Utilization of the microalga Pavlova sp. in marine fish nutrition · List of Tables V List of Tables Table I-1: Final rotifer density, final egg density, final egg ratio and instantaneous

Aus dem Institut für Tierzucht und Tierhaltung

der Agrar- und Ernährungswissenschaftlichen Fakultät

der Christian-Albrechts-Universität zu Kiel

Utilization of the microalga Pavlova sp. in marine fish

nutrition

Dissertation

zur Erlangung des Doktorgrades

der Agrar- und Ernährungswissenschaftlichen Fakultät

der Christian-Albrechts-Universität zu Kiel

vorgelegt von

Diplom-Biologin

SABINE REHBERG-HAAS

aus Miltenberg am Main

Kiel, 2014

Dekan: Prof. Dr. Eberhard Hartung

Erster Berichterstatter: Prof. Dr. Carsten Schulz

Zweiter Berichterstatter: Prof. Dr. Rüdiger Schulz

Tag der mündlichen Prüfung: 12. November 2014

Diese Dissertation wurde mit dankenswerter finanzieller Unterstützung der Deutschen

Bundesstiftung Umwelt (DBU) angefertigt.

Gedruckt mit Genehmigung des Dekans der Agrar- und Ernährungswissenschaftlichen Fakultät der

Christian-Albrechts-Universität zu Kiel.

„Wenn man Fische studieren will, wird man am besten selbst zum Fisch.“

Jacques-Yves Cousteau (1910-1997)

Table of Contents

IV

Table of Contents

GENERAL INTRODUCTION ....................................................................................................................................... 1

CHAPTER I. A COMPARISON AMONG DIFFERENT PAVLOVA SP. PRODUCTS FOR CULTIVATION OF

BRACHIONUS PLICATILIS ............................................................................................................. 11

CHAPTER II. USE OF THE MICROALGA PAVLOVA VIRIDIS AS ENRICHMENT PRODUCT FOR THE FEEDING OF

ATLANTIC COD LARVAE (GADUS MORHUA) ................................................................................ 26

CHAPTER III. MARINE MICROALGAE PAVLOVA VIRIDIS AND NANNOCHLOROPSIS SP. AS N-3 PUFA SOURCE IN

DIETS FOR JUVENILE EUROPEAN SEA BASS (DICENTRARCHUS LABRAX) .................................... 48

GENERAL DISCUSSION ........................................................................................................................................... 68

SUMMARY ............................................................................................................................................................. 83

ZUSAMMENFASSUNG ........................................................................................................................................... 86

DANKSAGUNG ....................................................................................................................................................... 89

LEBENSLAUF .......................................................................................................................................................... 90

List of Tables

V

List of Tables

Table I-1: Final rotifer density, final egg density, final egg ratio and instantaneous growth rate G

(mean±SD, n=4) of rotifer cultures fed with the experimental diets (Baker´s yeast, Nannochloropsis

sp. concentrate, Pavlova viridis concentrate, Pavlova viridis fresh culture and Pavlova sp. fresh

culture). Values with the same superscript are not significantly different (p<0.05). ........................... 17

Table I-2: Final rotifer density, final egg density, final egg ratio and instantaneous growth rate G

(mean±SD, n=5) of rotifer cultures fed with the experimental diets (Nannochloropsis sp. concentrate,

Pavlova viridis concentrate, Pavlova viridis fresh culture, Pavlova viridis frozen concentrate and

Pavlova viridis freeze-dried). Values with the same superscript are not significantly different (p<0.05).

............................................................................................................................................................... 19

Table II-1: Experimental settings of first feeding experiment (D= 24 h dark, L= 24 h light). ................ 29

Table II-2: Nutrient composition of experimental treatments (N, P and CP) in g kg-1 dry matter (DM).

............................................................................................................................................................... 29

Table II-3: Growth performance and survival of cod larvae of the three experimental groups N, P and

CP (mean±SD, n=3). Values with the same superscript are not significantly different (p<0.05). ......... 33

Table II-4: Diversity indices for the microbiota of larvae (L), feed (F) and water (W) samples of the

three treatment groups N, P and CP (mean±SD, n=2). Different superscripts indicate significant

differences (p<0.05). ............................................................................................................................. 39

Table II-5: Bray-Curtis-index - Comparison of larvae vs. feed and water of the treatments N, P and CP

(mean±SD, n=2). .................................................................................................................................... 40

Table III-1: Ingredients, nutrient composition and amino acid composition [g kg-1 dry matter] of the

experimental diets C, B, P50, P100, N50 and N100. ............................................................................. 52

Table III-2: Fatty acid composition of the experimental diets C, B, P50, P100, N50 and N100. Σ SFA is

the sum of saturated fatty acids, Σ MUFA is the sum of monounsaturated fatty acids, Σ PUFA is the

List of Tables

VI

sum of n-6 and n-3 polyunsaturated fatty acids, Σ n-3 is the sum of n-3 polyunsaturated fatty acids,

Σ n-6 is the sum of n-6 polyunsaturated fatty acids. ............................................................................. 53

Table III-3: Growth performance, feed intake and feed efficiency and biometric parameters

(mean±SD, n=3) of sea bass fed with the experimental diets C, B, P50, P100, N50, and N100. Values

with the same superscript are not significantly different (p<0.05). ..................................................... 56

Table III-4: Body composition (in g kg-1 OM; gross energy in MJ kg-1 OM) of sea bass fed with the

experimental diets C, B, P50, P100, N50 and N100 (mean±SD, n=3). Values with the same superscript

are not significantly different (p<0.05). Initial body composition was analyzed as dry matter

270 g kg-1; crude ash 44.3 g kg-1 OM; crude protein 164 g kg-1 OM; crude lipid 61.2 g kg-1 OM; energy

21.34 MJ kg-1 OM. .................................................................................................................................. 56

Table III-5: Fatty acid composition of sea bass fed with the experimental diets C, B, P50, P100, N50

and N100 (mean±SD, n=3). Values with the same superscript are not significantly different (p<0.05).

Lipid acronyms are defined in legend of Table III-2. ............................................................................. 58

Table-General discussion 1: Evaluation summary of Pavlova sp. for the use in marine fish nutrition. ...

............................................................................................................................................................... 77

List of Figures

VII

List of Figures

Figure I-1: Culture performance of the first experiment shown as rotifer density, egg density and egg

ratio during the experimental period (mean±SD, n=4). Values with the same superscript are not

significantly different (p<0.05). ............................................................................................................. 17

Figure I-2: Culture performance of the second experiment shown as rotifer and egg density and egg

ratio during the experimental period (mean±SD, n=5). Values with the same superscript are not

significantly different (p<0.05). ............................................................................................................. 18

Figure I-3: Filtration and ingestion rate (mean±SD, n=5) at day 5 and day 12 over a period of 20 h. .. 19

Figure I-4: Total organic carbon (TOC) content and dissolved organic carbon (DOC) content in culture

water samples at day 1, 7 and 14 (mean±SD, n=5). .............................................................................. 20

Figure II-1: Mortality depicted as cumulative mortality of absolute numbers of dead larvae being

removed once a day (upper panel) and depicted as absolute number of dead larvae (lower panel)

(mean±SD, n=3). + stands for significant differences: Na, Pab, CPb and ++ stands for significant

differences Na, Pa, CPb. Values with the same superscript are not significantly different (p<0.05). ..... 34

Figure II-2: Body size and instantaneous growth rate (G) of cod larvae fed with differently enriched

live feed (N, P, CP) over the experimental period of 42 days (mean±SD, n=3). + stands for Na, Pab, CPb

and ++ stands for Na, Pb, CPb. Values with the same superscript are not significantly different (p<0.05).

............................................................................................................................................................... 35

Figure II-3: Standardized RNA-DNA ratio (sRD) shown as one boxplot for each treatment (N, P, CP)

over the experimental period of 42 days (n=3). + stands for significant differences: Na, Pb, CPb. Values

with the same superscript are not significantly different (p<0.05). ..................................................... 36

Figure II-4: Dry weight-specific instantaneous rates of growth of cod larvae of the different treatment

groups (N, P, CP) plotted against replicate tank average sRD values. The black line represents the

predicted instantaneous growth rate (Gpr) according to the sRD-T-G model (Buckley et al., 2008).

Depicted are values of the sampling phase of constant temperature (12°C). ...................................... 37

List of Figures

VIII

Figure II-5: Residuals of observed instantaneous growth (G) – predicted instantaneous growth (Gpr)

calculated for the three experimental groups (N, P, CP). Depicted are values of the sampling phase of

constant temperature (12°C). The predicted instantaneous growth was calculated according to the

sRD-T-G model of Buckley et al. (2008). ................................................................................................ 37

Figure II-6: Ingestion by means of gut fullness index (GFI) depending on the different experimental

enrichment products (N, P, CP) at 4, 15 and 27 dph (mean±SD, n=3). Values with the same

superscript are not significantly different (p<0.05). ............................................................................. 38

Figure II-7: Standardized RNA-DNA ratio (sRD) of larvae from the different experimental groups (N, P,

CP) in the feed depletion experiment (mean±SD, n=3). The trial was repeated three times starting at

dph 5 (A), dph 16 (B) and dph 28 (C). The arrows indicate the day of re-feeding. Different superscripts

indicate significant differences (p<0.05). .............................................................................................. 38

Figure II-8: Non-metric multidimensional scaling (NMDS) based on Bray-Curtis distance for larval

samples only (4 larvae per tank, n=2). .................................................................................................. 39

Figure III-1: Liver of initially sampled sea bass (Figure A) (representing low vacuolization level) and

liver of sea bass fed with diet C sampled after 56 days (Figure B) (representing high vacuolization

level) (H&E, 200x). The vacuolated cytoplasm is seen light because of high lipid content. Black arrows

mark basophilic nuclei. .......................................................................................................................... 59

Figure III-2: Level of vacuolization of liver cells depending on the different experimental diets C, B,

P50, P100, N50 and N100 (mean±SD, n=3). No significant differences were found (p<0.05). ............. 59

Figure III-3: Intestine epithelium of initially sampled sea bass (Figure A) and of sea bass fed with diet

C (Figure B) and diet P100 (Figure C) sampled after 56 days (H&E, 200x). Continuous muscularis

externa (ME); submucosa (SM); lined villi with columnar epithelium (EP) are marked. ...................... 60

General introduction

1

GENERAL INTRODUCTION

Ocean diversity

The ocean presents a manifold diversity of organisms, which hold a great potential for a wide range

of purposes benefitting human society. There are for example medical and pharmaceutical

achievements like the detection of anti-cancer agents in corals and sponges (Cryptothetia crypta)

(Bergmann, Feeney, 1951). Furthermore, there is a range of cosmetic products containing ocean

derived substances like minerals or vitamins and there is a long list with further environmental and

industrial applications like marine biofuels or anti-fouling agents. And also a great nutritional

potential can be found in marine organisms. Major relevance as food and feed supplements can be

attributed to the group of microalgae.

Microalgae

The group of microalgae includes mainly unicellular, photo- or mixotrophic organisms, which live in

the aquatic habitat. Their size ranges from a few to a few hundreds of micrometers. This group is

highly diverse and encompasses around 800.000 species, of which only 50.000 have been described

and around 15.000 new compounds have been accessed so far (Cardozo et al., 2007). Microalgae are

known to contain high amounts of important macronutrients like protein and lipid, as well as

micronutrients like pigments (β-carotenoids and astaxanthin in Dunaliella sp. and Haematococcus

sp.) (Choubert, Heinrich, 1993; Rowan, 1989; Ye et al., 2008), vitamins (e.g. A, B1, B2, B6, C, E) (Becker,

2004; Brown et al., 1999), essential amino acids (Brown et al., 1997; Guil-Guerrero et al., 2004) and

essential long chain polyunsaturated fatty acids (PUFA), like the important eicosapentaenoic acid

(EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) (Brown, 2002; Patil et al., 2007; Volkman et

al., 1989). All these highly interesting features have aroused the interest of industry and the

biotechnology sector. The first large-scale commercialization of microalgae started in the beginning

of the 1960s with the large scale production of Chlorella sp. by Nihon Chlorella Inc. in Taiwan

(Iwamoto, 2004). The popularity of Chlorella sp. is based on nutritional and health promoting

substances like immunostimulants (β-1,3-glucan; Kojima et al. (1971)) or anti-tumor agents

(polysaccharide; Nomoto et al. (1983)). Further commercial production of other species like

Arthrospira sp. or Dunaliella sp. followed soon. Again the use of these products focused on human

nutrition (food additives) and pharmaceutical purposes. In addition, the incorporation of microalgal

products in animal feed got more and more important at the same time (Combs, 1952; Grau, Klein,

1957; Hintz, Heitman, 1967). Today around 30 % of the total microalgal production is used by the

animal feed production sector (Becker, 2004). Of course microalgae were also used in aquaculture

feed production, which is a natural link as microalgae are the basis of all aquatic food chains and are


2

therefore the natural feed for many aquaculture species like bivalves, larval stages of many aquatic

species and herbivorous zooplankton, which is used as live feed in aquaculture.

Traditionally microalgae are produced under autotrophic conditions depending on natural or artificial

light. They are either cultivated in open ponds, greenhouses or indoor cultivation systems with

culture volumes ranging from 10 to 5000 L (Brown, 2002). One factor limiting algal biomass yield is

light penetration, which is hampered with increasing cell density. Handling of extremely large culture

volumes demands increased amount of work, holding area and energy. Some species can also be

cultivated under heterotrophic conditions (Hellebust, Lewin, 1977) in biofermenters, which then

eliminates the need for light and can therefore help to enhance the biomass production. Another

factor impairing a successful microalgae production is further processing and harvesting of the

products. Different methods like sedimentation, centrifugation, flotation or skimming are commonly

used, whereas highly sensitive microalgal cells can be easily damaged due to shear force caused by

these procedures.

The required specifications of potential microalgal species for a successful production and application

in aquaculture nutrition are manifold. First of all a high growth potential of the specific microalgal

species is of relevance in order to facilitate mass culture and to limit energy and production costs.

Also the absence of toxins, which can be found in several algal species (Gates, Wilson, 1960; Gunter

et al., 1948; Shilo, 1967) is of great importance in order to avoid harmful effects on aquaculture

species or humans as end consumers. Furthermore, the microalgal species need to be tolerant

towards handling and harvest and preservation methods, as well as changes of the biotic and abiotic

culture conditions like nutrient supply, light intensity, temperature, salinity or pH. This is of special

relevance in case of outdoor or greenhouse cultivation. And of course a high content of the

substances of interest like the aforementioned macro- and micronutrients determines the potential

of a given microalgal species for the use in aquaculture.

Some of the most important microalgae candidates that have been used for aquaculture feed are

Nannochloropsis sp., Isochrysis galbana, Tetraselmis sp., Chaetoceros muelleri and Skeletonema

costatum, Dunaliella sp., Haematococcus sp., Spirulina sp. (Benemann, 1992; Borowitzka, 1997;

Brown, 2002; Brown et al., 1997; Muller-Feuga, 2000). In the past decades there has been a lot of

research about the biochemical composition of potential microalgal species for the use in

aquaculture (Brown, 1991; Brown et al., 1997; Brown et al., 1999; Dunstan et al., 1993; Mazur,

Clarke, 1938; Patil et al., 2007; Volkman et al., 1993; Volkman et al., 1989; Volkmann et al., 1991),

whereas the content of essential long chain n-3 PUFA has been of special interest. These substances

are not only essential for almost all animals (also for marine fish), but for humans as well. Especially

DHA and EPA play an important role in many biological processes. For example they are incorporated

in cell membrane phospholipids and therefore enhance membrane fluidity and they also play an


3

important role in the early development of brain and retina (Anderson et al., 1990). Based on the

various functions in biological processes, DHA and EPA have numerous health promoting and

protective effects, like anti-inflammatory effects (Yan et al., 2013), effects against atherosclerosis,

coronary heart disease, cancer and type 2 diabetes (Doughman et al., 2007).

However, most of the microalgal species commonly used in aquaculture so far, contain only one of

both essential PUFA, EPA or DHA, in distinct amounts. The microalgal species Isochrysis galbana

contains for example 0.8 mg g-1 dry weight (DW) of EPA and 15.8 mg g-1 DW DHA. In contrast

Nannochloropsis sp. contains no detectable amount of DHA and 23.4 mg g-1 DW EPA (Patil et al.,

2007). Hence, in order to meet the nutritional requirements and to provide both, DHA and EPA to the

target species, algal mixtures have to be used. As a result several algal species often have to be

cultivated, which of course leads to increased amount of work, energy and costs.

On that account the marine phytoflagellate Pavlova sp. (Butcher, 1952) belonging to the class of

prymnesiophyceae is of special interest, because it is known to be able to synthesize DHA and EPA in

larger amounts (Kato et al., 1995; Tonon et al., 2002; Volkmann et al., 1991). According to Patil et al.

(2007) Pavlova sp. comprises around 13 mg g-1 DHA and 18 mg g-1 EPA. However, these values can

vary strongly and can even be enhanced mainly by adjusting abiotic (e.g. salinity, pH, temperature)

and biotic (e.g. nutrient supply) culture conditions. Therefore, there is a strong research focus on the

optimal culture conditions and requirements of vitamins (thiamine (B1) and cyanocobalamin (B12)),

trace elements (Mn, Zn, B, Co, Cu and Mo) or choice of nitrogen source for Pavlova sp. in order to

increase the biomass and EPA and DHA yield (Carvalho et al., 2009; Carvalho et al., 2006; Dunstan et

al., 1993; Hu et al., 2007). Although the PUFA content of Pavlova sp. is very promising and predicts a

high potential for the use in aquaculture diets there is still no considerable commercial production of

this species. This is mainly attributed to the high sensitivity of Pavlova sp. cells to shear forces, which

occur during handling, harvest and processing (Heasman et al., 2000). The sensitivity is mainly due to

the cell appendices found in members of the order Pavlovales. There are mostly two flagella (short

flagellum – position posterior and long flagellum – position anterior) and a haptonema (Green, 1980),

which can easily break and damage the cells during handling and processing. Pavlova sp. is also often

intolerant against high temperatures, which appear especially under the open pond- or greenhouse

cultivation conditions. These characteristics lead to low production rates, difficulties during handling

and harvest processes and therefore to low quality products with low storability. In order to make

use of this highly interesting microalga for nutritional purposes it is still a major task to improve the

process technology in order to realize the commercial production for the use in aquaculture.


4

Aquaculture

The global aquaculture production is still growing. In 2012 the total aquaculture food fish production

of 66.6 million tonnes was valued at 137.7 billion US$ and represented 42 % of the total fish

production by fisheries and aquaculture (FAO, 2014). Going along with the increasing importance of

aquaculture for the global supply of fish for human consumption is the need to maintain or even

enhance the nutritive quality of aquaculture products. Fish and seafood are still major sources of

energy, protein and especially essential substances like the long chain n-3 PUFA, DHA and EPA.

Although these marine organisms are the main DHA and EPA source for human nutrition, they are

not able to convert C18 fatty acids to C20 and C22 PUFA in sufficient amounts. This is especially true

for marine fish, which need to be supplied with the essential PUFA via feed items. In contrast

freshwater fish are able to convert short chained n-3 fatty acids (linolenic acid, 18:3n-3) into EPA and

DHA due to a higher elongase and desaturase activity (Henderson, 1996). In natural marine

environments microalgae are the origin and main producers of the essential fatty acids, which are

transferred along the food chain to higher trophic levels. However, in aquaculture marine fish and

especially fish larvae need to be provided with these essential substances. And still the provision of

sustainably sourced feed raw materials rich in essential PUFA and other essential compounds is a

bottleneck in marine aquaculture.

Nutrition of fish larvae

There is a large number of studies confirming the positive effects of essential PUFA on survival,

development, growth, pigmentation and stress resistance of marine larval fish (Copeman et al., 2002;

Hamre et al., 2013; Izquierdo, 1996; Mourente et al., 1993; Sargent et al., 1999; Villalta et al., 2005;

Watanabe et al., 1983). Despite the considerable effort of the last decades there is still a lack in the

understanding of fish larval nutrition and species specific PUFA requirements (Conceição et al., 2010;

Hamre, 2006; Hamre et al., 2013). Of course this understanding and knowledge is of great

importance for the improvement of larval feeding protocols and regimes helping to enhance the

quality of juvenile aquaculture organisms. Until today the common practice for fish larvae feeding is

the use of rotifers (Brachionus sp.) and brine shrimp (Artemia sp.) as live prey, since application of

artificial microdiets as starter diet for most marine fish larvae is still in its infancy. However, also the

cultivation of live feed organisms is costly in terms of work and energy. For the cultivation of rotifers

mainly microalgae in combination with baker´s yeast (Saccharomyces cerevisiae) are used (Lubzens,

1987; Lubzens et al., 1995). In this combination yeast serves as cheap nutrient source, though lacking

the important fatty acids. For this reason microalgae or mostly microalgae mixtures rich in DHA and

EPA are added. However, the hatchery on-site-production of one or several microalgal species is very

cost- and time consuming and needs to be substituted by other storable products. Therefore,


5

Pavlova sp. products containing high amounts of both essential PUFA, DHA and EPA, seem to be

promising feed sources and the following research question will be answered in this work:

I) What are the effects of Pavlova sp. and Pavlova viridis products on the culture

performance of rotifers?

It was the aim of the first part of this work to evaluate the potential of different Pavlova strains

(Pavlova sp. and Pavlova viridis) for the cultivation of rotifers (Brachionus sp.). In order to further test

different storable products, fresh algae cultures were applied besides different preserved forms like

microalgal concentrate, frozen concentrate and freeze-dried meal and the effects on the culture

performance were investigated.

(Chapter I: A comparison among different Pavlova sp. products for cultivation of Brachionus

plicatilis)

Rotifers and Artemia do not meet the nutrient requirements of marine fish larvae as they are not

their natural prey and they are mainly lacking the essential fatty acids, DHA and EPA. Therefore, their

nutritional quality needs to be improved according to the specific requirements of the target species

(Ben-Amotz et al., 1987; Dhert et al., 2001; Watanabe et al., 1983). This nutritional manipulation is

achieved by enriching these organisms with special emulsions or concentrates rich in the important

macro- and micronutrients (Fernandez-Reiriz et al., 1993; Harel et al., 2002). Often a long-term

enrichment of more than 24 h is applied in order to not only fill the gut with the enrichment product,

but also to modify the whole biochemical composition of the organisms (Dhert et al., 2001; Sorgeloos

et al., 2001). Besides formulated enrichment products (e.g. Larviva Multigain®, Biomar, Brande,

Denmark or easy DHA selco®, INVE, Dendermonde, Belgium) mainly microalgal pastes, concentrates

or freeze-dried meals are used. However, the optimal composition of enrichment has not been found

and still fish larvae nutrition depicts a major bottleneck in marine fish production. Against this

background a second research question was answered:

II) Is Pavlova viridis a suitable live feed enrichment product for the feeding of marine fish

larvae?

The effects of Pavlova viridis as live feed enrichment product on the survival, growth and feeding

performance of Atlantic cod (Gadus morhua) larvae and on the associated bacterial community

structure were investigated in the second part of this work.

(Chapter II: Use of the microalga Pavlova viridis as enrichment product for the feeding of

Atlantic cod larvae (Gadus morhua))

Nutrition of adult fish

Although DHA and EPA play a major role in the early development of fish larvae, they are also

essential for juvenile and adult fish. So far the DHA and EPA supply of juvenile and adult fish in


6

aquaculture has been accomplished by the use of fish oil in dry diets. Fish oil is still the only

economically feasible long chain n-3 PUFA source for nutritional purposes containing about 10-20 %

DHA and EPA of total fatty acids (Gruger, 1967). However, as production rates are stagnating with

simultaneously increasing request for fish oil (and fish meal), the market prices are heavily influenced

by this development. In order to minimize the dependency on the fish oil production there is a strong

need for sustainable alternative PUFA sources in aquaculture. So far vegetable oils can successfully

substitute up to 60 % of fish oil in dry feed for various marine finfish species (Bell et al., 2003a;

Figueiredo-Silva et al., 2005; Richard et al., 2006). However, there is a lack of essential PUFA in

vegetable oils and therefore a total fish oil replacement is seldom viable (Bell et al., 2003b; Bell et al.,

2001). Against this background the use of microalgal products as n-3 PUFA sources in dry diets is of

increasing interest. For example mixtures of Nannochloropsis sp. and Schizochytrium sp. could

replace 100 % fish oil in diets for olive flounder (Paralichthys olivaceus) without negative effects on

growth, feed efficiency or nutritive quality (Qiao et al., 2014). The evaluation of Pavlova sp. as n-3

PUFA sources was addressed in this work:

III) Is Pavlova viridis a useful n-3 PUFA source in dry feed for juvenile and adult marine fish?

In order to answer this research question the third part of this work focused on the potential of

freeze-dried Pavlova viridis as n-3 PUFA source in dry feed for juvenile sea bass (Dicentrarchus

labrax) compared to Nannochloropsis sp. Besides the growth performance the body composition, the

fatty acid composition and liver and intestine histology were investigated.

(Chapter III: Marine microalgae Pavlova viridis and Nannochloropsis sp. as n-3 PUFA source in

diets for juvenile European sea bass (Dicentrarchus labrax))

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Chapter I

11

CHAPTER I. A COMPARISON AMONG DIFFERENT PAVLOVA SP. PRODUCTS

FOR CULTIVATION OF BRACHIONUS PLICATILIS

Rehberg-Haas, Sabine a,c; Meyer, Stefan a; Lippemeier, Sebastian b; Schulz, Carsten a,c

a GMA – Gesellschaft für Marine Aquakultur mbH, Büsum, Germany

b BlueBioTech GmbH, Büsum, Germany

c Institute of Animal Breeding and Husbandry, Christian-Albrechts-University, Kiel, Germany

Chapter I

12

Abstract

In the present study the potential of different products of the marine microalga Pavlova sp. for the

cultivation of rotifers (Brachionus plicatilis) was tested. Two growth performance trials were

conducted: In a first laboratory scale experiment rotifers were cultivated for 14 days with Pavlova

viridis concentrate, Pavlova viridis fresh culture, Pavlova sp. fresh culture, baker´s yeast

(Saccharomyces cerevisiae) and Nannochloropsis sp. concentrate. The Pavlova viridis fresh culture

group resulted in the significantly highest rotifer density (109.2 rotifers ml-1) and instantaneous

growth rate (G=0.14±0.02 d-1). There were no significant differences of G found between the Pavlova

viridis concentrate group and the Pavlova sp. fresh culture group. The baker´s yeast group showed

the significantly lowest rotifer numbers and growth rate. Based on the high growth rate of the

Pavlova viridis fresh culture group in the first experiment, different Pavlova viridis products

(concentrate, fresh culture, frozen concentrate, freeze-dried meal) were examined in the second

experiment and compared to Nannochloropsis sp. concentrate. The highest rotifer growth rate in

experiment 2 was determined for the frozen Pavlova viridis group (G=0.09±0.03 d-1), although it was

only significantly higher in comparison to the growth rate of the rotifers fed with freeze-dried

Pavlova viridis. Hence, most Pavlova groups showed reasonable growth performances. The frozen

product seems the most suitable Pavlova viridis product for the cultivation of live feed and it

provides advantages of storability and application.

Keywords: Brachionus plicatilis, Pavlova sp., live feed cultivation

Introduction

Despite the intensive effort in form of research on the development of larval microdiets, so far no

relevant success has been achieved (Conceição et al., 2010). Hence, the utilization of rotifers

(Brachionus plicatilis) as starter feed for the rearing of marine fish larvae and crustaceans is still

essential for commercial marine hatchery procedures (Bengston, 2003).

However, the stable rotifer cultivation is difficult and time consuming. The provision of sufficient

amounts of rotifers during the crucial phase of first feeding is still challenging, as the use of rotifers

includes biomass production and the boosting with essential nutrients, too. The successful rotifer

cultivation depends on a range of complex interactions, such as water quality and nutrition.

Moreover, a stable body composition is necessary to obtain a steady rotifer culture (Dhert et al.,

2001). Hirayama et al. (1979) and Okauchi and Fukusho (1984) displayed positive nutritional effects

of microalgae in rotifer cultivation. Further important factors describing the overall rotifer culture

performance, like growth rate and filtration and ingestion rates (Savas, Guclu, 2006), are affected by

Chapter I

13

the diet, too. Renaud et al. (2002) reported positive effects of diets containing microalgae rich in

proteins and essential fatty acids on growth rates of live feed species.

The expensive and time-consuming biomass production of rotifers is tried to achieve by the use of

cheap sources like baker´s yeast. Although the cultivation of rotifers on baker´s yeast only can be

successful over several weeks (Lubzens et al., 1995), these cultures are often instable and can crush

spontaneously. Additionally, the rotifers fed with yeast have a lack of the essential nutrients

(Hirayama, 1987). Thus the use of microalgae is an alternative to provide a wider range of needed

nutrients, especially fatty acids, (Ben-Amotz et al., 1987) and to secure a more stable cultivation. In

the past a few microalgal species have been used to cultivate and enrich rotifers. Still, these species

do not feature the optimal essential fatty acid composition. Tetraselmis sp. and Nannochloropsis sp.

contain considerable amounts of eicosapentaenoic acid (EPA, 20:5n-3) and nearly no

docosahexaenoic acid (DHA, 22:6n-3) (Hu, Gao, 2003; Koven et al., 1990; Patil et al., 2007; Watanabe

et al., 1983), whereas Isochrysis sp. contains substantial amounts of DHA and only little amounts of

EPA (Ben-Amotz et al., 1987; Lubzens et al., 1985; Patil et al., 2007). Hence, a great potential can be

attributed to the microalga Pavlova sp., as it contains both EPA (18.0 mg g DW-1; Patil et al. (2007))

and DHA (13.2 mg g DW-1; Patil et al. (2007)) in distinct amounts. However, it has not been possible

to produce Pavlova sp. in a larger scale of industrial relevance so far. The most common strains of

Pavlova are known to be sensitive against shear force and high temperature (>28°C) and they are

very sensitive to downstreaming processes and have a very short shelf-life, limiting their applicability

for the industrial purpose. For this project new production techniques for cultivation, harvest and

preservation of chosen Pavlova strains have been developed and the large-scale production has been

successfully implemented, to provide different Pavlova products. In order to provide “off the shelf

algal products” the aim of the present study was to evaluate the effects of different Pavlova strains

(Pavlova sp. and Pavlova viridis (Tseng et al., 1992)) and different Pavlova products (fresh algae

culture, concentrate, frozen concentrate, freeze-dried meal) on the culture performance by

investigating the culture growth, filtration and ingestion rates of the rotifers and the culture water

quality (total organic carbon-TOC; dissolved organic carbon-DOC).

Material and Methods

Experiment 1

Rotifer culture

In the pre-experimental period rotifers (Brachionus plicatilis, L-strain, mean lorica length 199.8 µm)

were cultivated regularly in 30 L circular tanks on a combination of baker´s yeast (Saccharomyces

cerevisiae) and Nannochloropsis sp. concentrate (12 x 109 cells ml-1). At the beginning of the first

Chapter I

14

laboratory scale cultivation experiment 1 L flasks were stocked with rotifers of about 30 ind ml-1.

Over the experimental period of 14 days the salinity (20 PSU) and the temperature (22.4±0.7 °C)

were maintained at a constant level. Light was set on a 24 h photoperiod. Water quality parameters

were maintained in a safe range and were measured regularly (7.0-7.3 pH; GMH 3530, Digital pH-

/mV-/Thermometer, Greisinger electronic, Germany; 9.7±0.1 mg L-1 O2; Handy Polaris; Oxy- Guard

International A/S, Birkerod, Denmark).

Experimental treatments

After previous tests of the cultivation methods for different Pavlova strains two strains were chosen

for further experiments. In this first experiment the potential of these two different Pavlova strains -

Pavlova viridis and Pavlova sp. (CCMP 1228) - as cultivation products were evaluated. In contrast to

other Pavlova strains both Pavlova viridis and Pavlova sp. showed higher temperature tolerance and

were suitable for cultivation.

The experimental treatments for the first trial were 1) Baker´s yeast, 2) Nannochloropsis sp.

concentrate (144 µg DW µl-1), 3) Pavlova viridis concentrate (35 µg DW µl-1), 4) Pavlova viridis fresh

culture (0.8 µg DW µl-1) and 5) Pavlova sp. fresh culture (1.0 µg DW µl-1). Each product was tested in a

four time replication and was applied at a daily ration of 0.8 g DW per 1 x 106 rotifers according to

the rotifer density at the beginning of the trial.

Sampling

A volume of 100 ml water was removed from the culture on a daily basis and five 1 ml sub-samples

were preserved with Lugol´s solution for further counting of population and eggs under a stereo-

microscope. The egg ratio (ER; eggs ind-1) was calculated as:

(1) ER = eggs ml-1 / female rotifers ml-1

The instantaneous growth rate (G in d-1) of rotifers was calculated according to Theilacker, McMaster

(1971):

(2) G = ((ln Nt) – (ln N0)) / t

where Nt is the number of rotifers at time t [d], t stands for the experimental period, N0 is the

number of rotifers at the start of the experiment.

After sampling the culture volume was filled up to 1 L including the daily feed ration.

Experiment 2

Rotifer culture

In the second experiment 10 L tubular containers were stocked with rotifers (pre-treatment like

experiment 1, see above) at a density of about 50-100 ind ml-1. The salinity (20 PSU) and the

temperature (21.5±0.2 °C) were maintained at a constant level during the experimental period of 14

Chapter I

15

days. Lighting was set on a 24 h photoperiod. Water quality parameters were maintained in a safe

range for the rotifers and measured regularly (7.0-7.4 pH; GMH 3530, Digital pH/mV-/Thermometer,

Greisinger electronic, Germany; 9.6±0.1 mg L-1 O2; Handy Polaris; Oxy- Guard International A/S,

Birkerod, Denmark).

Experimental treatments

Concerning the findings from experiment 1 and from further tests of harvest and storage methods

for the two Pavlova strains only Pavlova viridis was chosen for the second rotifer trial. Although both

strains could be harvested by means of centrifugation, the Pavlova sp. showed severe issues

concerning durability and storability. Therefore, this strain had to be excluded from further

application trials.

The algal products examined in the second experiment were: 1) Nannochloropsis sp. concentrate

(144 µg DW µl-1), 2) Pavlova viridis concentrate (35 µg DW µl-1), 3) Pavlova viridis fresh culture

(0.8 µg DW µl-1), 4) Pavlova viridis frozen concentrate (35 µg DW µl-1), 5) Pavlova viridis freeze-dried

meal. Each product was tested in a five time replication and was applied at a daily ration of

0.75 g DW per 1 x 106 rotifers according to the rotifer density at the beginning of the trial.

Growth performance

A volume of 1.5 L was removed daily from each container. Five 1 ml sub-samples were preserved

with Lugol´s solution for further counting of population and eggs under a stereo-microscope. The ER

and G were calculated as shown in equation (1) and (2).

After sampling the culture volume was filled up to 10 L including the daily microalgal feed ration.

Filtration and ingestion

Additionally, the product concentration in the cultures was measured every hour for 7 h and again

20 h after feeding at day 5 and 12.

Filtration and ingestion rates were calculated as follows (Yúfera, Pascual, 1985):

(3) F = ((lnC0) – (lnCt))/N x t

where F is the filtration rate in ml ind-1 min-1, C0 is the initial cultivation product concentration in

µg DW ml-1, Ct is the final cultivation product concentration in µg DW ml-1, N is the rotifer density in

ind ml-1 and t is the duration of the treatment in min.

(4) I = F x C0 x Ct

where I stands for the ingestion rate in µg DW ind-1 min-1.

In order to account for sedimentation of cultivation products, additional concentration

measurements of the diluted products were recorded in a separate setup without rotifers. The

filtration and ingestion values were corrected for sedimentation.

Chapter I

16

TOC and DOC analysis

At day 1, 7 and 14 samples for total and dissolved organic carbon (TOC and DOC) analyses were

taken. Culture water samples were filtered (30 µm mesh size) in order to remove rotifers. The

analyses were conducted by means of a Shimadzu TOC-L Total Organic Carbon Analyzer (Shimadzu,

Kyōto, Japan) using the 680°C combustion catalytic oxidation method.

Statistical analysis

The statistical analyses were performed using SPSS 18.0 for Windows (SPSS Inc., Chicago, USA). The

significance of differences of means (presented as mean ± standard deviation (SD)) depending on the

test treatments was analyzed. The Kolmogorov-Smirnov test was used to check data for normal

distribution (p<0.05). If test for normal distribution failed non-parametric Kruskal-Wallis One–Way

Analysis of Variance was carried out (p<0.05). In case of normal distribution One-Way Analysis of

Variance (ANOVA) was applied. Data was analyzed for variance homogeneity by Levene (confirmed if

p<0.05) and post-hoc multiple comparison was carried out by parametric Tukey-HSD (if test for

homogeneity was confirmed) (p<0.05) or non-parametric Dunnett-T3 test (if test for homogeneity

failed).

Results

Experiment 1

All groups in the first experiment that were cultivated with Pavlova showed reasonable culture

performances (Figure I-1 and Table I-1). The Pavlova viridis fresh culture group showed the highest

final rotifer density (160.7±8.5 rotifers ml-1) and the significantly highest G (0.14±0.02 d-1) followed by

the Pavlova sp. fresh culture group and the Pavlova viridis concentrate group. All groups resulted in

significant higher values than the baker´s yeast group, which showed a negative G. A sharp decline of

the egg density was recorded at day 6 in all experimental groups, which is also included in the

calculation of the ER.

Chapter I

17

Figure I-1: Culture performance of the first experiment shown as rotifer density, egg density and egg ratio during the experimental period (mean±SD, n=4). Values with the same superscript are not significantly different (p<0.05).

Table I-1: Final rotifer density, final egg density, final egg ratio and instantaneous growth rate G (mean±SD, n=4) of rotifer cultures fed with the experimental diets (Baker´s yeast, Nannochloropsis sp. concentrate, Pavlova viridis concentrate, Pavlova viridis fresh culture and Pavlova sp. fresh culture). Values with the same superscript are not significantly different (p<0.05).

Final rotifer density [ind ml-1]

Final egg density [eggs ml-1]

Final egg ratio [eggs ind-1]

G [d-1]

Baker´s yeast 4.3±1.7a

3.8±1.0a 0.96±0.39

a -0.12±0.03

a

Nanno.sp. conc. 79.9±14.7b

15.0±1.6b 0.19±0.03

c 0.11±0.02

b

P.viridis conc. 87.8±3.0b

21.4±1.0c 0.24±0.02

b 0.12±0.03

c

P.viridis fresh 160.7±8.5c

28.7±1.9d 0.18±0.01

c 0.14±0.02

c

P.sp. fresh 109.2±8.7d

22.3±3.5c 0.20±0.03

c 0.12±0.01

c

Chapter I

18

Experiment 2

Growth performance

The highest rotifer density at the end of experiment 2 was determined in the Nannochloropsis sp.

group (282.9±73.6 rotifers ml-1), although it was not significantly different from the results of the

Pavlova viridis frozen concentrate and fresh culture group. The density of the Pavlova concentrate

was lower, though not significantly different to the two aforementioned products. The lowest final

rotifer numbers were found for the group that was fed with the freeze-dried Pavlova viridis

(93.1±23.9 rotifers ml-1) (Figure I-2). The instantaneous growth rate exhibited no significant

differences between the Nannochloropsis sp. group and the Pavlova viridis groups, except for the

freeze-dried Pavlova viridis group (Table I-2).

Figure I-2: Culture performance of the second experiment shown as rotifer and egg density and egg ratio during the experimental period (mean±SD, n=5). Values with the same superscript are not significantly different (p<0.05).

Chapter I

19

Table I-2: Final rotifer density, final egg density, final egg ratio and instantaneous growth rate G (mean±SD, n=5) of rotifer cultures fed with the experimental diets (Nannochloropsis sp. concentrate, Pavlova viridis concentrate, Pavlova viridis fresh culture, Pavlova viridis frozen concentrate and Pavlova viridis freeze-dried). Values with the same superscript are not significantly different (p<0.05).

Final rotifer density [ind ml-1]

Final egg density [eggs ml-1]

Final egg ratio [eggs ind-1]

G [d-1]

Nanno.sp. conc. 282.9±73.6a 50.3±17.3

a 0.18±0.03

a 0.07±0.01

a

P.viridis conc. 197.8±54.8a,b

24.2±7.3b 0.12±0.03

a,b 0.06±0.03

a,b

P.viridis fresh 191.6±72.0a,b

22.5±10.4b 0.11±0.02

b 0.04±0.03

a,b

P.viridis frozen 188.9±46.7a,b

28.9±9.0a,b

0.15±0.02a,b

0.09±0.03a

P.viridis freeze-dried 93.1±23.9b 10.7±5.9

b 0.11±0.04

b 0.01±0.02

b

Filtration and ingestion

The significantly highest filtration (day 5: 5.615 x 10-6±3.562 x 10-7 ml ind-1 min-1; day 12:

3.289 x 10-6±2.051 x 10-7 ml ind-1 min-1) and ingestion (day 5: 4.188 x 10-4±2.755 x 10-5

µg DW ind-1 min-1; day 12: 1.917 x 10-4±2.680 x 10-5 µg DW ind-1 min-1) values were observed in the

Pavlova viridis fresh culture group. The Pavlova viridis freeze-dried group resulted in negative values

for both parameters at both sampling days. This is due to lower concentration values measured at

starting point (Figure I-3).

Figure I-3: Filtration and ingestion rate (mean±SD, n=5) at day 5 and day 12 over a period of 20 h.

TOC and DOC analysis

The total organic carbon (TOC) content ranged from 42.5 mg L-1 (group Nannochloropsis sp., day 1) to

141.2 mg L-1 (group Pavlova viridis fresh culture, day 14). For dissolved organic carbon (DOC) the

values ranged from 30.9 mg L-1 (group Nannochloropsis sp., day 1) to 126.8 mg L-1 (group Pavlova

viridis fresh culture, day 14). Both parameters measured in the Nannochloropsis sp. group and the

Pavlova viridis freeze-dried group ranked at low levels at all sampling days, whereas the values

increased continuously in the other experimental groups (P.vir.conc., P.vir. fresh and P.vir. frozen).

Chapter I

20

The highest values were found at all sampling days for the Pavlova viridis fresh culture group (Figure

I-4).

Figure I-4: Total organic carbon (TOC) content and dissolved organic carbon (DOC) content in culture water samples at day 1, 7 and 14 (mean±SD, n=5).

Discussion

The present work shows that the cultivation of rotifers on various Pavlova sp. products is possible

and reveals reasonable growth performances in comparison to the commonly used microalga

Nannochloropsis sp. In the first experiment the Pavlova viridis fresh culture resulted in the best

growth performance of rotifers. This Pavlova strain was therefore used for the subsequent

experiment. In the second experiment the best result for the final rotifer number was found in the

rotifer group, which was fed with Nannochloropsis sp. concentrate. This might be caused by the

favorable size of these microalgal cells (2-4 µm), which can be ingested more easily by the rotifers

than Pavlova sp. cells (4-6 µm). The smaller cell size might also be more suitable for the processing in

the mastax of the rotifers. It was shown by Baer et al. (2008) that rotifers ingest particles selectively.

Larger particles for example were found to be captured, but not ingested. Similarly Rothhaupt (1990)

described preferences of different rotifer strains for certain particle size ranges. Still, the highest

instantaneous growth rates in experiment 1 were calculated for all three Pavlova treatments (highest

value 0.14±0.019 for Pavlova viridis fresh culture) and in experiment 2 the highest growth rate was

found in the frozen Pavlova viridis group, although not significantly different from the other groups

except for the freeze-dried Pavlova viridis group. A comparison of the observed growth rates with

results of other studies is difficult, as the growth rate depends strongly on the specific rotifer strain,

food type and food concentration (Stemberger, Gilbert, 1985). Certainly the different starting

densities must be considered, as it was not possible to adjust the same numbers in all experimental

vessels. Hence, especially in experiment 2 the lower starting density of the frozen Pavlova viridis

group allowed a greater potential to grow than in the other groups.

Chapter I

21

The ER in both experiments are in agreement with results of other studies (Kostopoulou, Vadstein,

2007; Yúfera, 1987). In the first experiment the Pavlova viridis fresh culture group showed the

highest ER value (1.1±0.3, day 2). The highest value in the second experiment was found for the

Pavlova viridis fresh culture group at day 2, too, though it was not significantly higher than the other

groups, except for the freeze-dried Pavlova viridis group. Furthermore, all groups displayed a peak at

day 2, except for the freeze-dried Pavlova viridis group in experiment 2, which showed the peak at

day 4 indicating a delay in the process of events, as well as a slower and lower culture growth. The

decline of the ER values derived by the decline of egg density at day 6 of experiment 1 is an example

of the unpredictability of rotifer culture development. Often changes or unfavorable environmental

conditions (temperature, water quality, water pollution) are the reasons for culture growth declines

(Lubzens, Zmora 2003). However, in this case no clear indications, like changes of environmental

conditions, were recorded. It might be referred to a population dynamical compensation of the

increasing rotifer density.

The filtration values observed in experiment 2 were highest for the fresh culture Pavlova viridis

group. The live cells of this product probably feature the best distribution qualities in the culture

water and can therefore be easily ingested by the rotifers. Overall the rotifers accepted the Pavlova

products as good as the Nannochloropsis sp. concentrate. The negative filtration and ingestion rate

values of the Pavlova viridis freeze-dried group derive from the lowest concentration values

measured at the starting point. This product was difficult to distribute in the water column. Although

the other products were homogenously mixed at the time of concentration measurement, this might

not have been the case for the freeze-dried Pavlova viridis product. In this case the complete product

distribution might have occurred not until the second measurement leading to negative filtration and

ingestion values.

The TOC and DOC content are defined as the measure of the total (TOC) and dissolved (DOC) organic

carbon load derived from metabolism and decomposition of organisms and bacterial growth in water

samples. The low TOC and DOC values of the freeze-dried Pavlova viridis fed cultures can be

explained by the fast sinking of the particles. On that account the major amount of the organic

material sedimented quickly and therefore the time of decomposition in the water column was

shortened. Furthermore, the freeze-dried Pavlova viridis group remained at very low rotifer densities

throughout the experiment. Hence, only low metabolism and decomposition of organisms occurred

leading to low TOC and DOC values. Similarly in the Nannochloropsis sp. groups low TOC and DOC

values were measured at all sampling days. However, in contrast to the freeze-dried Pavlova viridis,

the Nannochloropsis sp. cells did not feature fast sinking or increased sedimentation. This algal

product seems to be ingested by rotifers quickly and it maintained a stable culture. Therefore, there

is no increased bacteria growth or increased rotifer mortality and degradation. The highest and also

Chapter I

22

increasing values of TOC and DOC determined at day 1, 7 and 14 were recorded for the Pavlova

viridis fresh culture. This is probably due to the high load of organic carbon that is brought with the

culture medium. Also the bacterial growth might have been stimulated by this additional nutrient

load coming from the culture medium. A high microfloral growth might also have been possible for

the Pavlova viridis concentrate and frozen concentrate groups, which also revealed high TOC and

DOC values. Furthermore, an increase of these values was recorded for these three experimental

groups, whereas the TOC and DOC concentrations of the other two groups (Nannochloropsis sp. and

Pavlova viridis freeze-dried) stayed at equal levels at all sampling dates. The increase of TOC and DOC

values for the Pavlova viridis fresh culture, concentrate and frozen concentrate group, is probably

due to increasing microbial growth rather than to accumulation processes, which otherwise might

have occurred in the other two remaining groups, as well. Although most common bacterial species

do not necessarily have a negative effect on rotifer cultures, the microflora needs to be controlled

because of possible introduction into the fish larvae system by rotifers (Støttrup, McEvoy, 2003). For

example Vibrionaceae, which have been identified in rotifer cultures (Nicolas et al., 1989), can be

harmful to fish larvae by hampering intestinal passage and assimilation. However, not only harmful

microbiota, but also probiotic bacteria strains can occur in rotifer cultures. These probiotic strains

can also enhance rotifer cultures (Douillet, 2000). Therefore, further research on the impact of

Pavlova viridis products on the microbial flora is needed.

The results of the present work show differences in the suitability of the tested algal products. First

of all the Pavlova viridis fresh culture products (experiment 1 and 2) seem to be qualified for the

cultivation of rotifers, mainly because the algal cells were completely intact and the cells stayed in

the water column and were therefore available for the rotifers. However, as mentioned before, the

production of large algae biomass is cost- and time consuming and needs to be substituted by other

storable products. The tested freeze-dried Pavlova viridis meal resulted in the lowest growth rates,

although the quality of the product and the composition remains unaltered, even after the process of

concentration and lyophilization (Lippemeier, unpublished results). The adverse culture development

is probably due to the unfavorable applicability, as it is difficult to mix the meal homogeneously in

water. Furthermore, the particles and cells sink fast and the availability for the rotifers is therefore

shortened compared to the other products. Similar effects were also found for other dried algal

products by Lubzens et al. (1995). The Pavlova viridis concentrate is another product, which can be

provided as commercial product. The quality of the single cells was unaltered and the cells stayed in

the water column and were available for the rotifers (Lippemeier, unpublished results). However, this

product displayed a low durability expressed by a slight color change from yellow-green to brown

and was therefore storable only for a few days to one week. This problem can be avoided by freezing

the algal concentrate and thawing the needed amount right before use. The frozen product was

Chapter I

23

stored at -20 °C. There was no loss in quality compared to the Pavlova viridis concentrate which was

stored at 4 °C and it also led to the same or even better growth of the rotifer cultures (Table I-2). Also

Lubzens et al. (1995) stated that frozen Nannochloropsis sp. concentrate could be stored at -20 °C

or -80 °C for four weeks and the thawed material maintained its quality and could be used for

enrichment for one week and for cultivation for two weeks.

In conclusion Pavlova viridis products, especially the frozen concentrate, were found to be

reasonable cultivation products for rotifers as an alternative to live algae. The frozen product

features the advantages of a long storability and the preservation of product quality. Furthermore,

higher algal concentrations can be applied to the rotifer cultures than with live algal cultures, which

are required for the resting egg production of rotifers. However, there are still some advantages of

Nannochloropsis sp. over the Pavlova viridis products as long-term rotifer cultivation product, like

more suitable cell size for ingestion and digestion in the mastax, as well as better effects on water

quality. On that account the short-term enrichment of live feed with Pavlova viridis might be the

more suitable application form for this alga.

Acknowledgments

This work was funded by the DBU-Deutsche Bundesstiftung Umwelt under grant agreement n° AZ

28183-34.

References

Baer, A., Langdon, C., Mills, S., Schulz, C., Hamre, K., 2008. Particle size preference, gut filling and

evacuation rates of the rotifer Brachionus “Cayman” using polystyrene latex beads. Aquaculture. 282,

75-82.

Ben-Amotz, A., Fishler, R., Schneller, A., 1987. Chemical composition of dietary species of marine

unicellular algae and rotifers with emphasis on fatty acids. Marine Biology. 95, 31-36.

Bengston, D.A., 2003. Status of marine aquaculture in relation of live prey: past, present and future.

in: Støttrup, J.G., McEvoy, L.A. (Eds.), Live Feeds in Marine Aquaculture. Blackwell Science Ltd., pp.

1-16.



Dhert, P., Rombaut, G., Suantika, G., Sorgeloos, P., 2001. Advancement of rotifer culture and

manipulation techniques in Europe. Aquaculture. 200, 129-146.

Douillet, P.A., 2000. Bacterial additives that consistently enhance rotifer growth under synxenic

culture conditions: 2. Use of single and multiple bacterial probiotics. Aquaculture. 182, 241-248.

Chapter I

24

Hirayama, K., 1987. A consideration of why mass culture of the rotifer Brachionus plicatilis with

baker’s yeast is unstable. in: May, L., Wallace, R., Herzig, A. (Eds.), Rotifer Symposium IV. Springer

Netherlands, pp. 269-270.

Hirayama, K., Takagi, K., Kimura, H., 1979. Nutritional effect of eight species of marine phytoplankton

on population growth of the rotifer, Brachionus plicatilis. Bulletin of the Japanese Society of Scientific

Fisheries. 45, 11-16.

Hu, H., Gao, K., 2003. Optimization of growth and fatty acid composition of a unicellular marine

picoplankton, Nannochloropsis sp., with enriched carbon sources. Biotechnology Letters. 25, 421-

425.

Kostopoulou, V., Vadstein, O., 2007. Growth performance of the rotifers Brachionus plicatilis, B.

‘Nevada’ and B. ‘Cayman’ under different food concentrations. Aquaculture. 273, 449-458.

Koven, W.M., Tandler, A., Kissil, G.W., Sklan, D., Friezlander, O., Harel, M., 1990. The effect of dietary

(n−3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus

aurata larvae. Aquaculture. 91, 131-141.

Lubzens, E., Marko, A., Tietz, A., 1985. De novo synthesis of fatty acids in the rotifer, Brachionus

plicatilis. Aquaculture. 47, 27-37.



Lubzens, E., Zmora, O., 2003. Production and nutritional value of rotifers. in: Støttrup, J.G., McEvoy,

L.A. (Eds.), Live feeds in marine aquaculture. Blackwell Science Ltd., pp. 300-303.

Nicolas, J.L., Robic, E., Ansquer, D., 1989. Bacterial flora associated with a trophic chain consisting of

microalgae, rotifers and turbot larvae: Influence of bacteria on larval survival. Aquaculture. 83, 237-

248.

Okauchi, M., Fukusho, K., 1984. Food value of a minute alga, Tetraselmis tetrahele, for the rotifer

Brachionus plicatilis culture. 1: Population growth with batch culture. Bulletin National Research

Institute of Aquaculture. 5, 13-18.



Renaud, S.M., Thinh, L., Lambrinidis, G., Parry, D.L., 2002. Effect of temperature on growth, chemical

composition and fatty acid composition of tropical Australian microalgae grown in batch cultures.


Rothhaupt, K.O., 1990. Population growth rates of two closely related rotifer species: effects of food

quantity, particle size, and nutritional quality. Freshwater Biology. 23, 561-570.

Savas, S., Guclu, Z., 2006. Filtration and ingestion rates of the rotifer Brachionus plicatilis fed five

species of microalgae at different cell densities. Israeli Journal of Aquaculture-Bamidgeh. 58, 39-45.

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25

Stemberger, R.S., Gilbert, J.J., 1985. Body size, food concentration, and population growth in

planktonic rotifers. Ecology. 66, 1151-1159.

Theilacker, G.H., McMaster, M.F., 1971. Mass culture of the rotifer Brachionus plicatilis and its

evaluation as a food for larval anchovies. Marine Biology. 10, 183-188.

Tseng, C.K., Chen, J., Zhang, Z., 1992. On a new species of Pavlova (Prymnesiophyceae) from China.

Chinese Journal of Oceanology and Limnology. 10, 23-30.

Watanabe, T., Kitajima, C., Fujita, S., 1983. Nutritional values of live organisms used in Japan for mass

propagation of fish: A review. Aquaculture. 34, 115-143.

Yúfera, M., 1987. Effect of algal diet and temperature on the embryonic development time of the

rotifer Brachionus plicatilis in culture. in: May, L., Wallace, R., Herzig, A. (Eds.), Rotifer Symposium IV.

Springer Netherlands, pp. 319-322.

Yúfera, M., Pascual, E., 1985. Effects of algal food concentration on feeding and ingestion rates of

Brachionus plicatilis in mass culture. Hydrobiologia. 122, 181-187.

Chapter II

26

CHAPTER II. USE OF THE MICROALGA PAVLOVA VIRIDIS AS ENRICHMENT

PRODUCT FOR THE FEEDING OF ATLANTIC COD LARVAE (GADUS MORHUA)

Rehberg-Haas, Sabine a,f; Meyer, Stefan a; Tielmann, Moritz a; Vadstein, Olav b; Evjemo, Jan

Ove c,d; Lippemeier, Sebastian e; Schulz, Carsten a,f


b NTNU Norwegian University of Science and Technology, Department of Biotechnology,

Trondheim, Norway

c NTNU Norwegian University of Science and Technology, Department of Biology, Trondheim,

Norway

d SINTEF Fisheries and Aquaculture, Trondheim, Norway

e BlueBioTech GmbH, Büsum, Germany

f Institute of Animal Breeding and Husbandry, Christian-Albrechts-University, Kiel, Germany

Chapter II

27

Abstract

A feeding experiment with Atlantic cod larvae (Gadus morhua) was carried out to compare three

different live feed enrichment products for Brachionus ibericus and Artemia salina:

1) Nannochloropsis sp. microalgal concentrate (frozen) (N), 2) Pavlova viridis microalgal concentrate

(frozen) (P), 3) Larviva Multigain® (BioMar, Denmark) - formulated commercial product (CP). Over the

experimental period of 42 days larvae of group P and CP showed significantly higher instantaneous

growth rates (G: 0.074±0.001 d-1 and 0.079±0.009 d-1) than larvae of group N (G: 0.040±0.001 d -1). At

the end of the trial survival of group CP was highest (19.65±7.51 %), whereas significantly lower

survival was observed for group P (6.47±2.02 %) and N (0.04±0.00 %). Live feed ingestion by means of

gut fullness index (GFI), evaluated at 4, 15 and 27 days post hatch (dph), was high for larvae of group

CP (GFI: 1.20±0.09, 1.42±0.03, 1.67±0.19, respectively) and group P (GFI: 1.37±0.06, 1.25±0.05,

1.80±0.15, respectively). Furthermore, larvae fed with CP enriched live feed were able to withstand

an extended period of feed deprivation (of 4 or 5 days), whereas group N and P were not able to re-

initiate growth beyond the point-of-no-return, assessed from 5, 16 and 28 dph on. Bacterial

community diversity of tank water, feed and larvae of the three groups revealed that treatments N

and P lead to similar, highly diverse microbial communities in comparison to group CP. Microalgae

enrichment using Pavlova viridis was not able to sustain the same survival rates as the commercial

product, but resulted in similar growth performance, feed ingestion rates and had a significant effect

on microbial community diversity. Potential mechanisms causing these effects are discussed.

Keywords: Cod larvae (Gadus morhua), microalgae, Pavlova viridis, live feed enrichment, EPA, DHA

Introduction

A major bottleneck in marine aquaculture is the provision of suitable larval diets. Our knowledge

about nutritional requirements of marine fish larvae is very limited (Holt, 2011). The dramatic

morphological and physiological changes in larval ontogenesis, calling for specific nutrients, feeding

behavior and digestion, need to be respected by any given larval feeding regime.

Up to this point conventional dry feed diets can hardly be used to cover the early feeding stages of

many marine fish larvae, even though significant progress was made regarding the formulation of

microdiets (Conceição et al., 2010). Hence, the use of live feed (i.e. Brachionus sp., Artemia sp.) as

initial feed for the rearing of marine fish larvae and crustaceans is still common practice. However,

cultured rotifers or Artemia are deficient in eicosapentaenoic acid (EPA, 20:5n-3) and

docosahexaenoic acid (DHA, 22:6n-3), which are essential for marine fish larval development, and

therefore the enrichment of these live feed organisms is necessary. It was shown that marine larval

growth, survival, brain-, eye-and skeletal development are effected by the amount of

Chapter II

28

polyunsaturated fatty acids (PUFA) (Bell et al., 1995; Izquierdo, 1996; Sargent et al., 1997; van Anholt

et al., 2004).

Marine microalgae in combination with live feed organisms are considered as ideal diet for marine

fish larvae (Brown et al., 1997). Not only the provision of essential PUFA (e.g. EPA and DHA) but also

of other key nutrients such as vitamins, minerals and proteins (essential amino acids), which are

transferred through the food chain, can be secured and adjusted by specifically enriched live feed. As

shown in a previous study (Rehberg-Haas et al., 2015) the microalga Pavlova viridis revealed a high

potential as (frozen) concentrate for the cultivation of rotifers. Pavlova sp. is characterized by a high

content of both DHA and EPA (Volkmann et al., 1991) in contrast to many other commonly used

microalgae, which are only able to synthesize either DHA or EPA in relevant amounts.

The present study aims to evaluate the effects of Pavlova viridis for the enrichment of rotifers

(Brachionus ibericus) and Artemia salina nauplii and their impact on the survival, growth and feeding

performance of Atlantic cod (Gadus morhua) larvae. Furthermore, influences on bacterial community

structure in the culture system, the larvae and the enriched live feed were investigated. The

influences of Pavlova viridis was compared to a commercially available formulated enrichment

product and another commonly used marine microalga, Nannochloropsis sp., that is known for its

green water suitability but also its lack of significant amounts of DHA.

Material and Methods

Experiments were conducted at the Norwegian University of Science and Technology (NTNU), Centre

of Fisheries and Aquaculture (Sealab-CodTechLab) in Trondheim. The analytical work was carried out

at the Gesellschaft für Marine Aquakultur mbH in Büsum, Germany. The analyses of the microbial

communities were done at NTNU, Department of Biotechnology.

Feeding experiment

Cod larvae were reared after standard protocol shown in Table II-1. For each treatment three

replicate tanks (150 L) were used. Rotifers were added to the tanks 4 times d-1 at a density of

5000-8000 ind L-1. All groups were weaned onto Artemia sp. (3000 ind L-1) from 19 dph (days post

hatch) on. From 34 dph on all groups were weaned onto dry feed (Gemma micro 300, Skretting,

Stavanger, Norway) with 8-10 g dry feed tank-1 d-1. Feeding was conducted by means of a feeding

robot (Storvik Aqua AS, Sunndalsøra, Norway). The experiment was terminated after 42 days.

Chapter II

29

Table II-1: Experimental settings of first feeding experiment (D= 24 h dark, L= 24 h light).

Dph 0 1 2 3 4 5 6 7 8 9 10 11-18 19 20

Temp [°C] 7 7 8 8 9 9 9 10 10 11 11 12 12 12

Light D D D L L L L L L L L L L L

Aeration Weak Strong

Water exchange rate [d-1

] 2 2 2 2 2 4 4 4 4 4 4 4 6 6

Rotifer feed [meals d-1

] 2 2 4 4 4 4 4 4 4 4 4

Artemia feed [meals d-1

] 1 1

Dry feed [g tank-1

d-1

]

Clay X X X X X X X X X X X

Dph 21 22 23 24 25 26-32 33 34 35 36 37 38 39-42

Temp [°C] 12 12 12 12 12 12 12 12 12 12 12 12 12

Light L L L L L L L L L L L L L

Aeration Strong

Water exchange rate [d-1

] 6 6 8 8 8 8 8 8 8 10 10 10 10

Rotifer feed [meals d-1

] 4 4 2

Artemia feed [meals d-1

] 1 1 2 4 4 4 4 4 4 3 2 1

Dry feed [g tank-1

d-1

] 3 3 6 6 8 8 10

Clay X X X

All live feed was enriched with one of three different products: 1) Nannochloropsis sp. concentrate

(N), 2) Pavlova viridis concentrate (P), 3) Larviva Multigain® (BioMar, Brande, Denmark)/commercial

product (CP). Both microalgae species were cultivated by BlueBioTech (BBT, Büsum, Germany) under

standard greenhouse conditions (modified F-media, 32 PSU, 23-25°C, pH 8.5) and were concentrated

by flow-through centrifugation.

Algal concentrates were stored at -20 °C and daily portions were thawed each day. The gross nutrient

composition of the experimental treatments is shown in Table II-2.

Table II-2: Nutrient composition of experimental treatments (N, P and CP) in g kg-1

dry matter (DM).

N P CP

Crude ash 61 100 67

Crude protein 230 398 128

Crude lipid 395 196 361

NfE* 314 306 444

Energy (MJ/kg) 26.63 23.26 26.03

*NfE: Nitrogen free extract = 1000 – (crude protein + crude fat + crude ash)

Live feed production and enrichment

Rotifers

Rotifers (Brachionus ibericus) were cultivated in 250 L flow through (100 % d-1) glass fibre tanks with

conical bottoms and 34 ppt seawater at 22 °C. Oxygen saturation (70-110 %) was checked twice a

day. The rotifers were fed continuously with a mixture of baker´s yeast and rotifer diet (Instant

Chapter II

30

Algae®, Reed Mariculture, Campbell, CA, USA). The rotifers were enriched with either

Nannochloropsis sp. concentrate (144 g dry weight L-1), Pavlova viridis concentrate (35 g dry

weight L-1) or with the commercial product for 2 h on a basis of 0.15 g dry weight per 1 x 106

individuals. After enrichment the rotifers were washed and loaded into a cooled holding tank for

delivery by the feeding robot.

Artemia

The Artemia cysts (EG®, INVE Aquaculture, Dendermonde, Belgium) were decapsulated according to

the protocol of Sorgeloos et al. (1977), hatched and enriched in 60 L tanks with heavy aeration at

temperatures of 25-29 °C in 34 ppt seawater for 24 h. The enrichment products were added twice

within 24 h (total amount of 1.5 g dry weight per 1 x 106 individuals). After enrichment the Artemia

sp. were washed and loaded into a cooled holding tank for delivery by the feeding robot.

Larval rearing

The cod eggs originated from group spawning at the Norwegian Cod Breeding Centre (Tromsø,

Norway). The eggs were disinfected with 400 ppm glutaraldehyd for 10 min and rinsed before they

were transferred to a 270 L egg incubator with 34 ppt seawater (4-5 °C, flow rate 3 L h-1). Two days

prior to hatching the eggs were transferred to the experimental tanks of 150 L. The mean egg

diameter was used to calculate the requested volume of eggs to achieve an egg density of

100 eggs L-1. The hatching rate was 90 %.

The temperature was gradually raised from 7 °C at 0 dph to 12 °C at 11 dph. The water exchange rate

was gradually increased from 2 times d-1 to 10 times d-1 at 37 dph (Table II-1).The feeding density was

kept at the given level, without adaption to mortality or growth. Seawater was sand filtered,

microbially matured and filtered through a 1 µm mesh before being used.

Sampling

Samples of ten individual cod larvae for analyses of standard length (SL) and dry weight (DW) were

taken at 0, 3, 6, 12, 18, 24, 30, 36 and 42 dph from each replicate tank. The first five sampled larvae

were used for individual RNA-DNA ratio analyses. Samples of water, live feed and larvae

(4 individuals per tank) for characterization of the bacterial community structure were taken at

18 dph.

All sampled larvae were anesthetized in 3-aminobenzoic acid ethyl ester (MS222), rinsed in purified

water and stored at -20 °C.

For measuring the standard length of the larvae a stereomicroscope M-80 (Leica, Wetzlar, Germany)

and a DFW-SX900 camera (Sony, Minato, Japan) were used.

For dry weight and RNA-DNA analyses larvae were freeze-dried and stored at -80 °C before further

analyses were carried out.

http://en.wikipedia.org/wiki/Troms%C3%B8

Chapter II

31

Growth and survival

The instantaneous growth rate (G in d-1) was calculated according to Ricker (1979):

(1) G = ((ln wt)-(ln wi)) / (tf-ti)

where wt is the dry weight [mg] at time point t [d], wi is the initial dry weight [mg] and tt and ti stand

for the sampling day t and initial sampling day, respectively.

The observed instantaneous growth rates were compared to predicted instantaneous growth rates

(Gpr) calculated according to a sRD-T-G model (Buckley et al., 2008):

(2) Gpr = 0.0145 x sRD + 0.0044 x sRD x T + (-0.078)

where sRD is the standardized RNA-DNA ratio and T is the temperature in °C.

Mortality was estimated based on the number of dead larvae removed from tanks on a daily base

from 21 dph on. At the final sampling day (after 42 days) all remaining larvae were counted and total

survival was calculated.

RNA-DNA analysis

The RNA and DNA contents were measured following the protocol by Caldarone et al. (2003). In

brief, whole body samples of cod larvae were freeze-dried and stored at -80°C until the analysis were

carried out. Nucleic acid content was determined by means of the intercalating fluorescent dye

ethidium bromide (Caldarone et al., 2001; Clemmesen, 1987) in crude homogenate. After addition of

RNAse and DNAse in two consecutive steps with intermediate fluorescence readings, the nucleic

acids were quantified from the difference between first and second reading and between second and

third reading, respectively. RNA-DNA ratios were standardized (sRD) to a reference slope ratio based

on the assay-specific ratio of the slopes of the standard curves (Caldarone et al., 2006).

Ingestion

To estimate the ingestion depending of different enriched live feed 50 larvae from each replicate

tank (three tanks per treatment) were transferred to 10 L tanks. Here they were kept for 4 h to allow

for complete gut evacuation. Afterwards rotifers (6000 ind L-1) or Artemia sp. (3000 ind L-1) enriched

with the experimental products were added to each 10 L tank according to the original treatment.

The larvae fed on the respective live feed for 1 h, before they were removed from the tanks,

snap-frozen and gut content (number of ingested live feed organisms) was analyzed. This procedure

was repeated at 4, 15 and 27 dph. The gut fullness index (GFI) was estimated by classifying individual

larvae by the number of ingested live organisms into three groups: 1) low ingestion (0-33% of

maximal number of live feed), 2) medium ingestion (34-66%) and 3) high ingestion (67-100%)

referring to the maximal number of live feed that was ingested at the specific sampling day. An

average value was calculated for each replicate tank according to van der Meeren et al. (2007):

(3) GFI = N1 + 2N2+ 3N3 / (N1 + N2 + N3)

Chapter II

32

where N1, N2 and N3 are the numbers of larvae referring to gut fullness groups 1, 2 and 3.

Feed depletion

In an additional side experiment condition of larvae under feed depletion and re-feeding was

observed (Clemmesen, 1987; Meyer et al., 2012). Groups of 200 larvae were loaded from respective

rearing tanks of the growth experiment (see above) into separate 10 L tanks in a temperature

controlled environment. No feed was provided until the point-of-no-return (PNR; Blaxter, Hempel,

1963) was reached, which was determined in an associated study. For that purpose four triplicate

groups of 100 cod larvae each were feed depleted and re-fed after 1, 2, 3 or 4 days, respectively. The

PNR was defined at that point in time, when less than 50 % of the larvae were able to re-start

feeding. In the feed depletion trial five individual larvae were sampled from each tank every second

day during the feed depletion phase and at the PNR in order to assess the change of body weight and

RNA-DNA ratio of fasting larvae. Before starvation-induced mortality (PNR) occurred, larvae were

offered feed items of their respective treatment group and the growth and the RNA-DNA ratio were

assessed again after 4 more days. The feed depletion trial was repeated three times (run A, B, C)

during the whole experimental period starting at 5, 16 and 28 dph, respectively.

Analysis of bacterial community structure by PCR/DGGE

DNA extraction, PCR (polymerase chain reaction) and DGGE (denaturing gradient gel electrophoresis)

analysis were performed as described by Bakke et al. (2013). DNA was extracted using the DNeasy

Blood and Tissue Kit (Qiagen, Venlo, Netherlands), a fragment of variable region 3 of the 16S rRNA

gene was amplified using primers 338f and 518r, and DGGE was run on the INGENYphorU DGGE

system (Ingeny, Netherlands) with a denaturing gradient of 35–55 %. For larvae and live feed a

nested PCR protocol was used to exclude possible co-amplification of eukaryotic 18S rDNA (Bakke et

al., 2011). The DGGE images were analyzed using the Gel2K program to make a peak area versus

sample spreadsheet (developed by Svein Nordland, University of Bergen), where the peak areas

reflect the intensities of the bands. Prior to further treatment the peak areas were normalized by

converting them to a percentage of the sum of all peaks for each DGGE profile. To avoid gel-to-gel

comparisons which introduce bias, samples were taken only from two of three tanks per treatment.

In the selection tanks with untypical behavior in terms of growth and survival were avoided.







Chapter II

33




failed).

To describe the diversity of the microbial community the following indices were calculated:

(4) Band richness S = number of bands

(5) Dominance D = ∑i (ni/n)2 where n is the intensity od band i

(6) Simpson = 1-D

(7) Shannon H = - ∑i (ni/n) x (ln (ni/n)

(8) Equitability J = H/ln S

For visualization of similarity in the bacterial communities between samples, ordination based on

Bray–Curtis similarities was performed using non-metric multidimensional scaling (NMDS). To test for

differences in Bray-Curtis similarities between groups of samples Permutational Multivariate Analysis

of Variance (PERMANOVA, Anderson (2001)) was used. The program package PAST version 2.04

(Hammer et al., 2001) was used for statistical analysis of the bacterial communities.

Results

Growth and survival

Enrichment of live feed with the commercial product resulted in the best growth performance of cod

larvae, though final body weight and instantaneous growth rate were not significantly different to

those of group P, whereas significantly lower numbers were found for group N. The survival was

significantly higher in the CP group in comparison to the other groups (Table II-3).

Table II-3: Growth performance and survival of cod larvae of the three experimental groups N, P and CP (mean±SD, n=3). Values with the same superscript are not significantly different (p<0.05).

IBW1 FBW

2 G

3 Survival

4

N 0.056±0.017 0.305±0.080 a 0.04±0.001

a 0.04±0.00

a

P 0.057±0.012 1.278±0.346 a,b

0.074±0.001 b 6.47±2.02

a

CP 0.058±0.010 1.716±0.819 b 0.079±0.009

b 19.65±7.51

b

1 Initial body dry weight observed at 0 dph [mg] 2 Final body dry weight observed at 42 dph [mg]

3 Instantaneous growth rate [d-1

] 4 Survival assessed on 42 dph [%]

Chapter II

34

Figure II-1: Mortality depicted as cumulative mortality of absolute numbers of dead larvae being removed once a day (upper panel) and depicted as absolute number of dead larvae (lower panel) (mean±SD, n=3). + stands for

significant differences: Na, P

ab, CP

b and ++ stands for significant differences N

a, P

a, CP

b. Values with the same

superscript are not significantly different (p<0.05).

The cumulative mortality (Figure II-1) was highest in group N and group P, whereas the mortality of

group CP was significantly lower. Daily mortality in CP was initially as higher than in the other

treatments (around 200 per day) and then decreased and remained on a very low level from day 25

post hatch onward. Mortality in N and P was consistently high throughout the trial, with occasional

peaks.

No significant differences in dry weight or standard length between the treatment groups were

recorded until 30 dph (Figure II-2). However, from this sampling date onward larvae in group N

showed no further biomass increase in comparison to larvae of group P and CP. The development of

the instantaneous growth rate (G; calculated for the period between consecutive sampling events)

shows increasing values for all groups until 30 dph, followed by a distinct decrease. In the end of this

trial the CP and P larvae had increased their dry weight almost 30-fold and 22-fold, respectively,

showing significantly higher values for standard length and body dry weight, whereas the larvae of

the N group have only quintupled their dry weight.

Chapter II

35

Figure II-2: Body size and instantaneous growth rate (G) of cod larvae fed with differently enriched live feed (N, P, CP) over the experimental period of 42 days (mean±SD, n=3). + stands for N

a, P

ab, CP

b and ++ stands for N

a,

Pb, CP

b. Values with the same superscript are not significantly different (p<0.05).

RNA-DNA analysis

Standardized RNA-DNA ratios (sRD) were high in yolk sac larvae (around 5, 0 dph) and decreased

continuously to values around 1 at the end of the trial, with all three treatments exhibiting similar

trajectories (Figure II-3). Altogether in the end of the trial sRD values of all three groups were

significantly different showing the highest value in the N (1.65±0.09) group and the lower values in

group P (0.79±0.09) and CP (0.53±0.14).

Chapter II

36

Figure II-3: Standardized RNA-DNA ratio (sRD) shown as one boxplot for each treatment (N, P, CP) over the experimental period of 42 days (n=3). + stands for significant differences: N

a, P

b, CP

b. Values with the same

superscript are not significantly different (p<0.05).

In Figure II-4 the relationship between sRD and instantaneous growth rate G is shown. The predicted

instantaneous growth rate (Gpr) was calculated from a previously published sRD-T-G model (Buckley

et al., 2008), which is used to evaluate observed G in cod larvae. Due to the high impact of

temperature only values of the sampling period with constant temperature (12°C, see Table II-1) are

included in this graph. The figure shows that realized G is mostly higher than calculated Gpr with sRD

values smaller than 2.5 in group P and CP. In contrast realized G of group N is consistently lower than

Gpr. The G-Gpr residuals show that these effects occur from sampling day 24 post hatch onwards,

whereas during the first sampling days all groups revealed lower G than predicted by the sRD-T-G

model (Figure II-5).

Chapter II

37

Figure II-4: Dry weight-specific instantaneous rates of growth of cod larvae of the different treatment groups (N, P, CP) plotted against replicate tank average sRD values. The black line represents the predicted

instantaneous growth rate (Gpr) according to the sRD-T-G model (Buckley et al., 2008). Depicted are values of the sampling phase of constant temperature (12°C).

Figure II-5: Residuals of observed instantaneous growth (G) – predicted instantaneous growth (Gpr) calculated

for the three experimental groups (N, P, CP). Depicted are values of the sampling phase of constant temperature (12°C). The predicted instantaneous growth was calculated according to the sRD-T-G model of

Buckley et al. (2008).

Chapter II

38

Ingestion

A higher feeding of the larvae of group P and CP at 4 dph and 27 dph was observed, whereas at

15 dph for both the P and N group a lower GFI was recorded in contrast to the value of group CP

(Figure II-6).

Figure II-6: Ingestion by means of gut fullness index (GFI) depending on the different experimental enrichment products (N, P, CP) at 4, 15 and 27 dph (mean±SD, n=3). Values with the same superscript are not significantly

different (p<0.05).

Feed depletion

Figure II-7: Standardized RNA-DNA ratio (sRD) of larvae from the different experimental groups (N, P, CP) in the feed depletion experiment (mean±SD, n=3). The trial was repeated three times starting at dph 5 (A), dph 16 (B) and dph 28 (C). The arrows indicate the day of re-feeding. Different superscripts indicate significant differences

(p<0.05).

Chapter II

39

The results of the feed depletion experiment are summarized in Figure II-7. Altogether only larvae

from group CP survived the feed depletion phase and re-feeding phase of run B and C. No live larvae

from group N and P were found at the final sampling days in run B and C. The sRD values of all groups

were highest during run A and were ranging at the same level during the feed depletion phase. The

sRD of run B started at similar values like run A, even so the values declined clearly during the feed

depletion phase. The condition of CP larvae rose only slightly during the days of re-feeding. Starting

at lower sRD levels in the last run (C) all groups showed sRD values ranging at constant levels.

DGGE analysis

The diversity indices for the microbiota are shown in Table II-4. The feed samples were similar, so

that they were pooled. CP larvae had lower richness and Shannon diversity, but this was not

reflected in the microbiota of the water. It is a tendency that there are more dominating bands in CP

larvae, indicated by Dominance and Equitability. This is the only consistent difference in the diversity

indices.

Table II-4: Diversity indices for the microbiota of larvae (L), feed (F) and water (W) samples of the three treatment groups N, P and CP (mean±SD, n=2). Different superscripts indicate significant differences (p<0.05).

Band Richness Dominance Simpson Shannon Equitability

L-N 25.3±2.1a 0.11±0.04

a 0.89±0.04

a 2.58±0.25

a 0.80±0.07

a

L-P 26.5±3.0a 0.12±0.03

a 0.88±0.03

a 2.53±0.22

a 0.77±0.05

ab

L-CP 17.0±1.4b 0.24±0.04

b 0.76±0.04

b 1.89±0.18

b 0.66±0.06

b

W-N 24.0±2.8ab

0.11±0.01a 0.89±0.01

a 2.560±0.16

a 0.80±0.02

a

W-P 20.5±2.1ab

0.11±0.00a 0.88±0.00

a 2.51±0.10

a 0.83±0.00

a

W-CP 23.5±2.1ab

0.10±0.01a 0.90±0.01

a 2.68±0.09

a 0.85±0.00

a

F-All 25.0±2.6a 0.07±0.01

a 0.93±0.01

a 2.81±0.09

a 0.88±0.01

a

Figure II-8: Non-metric multidimensional scaling (NMDS) based on Bray-Curtis distance for larval samples only (4 larvae per tank, n=2).

Chapter II

40

A 2D ordination based on Bray-Curtis distance using non-metric multidimensional scaling (NMDS)

indicated less individual-to-individual variability in the microbiota of the CP larvae than for the other

two treatments, and for all three treatments there was an overlap between replicate tanks (data not

shown). Larvae from the CP group seemed to cluster without overlap with the larvae from group N

and P, which were overlapping (Figure II-8). Statistical analysis (PERMANOVA) using the tanks as the

unit for comparison confirm this. Multiple comparison (sequencial Bonferroni significance) revealed

that the replicate tanks were not significantly different. The microbiota of larvae from the CP

treatment was significantly different from the other two, which had similar microbiota.

The effect of the microbiota in the water (W) and the feed (F) on the microbiota of the larvae (L) can

be quantified by comparing individual larvae versus water and feed, respectively using Bray-Curtis

similarities (Table II-5). In general the Bray-Curtis similarities were low, 0.15-0.20 and 0.10-0.15 for

comparisons with feed and water, respectively. A paired t-test revealed that larvae had a microbiota

that was significantly more similar to the feed than to the water microbiota (p < 0.03).

Table II-5: Bray-Curtis-index - Comparison of larvae vs. feed and water of the treatments N, P and CP (mean±SD, n=2).

N P CP

L vs. F 0.20±0.03 0.20±0.03 0.15±0.04 L vs. W 0.16±0.04 0.14±0.03 0.10±0.03 Paired t-test 0.031 0.003 0.024

Discussion

The evaluation of the microalga Pavlova viridis as enrichment product revealed promising results

concerning growth and development of cod larvae. The final standard length and dry weight of larvae

of group P ranged from 8.4 to 12.4 mm and from 0.6 to 1.9 mg and of group CP from 9.8 to 14.2 mm

and from 0.8 to 3.4 mg. These values are in accordance with findings for optimal cod larvae grown

under similar conditions (Kortner et al., 2011; O'Brien-MacDonald et al., 2006; Puvanendran, Brown,

1999). However, the significantly poorer growth rate of the N larvae is below the limits for a decent

growth of Atlantic cod larvae. A similar trend can be found in the survival data. The highest value of

19.7±7.5 % survival was recorded for the CP group. Both significantly lower were the survival data for

the P and N group of 6.5±2.0 % and 0.04±0.0 %, respectively. The almost total mortality of the N

group is probably due to the lack of the essential fatty acid DHA in this enrichment product (Patil et

al., 2007; Lippemeier, unpublished results). It has been found that dietary essential fatty acids

derived by marine microalgae either as green water application or live feed enrichment are

important for positive larval survival and growth (Howell, 1979; Reitan et al., 1997). In general the

lowest mortality was recorded in the CP group, which was even decreasing towards the end of the

experiment. In the N group the highest mortality occurred during the midtime of the experiment,

Chapter II

41

leading to strongly reduced larval numbers remaining in the tanks and therefore decreasing numbers

of additional dead larvae. The highest numbers of dead P group larvae taken out during the period

between 21 dph and 42 dph were counted around 36 dph. This coincides with the weaning process

indicating difficulties in the dry feed utilization and digestion of the P group larvae. However, higher

survival rates of around 30 % can be expected even with earlier weaning processes around 8 dph as it

was shown by Baskerville-Bridges and Kling (2000). The high mortality rates in all three groups could

not be explained by the recorded environmental or feeding conditions. However, in general low

survival rates of less than 30 % from larvae to fingerling stages are still one of the major bottlenecks

in marine aquaculture.

The sRD ratio gives also information about the larval performance. The development of the DNA

content is mainly in accordance with the growth progress of the three experimental groups and

therefore reflects the biomass and the cell number of the larvae. On the contrary the RNA content is

determined not only by the nutritional condition of the larvae, but also by the temperature.

Especially the rRNA activity increases with temperature (Buckley et al., 1999). This can be seen by the

increase of sRD from 12 dph on, which coincides with the rise of temperature to the highest applied

temperature of 12 °C during this experiment (Table II-1 and Figure II-3). However, the decrease of

sRD, going along with a temperature rise from 7 °C up to 9 °C from 0 dph to 6 dph is not in

accordance with this explanation. This sRD decrease and the low sRD values at 6 dph can be rather

explained by the coinciding exhaustion of the yolk-sac (Caldarone et al., 2003). Furthermore, the sRD

content shows a distinct drop for all three test groups at 30 dph and all three groups showed almost

stagnating sRD values at the last three sampling days, whereas group N revealed the highest sRD

values from 30 dph on. The increasing instantaneous growth rates in all groups up to 24 dph going

along with decreasing sRD values cannot be explained by the sRD-T-G model of Buckley et al. (2008).

Especially from 24 dph on the instantaneous growth rates of group P and CP are higher than

predicted by the model, whereas the values of group N are lower than predicted. The relationship of

sRD, T and G in this model is described to be independent of feeding type (Buckley, 1982; Caldarone,

2005), because in field studies feed type is assumed to be optimal. However, in this study distinct

differences of G have been found depending on the different feeding groups P, CP and N. Opposed

development of larval growth and sRD values was observed in this study especially in group CP and P,

which is not in accordance with common notion describing increasing G with larval size and sRD

(Buckley, 1984; Buckley et al., 1999; Buckley et al., 2008; Caldarone et al., 2006). The increase of

growth in this study particularly in group P and CP might rather be accomplished by a high RNA

activity than by an increase of RNA amount, especially from 30 dph on. This might be indicated by a

change to a more efficient synthesis of proteins by ribosomes and/or more efficient protein retention

as it was also hypothesized by Mathers et al. (1994) for larval herring (Clupea harengus). The

Chapter II

42

decreasing sRD values, especially from 30 dph onwards, based on increasing DNA content might also

be a sign for increased cell proliferation (hyperplasia). Also in early development of Japanese

flounder (Paralichthys olivaceus) (Gwak, 2001) and larval coregonid fish (Malzahn et al., 2003) a

change of phases of hypertrophy (cell enlargement, RNA-DNA ratio is rather high) and hyperplasia

(high DNA content) was described. Both enrichment products, the natural microalga Pavlova viridis

and the commercial product Larviva Multigain, seem to provide highly available nutrients for the cod

larvae and therefore leading to high biomass gain, although RNA amount and sRD values were

decreasing or stagnating. The Pavlova viridis enrichment even lead to similar growth rates of cod

larvae like the commercial product, although it provided less energy and lipid than the Larviva

Multigain enrichment (Table II-2). The high protein content (398 g kg-1 DM) of the Pavlova viridis

enrichment might have been favorable for the larval growth as well, as it was also found for turbot

(Scophthalmus maximus) larvae, where rotifers rich in protein (364 g kg-1 DM) could promote growth

and survival (Øie et al., 1997). However, it is still a complex task to investigate the digestive capacities

and feed efficiency in fish larvae and although novel methodologies (e.g. tracer studies) can help to

learn more about digestibility of different nutrients and nutritional effects these methods are still

time consuming and expensive.

Larval performance was also investigated using the gut fullness index (GFI) observed in the ingestion

trial. The GFI shows higher values for the CP and P group during all three runs (4, 15 and 27 dph). On

the one hand these findings might be due to a higher preference of larvae for P and CP enriched live

feed, maybe because of favorable olfactory effects. It was found by Døving et al. (1994) that newly

hatched cod larvae indeed possess olfactory organs that allow direct reactions to chemical stimuli,

which are send out by prey organisms (Dempsey, 1978). It was also found that cod larvae react more

strongly to increasing intensities of chemical stimuli like increasing arginine concentrations. On the

other hand the results again indicate a better performance of the P and CP group in comparison to

larvae of group N. Therefore, the lower levels of GFI and lower number of prey capture events might

also be due to the low DHA content in the feed of these larvae. In this context it is known that

successful capture of prey by fish larvae is mainly driven by vision (Bell et al., 1995) and that dietary

deficiencies of DHA can hamper visual abilities strongly.

Furthermore, the ability of the different test groups to cope with phases of feed deprivation was

validated. The experimental groups showed decreased sRD values after two days of feed depletion in

all three runs (A, B, C), as it was also shown in other studies (Buckley et al., 1999; Caldarone, 2005).

Only in group CP in run A and in group P in run C, higher sRD values were found at the second day of

feed deprivation. Altogether the highest condition of larvae by means of sRD was observed in the CP

group. This is also underlined by the fact that during run B and C only larvae of group CP survived

until the last sampling day. Furthermore, in run B group CP showed slightly increased sRD values

Chapter II

43

after the re-feeding phase indicating that re-initiation of nutrient utilization and therefore growth

and biosynthesis beyond the point-of-no-return might be possible. Hence, the highest ability to cope

with feed depletion was observed for larvae of group CP. Larvae of this group might have had better

and more energy resources, due to a more efficient nutrient utilization, which lead to a higher

resistance to feed deprivation. The ability of fish larvae to overcome phases of starvation is

dependent on the quality and amount of energy stores, which contain mainly protein, carbohydrate

and less lipid in early stage fish larvae (Clemmesen, 1987; Ehrlich et al., 1976). However, it has to be

considered that severe damage of the epithelial cells of the digestive tract can occur due to

starvation especially in early developmental stages of cod larvae (Kjørsvik et al., 1991). Therefore, a

re-initiation of growth after re-feeding can be hampered independent of feed type.

Another variable that could have had an effect on the performance of the larvae is induction of

differences in the microbiota of the larval gut due to the different enrichment products. We found in

fact that the larvae of the CP group revealed a different microbial community than the other two

groups. The highly available nutrients from the CP treatment might have increased the carrying

capacity and the growth of heterotrophic microflora (Attramadal et al., 2012). The different findings

between the algal groups and the CP group could have contributed to the differences in

performances during the feed depletion trial, because bacteria associated with larvae can induce

positive, as well as negative effects. However, the feed samples were highly similar

(Bray-Curtis > 0.78), as were the microbial communities of the water samples between treatments

(Bray-Curtis > 0.61, average 0.69). Thus the significantly different microbial communities of the

larvae from the CP treatment cannot be explained by a passive colonization, but rather a selective

colonization based on the available species pool. However, SIMPER analyses suggest that for the

seven bands contributing to more than 50 % of the differences in the microbial community of the CP

larvae versus the other larvae, only one of the seven bands were consistently different between the

feed samples. The difference in microbial communities might be due to the fact that both

Nannochloropsis sp. and Pavlova viridis as pure algae products promote different bacterial

communities than the commercial product. Furthermore, Nicolas et al. (1989) found that rotifers

grazed more on bacteria to compensate for possible nutritive deficiencies when being cultured with

Pavlova and other algae. However, mainly advantages (e.g. antibiotic effects) are attributed to the

use of microalgae as greenwater product or enrichment product (Skjermo, Vadstein, 1993). The

comparison by Bray-Curtis similarities for individual larvae versus water and feed, respectively,

revealed that larvae had a microbiota that was significantly more similar to the feed than to the

water microbiota. This is opposite to previous studies (Bakke et al., 2013). The fact that similarities in

general were low and that between treatments comparisons of microbiota of larvae versus

feed/water revealed the same tendency suggests that these differences are of limited biological

Chapter II

44

relevance. So far the impact of microalgae on the microbiota of live feed is not fully understood.

However, it is hypothesized that microalgae can change the microbial composition by production of

antibacterial substances (Austin, Day, 1990; Kellam, Walker, 1989).

Altogether the present study shows a high potential of the natural microalga Pavlova viridis as

enrichment product expressed by a positive larval performance in contrast to the microalga

Nannochloropsis sp. Further information about the feed efficiency and digestibility would help to

evaluate the use of Pavlova viridis in fish larvae nutrition more precisely. However, the product as

raw material is not yet a realistic competitor to an optimized commercial product. Nevertheless, the

results of this study can suggest further application of Pavlova viridis for example as ingredient in

enrichment formulations.

Acknowledgments


28183. The access to the NTNU Sealab was funded by the European Union‘s Seventh Framework

Programme (FP7/2007-2013) under grant agreement n° 262336 via the Aquaexcel project. We would

like to thank the members of the NTNU Sealab for their support. We thank Elin Kjørsvik for her

support and advice during the experimental period in Trondheim. Furthermore, we thank Ingrid

Bakke and Thi Truc Ly Trinh for analytical help.

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Kjørsvik, E., van der Meeren, T., Kryvi, H., Arnfinnson, J., Kvenseth, P.G., 1991. Early development of

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Kortner, T.M., Overrein, I., Øie, G., Kjørsvik, E., Arukwe, A., 2011. The influence of dietary

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Mathers, E.M., Houlihan, D.F., Burren, L.J., 1994. RNA, DNA and protein concentrations in fed and

starved herring Clupea harengus larvae. Marine Ecology-Progress Series. 107, 223-223.

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Holt, G.J., Høie, H., Kanstinger, P., Malzahn, A., Moran, D., Petereit, C., Støttrup, J.G., Peck, M.A.,

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Øie, G., Makridis, P., Reitan, K.I., Olsen, Y., 1997. Protein and carbon utilization of rotifers (Brachionus

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rearing. Aquaculture. 265, 206-217.



Chapter III

48

CHAPTER III. MARINE MICROALGAE PAVLOVA VIRIDIS AND

NANNOCHLOROPSIS SP. AS N-3 PUFA SOURCE IN DIETS FOR JUVENILE

EUROPEAN SEA BASS (DICENTRARCHUS LABRAX)

Rehberg-Haas, Sabine a,e; Bauer, Jonas L. b; Adakli, Aysun c; Meyer, Stefan a; Lippemeier,

Sebastian d; Schwarz, Karin b; Schulz, Carsten a,e


b Department of Food Technology, Institute of Human Nutrition and Food Science,

Christian-Albrechts-University, Kiel, Germany

c Fisheries Faculty, Department Aquaculture, Cukurova University, Adana, Turkey

d BlueBioTech GmbH, Büsum, Germany

e Institute of Animal Breeding and Husbandry, Christian-Albrechts-University, Kiel, Germany

Chapter III

49

Abstract

In the present study the potential of the microalga Pavlova viridis as n-3 polyunsaturated fatty acid

(PUFA) source was evaluated and compared to Nannochloropsis sp. in diets for juvenile European sea

bass (Dicentrarchus labrax L.) (initial weight ~12.8±1.7 g) in an eight-week lasting feeding trial. Six

different isoenergetic and isonitrogenous test diets were used: 1) Control diet (C): Major lipid source

fish oil (100 %), 2) Basal diet: 40 % fish oil and 60 % vegetable oil (in equal parts rapeseed-,

sunflower- and linseed oil), 3) Pavlova 50 % (P50): 50 % of the remaining fish oil of the basal diet was

substituted by lipid content of Pavlova viridis meal, 4) Pavlova 100 % (P100): 100 % of the remaining

fish oil of the basal diet was substituted by lipid content of Pavlova viridis meal, 5) Nannochloropsis

50 % (N50): 50 % of the remaining fish oil of the basal diet was substituted by lipid content of

Nannochloropsis sp. meal, 6) Nannochloropsis 100 % (N100): 100 % of the remaining fish oil of the

basal diet was substituted by lipid content of Nannochloropsis sp. meal. The specific growth rate was

highest and feed conversion ratio was lowest in the P100 group (1.77±0.10 % d-1 and 1.17±0.01),

although not significantly different to the results for the control and the other algae-groups.

Furthermore, the sum of PUFA was also highest in fish samples of group P100, followed by the P50,

N100, N50 and B group, with the lowest levels in the control group. The highest amounts of

docosahexaenoic acid (DHA) of total fatty acids were found in fish of the control and basal group,

although not significantly higher than the values of groups P50 and P100. The significantly highest

amount of eicosapentaenoic acid (EPA, % of total fatty acids) was measured in the P100 fish samples

and the lowest amount was measured in samples of the basal group. The histological analyses of liver

and intestine samples did not reveal any negative effects caused by the experimental treatments.

Keywords: Sea bass (Dicentrarchus labrax), microalgae, Pavlova viridis, Nannochloropsis sp., n-3 PUFA

Introduction

The identification and utilization of alternative feed sources is still of highest interest in aquaculture

development. One major topic is the use of alternatives to fish meal and fish oil as stagnating

production rates with simultaneously increasing request for these products are heavily influencing

market prices. A lot of research has been addressed to the utilization of alternative energy sources

(Tacon, Jackson, 1985) and a diverse array of ingredients has been used so far for fish feed in order to

meet the species specific nutritional requirements and still considering the cost efficiency. For

example plant processed products like meals, brans and grains, as well as animal-processing by-

products, like feather or blood meal or other animal based products like insect meals have been used

(FAO, 2005). However, both plant and animal derived feed ingredients still feature some difficulties

like low palatability, low digestibility or antinutritives, such as phytate and protease inhibitors

Chapter III

50

(Francis et al., 2001; Tacon, Jackson, 1985; Tusche et al., 2011). Additionally the application of some

animal-processing by-products is limited by consumer´s reservations against some product classes

(Naylor et al., 2009). Microalgal products are another alternative energy source for the use in fish

diets. They have a high potential as they contain high amounts of important macro- and

micronutrients. They are the natural feed of herbivorous zooplankton and bivalves and provide the

basis for almost all aquatic food chains. So far microalgae have been used successfully as enrichment

for live feed organisms for feeding of fish larvae, but first studies also showed positive results of

microalgae used as ingredients for dry fish feed (Benemann, 1992; Tartiel et al., 2008). Especially the

high amounts of essential polyunsaturated fatty acids (PUFA), like docosahexaenoic acid (DHA,

22:6n-3) or eicosapentaenoic acid (EPA, 20:5n-3), characterize marine microalgae as promising

alternative to fish oil, which usually serves as main PUFA source in fish diets.

As marine finfish species are not able to convert C18 fatty acids to C20 and C22 PUFA in higher

amounts, they have a high requirement for long chain n-3 PUFA, especially for EPA and DHA (Sargent

et al., 2002). Therefore, these essential PUFA need to be supplied via feed items. Furthermore, the

supply with the essential PUFA is not only important for the health and condition of the fish, but the

high PUFA content is characterizing the nutritive quality of fish as food for human consumption

(Simopoulos, 1991).

In this context the microalga Pavlova viridis is of special interest, because it is able to synthesize both

essential PUFA, DHA and EPA, in considerable amounts (Pereira et al., 2004; Volkmann et al., 1991),

in contrast to most other microalgae, which are able to synthesize either DHA or EPA in higher

concentrations (Volkman et al., 1989). For example the microalga Nannochloropsis sp. is known to

contain mainly EPA and nearly no DHA (Patil et al., 2007).

In the present study the potential of the microalgae Pavlova viridis and Nannochloropsis sp. as n-3

PUFA sources in nutrition of marine finfish was evaluated. In a feeding trial utilizing these microalgae

as n-3 PUFA source as alternative to fish oil in dry diets for juvenile European sea bass (Dicentrarchus

labrax) the growth performance, body composition, fatty acid composition and liver and intestine

histology were investigated.

Material and methods

Experimental setup

The trial was conducted at the experimental facilities of the Gesellschaft für Marine Aquakultur

(GMA) in Büsum, Germany. For the feeding trial 540 juvenile sea bass (12.84±1.67 g) were

acclimatized for 28 days in the experimental system. The tanks were arranged in a recirculation

system (4 m3 total water volume, water turnover rate 4 h -1). The water purification system consisted

of a mechanical filter and a moving bed filter as biological treatment unit. Water quality parameters

Chapter III

51

were determined daily (20.9±0.8 °C, 7.2±0.6 mg L-1 O2; Handy Polaris; Oxy-Guard International A/S,

Birkerod, Denmark; 0.4±0.3 mg L-1 NH4–N; 0.5±0.2 mg L-1 NO2–N; Microquant test kit for NH4 and

NO2; Merck KGaA, Darmstadt, Germany). The light conditions were set to 12h/12h light/dark cycle.

Prior to the experiment six triplicate groups of 30 sea bass each were randomly stocked in 18 tanks

(150 L each). Over the entire experimental period of 56 days, fish were fed by hand to apparent

satiation twice per day and daily feed intake (DFI) was recorded.

Experimental diets

Six different isonitrogenous and isoenergetic diets were formulated as shown in Table III-1. The

control diet (C) contained fish oil as major lipid source. In the basal diet (diet B) 60 % of fish oil was

substituted by a vegetable oil mixture (in equal parts rapeseed-, sunflower- and linseed oil). Diet B

served as an example for commercially available sea bass diets. Four additional test diets were

formulated substituting 50 % and 100 % of fish oil, based on the B diet, by the lipid content of the

microalga Pavlova viridis (P50 and P100) and Nannochloropsis sp. (N50 and N100). The microalgae

were cultivated using greenhouse technique (modified F-media, 32 PSU, 23-25°C, pH 8.5) by

BlueBioTech (BBT, Büsum, Germany). They were harvested by means of flow-through centrifugation.

The raw material was then lyophilized and homogenized to a mesh size of 300 µm (GM 200, Retsch,

Haan, Germany). All diets were pressed into 4 mm pellets using a feed press L14-175 (Amandus Kahl,

Reinbek, Germany).

In order to meet species specific amino acid (AA) requirements limiting methionine was

supplemented (Table III-1). Inert filler was included to maintain the isonitrogenous and isoenergetic

approach. The fatty acid composition of the test diets is shown in Table III-2.

Chapter III

52

Table III-1: Ingredients, nutrient composition and amino acid composition [g kg-1

dry matter] of the experimental diets C, B, P50, P100, N50 and N100.

C B P50 P100 N50 N100

Ingredients [g kg−1

]

Fish meal a 200 200 200 200 200 200

Soy protein

b 170 170 170 170 170 170

Pea protein

c 120 120 120 120 120 120

Wheat starch

d 161.2 160.9 138.4 114.9 148.9 137

Wheat gluten

d 240.7 240.6 192.5 144.5 228.5 216.3

Vitamin-Mineral Mix

e 10 10 10 10 10 10

Fish oil

a 79.5 31.8 15.9

15.9

Sunflower oil

f

16 16 16 16 16

Rapeseed oil

f

16 16 16 16 16

Linseed oil

f

16 16 16 16 16

Pavlova viridis

g

96.3 192.5

Nannochloropsis sp.

g

42.2 84.4

Methionine

h 1.1 1.1

1.2 1.4

Inert filler

i 17.6 17.6 8.8

15.2 12.9

Nutrient composition [g kg

−1]

Dry matter 927 929 931 937 934 934

Crude protein 559 564 562 561 563 568

Crude lipid 128 132 131 131 130 128

Crude ash 74 74 75 76 74 76

NfE* 239 230 232 232 233 228

Gross energy [MJ kg

−1 DM]

22.68 22.53 22.51 22.70 22.50 22.60

Essential amino acids [g kg−1

]** Req.***

Arginine 27.4 27.4 32.0 36.6 27.1 26.7 23.0

Histidine 12.0 12.0 12.7 13.5 11.8 11.6 8.0

Isoleucine 19.5 19.5 21.7 24.0 19.1 18.7 13.0

Leucine 36.8 36.8 40.0 43.2 36.0 35.3 21.5

Lysine 23.4 23.4 27.2 31.0 23.2 23.0 23.0

Methionine 10.0 10.0 10.1 11.3 10.0 10.0 10.0

Cysteine 7.5 7.5 7.0 6.4 7.3 7.0 5.0

Phenylalanine 23.8 23.8 26.6 29.4 23.3 22.8 13.0

Threonine 16.0 16.0 18.9 21.8 15.8 15.6 13.5

Valine 23.1 23.1 26.5 29.9 22.7 22.3 14.5 aVereinigte Fischmehlwerke Cuxhaven GmbH & Co. KG, Cuxhaven, Germany;

bHP 350, Hamlet Protein A/S,

Horsens, Denmark, Germany; cEmsland Aller-Aqua GmbH, Golßen, Germany;

dKröner Stärke GmbH,

Ibbenbüren; eVitamin and Mineral mixture 517158 and 508240, Vitfoss, Gråsten, Denmark;

fDifferent food

stores, Germany; gBlueBioTech GmbH, Büsum, Germany;

hCarl Roth GmbH, Karlsruhe, Germany;

iCaPO4

Lehmann & Voss Co. KG, Hamburg, Germany *NfE: Nitrogen free extract = 1000 – (crude protein + crude fat + crude ash) **The amino acid compositions were calculated based on previous analysis of ingredients, which were performed in accordance to Llames, Fontaine (1994). ***Requirements according to Tibaldi and Kaushik (2005).

Chapter III

53

Table III-2: Fatty acid composition of the experimental diets C, B, P50, P100, N50 and N100. Σ SFA is the sum of saturated fatty acids, Σ MUFA is the sum of monounsaturated fatty acids, Σ PUFA is the sum of n-6 and n-3 polyunsaturated fatty acids, Σ n-3 is the sum of n-3 polyunsaturated fatty acids, Σ n-6 is the sum of n-6 polyunsaturated fatty acids.

[% of FAMEs*] C B P50 P100 N50 N100

8:0 n.d.** n.d. n.d. n.d. tr n.d.

10:0 n.d. n.d. n.d. n.d. tr n.d.

12:0 0.10 tr*** tr tr tr tr

13:0 n.d. n.d. tr 0.41 tr tr

14:0 5.11 2.70 3.18 3.60 2.64 2.41

15:0 0.35 tr tr tr tr tr

16:0 13.35 10.65 10.57 10.62 12.68 14.11

17:0 tr tr tr tr tr tr

18:0 2.01 2.53 2.45 2.43 2.60 2.21


22:0 n.d. tr tr tr 0.31 tr

23:0 0.30 tr tr n.d. tr n.d.

24:0 0.90 0.51 0.37 tr 0.34 tr

Σ SFA 22.53 17.45 17.65 18.14 19.40 20.16

14:1 tr n.d. tr tr n.d. n.d.

15:1 n.d. tr tr tr 0.11 tr

16:1 3.93 2.03 3.36 4.70 4.98 7.95

18:1 n-9t 0.37 tr tr tr tr tr

18:1 n-9c 15.30 23.60 23.32 23.00 23.98 23.07

20:1 n-9 7.65 4.33 3.13 2.05 3.06 1.84

22:1 n-9 n.d. n.d. 0.30 0.16 0.22 n.d.

24:1 n-9 0.53 0.33 tr tr tr tr

Σ MUFA 28.51 31.06 30.64 30.11 32.75 33.37

18:2 n-6c 13.91 23.98 23.98 22.71 23.71 23.38

18:3 n-3 2.58 10.44 10.68 10.48 9.93 10.46

20:2 n-6 0.32 tr tr n.d. tr tr

20:3 n-6 n.d. tr n.d. n.d. tr tr

20:4 n-6 0.32 tr 0.46 0.70 0.43 0.64

20:3 n-3 tr n.d. n.d. n.d. n.d. n.d.

20:5 n-3 5.69 2.70 4.21 5.90 3.31 4.09

22:6 n-3 5.92 3.15 2.81 2.60 2.24 1.38

Σ PUFA 28.76 40.66 42.15 42.39 39.70 40.07

Total 79.80 89.17 90.44 90.64 91.85 93.61

Σ n-3 14.21 16.28 17.70 18.98 15.48 15.94

Σ n-6 14.54 24.38 24.44 23.41 24.24 24.14

Σ n-3/Σ n-6 0.98 0.67 0.72 0.81 0.64 0.66

DHA/EPA 1.04 1.17 0.67 0.44 0.68 0.34

EPA [%DM]**** 0.46 0.24 0.32 0.44 0.27 0.30

DHA [%DM]**** 0.52 0.30 0.24 0.22 0.20 0.11

*FAME = Fatty acid methyl ester **n.d. = not detected ***tr = traces; values < 0.3 ****Long chain PUFA requirement: 0.7 % DM of feed (Skalli, Robin, 2004)

Chapter III

54

Sampling

Prior to the start of the experiment, ten fish were sampled to assess initial whole body composition

and they were stored at −20 °C. Two liver samples and proximal intestine samples were preserved in

4 % phosphate-buffered formalin for histological analyses. At the end of the experiment again liver

and intestine samples of two fish per replicate were taken. Also the final biomass of each replicate

group was recorded. Additionally, ten fish per tank were sampled for determination of whole body

composition. The samples were stored at −20 °C. For each treatment growth and feed utilization

parameters as specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER),

protein retention efficiency (PRE) and Fulton condition factor (FCF) were calculated, according to the

formulas in Table III-3.

Body composition

For the analysis of whole body composition the pooled samples consisting of ten fish per tank taken

at the end of the experimental period were freeze-dried for 168 h, homogenized using a cutting mill

(GM 200, Retsch, Haan, Germany) and stored at 3 °C. Analysis for dry matter (DM), crude ash, crude

protein and crude lipid was carried out according to EU guideline (EC) 152/2009 (European Union,

2009a). Crude protein content (N×6.25) was determined by the Kjeldahl method (InKjel 1225M, WD

30; Behr, Düsseldorf, Germany), crude lipid content was analyzed by means of hydrolysis with

hydrochloric acid followed by a petroleum ether extraction with a Soxleth extraction system

(Soxtherm, Hydrotherm, Gerhardt, Königswinter, Germany). Dry matter of samples was determined

after drying at 103 °C until constant weight remained stable and ash content was analyzed after 4 h

incineration at 550 °C with a combustion oven (P300; Nabertherm, Lilienthal, Germany).

Furthermore, gross energy was measured in a bomb calorimeter (C 200; IKA, Staufen, Germany).

Fatty acid composition

The total lipids were extracted according to the method described by Folch et al. (1957) with slight

modifications. Dichloromethane/ methanol (2+1, v/v) was used as extraction medium instead of

chloroform/methanol mixture. The fatty acid composition was determined by gas chromatography.

The sample preparation (modified after AOAC official method 991.39) was based on saponification of

the lipid samples with methanolic NaOH and methylation by addition of Boron trifluoride methanol

complex solution (Sigma Aldrich GmbH). Heneicosanoic acid (C21:0; Sigma Aldrich GmbH) was used

as internal standard to calculate the recovery of fatty acids during methylation. Fatty acid methyl

esters (FAMEs) were determined with a gas chromatograph (Hewlett Packard HP Agilent 6890 Series)

using hydrogen (40 ml min-1) and nitrogen (45 ml min-1) as carrier gases. The standard-mix Supelco 37

component fatty acid-methyl-ester mix (FA Supelco Analytical, USA) was applied for fatty acid

identification. The fatty acid distribution was calculated as percent of fatty acid methyl ester relative

Chapter III

55

to total fatty methyl esters. DHA and EPA were quantified using external calibration with reference

compounds. The recovery of C21:0 was used as correction factor. The content was expressed as

milligram fatty acids per gram dry matter.

Histological analysis

After fixation in 4% phosphate-buffered formalin, the samples of liver and proximal intestine were

dehydrated in a graded series of ethanol and embedded in paraffin and cut in thin sections. These

sections were stained with haematoxylin-eosin (HE). The analysis was carried out with a CKX 41

microscope (Olympus, Shinjuku, Japan) equipped with a Moticam 10.0 MP digital camera (Motic,

Hongkong). Sections were typified and compared between treatments. Four cross sections per

individual were used for observation. The level of vacuolization in liver samples was determined by

recording the occurrence of vacuolization within a fixed area. For that purpose ten random sections

per sample were observed. The percentage of vacuoles within each section was recorded and the ten

values per sample were calculated as mean value. The proximal intestine samples were analyzed on a

qualitative approach.










failed).

Results

Growth performance and feed efficiency

All test groups almost tripled their weight (2.5-2.8 fold) within the experimental period. The specific

growth rate of the experimental groups revealed the highest value for the P100 group

(1.77±0.10 % d-1) although not significantly different to the SGR of the control group and the other

algae groups (Table III-3). Only the group fed with the basal diet showed a significantly lower SGR

(1.56±0.08 % d-1). Also the feed conversion ratio (FCR) was best in the P100 group. However, again

there were no significant differences to the values of the control and algae groups. Furthermore, the

protein retention efficiency (PRE) was significantly higher in the P100 than in the basal and N50

Chapter III

56

group. There were no significant differences found in the daily feed intake (DFI), protein efficiency

ratio (PER), hepatosomatic index (HSI) and Fulton condition factor (FCF) between all groups.

Table III-3: Growth performance, feed intake, feed efficiency and biometric parameters (mean±SD, n=3) of sea bass fed with the experimental diets C, B, P50, P100, N50, and N100. Values with the same superscript are not significantly different (p<0.05).

C B P50 P100 N50 N100

IBW 1 12.85±0.01 12.84±0.01 12.85±0.01 12.85±0.01 12.84±0.01 12.84±0.01

FBW 2 35.51±0.93

ab 31.94±1.80

a 34.95±1.26

ab 36.08±1.92

b 32.29±1.09

a 33.22±0.34

ab

DFI 3 2.12±0.03 1.97±0.05 2.00±0.12 2.07±0.11 1.93±0.10 1.91±0.04

SGR 4 1.76±0.06

a 1.56±0.08

b 1.69±0.07

ab 1.77±0.10

a 1.59±0.06

ab 1.63±0.04

ab

FCR 5 1.20±0.03

ab 1.27±0.03

a 1.18±0.03

b 1.17±0.01

b 1.22±0.02

ab 1.17±0.02

b

PER 6 1.49±0.03 1.40±0.04 1.51±0.02 1.53±0.02 1.46±0.02 1.50±0.02

PRE 7 27.3±0.4

ab 25.5±0.7

a 27.1±1.2

ab 30.6±3.0

b 26.6±0.8

a 27.7±0.3

ab

HSI 8 2.40±0.45 2.39±0.24 1.84±0.20 1.87±0.15 2.18±0.35 2.18±0.35

FCF 9 1.27±0.03 1.23±0.05 1.23±0.06 1.22±0.02 1.15±0.10 1.17±0.01

1 Initial body weight [g] 2 Final body weight [g] 3 Daily feed intake [% d

-1]

4 Specific growth rate [% d-1

] = [ln (FBW)−ln (IBW)]/feeding day × 100 5 Feed conversion ratio = feed intake [g]/weight gain [g] 6 Protein efficiency ratio = weight gain [g]/protein intake [g] 7 Protein retention efficiency = 100 x [(final body protein x final body weight) – (initial body protein x initial body weight)]/(protein intake) 8 Hepatosomatic index = (liver weight/final body weight) × 100 9 Fulton condition factor = 100 × final body weight × final body length

-3

Body composition

The analysis of the whole body composition revealed no significant differences between the

experimental groups, except for the energy content (Table III-4). Here the control and basal group

showed the highest energy content (25.26±0.08 and 25.22±0.8 MJ kg−1 OM), although only

significantly higher than the energy content of group N100 (23.59±0.4 MJ kg−1 OM).

Table III-4: Body composition (in g kg-1

OM; gross energy in MJ kg-1

OM) of sea bass fed with the experimental diets C, B, P50, P100, N50 and N100 (mean±SD, n=3). Values with the same superscript are not significantly different (p<0.05). Initial body composition was analyzed as dry matter 270 g kg

-1; crude ash 44.3 g kg

-1 OM;

crude protein 164 g kg-1

OM; crude lipid 61.2 g kg-1

OM; energy 21.34 MJ kg-1

OM.

C B P50 P100 N50 N100

Dry matter 334±0.6 330±7.4 322±2.6 340±22.9 328±13.3 316.3±7.1 Crude ash 38.6±0.9 39.2±1.4 38.4±1.3 42.5±5.3 39.4±1.7 39.0±0.7 Crude protein 175±0.6 173±0.8 173±2.2 186±13.6 173±2.6 175±0.3 Crude lipid 116±0.8 113±9.0 106±6.5 111±6.1 108.4±10.3 96.7±6.3 Gross energy 25.3±0.08

a 25.2±0.8

a 24.5±0.7

ab 24.2±0.3

ab 24.0±0.4

ab 23.6±0.4

b

Fatty acid composition

The fatty acid composition of the juvenile sea bass fed with the experimental diets for 56 days is

shown in Table III-5. The sum of total saturated fatty acids (especially palmitic acid, C16:0) was

highest in the control group and lowest in group P50. No significant differences of the sum of

Chapter III

57

monounsaturated fatty acids between the test groups were found. The highest amounts of PUFA

were detected in the P100 group, followed by the P50, N100, N50 and B group, with the lowest PUFA

content in the control group. However, the highest amounts of DHA were analyzed for the control

and basal group, although they were not significantly higher than the values of groups P50 and P100.

The significantly highest EPA content was measured in the P100 samples and the lowest amount was

found in samples of the basal group.

Chapter III

58

Table III-5: Fatty acid composition of sea bass fed with the experimental diets C, B, P50, P100, N50 and N100 (mean±SD, n=3). Values with the same superscript are not significantly different (p<0.05). Lipid acronyms are defined in legend of Table III-2.

[% of FAMEs*] C B P50 P100 N50 N100

12:0 tr** n.d.*** n.d. n.d. n.d. tr

13:0 tr n.d. n.d. n.d. n.d. n.d.

14:0 4.00±0.03a 2.84±0.05

b 3.13±0.07

c 3.52±0.06

d 2.81±0.01

b 2.81±0.07

b

15:0 0.30±0.00 tr tr tr tr tr

16:0 19.03±0.10a 17.18±0.16

b 16.20±0.10

c 16.13±0.09

c 17.47±0.19

b 18.00±0.43

abc


18:0 3.31±0.04a 3.77±0.08

b 3.55±0.03

c 3.63±0.02

bc 3.79±0.13

b 3.77±0.10

b


22:0 tr n.d. tr tr n.d. n.d.


24:0 0.81±0.03a 0.56±.0.02

b 0.48±0.04

bc 0.41±0.05

c 0.47±0.01

bc 0.44±0.06

bc

Σ SFA 28.18±0.13a 25.19±0.15

cd 24.26±0.21

e 24.60±0.18

de 25.60±0.28

bc 25.93±0.30

b



16:1 5.59±0.04a 4.24±0.16

b 4.51±0.05

c 5.29±0.16

d 5.65±0.04

e 7.04±0.05

f

17:1 n.d. n.d. 0.30±0.05 0.32±0.03 tr n.d.

18:1 n-9t 1.39±0.04a 0.93±0.06

b 0.80±0.10

bc 0.63±0.08

c 0.77±0.03

bc 0.65±0.04

c

18:1 n-9c 22.93±0.49a 27.40±0.60

b 26.71±0.15

b 26.85±0.63

b 27.41±0.36

b 26.47±0.46

b

20:1 n-9 4.93±0.11a 3.35±0.09

b 3.02±0.09

b 2.49±0.13

c 2.04±1.42

abc 2.37±0.15

c

22:1 n-9 0.49±0.05a 0.36±0.05

b 0.31±0.07

b tr 0.34±0.03

b 0.32±0.02

b

24:1 n-9 0.39±0.02a 0.33±0.03

b tr tr tr tr

Σ MUFA 35.95±0.50 36.81±0.76 36.01±0.22 36.26±0.69 36.86±0.99 37.29±0.31

18:2 n-6t tr tr tr tr 0.31±0.33a 0.68±0.04

b

18:2 n-6c 9.17±0.03a 14.70±0.10

ab 15.21±0.19

b 15.20±0.36

b 14.62±0.32

ab 14.67±0.26

ab

18:3 n-6 tr 0.39±0.05 0.37±0.06 0.38±0.01 0.45±0.04 0.41±0.03

18:3 n-3 1.74±0.05a 5.60±0.10

b 6.04±0.14

b 6.22±0.19

b 5.33±0.12

b 5.65±0.09

b

20:2 n-6 0.43±0.01a 0.50±0.04

b 0.55±0.05

b 0.59±0.06

b 0.50±0.03

b 0.52±0.02

b

20:3 n-6 tr tr tr tr tr tr

20:4 n-6 0.32±0.04a tr 0.39±0.02

b 0.55±0.01

c 0.35±0.01

ab 0.50±0.03

c

20:3 n-3 tr tr n.d. tr tr tr

20:5 n-3 4.01±0.03a 2.53±0.13

b 3.35±0.07

d 4.27±0.14

c 2.97±0.02

e 3.37±0.08

d

22:6 n-3 5.30±0.14a 3.70±0.18

ab 3.65±0.23

ab 3.48±0.24

ab 3.38±0.01

b 3.15±0.25

b

Σ PUFA 21.50±0.23a 27.91±0.21

ab 29.76±0.14

c 30.89±0.26

d 28.04±0.71

ab 29.07±0.30

b

Total 85.63±0.57a 89.91±0.44

b 90.04±0.23

b 91.75±0.81

bc 90.30±1.40

bc 92.28±0.52

c

Σ n-3 11.13±0.24a 11.87±0.27

b 13.04±0.16

c 13.99±0.28

d 11.72±0.16

ab 12.18±0.24

b

Σ n-6 10.37±0.11a 16.04±0.09

ab 16.72±0.24

b 16.90±0.32

b 16.32±0.60

ab 16.89±0.25

b

Σ n-3/Σ n-6 1.07±0.03a 0.74±0.02

c 0.78±0.02

bc 0.83±0.03

b 0.72±0.02

c 0.72±0.02

c

DHA/EPA 1.32±0.03a 1.46±0.04

b 1.09±0.07

c 0.81±0.03

d 1.14±0.01

c 0.94±0.05

e

EPA [%DM] 1.00±0.04a 0.60±0.02

c 0.73±0.09

abc 0.99±0.03

ab 0.66±0.04

bc 0.68±0.03

bc

DHA [%DM] 1.46±0.03a 0.97±0.01

ab 0.93±0.08

abc 0.88±0.05

bc 0.83±0.06

bc 0.72±0.05

c

*FAME =Fatty acid methyl ester **tr=traces; values < 0.3 ***n.d.=not detected

Chapter III

59

Histological analysis

Figure III-1: Liver of initially sampled sea bass (Figure A) (representing low vacuolization level) and liver of sea bass fed with diet C sampled after 56 days (Figure B) (representing high vacuolization level) (H&E, 200x). The vacuolated cytoplasm is seen light because of high lipid content. Black arrows mark basophilic nuclei.

The histology of the initially sampled fish served as reference to detect abnormal modifications of

liver or intestine. The liver tissue of all experimental groups showed an increased vacuolization of the

hepatocytes at the end of the experimental period (Figure III-1). However, there was no significant

difference in the level of vacuolization between the initial and final samples, as well as between the

samples of the different test groups (Figure III-2).

Figure III-2: Level of vacuolization of liver cells depending on the different experimental diets C, B, P50, P100, N50 and N100 (mean±SD, n=3). No significant differences were found (p<0.05).

Chapter III

60

Figure III-3: Intestine epithelium of initially sampled sea bass (Figure A) and of sea bass fed with diet C (Figure B) and diet P100 (Figure C) sampled after 56 days (H&E, 200x). Continuous muscularis externa (ME); submucosa

(SM); lined villi with columnar epithelium (EP) are marked.

No differences of the intestine sections of initially sampled sea bass in comparison to the finally

sampled sea bass were found. There were also no differences between samples of the different

experimental groups. In all samples the intestinal mucosa was lined by regularly-packed villi with

continuous basement membrane (Figure III-3).

Discussion

The present study revealed promising results of microalgae as n-3 PUFA source in dry diets for

juvenile sea bass. Fish oil substitution, especially by Pavlova viridis, lead to very good growth

performance and incorporation of the essential PUFA in the fish body in accordance to the contents

of PUFA in the diets.

The growth performance of the juvenile sea bass observed in this study can be compared to results

of similar experiments (Figueiredo-Silva et al., 2005; Skalli, Robin, 2004). The SGR values ranging

between 1.56 and 1.77 % d-1 were reasonable and indicated that the differences in the growth

performance can be attributed to the different test diets. The lowest SGR of 1.56±0.08 % d-1 was

Chapter III

61

recorded for group B, although other studies did not find any growth losses, when juvenile sea bass

were fed with comparable vegetable oil containing diets (Izquierdo et al., 2003; Mourente, Bell,

2006). However, varying growth performance can be explained by the different fatty acid

composition of the test diets, as it is well known that essential fatty acids (EFA) can stimulate growth

or vice versa that dietary EFA deficiencies will negatively affect growth (Glencross, 2009; Ruyter et

al., 2000; Watanabe, 1982). All in all the fatty acid composition of the fish body samples highly

resembles the fatty acid composition of the feed (Glencross, 2009; Watanabe, 1982). However, the

DHA amount of all fish samples, except for group C, is higher than in the respective diets. A high

deposition and retention of DHA in muscle lipids has also been described for Atlantic salmon (Salmo

salar) (Bell et al., 2001) and turbot (Scophthalmus maximus) (Regost et al., 2003). This effect might

be even enhanced when fish are supplied with low amounts of dietary DHA. It is hypothesized that

the deposition of DHA is supported by a high specificity of fatty acyl transferases for DHA and a

resistance of DHA to β-oxidation due to its complex catabolic pathway (Bell et al., 2001). However, in

this study all diets, even N50 and N100, contain distinct amounts of DHA, although it is known that

Nannochloropsis sp. contains no or only low amounts of this n-3 PUFA (Patil et al., 2007). This can be

attributed to the equal amount of fish meal in all test diets. Usually fish meal contains amounts of

about 5-10 % fish oil, which then leads to an extra supply of the included EFA. Still, the slight

undersupply of EPA+DHA not only in group N100 and N50, but also in group B and P50, might have

negatively influenced the growth performance. The requirement for long chain n-3 highly

unsaturated fatty acids of juvenile sea bass has been found at a level of 0.7 % DM (Skalli, Robin,

2004). The amount of EPA+DHA given in Table III-2 shows that this requirement is mainly met by

group C (1.0 % DM) and P100 (0.7 % DM), leading to the highest specific growth rates found in this

trial (group C: 1.76±0.06 % d-1, group P100: 1.77±0.10 % d-1).

The lower growth rate of group B might have been induced by the lower values of the n-6 PUFA

arachidonic acid (ARA), too. Although there are no generally approved findings about the

requirements for ARA in juvenile sea bass, it has been confirmed that ARA is important for the

reproductive success in sea bass (Bruce et al., 1999). Furthermore, Castell et al. (1994) reported

positive effects on the growth of juvenile turbot, when diets contained ARA as single PUFA in

comparison to diets containing DHA as single PUFA. They found an ARA requirement of 5 g juvenile

turbot of around 0.3 % of the diet (DM).

The ratio of n-3 to n-6 fatty acids was reduced when fish oil was replaced by vegetable oil. However,

Izquierdo et al. (2003) did not find negative effects on growth when vegetable oil containing diets

revealed a n-3/n-6 fatty acid ratio of about 0.8. In the present study only diet C and diet P100 had

n-3/n-6 fatty acid ratio of 0.8 or higher (diet C: 0.98; diet P100: 0.81).

Chapter III

62

Skalli and Robin (2004) also found that an oversupply with long chain n-3 PUFA did not further

promote growth and overall the n-3 PUFA supply did not affect the PER, PRE or protein and lipid

content of the juvenile sea bass. This is also in accordance with findings from the present study,

where the P100 group revealed the best growth performance (although not significantly higher),

whereas the control group received a higher dietary amount of EPA+DHA, but did not show a higher

growth rate. Only the PRE observed in this study revealed significant differences between the test

groups. These different values might be a matter of the different protein sources, because the algal

raw material also contains a certain amount of protein.

The best FCR was also found in group P100 (1.17±0.01), although again it was only significantly lower

than the FCR of group B (1.27±0.03). In this context high amounts of long chain n-3 PUFA could also

enhance the FCR in red seabream (Pagrus major) (Yone, Fujii, 1975). Also, Yu and Sinnhuber (1979)

found that FCR was best when juvenile coho salmon (Oncorhynchus kisutch) were fed with diets rich

in n-3 PUFA.

The fish body composition of all test groups was reasonable and mainly in accordance with findings

from related studies (Skalli, Robin, 2004). Except for the energy content no significant differences

between the six test groups were found. The significantly lowest energy content was recorded in the

N100 group (23.6±0.4 MJ kg-1 OM), which is probably due to the slightly lower lipid content

(96.7±6.3 g kg-1 OM) measured in this group, because lipid is known to be the most efficient nutrient

for energy intake (Bureau et al., 2002).

The histology of liver and intestine is an important parameter to validate the effects of different diets

on the health state, because both organs play the major role in digestion and absorption of ingested

nutrients. The histological analyses did not reveal histopathological effects on the liver and intestine

of the sea bass. Although the amount of lipid vacuoles in liver samples of all test groups was higher at

the final sampling than at the beginning of the trial, no significantly different effects between all

groups could be found. This can be explained by the unrestricted feeding and the natural disposition

of sea bass to deposit lipid mainly in the liver (Figueiredo-Silva et al., 2005). Also the similar whole

body lipid content and the HSI indicate that the different algal diets did not have any negative effects

on the lipid deposition in comparison to the control and basal diet. Similar results were found when

juvenile sea bass were fed with diets with soybean oil substituting 25 % and 50 % of fish oil

(Figueiredo-Silva et al., 2005), which also did not lead to histopathological changes of the liver.

Additionally, the intestine samples did not reveal any histological changes between the initial sample

taken at the start of the trial and the samples taken at the end of the trial. Also no effects depending

on the different test diets were observed. It can therefore be concluded that the algal material did

not cause histopathological changes in the intestine. Even in the developing digestive tract of larval

Chapter III

63

gilthead seabream (Sparus aurata) the use of a freeze-dried alga (Nannochloropsis sp.) did not lead

to any histopathological effects (Navarro, Sarasquete, 1998).

Microalgae have been used for a long time not only in aquaculture diets, but also in other animal

feed, due to the high nutritional value. Around 30 % of the global aquaculture algae yield is used for

animal feed production (Becker, 2004). High amounts of important macronutrients like protein

(20-40 %) and lipid (20-60 %), but also micronutrients like pigments, e.g. astaxanthin, which is

produced by Haematococcus sp. (Waldenstedt et al., 2003), vitamins (Brown et al., 1999) and

especially the aforementioned essential fatty acids can be found in microalgae. Besides, the

promising growth promoting nutritive contents of microalgae, also other aspects need to be taken

into account. The palatability is of high importance. The DFI was highest again in group C and group

P100, although there were no significant differences between all test groups. This indicates that the

microalgal raw material did not negatively influence the palatability of the diets. Positive effects were

described by Zeinhom (2004), where the DFI was significantly increased when diets contained dried

algal products. Furthermore, the digestibility of the algal raw material is a relevant factor indicating

the suitability of a feed ingredient. The results of the present study do not indicate hampered lipid

digestibility of the P50 and P100 diets and the Pavlova product. However, the lower lipid content and

therefore lower energy content of the body samples of the N50 and especially N100 group might be

due to lower digestibility of the Nannochloropsis sp. lipid source. Skrede et al. (2011) found

decreased lipid digestibility with increased amounts of freeze-dried Nannochloropsis sp. included in

diets for adult mink (Mustela vison), which was used as model organism due to the approved

relationship with digestibility in salmonid fish and other monogastric species. In diets including 6 %,

12 % and 24 % of Nannochloropsis sp., the apparent lipid digestibility declined from 96 % to 93 % to

79.8 %, respectively. This might be induced by the resistance of the hard cell wall structure of

Nannochloropsis sp. cells to the digestive enzymes. Another factor might be the possible presence of

lipase inhibitors, which have been found in some marine algal species (Bitou et al., 1999). However,

Zeinhom (2004) showed that the digestibility coefficient of dry matter (92.5 %), crude protein

(87.63 %) and energy (81.41 %) was increased by fish feed containing 15 % microalgae. Also the use

of whole-cell and homogenized Schizochytrium sp. and Crypthecodinium cohnii in microdiets for

gilthead seabream did not reveal any differences in the nutritional utilization of the important

substances like PUFA (Ganuza et al., 2008). This indicates that also developing fish larvae do not

show any problems digesting microalgal material and that the previous treatment of the raw

material (cracking of the cell walls) does not necessarily lead to a better utilization of the nutrients.

Therefore, also the processing including the lyophilization and homogenization of the microalgal

products used in the test diets seems to be suitable and sufficient to ensure optimal nutrient

utilization of the juvenile sea bass. However, no comparable digestibility studies were found for

Chapter III

64

Pavlova sp. Hence, a related digestibility study would help to further evaluate the potential of freeze-

dried Pavlova viridis and Nannochloropsis sp. as feed ingredients.

Altogether the use of the microalga Pavlova viridis as n-3 PUFA source revealed promising results of

specific growth rate, feed conversion, as well as nutrient and fatty acid composition of the fish body.

Also the results of both Nannochloropsis sp. groups revealed reasonable SGR and FCR values,

although slightly lower than the results of the control and Pavlova viridis groups. This can be mainly

attributed to the slight undersupply of essential long chain n-3 PUFA, which also leads to significantly

lower amounts of these fatty acids (EPA+DHA) in the fish body. Therefore, the utilization of Pavlova

products in dry fish diets seems to be superior to Nannochloropsis products. However, the

production of microalgae in general and also of Pavlova viridis is still very expensive and therefore

the use of Pavlova viridis as major feed ingredient is not feasible yet. Therefore, the cost-effective

production of Pavlova viridis is a future challenge in order to make use of it as natural algae product.

This microalga holds a great potential as quality enhancing and fish oil saving feed additive and it can

be a benefit for the marine aquafeed production.

Acknowledgments


28183.

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General discussion

68

GENERAL DISCUSSION

The aim of the present thesis was to evaluate the potential of the microalga Pavlova sp. as feed

ingredient in marine fish nutrition. The experimental work focused on three approaches: The use of

Pavlova sp. I) as nutrient source in rotifer cultivation, II) as live feed enrichment product for marine

fish larvae nutrition and III) as fish oil replacement in formulated diets for juvenile and adult marine

fish. In the following, the suitability of Pavlova sp. will be discussed in terms of nutritional quality,

production, processing and application and overall performance in aquaculture.

Nutritional quality

Microalgae have a high potential as nutrient source in fish larval nutrition, but also as ingredient in

formulated aquaculture diets for juvenile and adult fish. This promising potential is credited to the

high amounts of macro- and micronutrients of many microalgal species (Shields, Lupatsch, 2012).

Besides, proteins, lipids, pigments and vitamins, especially the essential PUFA, DHA and EPA, are of

high importance in marine fish nutrition.

Most freshwater fish are capable of synthesizing DHA and EPA from linolenic acid (18:3n-3), whereas

the desaturase and elongase activity in marine species is too low to synthesize sufficient amounts of

DHA and EPA (Sargent et al., 2002). So far the high amounts of n-3 PUFA in marine aquaculture

species are mainly achieved by the inclusion of fish meal and fish oil in formulated enrichment

products or dry diets. However, due to limited supply with fish oil, alternative n-3 PUFA sources are

needed. Compared to fish oil, which contains about 10-20 % DHA and EPA of total fatty acids

(Gruger, 1967), the microalga Pavlova sp. can provide similar amounts of these essential fatty acids

(10% DHA and about 20% EPA of total fatty acids; Volkmann et al., 1991). However, optimal diet

formulation including novel PUFA sources is still difficult, as the species specific essential fatty acid

requirements are still not known for many marine fish species. The differences in requirement levels

between species can be attributed to dietary adaptations to different environmental conditions and

habitats. Furthermore, fatty acid requirements can also change during ontogenetic development.

For larval stages of most aquaculture species the knowledge about exact nutrient and PUFA

requirements is incomplete. Due to the high sensitivity and fragility of the small early life stages

experimental handling is often difficult. However, it is commonly acknowledged that the lipid and

fatty acid amount and composition (Izquierdo, 1996; Tocher, 2010), the protein content and amino

acid composition (Conceição et al., 2003; Oie, Olsen, 1997) and other dietary components like

vitamins and minerals (Holt, 2011) are necessary to ensure normal ontogenetic development.

Especially the relevance of essential PUFA for fish larval nutrition has been underlined by several

studies (Copeman et al., 2002; Izquierdo, 1996; Sargent et al., 1999). These essential substances

General discussion

69

contribute to important biological and physiological processes and thereby promote growth and

survival (Izquierdo, 1996). There are several critical phases in marine fish larval rearing like

endogenous feeding, onset of exogenous feeding, change from rotifer to Artemia feeding and

weaning to dry diets. During the early life stages feed quality can have a major impact on the success

of larval performance to overcome these critical phases. Often microalgae are used to provide the

important PUFA in fish larvae during the early life stages. The marine microalga Pavlova sp. is of

special interest because of its favorable fatty acid composition, as it is able to synthesize high

amounts of DHA and EPA. The strain Pavlova viridis (used in this study) contains around 10 mg g-1 dry

matter (DM) DHA and around 40 mg g-1 DM EPA (Lippemeier, unpublished results). According to Patil

et al. (2007) other Pavlova species and strains can contain around 13 mg g-1 DM DHA and 18 mg g-1

DM EPA. Besides the total amount of essential PUFA it is suggested by some studies that also the

lipid fraction containing these fatty acids is of high relevance. It was observed that PUFA from dietary

phospholipids (PL) rather than from triacyglycerides (TAG) can be more effectively incorporated into

fish larvae (Olsen et al., 2014; Park et al., 2006; Sargent et al., 1997). In Pavlova lutheri PL can

amount for about 10% of total lipids (Meireles et al., 2003). In other algae, like Isochrysis galbana,

the PL fraction ranges from about 10 % to 39 % of the total lipids (depending on culture state, Fidalgo

et al. (1998)). The PL fraction in Pavlova lutheri contains 42 % of DHA and 7 % of EPA and the

glycolipid (GL) family, accounting for about 24 % of total fatty acid residues, contains 51 % of EPA and

only 15 % of DHA (Meireles et al., 2003). Glycolipids are, like phospholipids, membrane-forming

lipids, but do not contain phosphate. As GL are components of the chloroplast membrane and PL are

important structure parts of cytoplasm membranes, it might be possible to even enhance the PL and

GL amount and therefore PUFA profile by changing temperature, which has an effect on membranes

(Tatsuzawa, Takizawa, 1995). Furthermore, the lipid class composition of an enrichment product (e.g.

microalgae) can also affect the lipid and fatty acid composition of rotifers as a function of long- (24 h

to several days) or short-term (about 2 h) enrichment (Rainuzzo et al., 1994). The maximal DHA:EPA

ratio was found when rotifers received ethyl ester-based emulsions in long-term enrichment. The

nutritive quality of long-term enriched live feed (cultivation of rotifers in chapter I is also one form of

long-term enrichment; enrichment of Artemia in chapter II, too) is enhanced. Here, not only the gut

is filled with the important nutrients, but also the biochemical composition of the live prey is

modified. Further studies on the lipid class composition of Pavlova sp. in combination with live feed

are needed in order to improve the PUFA supply for fish larvae.

In contrast to fish larvae there are more indications available concerning fatty acid requirements of

juvenile and adult fish, due to easier experimental handling. For adult striped jack (Caranx vinctus) a

DHA requirement of 1.7 % of diet dry matter (DM) (Takeuchi et al., 1992), for juvenile gilthead

seabream (Sparus aurata) a long chain n-3 PUFA requirement of 1 % of diet DM (Ibeas et al., 1996)

General discussion

70

and for red seabream (Pagrus major) a DHA requirement of 0.5 and an EPA requirement of 1.0 % of

diet DM (Takeuchi et al., 1990) has been found. In chapter III the basal diet contained only 0.5 % DM

DHA+EPA. The best growth and feeding efficiency were observed in the control and P100 group in

this study, which contained 1.0 % and 0.7 % DHA+EPA of diet DM, respectively. This is in accordance

with results described by Skalli and Robin (2004), who determined a requirement of 0.7 % (DM)

dietary long chain PUFA for juvenile sea bass. Additionally, not only the absolute contents of

essential fatty acids or the composition of lipids, but also the ratio of the essential PUFA, like DHA

and EPA, play an important role. Ibeas et al. (1997) suggested a DHA:EPA ratio of 1:2 for juvenile

gilthead seabream, Sargent et al. (1999) suggested a ratio of 2:1 for larval turbot (Scophthalmus

maximus), halibut (Hippoglossus hippoglossus) and European sea bass (Dicentrarchus labrax) and

Skalli and Robin (2004) recommended a ratio of about 1.5:1 in diets for juvenile European sea bass.

However, in chapter III the DHA:EPA ratios of the test diets were lower than these suggestions for

European sea bass. The basal diet showed the highest ratio (1.2:1), but also revealed the lowest

specific growth rate and the highest feed conversion ratio. Although the DHA:EPA ratios were 1:1 and

0.4:1 in control diet and diet P100, fish fed with these diets showed the highest growth performance

and lowest feed conversion ratio. This indicates that PUFA content played a more crucial role than

DHA:EPA ratio in the present study (chapter III). Altogether more research is needed in order to

improve species specific formulated aquafeed including novel PUFA sources like Pavlova sp.

Besides the promotion of normal development, growth and health state of the fish, one of the major

reasons to enhance the fatty acid supply for marine fish is the production of fish biomass rich in n-3

PUFA for human nutrition. Like for most vertebrates the highly unsaturated fatty acids are also

essential for humans and need to be provided by food items and marine seafood is the most

important essential PUFA source for human nutrition. The Food and Agriculture Organization of the

United Nations (FAO) recommends a daily EPA and/or DHA intake of 0.25 g for adults and 0.3 g for

pregnant women (FAO, 2010). Therefore, it is suggested to consume marine fish two to three times

per week. For example Atlantic herring (Clupea harengus), Atlantic salmon (Salmo salar) and Atlantic

cod (Gadus morhua) contain 0.7, 1.2 and 0.2 g DHA per 100 g edible fish tissue and 1.0, 0.6 and 0.1 g

EPA per 100 g edible fish tissue, respectively (Nettleton, 1995; Spiller, 1995). The fatty acid analysis of

sea bass whole body samples in chapter III revealed DHA amounts between 0.7 and 1.5 % (DM) and

EPA amounts between 0.6 and 1.0 % DM. These findings are mainly in accordance with n-3 PUFA

contents of marine fish suggested as essential fatty acid source for human nutrition. Although

samples of cultivated fish mainly resemble the dietary fatty acid composition (Glencross, 2009; Hardy

et al., 1987), the relationship between the PUFA composition in the fish samples and diets is most

likely dependent on the relative essentiality of the specific fatty acids. Atlantic salmon showed an

increased elongation and desaturation of 18:3n-3 to EPA and DHA, when diets contained low

General discussion

71

amounts of these long chain PUFA (Bell et al., 2001). Furthermore, although the ability of marine fish

to convert C18 precursors to long chain PUFA, DHA and EPA, is limited, intermediates of elongation

and desaturation processes were found in turbot, when fed with linseed oil diets (Regost et al.,

2003). Also in chapter III higher levels of 20:2n-6 were found in fish tissue compared to experimental

diets which might be a sign of certain elongase activity. However, the underlying processes of how

varying compositions of dietary precursor 18:2n-6 and 18:3n-3 and DHA and EPA affect fatty acid

metabolism pathways are not fully understood. Therefore, it is advisable to further investigate the

metabolism and incorporation of PUFA in fish tissue in order to meet fatty acid requirements of fish

and ultimately humans as end consumers.

Altogether Pavlova viridis revealed a high potential as n-3 PUFA source in formulated diets, as it lead

to good growth performance, feed utilization and fatty acid composition of juvenile sea bass. Also

the positive growth of rotifer cultures and of cod larvae can be attributed to the favorable fatty acid

composition of Pavlova viridis. However, the incorporation of essential fatty acids derived by Pavlova

sp. products can be enhanced by detailed knowledge about digestive pathways in fish and other

marine organisms. On that account also the evaluation of different processing methods of Pavlova

sp. post harvesting can help to further improve the utilization of this microalga as nutrient source in

formulated diets.

Production, processing and application

The major bottleneck still hampering microalgal production is its expensive cultivation. Acién et al.

(2012) quantified the production cost of a Spanish microalgae production plant at 69 € kg-1 algal dry

matter and Coutteau and Sorgeloos (1992) reported values of 50 up to 300 € kg-1 DM for microalgae

production in aquaculture hatcheries. This means that the hatchery on-site-production of microalgae

can make up 30 to 70 % of the total production costs. The high costs are caused by several factors:

Often microalgae produced for nutritional purposes can only be cultivated in closed indoor systems

in order to avoid contamination and to provide the optimal environmental conditions. Therefore,

sophisticated culture systems have to be established and for example lighting by means of artificial

light as well as the optimal temperature (cooling or heating) have to be controlled leading to

increased energy consumption. Still unforeseen culture crashes can occur entailing high production

risk and varying product quality. Altogether microalgae production is very time- and labor intensive

and needs to be managed by qualified personnel resulting in high personnel costs. These aspects are

even more distinct in cultivation of “new” candidate species like Pavlova sp., where empirical values

and cultivation protocols have to be established in the first place. On that account new, but also well

established, microalgal products are still not competitively viable in comparison to the common

aquafeed ingredients, like fish meal and fish oil. Although fish meal and fish oil prices have been

General discussion

72

rising fivefold and fourfold, respectively, in the last decade, their use is still more cost-effective than

the use of microalgal products (FAO, 2014). Recent prices of fish meal and fish oil range around 1.5

and 1.6 US$ kg-1 (FAO, 2014), respectively, whereas preserved microalgae products amount between

50 and 300 US$ kg-1 (Muller-Feuga, 2000).

However, due to great research effort there is also a high potential of cost reduction for example by

improving cultivation facilities. The production of microalgae in photobioreactors allows an increased

yield due to a higher surface to volume ratio, an increased level of control and therefore improved

quality management (Tredici et al., 1991). In this way the production costs can be reduced to around

3 US$ kg-1 depending on the species and cultivation method (Chisti, 2007). Also the effort by

manpower can be reduced due to a higher level of automation (Borowitzka, 1997).

Although these live algae cultures are of high quality and are useful for example for the cultivation of

rotifers (chapter I), further processing is necessary in order to enhance storability (Hamada et al.,

1993) and handling of the products. The possibility of storing microalgal products alleviates the need

of hatcheries for “on time production” of large amounts of algal biomass for the cultivation and

enrichment of rotifers. The algal products can be produced, transported and distributed under

constant and high quality conditions (Lubzens et al., 1995). Common forms of processed algae are

cooled or frozen concentrates or pastes, next to dried algae meals. Concentrates are more suitable

for the cultivation or enrichment of live feed in order to feed fish larvae, whereas dried meals are

preferred for inclusion in formulated dry feed. For example when rotifers were provided fresh, frozen

and dried Nannochloropsis sp., the best specific growth performance was observed in the fresh algae

group and the slowest growth in the dried algae group (Lubzens et al., 1995). However, in that study

the application of frozen Nannochloropsis sp. led to the highest total fatty acid content and most

favorable fatty acid composition. Also in the chapter I frozen Pavlova viridis concentrates, as well as

live algae cultures revealed good rotifer culture performances in contrast to dried Pavlova viridis

meal. Lubzens et al. (1995) stated that frozen Nannochloropsis sp. concentrate could be stored

at -20 °C or -80 °C for four weeks and the thawed material maintained its quality and could be used

for enrichment and cultivation for up to two weeks. Furthermore, dried algae products can even be

stored for up to two years at -20 °C.

Besides high storability of processed algae products also the nutrient utilization can be improved by

processing procedures. A major constraint of the digestibility of microalgae is the cellulose found in

the cell walls. The cellulose content can amount up to 10 % of algae dry weight (Becker, 2007).

Hence, it is necessary to crack the cells walls by means of different processing methods. Chemical

methods like rupture of hydrogen bonds by phenol or formic acid can disrupt cell wall structures.

Alternatively physical methods including treatments with high temperatures and/or drying or

homogenization by means of mills or grinders can be applied. Becker (2004) summarized that freeze-

General discussion

73

dried Chlorella sp. resulted in higher protein efficiency ratio observed in rats (2.0, Saleh et al. (1985))

than drum-dried (1.31; Cheeke et al. (1977)) or autoclaved Chlorella sp. (0.84; Cheeke et al. (1977)).

In accordance to this finding the processing of the microalgal products in chapter III (freeze-drying

and homogenization via cutting mill) might have been suitable to crack the cells and hence,

digestibility could have been improved. In this context the homogenization seems to be a more

crucial step, because freeze-drying as such does not always lead to rupture of the cell walls (Becker,

2004). However, Pavlova sp. does not contain a common cell wall, but only cellulose scales and layers

of mucilage (Green, 1980). Therefore, cell-breaking and accessibility of nutrients for digestive

enzymes might be facilitated leading to a particularly good utilization of the Pavlova sp. diets by sea

bass juveniles (chapter III). In accordance to this the nutrient utilization of cod larvae fed with

Pavlova viridis enriched live feed, seemed to be adequate indicated by the high growth rates of the

larvae (chapter II). The ability for digestion of microalgae was also hypothesized for herring larvae

(Clupea harengus) (Hjelmeland et al., 1988). However, the digestive capacities of marine fish larvae

are rather poor due to the not fully developed digestive tract. Furthermore, there is still a lack of the

total understanding of the complexity of digestive processes in fish larvae. Even novel methodologies

like tracer studies, functional genomics or metabolic programming have improved the knowledge

about individual digestive performance, dietary regulation of metabolic processes or nutrition

physiology (Conceição et al., 2010), it is still difficult to test nutrient digestibility of novel feed

compounds, like Pavlova viridis, in fish larvae.

Besides the processing of whole microalgal cells another way to simplify the utilization of lipid and

essential fatty acids from microalgae is the extraction and purification of microalgal oil. So far the

well-established method according to Bligh, Dyer (1959) using chloroform, methanol and water for

lipid extraction, as well as saponification (Guil-Guerrero et al., 2000) or the supercritical fluid carbon

dioxide method (Mercer, Armenta, 2011) have been applied. However, these processes are still very

energy- and cost-intensive and the lipid yield is not always sufficient. Therefore, the commercial

microalgal oil production is still a major challenge and furthermore, the production of microalgal oil

for nutritive purposes is also in competition with the production of biofuel (Mercer, Armenta, 2011).

So far there is no commercial production of Pavlova sp. extracted oil and no studies on the use of

Pavlova sp. oil in animal feed have been found. Still, the cost effective production and application of

Pavlova sp. oil in fish diets might even result in higher fatty acid utilization and therefore a higher

incorporation of dietary fatty acids in fish.

Performance in aquaculture

The performance of Pavlova sp. products in aquaculture is not only characterized by the nutritive

potential, product quality or cost-efficiency, but by various other aspects, too.

General discussion

74

For example, the cell size, texture and palatability of Pavlova sp. products can affect the ingestion by

rotifers and larvae. Vadstein et al. (1993a) found that cell sizes of ≥2 µm were optimal for Brachionus

plicatilis, while Brachionus “Cayman” preferred particles of the size 4.5 µm (Baer et al., 2008).

Certainly the success of capture and ingestion of particles is also a matter of the individual mastax

size (Hino, Hirano, 1980). Although different preferred particle sizes were observed for different

rotifer species (e.g. B. angularis, B. calyciflorus, B. rubens), the texture and flavor of the different

microalgal cells might often have a certain influence, too (DeMott, 1986). In the present work

(chapter I) fresh Pavlova viridis and Pavlova sp. algal cells were favored by rotifers probably due to a

suitable cell size of about 4 µm and a suitable texture and flavor of the fresh cells. Additionally, in

chapter II ingestion was highest in cod larvae groups that were fed with Pavlova viridis enriched live

feed as well as with a commercial enrichment product. The high ingestion of Pavlova enriched live

feed might be caused by favorable olfactory effects. Newly hatched cod larvae possess olfactory

organs (Døving et al., 1994) and can therefore react directly to chemical stimuli sent out by prey

organisms (Dempsey, 1978).

Furthermore, growth, development and survival of the target species express the performance of

Pavlova sp. products in aquaculture. In chapter I rotifer culture growth rates were in a generally

acceptable range, when Pavlova sp. feeding products could be mixed homogenously in the culture

water. Therefore, low culture growth was mainly hampered by the applicability of the freeze-dried

Pavlova viridis product. In chapter III Pavlova viridis diets supported the growth of juvenile sea bass

considerably in contrast to the basal diet and growth rates were in accordance to findings of similar

studies (Figueiredo-Silva et al., 2005). Additionally, in chapter II cod larvae from the Pavlova viridis

group and the commercial product group reached SGR of more than 7 % per day, which was also in

accordance to findings from related studies (Garcia et al., 2008). In contrast, larvae, which were fed

with Nannochloropsis sp. enriched live feed, only reached a growth rate of about 4 % per day and a

survival rate of 0.04 %. In spite of promising growth rates the Pavlova viridis group revealed low

survival of about 6.5 %, in contrast to around 19.7 % in the commercial product group. However,

there is still lack of knowledge concerning the reasons for low survival rates and deficiencies in the

early development of fish larvae (Bell et al., 2003; Cahu et al., 2003). Also the maximal growth

potential of fish larvae in aquaculture is often not realized (Shields, 2001), as it was also found in

chapter II, where high mortality and distinct variations in the growth performance between the test

groups could be observed. However, it is difficult to identify distinct factors causing the low survival

rates under the specific conditions of the very experimental setup chosen in the present research.

Altogether Pavlova sp. diets supported the growth performance of aquaculture species, but the right

form (e.g. frozen concentrate, freeze-dried meal or single product, feed additive) needs to be chosen

for each specific feeding purpose.

General discussion

75

Another attempt to evaluate the suitability of Pavlova sp. for nutritional purposes is the analysis of

the current condition of fish larvae by means of RNA-DNA ratio analysis. It is assumed that the RNA

content is correlated with the protein biosynthesis in the cell, whereas the amount of DNA is

constant in somatic cells and is therefore correlated with the biomass of an organism. This parameter

gives information about the current nutritional state of the fish larvae and reacts to changes within

one to a few days (Martin, Wright, 1986). The RNA-DNA ratio has been intensively used since the

1980s to estimate the nutritional status of fish larvae (Buckley, 1984; Bulow, 1987). These studies

mainly focused on field applications aiming to investigate environmental and anthropogenic factors

affecting recruitment success (Buckley, 1984; Clemmesen, 1987). In these experimental studies often

the nutritional state e.g. in phases of feed supply and feed deprivation was investigated mainly

without focusing on the feed type and feed quality. However, the information which can be gained

from the RNA-DNA analysis can also contribute to a better understanding of the nutritional condition

of larvae reared in aquaculture and fed with different diet types. In chapter II distinct differences

between the growth rate and standardized RNA-DNA ratio (sRD) were found, which can be explained

by different suitability and nutritive efficiency of the feed types/enrichment products. In this case a

higher growth rate seemed to be caused by highly available nutrients in the commercial enrichment

product and natural microalga Pavlova viridis. The enhanced growth performance in both groups

might rather be accomplished by an increased specific RNA activity than by an increase of RNA

amount. Similar explanations were found by Buckley et al. (2008) for increasing growth rates with

increasing temperature, although no enhanced sRD was detected. In this case the results of the RNA-

DNA analysis need to be supported by further studies on the digestibility of the test diets. However,

it is still difficult to investigate the digestive capacities and feed efficiency in fish larvae. Although

novel methodologies can help to extend the knowledge about these processes these methods are

still time consuming and expensive.

Another characteristic of Pavlova sp. is its potential to influence microbiota. The use of other natural

microalgae, either as enrichment product or supplemented directly to the tank water (“greenwater

technique”), can affect the bacterial community of live feed cultures, tank environment (Nicolas et

al., 1989) and larvae (Skjermo, Vadstein, 1993). This feature can be of great importance as the

occurrence, especially of detrimental bacteria, can hamper the survival and normal development of

fish larvae (Vadstein et al., 1993b). This problem often occurs in closed aquaculture systems, due to

the high load of organic matter that is introduced by high amounts of feed and the accumulation of

waste. For example, commercial/artificial compounds with highly available nutrients can support the

carrying capacity for heterotrophic bacteria. To overcome this issue, means of chemical and

mechanical water treatment strive to control the bacterial community. However, this often leads to

occurrence of opportunistic microbes instead of stabilization of the microbiota (Attramadal et al.,

General discussion

76

2012). The use of microalgae can help to stabilize microbial communities. A helpful method to

distinguish different microbial communities is the denaturing gradient gel electrophoresis of PCR

amplified 16S rDNA (PCR/DGGE) (Muyzer et al., 1996). The composition of the bacterial community

of a sample can be evaluated by the presence of certain bands on the gel representing bacterial

species. In chapter II it was found by means of PCR/DGGE that larvae of the commercial enrichment

product group revealed indeed a different microbial community than larvae of both algal groups. The

difference in microbial communities might be due to the fact that both Nannochloropsis sp. and

Pavlova viridis as pure algae products promote and/or suppress different bacterial communities than

the commercial product. Olsen et al. (2000) found that the microbial community in the gut of live

feed (Artemia franciscana) was changed after enrichment with the microalga Tetraselmis sp. Here

the total number of bacteria decreased and the diversity of the microbial community was enhanced,

which altogether stabilized the microflora over the experimental period. Furthermore, Attramadal et

al. (2012) found less larval performance variability between replicate rearing tanks when the

bacterial community was more stable. Also in chapter II both natural microalgal treatments, Pavlova

viridis and Nannochloropsis sp., lead to less variances in larval growth between replicate tanks in

contrast to the commercial product treatment. It is hypothesized that microalgae can change the

microbial composition by production of antibacterial substances (Austin, Day, 1990; Kellam, Walker,

1989). Moreover, it was found that algae can harbor symbiotic bacteria (Phaeobacter strain), which

are able to produce a Vibrio anguillarum inhibiting substance (tropodithietic acid) (D'Alvise et al.,

2010). On that account microalgae can also support probiotic microflora. Altogether there is a

potential of stabilizing the microbial community in the fish larvae tanks also leading to more stable

development of the fish larvae.

Altogether the use of microalga Pavlova sp. in marine fish nutrition provides a broad range of

advantages. Due to the favorable nutrient and fatty acid composition, suitable palatability, as well as

the possibility to provide down-streamed products (e.g. frozen, freeze-dried), this alga has a high

potential as nutrient source for aquaculture purposes (Table-General discussion 1).

General discussion

77

Table-General discussion 1: Evaluation summary of Pavlova sp. for the use in marine fish nutrition.

Nutritional quality Production, processing and application

Performance in aquaculture

+Favorable DHA and EPA content +Adequate n-3 PUFA incorporation in sea bass (III) (Provision of n-3 PUFA for human nutrition) +High utilization and feed conversion of Pavlova derived nutrients by cod larvae and sea bass (II, III)

+Consistent quality of frozen and freeze-dried products (I, II, III) +Suitable application of freeze-dried meal in dry feed (III) -Limited storability of Pavlova concentrate (I) -Difficult application of freeze-dried product for rotifer cultivation (I) -Energy- and cost-intensive production

+Adequate growth rate, ingestion and filtration of rotifers (I) +Decent growth rate, feed deprivation resistance, ingestion rate of cod larvae (II) +Good growth performance of sea bass (III) -Low survival of cod larvae (II) ±Stabilization of microbiota (II)further studies needed

Further perspective

In order to further promote the use of Pavlova sp., enhance yields and reduce production costs, the

cultivation and processing methods for this “new” candidate species need to be improved. In

particular a stronger focus should be placed on the use of Pavlova sp. as ingredient in formulated live

feed enrichment, rather than as single product. By this way the advantages of Pavlova sp. can be

used and be complemented with further additives. Additionally, the application of Pavlova sp. in

formulated dry diets for adult fish still reveals a potential for optimization in terms of digestibility and

nutrient utilization of Pavlova sp. products.

Altogether the establishment of “new” species, like the microalga Pavlova sp., is an encouraging

approach to diversify aquaculture nutrition. If the aquaculture sector is to rise further, there will be a

limitation by fish meal and fish oil supply. In that case alternative nutrient sources will be of

increasing importance in order to maintain the provision of high quality fish and seafood for human

nutrition.

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Summary

83

SUMMARY

The great diversity of marine organisms holds a large potential for a wide range of purposes

benefitting human society. A great potential can be found in the group of marine microalgae.

Microalgae are known to contain important macronutrients like protein and lipid, as well as

micronutrients like pigments, vitamins and essential amino acids and polyunsaturated fatty acids

(PUFA). Therefore, microalgae are used in human nutrition, but primarily in animal nutrition,

especially in aquaculture. Some of the most important microalgal candidates that have been used for

aquaculture feed are Nannochloropsis sp., Isochrysis galbana and Tetraselmis sp. However, these

species contain only one of both essential n-3 PUFA, eicosapentaenoic acid (EPA; 20:5n-3) or

docosahexaenoic acid (DHA; 22:6n-3). In contrast to that, the microalga Pavlova sp. is known to be

able to synthesize DHA and EPA in larger amounts. The provision of sustainably sourced feed raw

materials rich in essential PUFA is still a bottleneck in marine aquaculture. Therefore, the potential of

the microalga Pavlova sp. as n-3 PUFA source in marine fish nutrition was evaluated in this thesis.

In chapter I the potential of different Pavlova sp. products for the cultivation of rotifers (Brachionus

plicatilis) was tested in two growth performance trials: Firstly in a laboratory scale experiment

Pavlova viridis concentrate, Pavlova viridis fresh culture, Pavlova sp. fresh culture, baker´s yeast and

Nannochloropsis sp. concentrate were fed to rotifers over an experimental period of 14 days. The

significantly highest rotifer density (109.2 rotifers ml-1) and instantaneous growth rate

(G=0.14±0.02 d-1) were observed in the Pavlova viridis fresh culture group. No significant differences

of the growth rate were found between the Pavlova viridis concentrate group and the Pavlova sp.

fresh culture group. The significantly lowest rotifer numbers and growth rate were observed in the

baker´s yeast group. On grounds of the high growth performance of the Pavlova viridis fresh culture

group in the first experiment, different Pavlova viridis products (concentrate, fresh culture, frozen

concentrate, freeze-dried meal) were examined in the second experiment and compared to

Nannochloropsis sp. concentrate. In experiment 2 the highest rotifer culture growth was observed in

the frozen Pavlova viridis group (G=0.09±0.03 d-1). Compared to that, only the growth of rotifers fed

with the Pavlova viridis freeze-dried was significantly lower. The other Pavlova viridis groups showed

reasonable growth performances. The most suitable product for the cultivation of live feed was the

frozen Pavlova viridis concentrate. It provided advantages of storability and application.

In chapter II frozen Pavlova viridis concentrate (P) was tested as enrichment product for live feed

organisms (Brachionus ibericus and Artemia salina) for feeding of Atlantic cod larvae (Gadus

morhua). This product was compared to Nannochloropsis sp. microalgal concentrate (N), and to a

commercially available enrichment product (CP; Larviva Multigain® BioMar, Brande, Denmark) in a

Summary

84

six-week feeding trial. Significantly higher instantaneous growth rates (G=0.074±0.001 d-1 and

G=0.079±0.009 d-1) were observed for larvae of group P and CP in comparison to larvae of group N

(G=0.04±0.001 d -1). The survival of group CP was highest (19.65±7.51 %) and significantly lower

survival was observed for group P (6.47±2.02 %) and N (0.04±0.0 %). The ingestion of live feed by cod

larvae was evaluated at 4, 15 and 27 days post hatch (dph) revealing the highest gut fullness index

for larvae of the CP and P group. The ability to resist feed depletion was tested at 5, 16 and 28 dph. It

was found that only cod larvae fed with CP enriched live feed were able to withstand an extended

period of feed depletion (of 4 or 5 days), in contrast to larvae of group P and N. Regarding the effects

of the different treatments on the diversity of the microbiota of the tank water, feed and larvae it

was observed that treatments N and P lead to similar, highly diverse microbial communities. In

contrast to that, treatment CP showed a low diversity. Altogether the microalga Pavlova viridis as live

feed enrichment lead to a high growth performance, stimulation of feed ingestion and it affected the

diversity of the microbial community. However, it was not able to sustain the same survival rates as a

commercial product.

In chapter III the microalga Pavlova viridis was used as n-3 polyunsaturated fatty acid (PUFA) source

and compared to Nannochloropsis sp. in diets for juvenile European sea bass (Dicentrarchus labrax L.)

(initial weight ~12.8±1.7 g). Six different isoenergetic and isonitrogenous experimental diets were

formulated and fed to juvenile sea bass in an eight-week lasting feeding trial. In the control diet (C)

the major lipid source was fish oil (100 %). The basal diet contained 40 % fish oil and 60 % vegetable

oil, whereas rapeseed-, sunflower- and linseed oil were included in equal parts. Two Pavlova diets

were formulated replacing 50 % and 100 % of the remaining fish oil of the basal diet by the lipid

content of freeze-dried Pavlova viridis meal (diet P50 and P100). According to that, two

Nannochloropsis sp. diets were formulated substituting 50 % and 100 % of the remaining fish oil of

the basal diet by the lipid content of the Nannochloropsis sp. meal (N50 and N100). The highest

specific growth rate and best feed conversion ratio were observed in the P100 group (1.77±0.10 % d-1

and 1.17±0.01), although there were no significant differences found in comparison to the results for

the control and the other algae groups. Also the sum of PUFA was highest in fish samples of the P100

group, followed by the P50, N100, N50 and B group, with the lowest levels in the control group. The

highest amounts of docosahexaenoic acid (DHA) were found in fish samples of the control and basal

group. However, there were no significant differences compared to the values of groups P50 and

P100. The significantly highest amount of eicosapentaenoic acid (EPA) was measured in the P100 fish

samples and the lowest amount was measured in samples of the basal group. The histological

analyses of liver and intestine samples did not reveal any negative effects of the experimental

treatments. Altogether the use of the microalga Pavlova viridis as n-3 PUFA source revealed

Summary

85

promising results in terms of specific growth rate, feed conversion, as well as nutrient and fatty acid

composition of fish bodies.

This study revealed a high potential of Pavlova sp. for the use in live feed cultivation, live feed

enrichment for the feeding of marine fish larvae and as n-3 PUFA source in marine fish dry diets. The

use of Pavlova sp. is an encouraging approach to make use of new and sustainable nutrient sources,

in order to advance aquacultural procedures and to provide high quality fish and seafood for human

nutrition.

Zusammenfassung

86

ZUSAMMENFASSUNG

Im marinen Lebensraum ist eine enorme Vielfalt an Organismen zu finden, die sich der Mensch auf

unterschiedliche Weise zu Nutze machen kann. Ein großes Potential ist dabei in der Gruppe der

marinen Mikroalgen zu finden. Diese enthalten oft große Mengen an wichtigen Makronährstoffen,

wie Proteinen oder Fetten, aber auch an Mikronährstoffen, wie Pigmenten, Vitaminen, essentiellen

Aminosäuren oder essentiellen, mehrfachungesättigten Fettsäuren. Deshalb werden Mikroalgen

erfolgreich in der Humanernährung als Nahrungsergänzungsmittel, aber vor allem in der

Tierernährung, besonders in der Aquakultur, eingesetzt. Nannochloropsis sp., Isochrysis galbana und

Tetraselmis sp. sind Mikroalgenarten, die bisher schon zur Ernährung von Fischen, v.a. von

Fischlarven, eingesetzt wurden. Allerdings enthalten sie meist nur eine der beiden essentiellen

omega-3 Fettsäuren, die Eicosapentaensäure (EPA, 20:5n-3) oder die Docosahexaensäure (DHA,

22:6n-3), die oft den nutritiven Wert der Mikroalgen ausmachen. Im Gegensatz dazu ist die

Mikroalge Pavlova sp. in der Lage beide Fettsäuren, EPA und DHA, in großen Mengen zu bilden. Da

die Versorgung von Fischen in Aquakultur mit essentiellen omega-3 Fettsäuren aus nachhaltigen

Quellen ein großes Problem darstellt, wird der Mikroalge Pavlova sp. ein großes Potential als

omega-3 Fettsäurequelle zugesprochen. Dieses Potential wurde in der vorliegenden Arbeit bewertet.

In Kapitel I wurde das Potential unterschiedlicher Pavlova sp.-Produkte als Nahrung für die

Kultivierung von Rädertierchen (Brachionus plicatilis) in zwei Experimenten getestet. Zunächst

wurden in einem Versuch im Labormaßstab Rädertierchen über einen Zeitraum von zwei Wochen

mit Pavlova viridis Konzentrat, Pavlova viridis Frischkultur, Pavlova sp. Frischkultur, Hefe

(Saccharomyces cerevisiae) oder Nannochloropsis sp. Konzentrat gefüttert. Die signifikant höchste

Dichte der Rädertierchen (109.2 Rädertierchen ml-1) und die höchste Wachstumsrate

(G=0.14±0.02 Tag-1) wurden in der Pavlova viridis Frischkultur-Gruppe beobachtet. Es konnten keine

signifikanten Unterschiede in der Wachstumsrate zwischen der Pavlova viridis Konzentrat-Gruppe

und der Pavlova sp. Frischkultur-Gruppe festgestellt werden. Die deutlich niedrigste Wachstumsrate

wurde in der Gruppe verzeichnet, die mit Hefe gefüttert wurde. Aufgrund der hohen Wachstumsrate

der Rädertierchenkultur, die mit frischen Pavlova viridis Mikroalgen gefüttert wurde, wurden

verschiedene Produkte aus diesem Algenstamm (Konzentrat, Frischkultur, gefrorenes Konzentrat und

gefriergetrocknetes Mehl) in einem zweiten Versuch eingesetzt und wiederum mit der Mikroalge

Nannochloropsis sp. verglichen. In diesem zweiten Versuch wurde das höchste Kulturwachstum in

der Rädertierchengruppe ermittelt, die mit gefrorenem Algenkonzentrat gefüttert wurde

(G=0.09±0.03 Tag-1). Alle Gruppen zeigten angemessene Wachstumsraten, wohingegen nur die

Gruppe, die mit getrockneten Algen gefüttert wurde, ein signifikant niedrigeres Wachstum zeigte.

Zusammenfassung

87

Zusammenfassend war das gefrorene Pavlova viridis Konzentrat am besten geeignet und zeichnete

sich auch durch Vorteile in der Haltbarkeit und Anwendung aus.

In Kapitel 2 wurde gefrorenes Pavlova viridis Konzentrat (P) als Anreicherungsprodukt für

Lebendfuttertiere (Brachionus ibericus und Artemia salina) zur Ernährung von Kabeljaularven (Gadus

morhua) eingesetzt. Dieses Produkt wurde mit einem Konzentrat der Algenart Nannochloropsis sp.

(N) und einem kommerziellen Anreicherungsprodukt (CP; Larviva Multigain® BioMar, Brande,

Dänemark) in einem sechswöchigen Fütterungsversuch verglichen. Die signifikant höchsten

Wachstumsraten (G=0.074±0.001 Tag-1 und G=0.079±0.009 Tag-1) wurden in den Gruppen P und CP

verzeichnet, wohingegen die Gruppe N ein niedrigeres Wachstum zeigte (G=0.04±0.001 Tag-1). Die

Überlebensrate war in der Gruppe CP am höchsten (19.65±7.51 %). Im Vergleich dazu waren die

Überlebensraten der anderen beiden Gruppen signifikant niedriger (P: 6.47±2.02 % und N:

0.04±0.00 %). Die Futteraufnahme wurde an Tag 4, 15 und 27 nach dem Schlupf ermittelt. Hierbei

war der Füllungsgrad des Verdauungstraktes an allen drei Versuchstagen in den Gruppen P und CP

am höchsten. Des Weiteren wurde beginnend an Tag 5, 16 und 28 nach dem Schlupf die Fähigkeit

der Larven getestet, eine Phase des Nahrungsentzuges zu überstehen. Dabei zeigte sich, dass nur

Larven der CP Gruppe eine Nahrungsentzugsdauer von bis zu 5 Tagen überstehen konnten und auch

danach ein erneutes Wachstum zeigten. Zusätzlich wurde die Auswirkung der unterschiedlichen

Anreicherungsprodukte auf die Diversität der Bakterien im Haltungswasser, im Futter und in den

Fischlarven untersucht. Dabei wurde beobachtet, dass die beiden reinen Algenprodukte zu einer

ähnlichen, hochdiversen mikrobiellen Zusammensetzung in den Larvenproben führten. Im Gegensatz

dazu zeigten die CP Proben eine niedrigere Vielfalt der Bakterienzusammensetzung in den

Fischlarven. Zusammenfassend kann die Mikroalge Pavlova viridis als durchaus geeignete

Komponente in Anreichungsprodukten bewertet werden, da sie nicht nur das Wachstum der Larven

begünstigte, sondern auch die Futteraufnahmerate der Fischlarven erhöhte, sowie die Diversität der

Mikroflora positiv beeinflussen konnte. Allerdings konnten nicht ähnlich hohe Überlebensraten

erreicht werden, wie durch Nutzung des kommerziellen Produktes.

In Kapitel III wurde die Mikroalge Pavlova viridis als omega-3 Fettsäurequelle in Trockenfuttermitteln

verwendet und wiederum mit der Mikroalge Nannochloropsis sp. verglichen. Beide Algen wurden in

gefriergetrockneter Form in Trockenfuttermitteln für juvenile Europäische Wolfsbarsche

(Dicentrarchus labrax L.) (Startgewicht ~12.8±1.7 g) eingesetzt. In einem achtwöchigen

Fütterungsversuch wurden sechs isonitrogene und isoenergetische Futtermittel getestet. Die

alleinige Hauptfettquelle im Kontrollfuttermittel war Fischöl (100 %). Die Basaldiät enthielt nur noch

40 % Fischöl und 60 % Pflanzenöle, wobei Raps-, Sonnenblumen- und Leinsamenöl zu gleichen Teilen

verwendet wurden. In den zwei Pavlova-Futtermitteln wurden 50 %, beziehungsweise 100 % des

Zusammenfassung

88

noch verbliebenen Fischölanteils der Basaldiät durch den Fettanteil im Pavlova viridis-Mehl ersetzt

(P50 und P100). Ebenso wurden zwei Nannochloropsis-Futtermittel formuliert. Hierbei wurden

wiederum 50 %, beziehungsweise 100 % des noch verbliebenen Fischölanteils der Basaldiät durch

den Fettanteil im Nannochloropsis sp.-Mehl ausgetauscht (N50 und N100). Das höchste spezifische

Wachstum und die beste Futterverwertung wurden nach Versuchsende in der P100 Gruppe

beobachtet (1.77±0.10 % Tag-1und 1.17±0.01). Allerdings waren diese Ergebnisse nicht signifikant

unterschiedlich zu denen der Kontrollgruppe oder der anderen Algengruppen. Der höchste Gehalt an

mehrfachungesättigten Fettsäuren wurde ebenfalls in Fischproben der P100 Gruppe ermittelt. Die

höchsten Gehalte an DHA wurden allerdings in der Kontroll- und Basalgruppe beobachtet, wenn auch

ohne signifikanten Unterschied zu den Gruppen P50 und P100. Die höchsten Werte an EPA wurden in

der Gruppe P100 gemessen. Die histopathologischen Untersuchungen von Leber- und Darmgewebe

zeigten keine Einflüsse der unterschiedlichen Futtermittelverabreichung. Insgesamt ließ die

Mikroalge Pavlova viridis ein hohes Potential als omega-3 Fettsäurequelle in Trockenfuttermitteln

erkennen. Sowohl das Wachstum, als auch die Futterverwertung, sowie die

Fettsäurezusammensetzung der Wolfsbarsche konnten durch Pavlova-Mehl positiv beeinflusst

werden.

Durch die vorliegende Studie kann der Einsatz der Mikroalge Pavlova sp. als Kultivierungsprodukt für

Lebendfuttermittel, als Anreichungsprodukt für Lebendfutter zur Ernährung mariner Fischlarven und

als omega-3 Fettsäurequelle in Trockenfuttermitteln als positiv bewertet werden. Die Nutzung dieser

Alge ist ein vielversprechender Ansatz eine neue Nährstoffquelle in der Aquakultur zu etablieren, um

so zum einen die Fütterungsverfahren in der Aquakultur weiterzuentwickeln und zum anderen eine

hohe Qualität von Fisch und Meeresfrüchten aus Aquakultur zu gewährleisten.

Danksagung

89

DANKSAGUNG

Ich möchte mich an dieser Stelle bei einigen Personen bedanken, die mich bei der Erstellung dieser

Arbeit sehr unterstützt haben:

Ich bedanke mich bei Prof. Dr. Carsten Schulz, der mir die Möglichkeit gegeben hat, diese Arbeit

durchzuführen. Vielen Dank für hervorragende Betreuung und die jederzeit gewährte und immer

umgehende Beratung und Unterstützung.

Vielen Dank an Dr. Stefan Meyer. Die konstruktiven Gespräche und Ratschläge haben nicht nur

deutlich zum Gelingen dieser Arbeit, sondern auch zur Weiterentwicklung meiner wissenschaftlichen

Arbeitsweise beigetragen.

Dr. Sebastian Lippemeier und Dr. Daniela Martensen-Staginnus möchte ich für die nette und

erfolgreiche Zusammenarbeit danken. Es war mir eine große Freude, dass ich durch diese Arbeit

einen Beitrag zum Pavlova-Projekt liefern konnte.

Ich möchte mich auch herzlich bei allen Kollegen der GMA bedanken. Ob durch Diskussionen,

tatkräftige Hilfe im Labor und bei Versuchsauflösungen oder einfach nette Gespräche am

Mittagstisch, habt ihr mich während meiner Doktorarbeit unterstützt.

Nicht nur die harmonische Stimmung im ganzen Team, sondern besonders auch die

Büronachbarschaft mit Anja und Christin war wundervoll. Anja, wir konnten zusammen immer

perfekt unser Zeitmanagement aufarbeiten, wir hatten ernste, aber noch viel mehr lustige

Gespräche. Dafür und auch für das ein oder andere Fischbrötchen, das du für mich „gesichert“ hast,

danke ich dir.

Ich bedanke mich an dieser Stelle auch bei vielen „alten und neuen“ Freunden, für ihre liebe

Unterstützung und das Interesse an meiner Arbeit.

Ein besonderer Dank gilt meiner Familie, vor allem meiner Schwester und meinen Eltern, die mich in

allen Phasen der Promotion mit jeder möglichen und uneingeschränkten Unterstützung bedacht

haben.

Schließlich und keineswegs zuletzt möchte ich mich bei meinem Mann Christopher bedanken. Nicht

nur während dieser Promotion, sondern immer, bist du mein Halt, motivierst mich und bist für mich

da. Danke!

Lebenslauf

90

LEBENSLAUF

Name Rehberg-Haas (geb. Rehberg)

Vorname Sabine

Geburtsdatum 1. Oktober 1985

Geburtsort Miltenberg am Main

Staatsangehörigkeit Deutsch

Familienstand Verheiratet

Ausbildung

Universität – Biologiestudium

Okt 2007 – Aug 2010 Universität Rostock Prüfungsfächer: Meeresbiologie (Hauptfach), Fischereibiologie,

Zoologie, Meereschemie; Abschluss: Diplom Diplomarbeit: („Validation on age determination of juvenile western Baltic cod (Gadus morhua L.)“); Institut für Hydrobiologie und Fischereiwissenschaften, Hamburg und Thünen-Institut für Ostseefischerei, Rostock

Apr 2006 – Sep 2007 Julius-Maximilians-Universität, Würzburg Abschluss: Vordiplom

Okt 2005 – Mrz 2006 Universität Regensburg

Schule

Sep 1996 – Jul 2005 Johannes-Butzbach-Gymnasium, Miltenberg am Main

Sep 1992 – Jul 1996 Grundschule, Freudenberg am Main

Berufliche Tätigkeit

Seit Sept 2014 Wissenschaftliche Mitarbeiterin am Institut für Tierzucht und Tierhaltung, Christian-Albrechts-Universität zu Kiel

Jan 2012 – Aug 2014 Wissenschaftliche Mitarbeiterin und Doktorandin an der Gesellschaft für Marine Aquakultur mbH, Büsum

Okt 2011 – Dez 2011 Wissenschaftliche Mitarbeiterin (Auftragsforschung) an der Gesellschaft für Marine Aquakultur mbH, Büsum

Documents

Utilization of the microalga Pavlova sp. in marine fish nutrition · List of Tables V List of Tables Table I-1: Final rotifer density, final egg density, final egg ratio and instantaneous