61 3 6324 3804. E-mail address: [email protected] (C.G. Carter). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.12.007
trade (Vincent, 1996). In many locations collection of wild seahorses is viewed as an unsustainable practice, leading to the development of seahorse aquaculture that has the opportunity to reduce or replace the reliance on wild caught animals and propagate endangered species (Vincent, 1996; Payne and Rippingale, 2000; Woods, 2000a,b). The big-bellied seahorse is endemic to southern Australia and New Zealand and can be successfully reared through multiple captive generations following well established production
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techniques based on the use of live feeds (Forteath, 1995; Woods, 2000a,b). A significant problem encountered when establishing economically viable and biologically sustainable seahorse aquaculture is providing sufficient quantities of nutritionally complete feed that will be consumed by seahorses. In commercial seahorse culture, there has been heavy reliance on cultured live feeds predominantly Artemia, but also wild caught mysid shrimp and amphipods (Woods, 2003a) either in the live or frozen form. There is a need to reduce the reliance on live feeds because Artemia varies in cost, availability and quality from year to year (Lavens and Sorgeloos, 2000) and collection of live organisms from the wild can be unpredictable (Woods and Valentino, 2003). Whilst the use of frozen feeds has supported good growth and survival, formulated pelleted feeds have not generally been successful because seahorses do not readily accept such feeds (Woods, 2003b; Woods and Valentino, 2003). Despite seahorses being very distinctive relatively little is known about their nutritional physiology including the digestibility of live and pelleted feeds, the influence of feed on ammonia and urea excretion and the efficiency with which nitrogen is retained. The construction of nitrogen budgets through the measurement of protein–nitrogen intake, digestive and excretory losses and nitrogen retention provides a highly informative approach to developing an understanding of fundamental processes of nutrient utilisation, growth and growth efficiency (Birkett, 1969; Kaushik and Cowey, 1991; Carter and Houlihan, 2001). The aims of this study were to investigate the growth response and nitrogen budgets of juvenile H. abdominalis fed three experimental feeds and tested against live enriched Artemia metanauplii as a standard: a mix of local frozen mysid shrimps, a pellet prepared from the mysid shrimps and a commercial pelleted larval feed. Ammonia and urea excretion were quantified and also assessed as non-destructive measures of nitrogen retention in order to predict the success of feeds.
2. Materials and methods 2.1. Fish and fish husbandry Three hundred and sixty juvenile big-bellied seahorses H. abdominalis (1.17 ± 0.07 g, mean ± SD) were randomly selected from a captive population held at the Aquaculture Centre, University of Tasmania. Animals were held in a seawater recirculation system, consisting of eight 25-l tanks, a sump (150-l), mechanical filtration via dacron matting and a trickle biological filter, housed in a temperature controlled room. Tank flow rate was maintained at 2.9 ± 0.9 l min− 1, constant aeration was supplied via an air-ring placed around the bottom of the tank outlet, which aided in keeping the feeds in suspension and plastic netting was supplied for seahorse attachment. Water quality parameters (mean ± SD) temperature (15.9 ± 0.4 °C), dissolved oxygen (DO, 8.0 ± 0.7 mg l− 1) and salinity (32.6 ± 1.4‰) were measured daily; total ammonia (0.02 ± 0.03 mg l− 1), nitrite (0.1 ± 0.1 mg l− 1), nitrate (6.4 ± 2.4 mg l− 1) and pH (8.1 ± 0.1) were measured weekly. These levels were maintained by a 25% water change per week. Photoperiod was 12 L : 12 D and light intensity at the water surface was 6.8 μE s− 1 m− 2. 2.2. Experimental feeds Four feeds were investigated: Artemia sp. instar II (INVE Thailand) were enriched with Super Selco (INVE Aquaculture) according to manufacturers instructions; a mix of mysid shrimps (Paramesopodopsis rufa, Tasmanomysis oculata, Tenagomysis sp.) collected along the north coast of Tasmania and frozen; a pelleted feed prepared using mysid shrimp and a commercial crumble, CRUM (NRD 5–8, 500–800 μm crumble, INVE Lansy, Primo Aquaculture Pty Ltd, NSW, Australia) (Table 1). Fish were fed a mixed feed of Artemia, frozen mysid shrimp, mysid pellet and CRUM for a 10 day period prior to the commencement of the 30 day experimental period, after which fish were fed solely on the allocated experimental feed.
Table 1 Composition experimental feeds
Dry matter (%) Crude lipid (% DM) Crude protein (% DM) Ash (% DM) Energy (kJ g− 1 DM) Mean ± SE, n = 3.
Artemia
Mysid
Mysid pellet
CRUM
19.78 ± 0.12 9.55 ± 1.38 45.53 ± 0.11 2.03 ± 0.03 22.56 ± 0.12
19.72 ± 0.40 15.34 ± 3.95 59.97 ± 0.22 3.07 ± 0.07 19.16 ± 0.03
89.40 ± 0.06 17.28 ± 2.05 60.77 ± 0.11 17.00 ± 0.06 16.59 ± 0.03
92.72± 0.22 17.53 ± 2.37 56.22 ± 1.67 13.20 ± 0.06 19.43 ± 0.03
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Artemia were hatched for 30 h at 27 °C and enriched with Super Selco for 18 h at 16 °C. The mysid pellet was prepared by freeze-drying frozen mysid shrimp to a constant weight. Following freeze-drying the mysids were ground to a fine powder using a hammer mill (1 mm mesh size). Gelatin (type A, ICN Biochemicals, OH, USA) was included at 10% of dry mysid weight and dissolved in hot water prior to addition, with water being added at 20% inclusion. The ingredients were mixed using a Kenwood mixer, extruded through a 10 ml syringe, spread out onto trays and dried at 35 °C for 48 h. Following drying, the mixture was ground and sieved through a 1 mm sieve, to obtain particles b1 mm. The mysid shrimp, mysid pellet and CRUM were frozen until used. The ration of frozen mysid shrimp required at each meal was thawed in seawater before being fed to the seahorses and following enrichment the Artemia were rinsed with fresh water before each meal. 2.3. Growth experiment The experiment was conducted over two 30 day time periods with two replicate tanks for each feed in each time block (n = 4). Each tank contained 20 seahorses and feeds were randomly allocated to tanks using a random number generator. Seahorses were fed daily at a ration of 5% DM BW− 1 d− 1 that was divided into two equal sized meals. Feeding was at 09:00 and 15:00 h and meal times were restricted to 2 h after which remaining food was siphoned from tanks, dried and feed consumption calculated by difference. Prior to each meal tanks were siphoned to remove faeces so as not to interfere with the measurement of feed consumption. Individual fish wet weight was measured on days 1, 16 and 31. Growth rate (G: % initial weight d− 1) was calculated as a proportion of the initial weight using G = 100(Wf − Wi)(Wit)− 1 (Hopkins, 1992); where Wi and Wf were mean individual initial and final wet weight (g) and t is the experimental period (d). Daily feed consumption (FC, % DM B− 1 d− 1) was calculated using FC = 100 feed consumed (g DM) B− 1; where B was initial tank biomass (g). Feed conversion ratio (FCR g g− 1) was calculated using FCR = total feed consumed (g dry weight)[(Wf − Wi) × carcass dry matter]− 1. Chemical composition was measured in initial samples from 20 seahorses taken at the start of each time-block and in all experimental seahorses following the final weight check (10 mg l− 1 Benzocaine). Seshorses and feed samples were frozen in liquid nitrogen and stored at − 80 °C prior to analysis.
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2.4. Excretion Ammonia and urea excretion were measured on day 15 and 30 of the growth experiment. This was conducted in a second set of tanks that were identical to the tanks used for growth and were first cleaned with hot water, filled with 20 l of 0.2 μm filtered seawater and supplied with gentle aeration (Verbeeten et al., 1999). The seahorses were then netted and moved into the tanks for 24 h, fed as normal and water samples taken after 24 h; all samples were frozen within 1 h of being taken. Ammonia and urea were measured in duplicate tanks containing each feed, but no seahorses, to account for the affects of feeds on ammonia and urea concentrations. In addition, positive (1 mg l− 1 NH3Cl) and negative (0 mg l− 1) ammonia chloride controls showed that no ammonia was gained or lost during the 24 h sampling period. Frozen water samples were thawed at room temperature prior to analysis, ammonia was measured using the salicylate–hypochlorite method (Bower and Holm-Hansen, 1980) and urea measured using the diacetyl–monoxime method (Rahmatullah and Boyde, 1980). 2.5. Faecal collection Faeces were collected on day 27–29 of the growth experiment from each replicate tank of the Artemia and mysid shrimp treatments, with faeces being siphoned onto a 25 μm screen and washed into 250 ml sample jars with fresh water and frozen. Following freeze-drying faecal samples from replicate tanks of each feed were pooled together to obtain sufficient sample for analysis. No faeces were collected from animals fed the mysid pellet or CRUM due to lack of faecal production. Calculating faecal nitrogen was not a major aim of this study and only an initial investigation due to the difficulty in collecting enough faecal material from small seahorses. Apparent nitrogen digestibility (ADN, %) was calculated directly for the Artemia and mysid shrimp feeds using ADN = 100 (R − F)R− 1 (Brafield, 1985), where R and F were the nitrogen (crude protein) contents of the feed and faeces, respectively. 2.6. Chemical analysis Moisture content of the feeds was determined by drying samples to a constant weight in an oven at 105 °C (AOAC, 1990, method 950.02). Seahorse carcasses were freeze-dried to a constant weight and then homogenised to a fine powder. Samples were analysed for nitrogen using Kjeldahl (selenium
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2.50
a ***
a ***
sured for the Artemia and frozen mysid treatments, but could not be determined for fish fed the mysid pellet or CRUM. Mean excretion values of AN and UN from day 15 and 30 were used. The unknown component (%) was calculated by difference using CN − (FN + AN + UN + PN).
Initial weight
Wet weight (g)
2.00
Final weight
1.50
b
b
2.8. Statistical analysis 1.00
Mean (± S.E.M.) values are reported. Levene's test and Shapiro–Wilk test were used to test for equality of error variances and normality, respectively. It was not necessary to transform any data prior to analysis. A paired sample t-test was conducted to determine if significant changes occurred between the initial and final weight of animals fed each feed. Two-way ANOVA was used to determine if a significant interaction existed between time and feed. No significant interaction occurred in any of the variables, therefore the data was pooled and a one-way ANOVA conducted. Means were compared using Tukey HSD test and significance was accepted as P b 0.05. Statistical analyses were performed using SPSS v. 10.0.
0.50
0.00 Artemia
Mysid shrimp Mysid pellet
CRUM
Diet Fig. 1. Initial and final wet weight of juvenile H. abdominalis fed experimental feeds (mean + SE, n = 4). Final weights with different superscripts are significantly different (P b 0.001, one-way ANOVA) between treatments, ⁎⁎⁎ indicates significant difference between initial and final weight (P b 0.001, paired sample t-test).
catalyst; N × 6.25 = crude protein), crude lipid by chloroform and methanol extraction (Bligh and Dyer, 1959), energy using a bomb calorimeter (Gallenkamp autobomb, calibrated with benzoic acid) and ash by combustion in a furnace at 550 °C for 16 h (AOAC, 1990, method 935.11). Nutrient retention efficiency for protein (PP, %) and energy (PE, %) were calculated as 100 (final carcass nutrient content (g)—initial carcass nutrient content (g)) total nutrient consumption (g)− 1.
3. Results 3.1. Growth performance There was no significant difference in seahorse wet weight at the start of the experiment (F = 0.018, df 3, 316, P N 0.05). After 30 days there was a significant difference in final wet weight (F = 62.003, df 3, 305, P b 0.001), with animals fed Artemia and mysid shrimp both having an almost 2 fold increase in weight (Fig. 1). These animals were significantly heavier than the seahorses fed the mysid pellet and CRUM (Fig. 1). Animals fed the mysid pellet lost weight and animals fed CRUM increased in weight, however changes in weight were small and there were no significant differences between the initial and final weights (Fig. 1). Consequently, feed had a significant effect on growth rate (Table 2), seahorses fed Artemia and mysid shrimp had positive growth rates where as the mysid pellet
2.7. Nitrogen budgets A nitrogen budget (Carter and Brafield, 1992; Carter and Houlihan, 2001) was calculated for each feed using the data from the growth and excretion experiments (as % of CN) according to CN = FN + AN + UN + PN, where CN is consumed nitrogen, FN is faecal nitrogen (FN = 100 − ADP), AN and UN are nitrogen excreted as ammonia and urea, respectively, and PN is retained nitrogen (calculated for protein retention efficiency). FN was meaTable 2 Growth performance of juvenile H. abdominalis fed experimental feeds Parameter
Artemia −1
Growth rate (% Wi d ) Feed consumption (% DM B d− 1) FCR (g g− 1) Survival (%)
Mysid
a
3.16 ± 0.13 2.12 ± 0.07a 3.33 ± 0.45b 95.0 ± 2.04
Mysid pellet
a
2.92 ± 0.10 1.60 ± 0.09b 2.68 ± 0.07b 97.5 ± 1.44
CRUM
− 0.11 ± 0.04 0.67 ± 0.03c − 14.36 ± 1.18c 96.25 ± 1.25 c
One-way ANOVA
b
0.35 ± 0.03 0.85 ± 0.05c 16.48 ± 1.48a 97.5 ± 1.44
Means in the same row with same letter are not significantly different (Tukey HSD) (mean ± S.E.M., n = 4).
F value
P (df)
258.005 106.088 168.410 0.579
⁎⁎⁎ (3, 12) ⁎⁎⁎ (3, 12) ⁎⁎⁎ (3, 12) NS (3, 12)
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Table 3 Chemical composition (% wet weight), energy content and nutrient retention efficiency for protein–nitrogen (PN) and energy (PE) of juvenile H. abdominalis fed experimental feeds Parameter
Moisture Crude protein Crude lipid Ash Energy (kJ g− 1 W) PN (%) PE (%)
Artemia
76.47 ± 0.76 13.90 ± 0.28 3.84 ± 0.37 3.96 ± 0.19 4.37 ± 0.20 46.38 ± 6.10a 33.66 ± 5.53a
Mysid
78.26 ± 0.70 14.10 ± 0.46 2.57 ± 0.15 3.56 ± 0.19 4.11 ± 0.14 45.72 ± 1.73a 45.11 ± 2.33a
Mysid pellet
78.66 ± 1.85 12.75 ± 0.85 2.15 ± 0.81 4.27 ± 0.17 3.69 ± 0.41 − 5.90 ± 7.33b 1.90 ± 11.65b
CRUM
79.24 ± 0.45 12.18 ± 0.59 2.24 ± 0.35 4.12 ± 0.05 3.54 ± 0.15 3.67 ± 4.64b 9.06 ± 3.45b
One-way ANOVA F value
P (df)
0.635 1.912 2.736 2.507 0.437 26.205 9.110
NS (3, 12) NS (3, 12) NS (3, 12) NS (3, 12) NS (3, 12) ⁎⁎⁎ (3, 12) ⁎⁎ (3, 12)
Means with different superscripts are significantly different (Tukey HSD) where NS = not significant, ⁎ P b 0.05, ⁎⁎ P b 0.01, ⁎⁎⁎P b 0.001) (mean ± S.E.M, n = 4).
resulted in a negative growth rate (Table 2). Despite the poor growth and feeding of seahorses fed the mysid pellet and CRUM they had high survival, which was not significantly different between any of the feeds (Table 2). Feed had a significant effect on feed consumption (FC) and feed conversion ratio (FCR) (Table 2). Feed consumption of Artemia was significantly higher than for the other feeds and mysids were consumed at a significantly higher rate than either the mysid pellet or CRUM feeds (Table 2). Animals fed Artemia and mysid had the most efficient FCR values of 3.33 and 2.68 g g− 1, respectively. The initial whole-body chemical composition (mean ± SD) was 77.75 ± 2.07% moisture, 12.54 ± 1.06% crude protein, 3.97 ± 0.55% crude lipid, 4.23 ± 0.42% ash and an energy value of 3.36 ± 0.61 kJ g− 1 W. There were no significant differences between initial and final samples or between treatments for any component of composition
(Table 3). Feed had a significant effect on nutrient retention efficiency, values were significantly higher for both protein and energy in animals fed Artemia and mysid (Table 3). 3.2. Nitrogen excretion Feed had a significant but different effect on nitrogenous excretion measured on days 15 and 30 (Table 4). On day 15, ammonia excretion on Artemia was significantly higher than on mysid pellets but there were no differences in urea excretion or the urea to total nitrogen ratio. Feed had a significant effect on total nitrogen excreted on day 15, with seahorses fed Artemia and mysid excreting the most nitrogen. The proportion of consumed nitrogen excreted as total nitrogen on day 15 was significantly different between Artemia and mysid pellet.
Table 4 Ammonia (AN), urea (UN) and total nitrogen (TN) excretion, the proportion of total nitrogen excreted as urea (UN : TN) and the proportion of consumed nitrogen excreted as total nitrogen (TN : CN) on day 15 and 30 of juvenile H. abdominalis fed experimental feeds Parameter
Artemia
Mysid
Mysid pellet
CRUM
One-way ANOVA F value
P (df)
Day 15 AN (mg N kg− 1 d− 1) UN (mg N kg− 1 d− 1) TN (mg N kg− 1 d− 1) UN : TN (%) TN : CN (%)
253.61 ± 9.48a 141.92 ± 26.50 395.53 ± 24.48a 35.15 ± 4.83 47.79 ± 3.09a
200.26 ± 16.34ab 158.17 ± 44.42 358.43 ± 34.05ab 41.59 ± 9.79 43.03 ± 1.90ab
142.79 ± 19.38b 101.17 ± 13.06 243.96 ± 21.99b 41.63 ± 4.86 35.11 ± 1.43b
185.63 ± 32.64ab 84.11 ± 16.63 269.74 ± 31.25b 31.87 ± 7.00 38.57 ± 2.18ab
4.874 1.525 6.401 0.494 6.064
⁎ (3, 12) NS (3, 12) ⁎⁎ (3, 12) NS (3, 12) ⁎⁎ (3, 12)
Day 30 AN (mg N kg− 1 d− 1) UN (mg N kg− 1 d− 1) TN (mg N kg− 1 d− 1) UN : TN (%) TN : CN (%)
209.25 ± 11.75a 229.94 ± 12.97a 439.19 ± 5.89a 52.33 ± 2.69 53.06 ± 3.43a
214.13 ± 11.03a 172.85 ± 13.62ab 386.97 ± 15.47ab 44.58 ± 2.61 46.45 ± 2.05ab
133.92 ± 10.07b 94.58 ± 23.89b 228.50 ± 13.94c 40.01 ± 7.66 32.88 ± 1.34c
152.96 ± 12.21b 124.55 ± 48.62ab 277.52 ± 52.64bc 39.87 ± 9.00 39.68 ± 2.24bc
12.675 4.263 11.603 0.889 13.249
⁎⁎⁎ (3, 12) ⁎ (3, 12) ⁎⁎ (3, 12) NS (3, 12) ⁎⁎⁎ (3, 12)
Means with different superscripts are significantly different (Tukey HSD) where NS = not significant, ⁎ P b 0.05, ⁎⁎ P b 0.01, ⁎⁎⁎P b 0.001) (mean ± S.E.M, n = 4).
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On day 30, ammonia and urea excretion were significantly affected by feed (Table 4). Seahorses fed Artemia and mysid excreted significantly higher amounts of ammonia than when fed the other two feeds whereas urea excretion was significantly higher only between Artemia and the mysid pellet (Table 4). However, the proportion of total nitrogen excreted as urea was not significantly different between treatments. The total excreted nitrogen was significantly affected by feed with the highest rates from animals fed Artemia and mysid, both significantly higher than for mysid pellets. The TN : CN ratio was highest for Artemia and mysid, with mysid pellets having a significantly lower ratio than Artemia and mysid but not CRUM (Table 4). 3.3. Nitrogen budgets Nitrogen budgets expressed as a proportion of consumed nitrogen are presented in Table 5. There was a high balance of nitrogen achieved for animals fed Artemia and mysid shrimp, 103.18 ± 7.30% and 101.24 ± 3.11%, respectively. However there was a poor recovery of nitrogen in animals fed the mysid pellet and CRUM feed, with 71.09 ± 7.00% and 58.93 ± 4.95% remaining unaccounted for (Table 5). This was primarily because faecal nitrogen could not be determined for fish fed the mysid pellet and CRUM, which increased the unknown component. The apparent nitrogen digestibility for the Artemia and mysid shrimp feeds was 90.73% and 86.13%, respectively, and faecal nitrogen loss represented 9.27 ± 0.90% and 13.69 ± 0.25% of consumed nitrogen, respectively. The proportion of consumed nitrogen excreted as ammonia remained relatively constant between feeds ranging from 20.25 to 26.17% (Table 5). Urea excretion represented a smaller proportion of CN although it tended to be higher in the two treatments where consumption and growth were Table 5 Nitrogen budgets (% of consumed nitrogen, CN) showing faecal (FN), ammonia (AN) and urea (UN) excretion and nitrogen retained as growth (PN) of juvenile H. abdominalis fed experimental feeds Parameter Artemia
Mysid
Mysid pellet CRUM
CN (%) FN (%) AN (%) UN (%) PN (%) Unknown (%) Balance (%)
100 13.69 ± 0.25 23.25 ± 0.38 18.58 ± 2.92 45.72 ± 1.73 (− 1.24)
100
100
20.25 ± 1.24 14.56 ± 2.17 −5.90 ± 7.33 71.09 ± 7.00
23.03 ± 2.27 14.36 ± 2.78 3.67 ± 4.64 58.93 ± 4.95
103.18 ± 7.30 101.24 ± 3.11 28.91 ± 7.00
41.07 ± 4.95
100 9.27 ± 0.90 26.17 ± 2.11 21.36 ± 1.08 46.38 ± 6.10 (−3.18)
Mean ± S.E.M., n = 4.
highest. The nitrogen retained as growth represented 46.38 ± 6.10% and 45.72% of CN in seahorses fed Artemia and mysid, respectively. However there was low retention of nitrogen in animals fed CRUM (3.67%) and those fed the mysid pellet lost nitrogen (− 5.90%). 4. Discussion There are few papers published where alternative feeds for seahorses have been investigated and this study demonstrated that 1 g juvenile seahorses can be successfully weaned onto frozen mysid shrimp. The use of mysid shrimp as a sole food source was demonstrated previously with larger H. abdominalis of 3 g (Woods and Valentino, 2003) and the SGR in both studies was around 3% d− 1. As in the present study final weights were not different between the frozen mysid and the live Artemia. In contrast, seahorses fed in the present study on mysid pellets or the commercial crumble did not show any significant change from their initial weight. The successful weaning of seahorses onto frozen mysid shrimp has significant implications for seahorse aquaculture since it reduces the reliance on enriched Artemia which is important because of the variable cost, availability and quality of Artemia cysts from year to year (Lavens and Sorgeloos, 2000). Although juvenile seahorses were successfully weaned onto frozen mysid shrimp further research is required to confirm the suitability of mysids as a long-term feed for seahorses. The use of pelleted feeds was not successful in the present study principally due to low feed intake. Reasons for the low intake and associated reduced growth compared to the intake of the Artemia and mysids in seahorses are unclear but contributing factors during weaning in other marine species include food form, diet colour, lack of specific movement, lack of chemical stimuli, lack of visual stimuli, inadequate fish size, inadequate digestive tract/enzyme development and environment (Person Le Ruyet et al., 1993; Kolkovski et al., 1997a,b; Rosenlund et al., 1997; Hamlin and Kling, 2001). Seahorses fed the mysid pellet lost weight after 30 days, but the CRUM feed was slightly more successful since seahorses were weaned onto it and maintained their weight. Woods (2003b) investigated the use of a formulated feed for newborn, one-month and twomonth H. abdominalis. One-month old juveniles fed formulated feed compared to juveniles fed live Artemia had significantly lower survival and lower weight gain, whereas, two-month old juveniles had similar survival to those fed live Artemia but again with lower weight gain. The high survival of the two-month old juveniles
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(Woods, 2003b) and of the animals used in the present study is most likely due to their larger size and greater ability to withstand periods of under-nutrition during weaning, compared to newborn and one-month old animals (Woods, 2003b). In the present study animals fed on the crumble but weaning was not successful since the increase in weight was not significantly different from the initial weight. The tanks were designed with an air-ring around the bottom of the tank outlet, which aided in keeping the feeds suspended in the water. The seahorses would observe the food as it moved around the tank but fewer strikes occurred against pelleted feeds (personal observation), which suggested these feeds did not trigger as active a feeding response. The resemblance and behaviour of pelleted feed compared to live feed is an important factor to consider in seahorse culture since seahorses are visual feeders and scrutinise feed before it is consumed (Woods, 2003b). The mixed feeding of pelleted and live feeds is common practice in improving growth and survival of larval marine fish (Kanazawa et al., 1989; Holt, 1993; Rosenlund et al., 1997). The successful use of pelleted feed as a sole food source in seahorse culture may require a longer weaning protocol than the 10 days of mixed feeding used in this study. Physiological and nutritional factors, principally water temperature, body size, ration and feed composition, influence nitrogenous excretion by fish (Jobling, 1981; Beamish and Thomas, 1984; Carter and Brafield, 1992). There is little information available on nitrogenous excretion of H. abdominalis and this study provides detailed information on nitrogen excretion in relation to nutritional factors. The amount of feed consumed and feed composition, including protein digestibility and content, were the main factors that affected ammonia and urea excretion in the present study. Rates of ammonia excretion were broadly similar to other marine fish and provided further evidence of a positive relationship between feed consumption and ammonia excretion (Kikuchi et al., 1991; Dosdat et al., 1995; Carter and Bransden, 2001). The proportion of consumed nitrogen excreted as ammonia varied according to amount of feed consumed, with a maximum of 26% excreted by animals fed Artemia, which is similar to other values reported in the literature (Porter et al., 1987; Kikuchi et al., 1992; Harris and Probyn, 1996; Verbeeten et al., 1999). The proportion of total nitrogen excreted as urea varied from 32% to 52% which is high since teleosts typically excrete b 20% of total nitrogen as urea (Wood, 1993; Korsgaard et al., 1995). There is evidence that urea excretion plays an important role in nitrogenous excretion
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by marine fish, and a number of marine species are now known to have higher urea excretion rates than previously predicted (Dosdat et al., 1996; Tanaka and Kadowaki, 1995; Carter et al., 1998). In addition, urea excretion rates increased as ingested food increased in seahorses as with other marine fishes (Dosdat et al., 1996; Carter et al., 1998; Verbeeten et al., 1999). The morphology of the gut may play a role in nitrogen excretion, as seahorses lack a true stomach (Woods, 2003b). Dosdat et al. (1995) hypothesised that urea could be produced along with faeces in the digestive tract of turbot, which may also be the case in seahorses and could explain the link between feed intake and urea excretion. The nitrogen budgets of growing seahorses fed Artemia and mysid shrimp, calculated from independently measured components accounted for 103% and 101%, respectively, of the consumed nitrogen and appeared to provide an accurate representation of nitrogen flux (Carter and Brafield, 1992; Owen et al., 1998). Although it should be noted that the measurement of each parameter of the nitrogen budget has its own margin of error, which must be taken into consideration when the accuracy of the budgets are assessed (Brafield, 1985; Carter and Brafield, 1991). Only 29% and 41% of the consumed nitrogen could be accounted for in the other two treatments. There may be several reasons for these poor budgets including no calculation of faecal nitrogen; not all routes of nitrogenous excretion were measured (although the excretion of nitrite and nitrate would only account for a very small fraction of consumed nitrogen (Owen et al., 1998)); nitrogen intake was overestimated due to leaching of nitrogenous compounds from the pelleted feed prior to consumption; budgets are harder to estimate when growth is low (Carter and Brafield, 1991). The total nitrogen excreted from fish in the form of ammonia, urea and faecal nitrogen, expressed as a proportion of consumed nitrogen allows an indirect estimate of retained nitrogen to be made. Although, it is only applicable if these components are measured accurately. The predicted nitrogen retention for seahorses fed Artemia and mysid shrimp calculated using the proportion of consumed nitrogen excreted from the fish was approximately 43% and 44%, respectively, which is very similar to the measured nitrogen retention, 46% and 45%, respectively. Therefore, indirectly assessing nitrogen retention in growing seahorses has potential to predict the relative success of a feed, particularly if used in combination with observations on feeding success and non-destructive measurements of feeding and growth rates.
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References AOAC, 1990. Official Methods of Analysis of the Association of Official Analytical Chemists. AOAC International, Arlington, VA. Beamish, F.W.H., Thomas, E., 1984. Effects of dietary protein and lipid on nitrogen losses in rainbow trout, Salmo gairdneri. Aquaculture 41, 359–371. Birkett, L., 1969. The nitrogen balance in plaice, sole and perch. J. Exp. Biol. 50, 375–386. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Bower, C.E., Holm-Hansen, T., 1980. A salicylate–hypochlorite method for determining ammonia in seawater. Can. J. Fish. Aquat. Sci. 37, 794–798. Brafield, A.E., 1985. Laboratory studies of energy budgets. In: Tytler, P., Calow, P. (Eds.), Fish Energetics: New Perspectives. Croom Helm, London, pp. 257–281. Carter, C.G., Brafield, A.E., 1991. The bioenergetics of grass carp, Ctenopharyngodon idella (Val.): energy allocation at different planes of nutrition. J. Fish Biol. 39, 873–887. Carter, C.G., Brafield, A.E., 1992. The bioenergetics of grass carp, Ctenopharyngodon idella (Val.): the influence of body weight, ration and dietary composition on nitrogenous excretion. J. Fish Biol. 41, 533–543. Carter, C.G., Bransden, M.P., 2001. Relationships between protein– nitrogen flux and feeding regime in greenback flounder, Rhombosolea tapirina (Günther). Comp. Biochem. Physiol. 130A, 799–807. Carter, C.G., Houlihan, D.F., 2001. Protein synthesis. In: Wright, P.A., Anderson, P.M. (Eds.), Nitrogen Excretion. Academic Press, Sydney, pp. 31–75. Carter, C.G., Houlihan, D.F., Owen, S.F., 1998. Protein synthesis, nitrogen excretion and long-term growth of juvenile Pleuronectes flesus. J. Fish Biol. 53, 272–284. Dosdat, A., Metailler, R., Tetu, N., Servais, F., Chartois, H., Huelvan, C., Desbruyeres, E., 1995. Nitrogenous excretion in juvenile turbot, Scophthalmus maximus (L.), under controlled conditions. Aquac. Res. 26, 639–650. Forteath, N., 1995. Seahorse, Hippocampus abdominalis, in culture. Austasia Aquac. 9 (6), 83–84. Hamlin, H.J., Kling, L.J., 2001. The culture and early weaning of larval haddock (Melanogrammus aeglefinus) using a microparticulate diet. Aquaculture 201, 61–72. Harris, S.A., Probyn, T., 1996. Nitrogen excretion and absorption efficiencies of white steenbras, Lithognathus lithognathus Cuvier (Sparidae), under experimental culture conditions. Aquac. Res. 27, 43–56. Holt, G.J., 1993. Feeding larval red drum on microparticulate feeds in a closed recirculating water system. J. World Aquac. Soc. 24, 225–230. Hopkins, K.D., 1992. Reporting fish growth: a review of the basics. J. World Aquac. Soc. 23, 173–179. Jobling, M., 1981. Some effects of temperature, feeding and body weight on nitrogenous excretion in young plaice Pleuronectes platessa L. J. Fish Biol. 18, 87–96. Kanazawa, A., Koshio, S., Teshima, S.I., 1989. Growth and survival of larval red sea bream Pagrus major and Japanese flounder Paralichthys olivaceus fed microbound feeds. J. World Aquac. Soc. 20, 31–37. Kaushik, S.J., Cowey, C.B., 1991. Dietary factors affecting nitrogen excretion by fish. In: Cowey, C.B., Cho, C.Y. (Eds.), Nutrition
Strategies and Aquaculture Waste. Fish Nutrition Research Laboratory, Ontario, pp. 3–17. Kikuchi, K., Takeda, S., Honda, H., Kiyono, M., 1991. Effect of feeding on nitrogen excretion of Japanese flounder Paralichthys olivaceus. Nippon Suisan Gakkaishi 57, 2059–2064. Kikuchi, K., Takeda, S., Honda, H., 1992. Nitrogenous excretion of juvenile and young Japanese flounder. Nippon Suisan Gakkaishi 58, 2329–2333. Kolkovski, S., Arieli, A., Tandler, A., 1997a. Visual and chemical cues stimulate microdiet ingestion in sea bream larvae. Aquac. Int. 5, 527–536. Kolkovski, S., Tandler, A., Izquierdo, M.S., 1997b. Effects of live food and dietary digestive enzymes on the efficiency of microdiets for seabass (Dicentrarchus labrax) larvae. Aquaculture 148, 313–322. Korsgaard, B., Mommsen, T.P., Wright, P.A., 1995. Nitrogen excretion in Teleostean fish: adaptive relationships to environment, ontogenesis and viviparity. In: Walsh, P.J., Wright, P.A. (Eds.), Nitrogen Metabolism and Excretion. CRC Press, New York, pp. 259–287. Lavens, P., Sorgeloos, P., 2000. The history, present status and prospects of the availability of Artemia cysts for aquaculture. Aquaculture 181, 397–403. Owen, S.F., Houlihan, D.F., Rennie, M.J., Van Weerd, J.H., 1998. Bioenergetics and nitrogen balance of the European eel (Anguilla anguilla) fed at high and low ration levels. Can. J. Fish. Aquat. Sci. 55, 2365–2375. Payne, M.F., Rippingale, R.J., 2000. Rearing West Australian seahorse, Hippocampus subelongatus, juveniles on copepod nauplii and enriched Artemia. Aquaculture 188, 353–361. Person Le Ruyet, J., Alexandre, J.C., Thébaud, L., Mugnier, C., 1993. Marine fish larvae feeding: formulated diets or live prey? J. World Aquac. Soc. 24, 211–224. Porter, C.B., Krom, M.D., Robbins, M.G., Brickell, L., Davidson, A., 1987. Ammonia excretion and total N budget for gilthead seabream and its effect on water quality conditions. Aquaculture 66, 287–297. Rahmatullah, M., Boyde, T.R.C., 1980. Improvements in the determination of urea using diacetyl monoxime; methods with and without deproteinisation. Clin. Chim. Acta 107, 3–9. Rosenlund, G., Stoss, J., Talbot, C., 1997. Co-feeding marine fish larvae with inert and live feeds. Aquaculture 155, 183–191. Tanaka, Y., Kadowaki, S., 1995. Kinetics of nitrogen excretion by cultured flounder Paralichthys olivaceus. J. World Aquac. Soc. 26, 188–193. Verbeeten, B.E., Carter, C.G., Purser, G.J., 1999. The combined effect of feeding time and ration on growth performance and nitrogen metabolism of greenback flounder. J. Fish Biol. 55, 1328–1343. Vincent, A.C.J., 1996. The International Trade in Seahorses. TRAFFIC International, Cambridge. 163 pp. Wood, C.M., 1993. Ammonia and urea metabolism and excretion. In: Evans, D.H. (Ed.), The Physiology of Fishes. CRC Press, Boca Raton, pp. 379–425. Woods, C.M.C., 2000a. Improving initial survival in cultured seahorses, Hippocampus abdominalis Leeson, 1827 (Teleostei: Syngnathidae). Aquaculture 190, 377–388. Woods, C.M.C., 2000b. Preliminary observations on breeding and rearing the seahorse Hippocampus abdominalis (Teleostei: Syngnathidae) in captivity. N.Z. J. Mar. Freshw. Res. 34, 475–485.
Z. Wilson et al. / Aquaculture 255 (2006) 233–241 Woods, C.M.C., 2003a. Effects of varying Artemia enrichment on growth and survival of juvenile seahorses, Hippocampus abdominalis. Aquaculture 220, 537–548. Woods, C.M.C., 2003b. Growth and survival of juvenile seahorse Hippocampus abdominalis reared on live, frozen and artificial foods. Aquaculture 220, 287–298.
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Woods, C.M.C., Valentino, F., 2003. Frozen mysids as an alternative to live Artemia in culturing seahorses Hippocampus abdominalis. Aquac. Res. 34, 757–763.
Nitrogen budgets for juvenile big-bellied seahorse Hippocampus abdominalis fed Artemia, mysids or pelleted feeds Z. Wilson, C.G. Carter ⁎, G.J. Purser School of Aquaculture, Tasmania Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia Received 2 September 2005; received in revised form 5 December 2005; accepted 6 December 2005
Abstract This study aimed to compare nitrogen budgets for juvenile (1.17 ± 0.07 g, mean ± S.D.) big-bellied seahorses fed live, frozen and pelleted feeds and to examine the potential of non-destructive measurements of excretion to predict feed performance. Three feeds were tested against live Artemia: frozen mysid shrimp species (Paramesopodopsis rufa, Tasmanomysis oculata,Tenagomysis sp.), a pellet prepared from the mysid shrimps and a commercial crumbled feed. Nitrogen budgets were constructed from nitrogen retention measured over a 30 day growth experiment, ammonia and urea excretion measured over 24 h on days 15 and 30, and nitrogen digestibility. After 30 days seahorses fed Artemia and frozen mysid had significantly (P b 0.001) higher final weights and growth than the other two feeds. Although seahorses successfully weaned onto the commercial feed, the final weight was not significantly different from their initial weight. Seahorses fed the mysid pellet were not successfully weaned and lost weight. Feed conversion ratio was significantly (P b 0.001) affected by feed and seahorses fed Artemia and frozen mysid had the most efficient conversion of feed to growth. Despite the poor growth performance of seahorses fed the mysid and commercial pellets survival over 30 days was above 95% and there was no significant difference between treatments. Ammonia excretion was significantly affected by feed on day 15 (P b 0.05) and day 30 (P b 0.001), excretion was higher for Artemia than for mysid pellets on both days. Urea excretion was significantly (P b 0.05) affected by feed only on day 30 and was higher for Artemia and frozen mysids. Nitrogen retention was above 45% on Artemia and frozen mysid treatments. An indirect method of assessing nitrogen retention was an accurate technique where seahorses were growing but not where growth was near or below maintenance. © 2005 Elsevier B.V. All rights reserved. Keywords: Excretion; Larval pelleted feed; Live feed; Nitrogen balance; Nitrogen budget; Seahorse
1. Introduction The demand for seahorses has resulted in many animals being taken from the wild, for example approximately 20 million seahorses were taken in 1995 for a variety of markets such as the aquarium and medicine ⁎ Corresponding author. Tel.: +61 3 63243823; fax: +61 3 6324 3804. E-mail address: [email protected] (C.G. Carter). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.12.007
trade (Vincent, 1996). In many locations collection of wild seahorses is viewed as an unsustainable practice, leading to the development of seahorse aquaculture that has the opportunity to reduce or replace the reliance on wild caught animals and propagate endangered species (Vincent, 1996; Payne and Rippingale, 2000; Woods, 2000a,b). The big-bellied seahorse is endemic to southern Australia and New Zealand and can be successfully reared through multiple captive generations following well established production
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techniques based on the use of live feeds (Forteath, 1995; Woods, 2000a,b). A significant problem encountered when establishing economically viable and biologically sustainable seahorse aquaculture is providing sufficient quantities of nutritionally complete feed that will be consumed by seahorses. In commercial seahorse culture, there has been heavy reliance on cultured live feeds predominantly Artemia, but also wild caught mysid shrimp and amphipods (Woods, 2003a) either in the live or frozen form. There is a need to reduce the reliance on live feeds because Artemia varies in cost, availability and quality from year to year (Lavens and Sorgeloos, 2000) and collection of live organisms from the wild can be unpredictable (Woods and Valentino, 2003). Whilst the use of frozen feeds has supported good growth and survival, formulated pelleted feeds have not generally been successful because seahorses do not readily accept such feeds (Woods, 2003b; Woods and Valentino, 2003). Despite seahorses being very distinctive relatively little is known about their nutritional physiology including the digestibility of live and pelleted feeds, the influence of feed on ammonia and urea excretion and the efficiency with which nitrogen is retained. The construction of nitrogen budgets through the measurement of protein–nitrogen intake, digestive and excretory losses and nitrogen retention provides a highly informative approach to developing an understanding of fundamental processes of nutrient utilisation, growth and growth efficiency (Birkett, 1969; Kaushik and Cowey, 1991; Carter and Houlihan, 2001). The aims of this study were to investigate the growth response and nitrogen budgets of juvenile H. abdominalis fed three experimental feeds and tested against live enriched Artemia metanauplii as a standard: a mix of local frozen mysid shrimps, a pellet prepared from the mysid shrimps and a commercial pelleted larval feed. Ammonia and urea excretion were quantified and also assessed as non-destructive measures of nitrogen retention in order to predict the success of feeds.
2. Materials and methods 2.1. Fish and fish husbandry Three hundred and sixty juvenile big-bellied seahorses H. abdominalis (1.17 ± 0.07 g, mean ± SD) were randomly selected from a captive population held at the Aquaculture Centre, University of Tasmania. Animals were held in a seawater recirculation system, consisting of eight 25-l tanks, a sump (150-l), mechanical filtration via dacron matting and a trickle biological filter, housed in a temperature controlled room. Tank flow rate was maintained at 2.9 ± 0.9 l min− 1, constant aeration was supplied via an air-ring placed around the bottom of the tank outlet, which aided in keeping the feeds in suspension and plastic netting was supplied for seahorse attachment. Water quality parameters (mean ± SD) temperature (15.9 ± 0.4 °C), dissolved oxygen (DO, 8.0 ± 0.7 mg l− 1) and salinity (32.6 ± 1.4‰) were measured daily; total ammonia (0.02 ± 0.03 mg l− 1), nitrite (0.1 ± 0.1 mg l− 1), nitrate (6.4 ± 2.4 mg l− 1) and pH (8.1 ± 0.1) were measured weekly. These levels were maintained by a 25% water change per week. Photoperiod was 12 L : 12 D and light intensity at the water surface was 6.8 μE s− 1 m− 2. 2.2. Experimental feeds Four feeds were investigated: Artemia sp. instar II (INVE Thailand) were enriched with Super Selco (INVE Aquaculture) according to manufacturers instructions; a mix of mysid shrimps (Paramesopodopsis rufa, Tasmanomysis oculata, Tenagomysis sp.) collected along the north coast of Tasmania and frozen; a pelleted feed prepared using mysid shrimp and a commercial crumble, CRUM (NRD 5–8, 500–800 μm crumble, INVE Lansy, Primo Aquaculture Pty Ltd, NSW, Australia) (Table 1). Fish were fed a mixed feed of Artemia, frozen mysid shrimp, mysid pellet and CRUM for a 10 day period prior to the commencement of the 30 day experimental period, after which fish were fed solely on the allocated experimental feed.
Table 1 Composition experimental feeds
Dry matter (%) Crude lipid (% DM) Crude protein (% DM) Ash (% DM) Energy (kJ g− 1 DM) Mean ± SE, n = 3.
Artemia
Mysid
Mysid pellet
CRUM
19.78 ± 0.12 9.55 ± 1.38 45.53 ± 0.11 2.03 ± 0.03 22.56 ± 0.12
19.72 ± 0.40 15.34 ± 3.95 59.97 ± 0.22 3.07 ± 0.07 19.16 ± 0.03
89.40 ± 0.06 17.28 ± 2.05 60.77 ± 0.11 17.00 ± 0.06 16.59 ± 0.03
92.72± 0.22 17.53 ± 2.37 56.22 ± 1.67 13.20 ± 0.06 19.43 ± 0.03
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Artemia were hatched for 30 h at 27 °C and enriched with Super Selco for 18 h at 16 °C. The mysid pellet was prepared by freeze-drying frozen mysid shrimp to a constant weight. Following freeze-drying the mysids were ground to a fine powder using a hammer mill (1 mm mesh size). Gelatin (type A, ICN Biochemicals, OH, USA) was included at 10% of dry mysid weight and dissolved in hot water prior to addition, with water being added at 20% inclusion. The ingredients were mixed using a Kenwood mixer, extruded through a 10 ml syringe, spread out onto trays and dried at 35 °C for 48 h. Following drying, the mixture was ground and sieved through a 1 mm sieve, to obtain particles b1 mm. The mysid shrimp, mysid pellet and CRUM were frozen until used. The ration of frozen mysid shrimp required at each meal was thawed in seawater before being fed to the seahorses and following enrichment the Artemia were rinsed with fresh water before each meal. 2.3. Growth experiment The experiment was conducted over two 30 day time periods with two replicate tanks for each feed in each time block (n = 4). Each tank contained 20 seahorses and feeds were randomly allocated to tanks using a random number generator. Seahorses were fed daily at a ration of 5% DM BW− 1 d− 1 that was divided into two equal sized meals. Feeding was at 09:00 and 15:00 h and meal times were restricted to 2 h after which remaining food was siphoned from tanks, dried and feed consumption calculated by difference. Prior to each meal tanks were siphoned to remove faeces so as not to interfere with the measurement of feed consumption. Individual fish wet weight was measured on days 1, 16 and 31. Growth rate (G: % initial weight d− 1) was calculated as a proportion of the initial weight using G = 100(Wf − Wi)(Wit)− 1 (Hopkins, 1992); where Wi and Wf were mean individual initial and final wet weight (g) and t is the experimental period (d). Daily feed consumption (FC, % DM B− 1 d− 1) was calculated using FC = 100 feed consumed (g DM) B− 1; where B was initial tank biomass (g). Feed conversion ratio (FCR g g− 1) was calculated using FCR = total feed consumed (g dry weight)[(Wf − Wi) × carcass dry matter]− 1. Chemical composition was measured in initial samples from 20 seahorses taken at the start of each time-block and in all experimental seahorses following the final weight check (10 mg l− 1 Benzocaine). Seshorses and feed samples were frozen in liquid nitrogen and stored at − 80 °C prior to analysis.
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2.4. Excretion Ammonia and urea excretion were measured on day 15 and 30 of the growth experiment. This was conducted in a second set of tanks that were identical to the tanks used for growth and were first cleaned with hot water, filled with 20 l of 0.2 μm filtered seawater and supplied with gentle aeration (Verbeeten et al., 1999). The seahorses were then netted and moved into the tanks for 24 h, fed as normal and water samples taken after 24 h; all samples were frozen within 1 h of being taken. Ammonia and urea were measured in duplicate tanks containing each feed, but no seahorses, to account for the affects of feeds on ammonia and urea concentrations. In addition, positive (1 mg l− 1 NH3Cl) and negative (0 mg l− 1) ammonia chloride controls showed that no ammonia was gained or lost during the 24 h sampling period. Frozen water samples were thawed at room temperature prior to analysis, ammonia was measured using the salicylate–hypochlorite method (Bower and Holm-Hansen, 1980) and urea measured using the diacetyl–monoxime method (Rahmatullah and Boyde, 1980). 2.5. Faecal collection Faeces were collected on day 27–29 of the growth experiment from each replicate tank of the Artemia and mysid shrimp treatments, with faeces being siphoned onto a 25 μm screen and washed into 250 ml sample jars with fresh water and frozen. Following freeze-drying faecal samples from replicate tanks of each feed were pooled together to obtain sufficient sample for analysis. No faeces were collected from animals fed the mysid pellet or CRUM due to lack of faecal production. Calculating faecal nitrogen was not a major aim of this study and only an initial investigation due to the difficulty in collecting enough faecal material from small seahorses. Apparent nitrogen digestibility (ADN, %) was calculated directly for the Artemia and mysid shrimp feeds using ADN = 100 (R − F)R− 1 (Brafield, 1985), where R and F were the nitrogen (crude protein) contents of the feed and faeces, respectively. 2.6. Chemical analysis Moisture content of the feeds was determined by drying samples to a constant weight in an oven at 105 °C (AOAC, 1990, method 950.02). Seahorse carcasses were freeze-dried to a constant weight and then homogenised to a fine powder. Samples were analysed for nitrogen using Kjeldahl (selenium
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2.50
a ***
a ***
sured for the Artemia and frozen mysid treatments, but could not be determined for fish fed the mysid pellet or CRUM. Mean excretion values of AN and UN from day 15 and 30 were used. The unknown component (%) was calculated by difference using CN − (FN + AN + UN + PN).
Initial weight
Wet weight (g)
2.00
Final weight
1.50
b
b
2.8. Statistical analysis 1.00
Mean (± S.E.M.) values are reported. Levene's test and Shapiro–Wilk test were used to test for equality of error variances and normality, respectively. It was not necessary to transform any data prior to analysis. A paired sample t-test was conducted to determine if significant changes occurred between the initial and final weight of animals fed each feed. Two-way ANOVA was used to determine if a significant interaction existed between time and feed. No significant interaction occurred in any of the variables, therefore the data was pooled and a one-way ANOVA conducted. Means were compared using Tukey HSD test and significance was accepted as P b 0.05. Statistical analyses were performed using SPSS v. 10.0.
0.50
0.00 Artemia
Mysid shrimp Mysid pellet
CRUM
Diet Fig. 1. Initial and final wet weight of juvenile H. abdominalis fed experimental feeds (mean + SE, n = 4). Final weights with different superscripts are significantly different (P b 0.001, one-way ANOVA) between treatments, ⁎⁎⁎ indicates significant difference between initial and final weight (P b 0.001, paired sample t-test).
catalyst; N × 6.25 = crude protein), crude lipid by chloroform and methanol extraction (Bligh and Dyer, 1959), energy using a bomb calorimeter (Gallenkamp autobomb, calibrated with benzoic acid) and ash by combustion in a furnace at 550 °C for 16 h (AOAC, 1990, method 935.11). Nutrient retention efficiency for protein (PP, %) and energy (PE, %) were calculated as 100 (final carcass nutrient content (g)—initial carcass nutrient content (g)) total nutrient consumption (g)− 1.
3. Results 3.1. Growth performance There was no significant difference in seahorse wet weight at the start of the experiment (F = 0.018, df 3, 316, P N 0.05). After 30 days there was a significant difference in final wet weight (F = 62.003, df 3, 305, P b 0.001), with animals fed Artemia and mysid shrimp both having an almost 2 fold increase in weight (Fig. 1). These animals were significantly heavier than the seahorses fed the mysid pellet and CRUM (Fig. 1). Animals fed the mysid pellet lost weight and animals fed CRUM increased in weight, however changes in weight were small and there were no significant differences between the initial and final weights (Fig. 1). Consequently, feed had a significant effect on growth rate (Table 2), seahorses fed Artemia and mysid shrimp had positive growth rates where as the mysid pellet
2.7. Nitrogen budgets A nitrogen budget (Carter and Brafield, 1992; Carter and Houlihan, 2001) was calculated for each feed using the data from the growth and excretion experiments (as % of CN) according to CN = FN + AN + UN + PN, where CN is consumed nitrogen, FN is faecal nitrogen (FN = 100 − ADP), AN and UN are nitrogen excreted as ammonia and urea, respectively, and PN is retained nitrogen (calculated for protein retention efficiency). FN was meaTable 2 Growth performance of juvenile H. abdominalis fed experimental feeds Parameter
Artemia −1
Growth rate (% Wi d ) Feed consumption (% DM B d− 1) FCR (g g− 1) Survival (%)
Mysid
a
3.16 ± 0.13 2.12 ± 0.07a 3.33 ± 0.45b 95.0 ± 2.04
Mysid pellet
a
2.92 ± 0.10 1.60 ± 0.09b 2.68 ± 0.07b 97.5 ± 1.44
CRUM
− 0.11 ± 0.04 0.67 ± 0.03c − 14.36 ± 1.18c 96.25 ± 1.25 c
One-way ANOVA
b
0.35 ± 0.03 0.85 ± 0.05c 16.48 ± 1.48a 97.5 ± 1.44
Means in the same row with same letter are not significantly different (Tukey HSD) (mean ± S.E.M., n = 4).
F value
P (df)
258.005 106.088 168.410 0.579
⁎⁎⁎ (3, 12) ⁎⁎⁎ (3, 12) ⁎⁎⁎ (3, 12) NS (3, 12)
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Table 3 Chemical composition (% wet weight), energy content and nutrient retention efficiency for protein–nitrogen (PN) and energy (PE) of juvenile H. abdominalis fed experimental feeds Parameter
Moisture Crude protein Crude lipid Ash Energy (kJ g− 1 W) PN (%) PE (%)
Artemia
76.47 ± 0.76 13.90 ± 0.28 3.84 ± 0.37 3.96 ± 0.19 4.37 ± 0.20 46.38 ± 6.10a 33.66 ± 5.53a
Mysid
78.26 ± 0.70 14.10 ± 0.46 2.57 ± 0.15 3.56 ± 0.19 4.11 ± 0.14 45.72 ± 1.73a 45.11 ± 2.33a
Mysid pellet
78.66 ± 1.85 12.75 ± 0.85 2.15 ± 0.81 4.27 ± 0.17 3.69 ± 0.41 − 5.90 ± 7.33b 1.90 ± 11.65b
CRUM
79.24 ± 0.45 12.18 ± 0.59 2.24 ± 0.35 4.12 ± 0.05 3.54 ± 0.15 3.67 ± 4.64b 9.06 ± 3.45b
One-way ANOVA F value
P (df)
0.635 1.912 2.736 2.507 0.437 26.205 9.110
NS (3, 12) NS (3, 12) NS (3, 12) NS (3, 12) NS (3, 12) ⁎⁎⁎ (3, 12) ⁎⁎ (3, 12)
Means with different superscripts are significantly different (Tukey HSD) where NS = not significant, ⁎ P b 0.05, ⁎⁎ P b 0.01, ⁎⁎⁎P b 0.001) (mean ± S.E.M, n = 4).
resulted in a negative growth rate (Table 2). Despite the poor growth and feeding of seahorses fed the mysid pellet and CRUM they had high survival, which was not significantly different between any of the feeds (Table 2). Feed had a significant effect on feed consumption (FC) and feed conversion ratio (FCR) (Table 2). Feed consumption of Artemia was significantly higher than for the other feeds and mysids were consumed at a significantly higher rate than either the mysid pellet or CRUM feeds (Table 2). Animals fed Artemia and mysid had the most efficient FCR values of 3.33 and 2.68 g g− 1, respectively. The initial whole-body chemical composition (mean ± SD) was 77.75 ± 2.07% moisture, 12.54 ± 1.06% crude protein, 3.97 ± 0.55% crude lipid, 4.23 ± 0.42% ash and an energy value of 3.36 ± 0.61 kJ g− 1 W. There were no significant differences between initial and final samples or between treatments for any component of composition
(Table 3). Feed had a significant effect on nutrient retention efficiency, values were significantly higher for both protein and energy in animals fed Artemia and mysid (Table 3). 3.2. Nitrogen excretion Feed had a significant but different effect on nitrogenous excretion measured on days 15 and 30 (Table 4). On day 15, ammonia excretion on Artemia was significantly higher than on mysid pellets but there were no differences in urea excretion or the urea to total nitrogen ratio. Feed had a significant effect on total nitrogen excreted on day 15, with seahorses fed Artemia and mysid excreting the most nitrogen. The proportion of consumed nitrogen excreted as total nitrogen on day 15 was significantly different between Artemia and mysid pellet.
Table 4 Ammonia (AN), urea (UN) and total nitrogen (TN) excretion, the proportion of total nitrogen excreted as urea (UN : TN) and the proportion of consumed nitrogen excreted as total nitrogen (TN : CN) on day 15 and 30 of juvenile H. abdominalis fed experimental feeds Parameter
Artemia
Mysid
Mysid pellet
CRUM
One-way ANOVA F value
P (df)
Day 15 AN (mg N kg− 1 d− 1) UN (mg N kg− 1 d− 1) TN (mg N kg− 1 d− 1) UN : TN (%) TN : CN (%)
253.61 ± 9.48a 141.92 ± 26.50 395.53 ± 24.48a 35.15 ± 4.83 47.79 ± 3.09a
200.26 ± 16.34ab 158.17 ± 44.42 358.43 ± 34.05ab 41.59 ± 9.79 43.03 ± 1.90ab
142.79 ± 19.38b 101.17 ± 13.06 243.96 ± 21.99b 41.63 ± 4.86 35.11 ± 1.43b
185.63 ± 32.64ab 84.11 ± 16.63 269.74 ± 31.25b 31.87 ± 7.00 38.57 ± 2.18ab
4.874 1.525 6.401 0.494 6.064
⁎ (3, 12) NS (3, 12) ⁎⁎ (3, 12) NS (3, 12) ⁎⁎ (3, 12)
Day 30 AN (mg N kg− 1 d− 1) UN (mg N kg− 1 d− 1) TN (mg N kg− 1 d− 1) UN : TN (%) TN : CN (%)
209.25 ± 11.75a 229.94 ± 12.97a 439.19 ± 5.89a 52.33 ± 2.69 53.06 ± 3.43a
214.13 ± 11.03a 172.85 ± 13.62ab 386.97 ± 15.47ab 44.58 ± 2.61 46.45 ± 2.05ab
133.92 ± 10.07b 94.58 ± 23.89b 228.50 ± 13.94c 40.01 ± 7.66 32.88 ± 1.34c
152.96 ± 12.21b 124.55 ± 48.62ab 277.52 ± 52.64bc 39.87 ± 9.00 39.68 ± 2.24bc
12.675 4.263 11.603 0.889 13.249
⁎⁎⁎ (3, 12) ⁎ (3, 12) ⁎⁎ (3, 12) NS (3, 12) ⁎⁎⁎ (3, 12)
Means with different superscripts are significantly different (Tukey HSD) where NS = not significant, ⁎ P b 0.05, ⁎⁎ P b 0.01, ⁎⁎⁎P b 0.001) (mean ± S.E.M, n = 4).
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On day 30, ammonia and urea excretion were significantly affected by feed (Table 4). Seahorses fed Artemia and mysid excreted significantly higher amounts of ammonia than when fed the other two feeds whereas urea excretion was significantly higher only between Artemia and the mysid pellet (Table 4). However, the proportion of total nitrogen excreted as urea was not significantly different between treatments. The total excreted nitrogen was significantly affected by feed with the highest rates from animals fed Artemia and mysid, both significantly higher than for mysid pellets. The TN : CN ratio was highest for Artemia and mysid, with mysid pellets having a significantly lower ratio than Artemia and mysid but not CRUM (Table 4). 3.3. Nitrogen budgets Nitrogen budgets expressed as a proportion of consumed nitrogen are presented in Table 5. There was a high balance of nitrogen achieved for animals fed Artemia and mysid shrimp, 103.18 ± 7.30% and 101.24 ± 3.11%, respectively. However there was a poor recovery of nitrogen in animals fed the mysid pellet and CRUM feed, with 71.09 ± 7.00% and 58.93 ± 4.95% remaining unaccounted for (Table 5). This was primarily because faecal nitrogen could not be determined for fish fed the mysid pellet and CRUM, which increased the unknown component. The apparent nitrogen digestibility for the Artemia and mysid shrimp feeds was 90.73% and 86.13%, respectively, and faecal nitrogen loss represented 9.27 ± 0.90% and 13.69 ± 0.25% of consumed nitrogen, respectively. The proportion of consumed nitrogen excreted as ammonia remained relatively constant between feeds ranging from 20.25 to 26.17% (Table 5). Urea excretion represented a smaller proportion of CN although it tended to be higher in the two treatments where consumption and growth were Table 5 Nitrogen budgets (% of consumed nitrogen, CN) showing faecal (FN), ammonia (AN) and urea (UN) excretion and nitrogen retained as growth (PN) of juvenile H. abdominalis fed experimental feeds Parameter Artemia
Mysid
Mysid pellet CRUM
CN (%) FN (%) AN (%) UN (%) PN (%) Unknown (%) Balance (%)
100 13.69 ± 0.25 23.25 ± 0.38 18.58 ± 2.92 45.72 ± 1.73 (− 1.24)
100
100
20.25 ± 1.24 14.56 ± 2.17 −5.90 ± 7.33 71.09 ± 7.00
23.03 ± 2.27 14.36 ± 2.78 3.67 ± 4.64 58.93 ± 4.95
103.18 ± 7.30 101.24 ± 3.11 28.91 ± 7.00
41.07 ± 4.95
100 9.27 ± 0.90 26.17 ± 2.11 21.36 ± 1.08 46.38 ± 6.10 (−3.18)
Mean ± S.E.M., n = 4.
highest. The nitrogen retained as growth represented 46.38 ± 6.10% and 45.72% of CN in seahorses fed Artemia and mysid, respectively. However there was low retention of nitrogen in animals fed CRUM (3.67%) and those fed the mysid pellet lost nitrogen (− 5.90%). 4. Discussion There are few papers published where alternative feeds for seahorses have been investigated and this study demonstrated that 1 g juvenile seahorses can be successfully weaned onto frozen mysid shrimp. The use of mysid shrimp as a sole food source was demonstrated previously with larger H. abdominalis of 3 g (Woods and Valentino, 2003) and the SGR in both studies was around 3% d− 1. As in the present study final weights were not different between the frozen mysid and the live Artemia. In contrast, seahorses fed in the present study on mysid pellets or the commercial crumble did not show any significant change from their initial weight. The successful weaning of seahorses onto frozen mysid shrimp has significant implications for seahorse aquaculture since it reduces the reliance on enriched Artemia which is important because of the variable cost, availability and quality of Artemia cysts from year to year (Lavens and Sorgeloos, 2000). Although juvenile seahorses were successfully weaned onto frozen mysid shrimp further research is required to confirm the suitability of mysids as a long-term feed for seahorses. The use of pelleted feeds was not successful in the present study principally due to low feed intake. Reasons for the low intake and associated reduced growth compared to the intake of the Artemia and mysids in seahorses are unclear but contributing factors during weaning in other marine species include food form, diet colour, lack of specific movement, lack of chemical stimuli, lack of visual stimuli, inadequate fish size, inadequate digestive tract/enzyme development and environment (Person Le Ruyet et al., 1993; Kolkovski et al., 1997a,b; Rosenlund et al., 1997; Hamlin and Kling, 2001). Seahorses fed the mysid pellet lost weight after 30 days, but the CRUM feed was slightly more successful since seahorses were weaned onto it and maintained their weight. Woods (2003b) investigated the use of a formulated feed for newborn, one-month and twomonth H. abdominalis. One-month old juveniles fed formulated feed compared to juveniles fed live Artemia had significantly lower survival and lower weight gain, whereas, two-month old juveniles had similar survival to those fed live Artemia but again with lower weight gain. The high survival of the two-month old juveniles
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(Woods, 2003b) and of the animals used in the present study is most likely due to their larger size and greater ability to withstand periods of under-nutrition during weaning, compared to newborn and one-month old animals (Woods, 2003b). In the present study animals fed on the crumble but weaning was not successful since the increase in weight was not significantly different from the initial weight. The tanks were designed with an air-ring around the bottom of the tank outlet, which aided in keeping the feeds suspended in the water. The seahorses would observe the food as it moved around the tank but fewer strikes occurred against pelleted feeds (personal observation), which suggested these feeds did not trigger as active a feeding response. The resemblance and behaviour of pelleted feed compared to live feed is an important factor to consider in seahorse culture since seahorses are visual feeders and scrutinise feed before it is consumed (Woods, 2003b). The mixed feeding of pelleted and live feeds is common practice in improving growth and survival of larval marine fish (Kanazawa et al., 1989; Holt, 1993; Rosenlund et al., 1997). The successful use of pelleted feed as a sole food source in seahorse culture may require a longer weaning protocol than the 10 days of mixed feeding used in this study. Physiological and nutritional factors, principally water temperature, body size, ration and feed composition, influence nitrogenous excretion by fish (Jobling, 1981; Beamish and Thomas, 1984; Carter and Brafield, 1992). There is little information available on nitrogenous excretion of H. abdominalis and this study provides detailed information on nitrogen excretion in relation to nutritional factors. The amount of feed consumed and feed composition, including protein digestibility and content, were the main factors that affected ammonia and urea excretion in the present study. Rates of ammonia excretion were broadly similar to other marine fish and provided further evidence of a positive relationship between feed consumption and ammonia excretion (Kikuchi et al., 1991; Dosdat et al., 1995; Carter and Bransden, 2001). The proportion of consumed nitrogen excreted as ammonia varied according to amount of feed consumed, with a maximum of 26% excreted by animals fed Artemia, which is similar to other values reported in the literature (Porter et al., 1987; Kikuchi et al., 1992; Harris and Probyn, 1996; Verbeeten et al., 1999). The proportion of total nitrogen excreted as urea varied from 32% to 52% which is high since teleosts typically excrete b 20% of total nitrogen as urea (Wood, 1993; Korsgaard et al., 1995). There is evidence that urea excretion plays an important role in nitrogenous excretion
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by marine fish, and a number of marine species are now known to have higher urea excretion rates than previously predicted (Dosdat et al., 1996; Tanaka and Kadowaki, 1995; Carter et al., 1998). In addition, urea excretion rates increased as ingested food increased in seahorses as with other marine fishes (Dosdat et al., 1996; Carter et al., 1998; Verbeeten et al., 1999). The morphology of the gut may play a role in nitrogen excretion, as seahorses lack a true stomach (Woods, 2003b). Dosdat et al. (1995) hypothesised that urea could be produced along with faeces in the digestive tract of turbot, which may also be the case in seahorses and could explain the link between feed intake and urea excretion. The nitrogen budgets of growing seahorses fed Artemia and mysid shrimp, calculated from independently measured components accounted for 103% and 101%, respectively, of the consumed nitrogen and appeared to provide an accurate representation of nitrogen flux (Carter and Brafield, 1992; Owen et al., 1998). Although it should be noted that the measurement of each parameter of the nitrogen budget has its own margin of error, which must be taken into consideration when the accuracy of the budgets are assessed (Brafield, 1985; Carter and Brafield, 1991). Only 29% and 41% of the consumed nitrogen could be accounted for in the other two treatments. There may be several reasons for these poor budgets including no calculation of faecal nitrogen; not all routes of nitrogenous excretion were measured (although the excretion of nitrite and nitrate would only account for a very small fraction of consumed nitrogen (Owen et al., 1998)); nitrogen intake was overestimated due to leaching of nitrogenous compounds from the pelleted feed prior to consumption; budgets are harder to estimate when growth is low (Carter and Brafield, 1991). The total nitrogen excreted from fish in the form of ammonia, urea and faecal nitrogen, expressed as a proportion of consumed nitrogen allows an indirect estimate of retained nitrogen to be made. Although, it is only applicable if these components are measured accurately. The predicted nitrogen retention for seahorses fed Artemia and mysid shrimp calculated using the proportion of consumed nitrogen excreted from the fish was approximately 43% and 44%, respectively, which is very similar to the measured nitrogen retention, 46% and 45%, respectively. Therefore, indirectly assessing nitrogen retention in growing seahorses has potential to predict the relative success of a feed, particularly if used in combination with observations on feeding success and non-destructive measurements of feeding and growth rates.
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