Journal of Fish Biology 2005 Peña

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Effect of delayed first feeding on development andfeeding ability ofParalabrax maculatofasciatus larvae

R. P E N A * A N D S. D U M A S

Unidad Piloto de Maricultivos. Centro Interdisciplinario de Ciencias Marinas(CICIMAR-IPN). Av. Instituto Politecnico Nacional s/n, Col. Playa Palo de Santa

Rita, La Paz, B.C.S. 23096, Mexico

(Received 14 June 2004, Accepted 7 March 2005)

The impact of delayed feeding early in development on late feeding ability and development of

spotted sand bass Paralabrax maculatofasciatus larvae was examined. Larvae were sampled

from hatching until day 19 after delayed feeding for zero (control), 1, 2, or 3 days. Feeding

incidence was evaluated as the percentage of larvae with food in the gut and feeding intensity

was measured by direct counting of prey after dissection of the gut. Delayed feeding effects due

to starvation were observed early in development. By day 5, significant differences (P < 005)

were observed between controls and larvae submitted to degrees of delayed feeding, including

total length (LT), eye diameter, and the size of the head, liver, gut, muscles and body.

Differences were still apparent inLT, and body, liver and muscle heights at the end of the studied

period. Larvae under total starvation did not survive beyond day 5. Initial feeding incidence was

35, 60, 90 and 10% for larvae first fed on day 2, 3, 4 or 5, respectively. Mean S.D. feeding

intensity was 36 08, 28 13, 52 03, and 102 15 rotifers per larva depending on

whether larvae were initially fed on day 2, 3, 4 or 5, respectively. The point of no return occurred

between 4 and 5 days after hatching. # 2005 The Fisheries Society of the British Isles

Key words: feeding ability;Paralabrax maculatofasciatus; spotted sand bass; starvation.

INTRODUCTION

At the onset of exogenous feeding, fish larvae face death from starvation iffirst feeding is delayed past the point of no return (Blaxter & Hempel, 1963).Along with predation, starvation is a major cause of mortality during the larvalperiod, affecting recruitment in natural populations and larval quality underaquacultural conditions (OConnell, 1980; Theilacker, 1986). The effects of totalor partial starvation during larval development have been evaluated in otherteleost species by methods of histology (Ehrlich et al., 1976; OConnell, 1976,1980; Theilacker, 1978; Oozeki et al., 1989), morphology (Ehrlich et al., 1976;Yufera et al., 1993) or biochemistry (Ueberscha r & Clemmesen, 1992).

Unlike other teleost species, the endogenous feeding period of the spotted sandbass larvae Paralabrax maculatofasciatus (Steindachner) is relatively short,

*Author to whom correspondence should be addressed. Tel.: 52 612 122 5344; fax: 52 612 122 5322;

email: [email protected]

Journal of Fish Biology (2005) 67, 640651

doi:10.1111/j.1095-8649.2005.00765.x, availableonlineat http://www.blackwell-synergy.com

640# 2005The Fisheries Society of theBritish Isles


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lasting 2 days at 25 C (Pen a et al., 2003). During this time, the eyes becomepigmented and the mouth and anus open for food ingestion and egestion (Pen aet al., 2004). Although survival during larval rearing of the spotted sand bass hasimproved in recent years (Alvarez-Gonza lez et al., 2001), high mortality rateshave occurred during the first-feeding phase. In previous studies, the impact ofother rearing conditions have been reported, such as light intensity (Pen a et al.,2004), white tank colour and prey density (unpubl. data). No attempt has beenmade to assess the effects of early delayed feeding.

Several authors have showed that larvae are most vulnerable to starvation atthe time of first feeding (McGurk, 1984; Martin & Wright, 1987; Yin & Blaxter,1987; Richardet al., 1991; Yuferaet al., 1993). It is likely that starving larvae inaquaculture reflect some inadequacy of the culture conditions and live feed at thebeginning of the exogenous feeding period. Therefore, to improve larval qualityand survival, characterization of starvation effects under culture conditions canbe a valuable tool for evaluating the adequacy of the aquacultural conditionsand finding methods for avoiding mortality caused by starvation. This studyattempted to describe the effects of early starvation on the development andfeeding ability of spotted sand bass larvae under cultural conditions as caused bydelayed first feeding.

MATERIALS AND METHODS

Fertilized eggs were obtained from spontaneously spawning spotted sand bass brood-stock under culture conditions (24 C, 35 salinity and 13L : 11D) (Rosales-Vela zquez,1997) and maintained until hatching in a 100 l conical tank. Newly hatched larvae were

placed in twelve 100 l cubic tanks at an initial density of 50 larvae l

1

(5000 larvae pertank) under the same culture conditions.Four treatments were tested in triplicate. The control groups were first fed at 2 days,

which is the normal time after hatching at which the mouth opens and the eye iscompletely pigmented (Pen a et al., 2003). Three treatments then delayed first feedingby 1 (D1), 2 (D2) or 3 (D3) days. An additional experiment was used to study larvae keptin a 100 l conical tank under identical culture conditions to the earlier treatments butwith larvae starved until they died.

The diet regime was the same for all treatments. Larvae were first fed with rotifersBrachionus rotundiformis at five rotifers ml1 for 2 days, followed by enriched (SelcoTM

Artemia Systems, Ghent, Belgium) rotifers B. rotundiformis at five rotifers ml1 for 10days, followed by Selco TM enrichedArtemia sp. nauplii at three to five nauplii ml1 for10 days. Prey densities were maintained throughout the period of study.

From hatching until day 19, 30 5 (mean S.D.) larvae from each replicate of everytreatment were sampled 1 h after feeding. The sampled larvae were anaesthetized andsacrificed with 35 mg l1 MS-222, and digitally photographed using Image-Pro Plus v.4.5 software (Media Cybernetics, Silver Spring, MD, U.S.A.) for measurement of totallength (LT); body depth at pectoral level excluding the fin folds (BD); eye diameter alongthe body axis (ED); head depth (HD); liver height (LH); gut height at intestinal valvelevel (DTH); muscular height at anus level (MH) (Fig. 1).

After measuring, feeding incidence was evaluated on each sampling day as the percent-age of sampled larvae with food in the gut. Feeding intensity was estimated by directcounting of prey after dissection of the gut. Both evaluations were made on the larvaepreviously sampled and photographed, using a dissection microscope (Olympus modelSZ40, Tokyo, Japan). The point of no return was established as the time in days when the

feeding incidence dropped to less than half the highest feeding incidence recorded amongtreatments at the time of first feeding. The 50% descent in feeding incidence has beenused in species like Clupea harengus L., Gadus morhua L., Platichthys flesus (L.) (Yin &

D E L A Y E D F I RS T F E E D I N G I N S P O T T E D S A N D B A SS 641

# 2005 The Fisheries Society of the British Isles, Journal of Fish Biology2005, 67, 640651


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Blaxter, 1987) and Sparus aurata L. (Yufera et al., 1993) and it is considered as the timewhen the larvae will not recover from delayed feeding, and eventually die due tostarvation.

Additionally, on each sampling day, 30 5 larvae from each replicate of every treat-ment were sampled, anaesthetized, fixed in Bouin solution for 24 h and then preserved in70% ethanol for histological preparation. They were dehydrated in increasing concentra-tions of alcohol and then embedded in paraffin. Sagital sections (5 mm) were obtained bya rotary microtome and stained with haematoxylin-eosin technique for general histolo-gical features. Microsections were digitally photographed with the Image Pro Plus v 4.5(Media Cybernetics) software for posterior measurement of hepatocytes nuclear area andenterocytes height (from the basement membrane to the luminal surface of the brush

border) in both intestines.The data were tested for normality and homogeneity of variances. One-way ANOVAwas performed with days of delayed feeding as the factor. When a significant differencewas found, Tukeys test was performed (P < 005).

RESULTS

The data followed a normal distribution (KolmogorovSmirnov test,D 01713, P < 005), and showed homogeneity of variances (Levenes test,F4,63, P < 005). The effects of delayed first feeding were detected early in

development. Significant differences (P < 005) were observed in all measure-ments by day 5 between the control group and all other treatments (Fig. 2). TheD1 and D2 larvae recovered from their initial starvation period and survivedthroughout the study, but significant differences (P < 005) were observed in theLT of D1 and D2 larvae at the end of the experiment, compared with controllarvae (Fig. 3). After day 14, significant differences (P < 005) were observed inall measurements between the control group and D2 larvae (Table I). Controland D1 larvae were significantly different (P < 005) only in MH (Table I) andLT(Fig. 3) at day 19. Non fed larvae and D3 larvae did not survive beyond days5 and 6, respectively.

The initial feeding incidence of control, D1, D2 and D3 larvae was 35, 60, 90and 10%, respectively (Fig. 4). The point of no return occurred between days 4and 5 after hatching. Feeding incidence of control, D1 and D2 larvae reached

LT

ED

BDHD

LH

MH

DTH

FIG. 1. Spotted sand bass larvae showing morphometric measurements. BD, body depth; DTH, gut

height; ED, eye diameter; HD, head depth; LH, liver height; MH, musculature height; LT, total

length.

642 R . P E N A A N D S . D U M A S

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07

06

05

04

03

02

0 1 2 3 4 5 6 0 1 2 3 4 5 6

0 1 2 3 4 5 6

a

a

b

c

b

a

b

b

0 1 2 3 4 5 6

0 1 2 3 4 5 6

0 1 2 3 4 5 6

Headdepth(m

m)

07

06

05

04

03

02

Bodydepth(mm)

a

b

a

b

(a) (b)

(d)

(f)(e)

c

(c)

022

020

018

016

014

012010

008

006

004

002

028

026

024

022

020

018

016

014

012

016

014

012

010

008

006

004

002

Age (days)

Musculatureheight(mm)

028

026

024

022

020

018

016

014

012

010

Gutheig

ht(mm)

Eyediameter(m

m)

Liverheight(mm)

FIG. 2. Early morphometrical development of spotted sand bass larvae after delayed first feeding

[*, 1 day (D1); ., 2 days (D2); ,, 3 days (D3); &, starved;, control]. Data are mean S.D.(n > 30). (a) Head depth, (b) eye diameter, (c) body depth, (d) muscular height, (e) liver height and

(f) gut height. Different lower case letters denote significant differences (P < 005).




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100% by day 9 and it remained at this level throughout the experiment. Thenumber of prey ingested at first feeding was also affected by the length of timelarvae were kept without food (Fig. 5). Mean S.D. initial feeding intensity ofcontrol, D1, D2 and D3 larvae was 36 08, 28 13, 52 03 and102 15 rotifers per larva, respectively.

Delayed feeding also affected the cellular structure of the liver and intestines.The average nuclear hepatocyte area after first feeding was significantly higher

(P < 005) in the control larvae (166 21 mm2

) compared to D1, D2 and D3larvae (mean S.D. 116 25, 104 26 and 101 20 mm2, respectively).After day 7, however, no significant difference (P > 005) was observed betweencontrol, D1 and D2 larvae [Fig. 6(a)]. Enterocyte height in the anterior intestineshowed no significant difference (P > 005) between control and D1 larvae(157 33 and 149 21 mm, respectively) at first feeding. A significant dif-ference (P < 005), however, was observed with D2 and D3 larvae (102 19and 72 17 mm, respectively) [Fig. 6(b)]. No significant differences (P > 005)were observed between control, D1 and D2 larvae after day 12 [Fig. 6(b)]. In theposterior intestine, a significant difference (P < 005) was observed between

control (144 13 mm) and D1 and D2 larvae after first feeding (113 15and 107 14 mm, respectively) [Fig. 6(c)]. Enterocyte height in D1 and D2larvae were significantly higher (P < 005) than in D3 larvae (80 12 mm)

10

10 12 14 16 18 20

a

b

b

8

8

6

6

4

4

2

2

0

0

Age (days)

LT(mm)

FIG. 3. Mean S.D. total length (n > 30) of spotted sand bass larvae after delayed first feeding (see

Fig. 2). Different lower case letters denote significant differences (P < 005).




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TABLE

I.Developmentofthespottedsandbasslarvaeafterdel

ayedfirstfeeding(C,

control;D1,

1day;D2,

2days).D

atarepresent

mean

(S.D.)

(n 30) of spotted sand bass

larvae after delayed first feeding (see Fig. 2). Different lower case letters denote significant differ-

ences (P < 005).




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35

30

25

20

15

10

5

30

25

20

15

10

5

0

24

22

20

18

16

14

12

10

8

6

0 2 4 6 8 10 12 14 16 18 20 22

0 2 4 6 8 10 12 14 16 18 20 22

0 2 4 6 8 10 12 14 16 18 20 22

Age (days)

Enterocyteheight(m)

Enterocyteheight(m)

Nucleararea(m)

(c)

(b)

(a)

a

a

a

b

a

b b

c

a

a

b

b

b

b

bb

FIG. 6. Development of liver and intestines of spotted sand bass larvae after delayed first feeding (see

Fig. 2). All measurements are mean S.D. (n > 30). (a) Nuclear hepatocyte area, (b) enterocyteheight in the anterior intestine and (c) enterocyte height in the posterior intestine. Different lower

case letters denote significant differences (P < 005). The absence of letters indicates that there is no

significant difference (P > 005).




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for larvae of several fish species (Mookerji & Ramakrishna, 1995; Hart, 1997;Parra & Yufera, 2000).

Although D3 larvae were capable of foraging and ingestion after 3 dayswithout food, they were unable to properly digest prey. In the spotted sandbass larvae, the nuclear hepatocyte area and the enterocytes height were signifi-cantly affected by the length of the non-feeding period. Both measurements havebeen successfully used to establish the effect of starvation in other teleost species(Inui & Egusa, 1967; Ehrlich et al., 1976; Oozeki et al., 1989; Theilacker &Watanabe, 1989; Stru ssmann & Takashima, 1990; Theilacker & Porter, 1995).The liver is involved with glycogen storage from early in development and theintestinal mucosa is related to digestion and absorption of nutrients (Ehrlichet al., 1976; Guyotet al., 1995; Pen aet al., 2003). According to Theilacker (1978)and OConnell (1980) the gut and the liver are the first organs to reflect star-vation conditions due to autolysis. Indeed, the use of body tissues as an energysource during a prolonged non-feeding period produces alterations of the gutand associated glands, including enzymatic activity (Yuferaet al., 1993).

Some authors have showed that histological criteria are better than morpho-metric measurements in establishing the starvation condition of fish larvae, eventhough the techniques are time consuming (Theilacker, 1986; Theilacker &Watanabe, 1989; Theilacker & Porter, 1995). Other criteria such as larval behav-iour have been used as an indicator of poor larval condition related tostarvation (Skiftesvik, 1992). In the spotted sand bass larvae, both morpho-logical and histological criteria were good indicators of the effect of the non-feeding period during the first few days of development. After c. day 14, however,morphological criteria were more suitable in establishing the effects of delayed

first feeding. Biochemical indexes, like trypsin activity and the RNA : DNAratio (Ueberscha r & Clemmesen, 1992) have been used to establish the effectof a non-feeding period over the nutritional condition of fish larvae. Biochemicalindexes proved to be a more accurate and less time-consuming tool for deter-mining the nutritional condition of larvae, since these approaches assess thephysiological state of larvae as an active response to starvation.

The National Council for Science and Technology of Mexico (CONACYT) (Project3166-B) and the Comisio n de Operacio n y Fomento de Actividades Acade micas(COFAA-IPN) provided financial support for this project.

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