Functional development of the digestive

Transcription

1 1 Functional development of the digestive system in Atlantic Al cod (Gadus morhua L) L.) larvae. Per-Arvid Wold * and Elin Kjørsvik Department of Biology, Norwegian University of Science and Technology (NTNU),Trondheim, Norway * Present address: Department of Hydraulic and Environmental Engineering, NTNU, Trondheim, Norway Functional development of the digestive system in Atlantic cod larvae

2 2 Aims of the study 1. To study functional development of the intestine and liver in cod larvae 2. To describe the effect(s) () of early weaning and dietary composition on functional development and to evaluate tissue changes 3. To establish quantitative histological parameters suitable for evaluating nutritional status of developing cod larvae

3 3 Methods- Start-feeding protocol All start feeding experiments were conducted according to the standard start feeding protocol established at NTNU/SINTEF 3-16 dph dph 23-3 dph 3-34 dph 34- dph Standard Formulated diet Enriched rotifers + micro algae Enriched Artemia Early weaning Enriched rotifers + micro algae Formulated diet Formulated diet 7,5 12ºC 12ºC Water exchange 1 7x 8x

4 4 Methods sample preparation Sampled larvae were fixed in glutharaldehyde and paraformaldhyde in cacodylic buffer. The intestine and liver were dissected out under a stereomicroscoope and then embedded inepon Liver Intestine Samples were sectioned according to a pre calculated protocol designed to give an unbiased estimate of the chosen parametres. Sections were made for LM and TEM. All measurements, and quantitative estimates by stereology, were calculated with the software CAST2

5 5 Functional development of the intestine size and growth The development of the intestinal i size was positively ii correlated to larval l standard length. 5x1 3 ume (mm 3 ) Midgut epithelium vol 1,,8,6,4,2, 12 dph 17 dph 24 dph 3 dph 3 dph co-fed Larval standard length (mm) Hyperplasia Hypertrophy tes mber of enterocyt Nu 4x1 3 3x1 3 2x1 3 1x1 3 Enterocyte volume (µm 3 ) Larval standard lenght (mm) 4 Midgut volume increased 4X on 18 days (12 3 dph) dry weight increased 28X. Larval standard length (mm) Data from Wold et al dph 17 dph 24 dph 3 dph 3 co-fed

6 6 Functional development of the intestine microvilli surface 12 days post hatching (2K) 3 25 c area (mm 2 ) Microvilli surface d 3 days post hatching (2K) 2 a a b co-feeding Days post hatching The microvilli surface increased more than 8 times from 12 to 3 days post hatching Data from Wold et al. 28

7 7 Functional development of the intestine mitochondria Estimated numbers of mitochondria per enterocyte increased from 53 (12 dph) to 99 (3 dph) Three types of mitochondria were observed in enterocytes according to their membrane structures......distribution changed during larval development Type1 Type 2 Type 3 (%) Mitoc condrial category dph 17 dph 24 dph 3 dph cf 3 dph Type 1 Type 2 Type 3 Data from Wold et al. 28

8 8 Functional development of the liver size and growth Growth and development of the liver showed similar pattern as observed for the intestine. 35x1 3 Liver volume (µm 3 ) 1,,8,6,4,2, Standard length (mm) Hyperplasia Hypertrophy Liver size increased 16X between 3 and 39 dph (dry weight 5X) Number of hepatocytes per liver 3x1 3 25x1 3 2x1 3 15x1 3 1x1 3 5x Hepatocyte volum me (mm 3 ) Standard length (mm) Standard length (mm) Data from Høvde et al. In prep

9 9 Functional development of the liver energy storage Glycogen was observed in hepatocytes from 17 days post hatching. 5 Glycogen storage seemed to be related to larval growth rates rapid growing larvae stored glycogen (DWI > 1%) Low levels of lipids were observed (<5%). gen volume pe er hepatocyte (% %) Control Early weaning Days post DWI DWI Early hatch Control weaning Glyco Days post hatch ± ± ± ± ±.5 Data from Høvde et al. In prep

10 1 Organ development vs. larval dry weight Larval characteristics SL-interval Increase Dry weight increase Regardless of onset (mm) of weaning or dietary treatment, functional development was rapid within the Wold larval et al. size 28 interval 6-14 mm SL Midgut volume * 4x 28x Enterocyte number 6.7x Microvilli surface Høvde et al in prep. 84x Liver volume x 8.7x Hepatocyte number Wold et al x Alkaline x 6.5x phosphatase activity No. of ossified vertebrae Kjørsvik et al from 2 to 44 vertebrae 6.5x

11 11 Larval response to dietary treatment The liver was the organ that responded most rapidly to dietary changes. Hepatocytenucleus Days post DWI size was closely DWI related Early hatch to larval growth Control rates by larger weaning nucelus in 5-17 faster growing larvae: 11.6 ±.5 a change from live feed to formulated feed ± ±.19 led to an reduction in growth rates and in heaptocyte nuclei size ± ±.5 In larvae suffering from malnutrition/starvation, enlarged, swollen hepatocyte mitochondria were observed. Ce ell nucleus volum me (μm 3 ) Volu ume of mitokondrium m (μm 3 ) 12 Control 1 a Early weaning Copyfeed ab ab ab b c b ab Control Early weaning Copyfeed 2 1 Days post hatch Data from Høvde et al. In prep Days post hatch

12 12 Larval response to dietary treatment In an early weaning experiment with three different formulated diets, containing different levels of marine phospholipids (PL); hepatocyte nucleus size were significantly larger in larvae fed PL diets than in larvae fed a diet with no marine PL (NL1). Dry weight (mg/lar rvae) 3,5 3, 2,5 2, 1,5 1 1, PL3 PL1 NL1 a b hepatocyte mitochondria in larvae fed the NL1 contained a more open intermembranous matrix with more visible space between the christae. volume (µm 3 ) 1 8 6,5, a Days after hatching hi a b Hep patocyte nucleus 4 2 Data from Wold et al. 29 PL3 PL1 NL1 Diet

13 13 Conclusions Cod larval digestive system is more related to larval size rather than age. The rapid larval development suggest a maturation period during the mixed-feeding period and furthermore a possible explanation why larvae are sensitive to suboptimal nutrition during the early days of development. The liver was more sensitive to dietary changes than the gut, and hepatocyte nucleus size was the most suitable parameter for evaluating larval nutrition status.

14 14 Acknowledgements All activity was related to the research programme CodTech (NRC /12) and the project Effects of phospholipid supplement in microdiets during co feeding of marine fish larvae (NRC 14225/12). Thanks to: Professor Yngvar Olsen (NTNU Norway) Dr. Katja Hoehne Reitan (NTNU Norway) Dr. Gunvor Øie (Sintef Fisheries and Aquaculture Norway) Dr. Jose Rainuzzo (Sintef Fisheries and Aquaculture Norway) Dr. Cantal Cahu (IFREMER France) Dr. Jose Zambonino Infante (IFREMER France) Chief Engineer Tora Bardal (NTNU) MSc Gunnar Høvde