in diets for carp, Cyprinus carpio Ayub Younis Anwar

174 Pages·2013·5.17 MB·English

by  Ayub Anwar

Copyright Statement
This copy of the thesis has been supplied on the condition that anyone who
consults it is understood to recognize that its copyright rests with its author and
that no quotation from the thesis and no information derived from it may be
published without the author’s prior consent.

I
Inclusion of lupin meal and effect of a commercial
feed supplement (Synergen™) in diets for carp,
Cyprinus carpio

Ayub Younis Anwar
A thesis submitted to the University of Plymouth
In partial fulfilment for the degree of

ResM (Research Masters) Degree

School of Biological Sciences
Faculty of Science and Technology

(July 2013)

II
Inclusion of lupin meal and effect of a commercial feed
supplement (Synergen™) in diets for carp, Cyprinus carpio
Ayub Younis Anwar
Abstract
Plant proteins are mainly used to formulate diets for carp. Soybean meal (SBM) is
one of the most nutritious of all plant protein sources in carp feeds. However, an
increase in the use of soybean meal for human consumption and animal feed in
both developed and developing countries has resulted in an increase market
price of soybean meal globally. Therefore, using other inexpensive plant protein
sources in carp feeds would be beneficial to reduce feed cost and contribute to
food security as well as to sustain aquaculture production. Anti-nutritional factors
(ANFs) are believed to be the most important factors limiting the use of plant
protein concentrates in fish feeds. To address this limitation, one option is to use
an exogenous (mixed- enzyme containing, solid state fermentation, SSF) dietary
supplement. Two nutritional trials were conducted in order to assess the
incorporation of extruded white lupin (Lupinus albus) and supplement Synergen™
in diets for juvenile carp. The first trial was designed to determine the effect of
including 12.5% and 25% of white lupin as a soybean meal replacement with the
addition of 0.05% of Synergen™ for common carp BSD (Basal skretting diet)
based diet on growth performance, feed utilization and general health of the fish.
All diets were formulated based on the summit dilution trial type. Supplementing
Synergen™ to the BSD based diet and the diet including 12.5% of white lupin
significantly (P<0.05) improved growth performance and feed utilization but this
trend was very slight with the diet that contained 25% of white lupin. On the other
hand, including 12.5% of white lupin significantly (P<0.05) improved growth
performance and feed utilization. On contrary, including 12.5% of white lupin
III
significantly (P<0.05) decreased growth performance and feed utilization. No
significant (P>0.05) differences were found in growth performance, carcass
composition or liver and gut histology between the BSD based diet and diet that
contained 25% of white lupin with Synergen™. The second trial was designed to
determine the effects of substituting 12.5% and 25% of the soya protein
concentrate (SPC) with white lupin seed meal and with the addition of 0.1% of
Synergen™ for the mirror carp (Cyprinus carpio) plant based diet on growth
performance, feed utilization and general health of the fish. All diets were
formulated to be iso-nitrogenous (38% crude protein) and isolipdic (8% crude
lipid). Supplementing Synergen™ to the soya protein concentrate based diet and
the diets substituting 12.5% and 25% of soya protein concentrate with white lupin
significantly improved growth performance, feed utilization and gut and liver
histology. Additionally, substituting up to 25% of soya protein concentrate with
white lupin in the complete diet for mirror carp did not have any significant
negative effects on growth performance, feed utilization, carcass composition and
fish health.
The results of this research program demonstrate that supplementing Synergen™
to plant based diets is beneficial to reduce the negative effect of anti-nutritional
factors and to improve growth performance and nutrient utilization for carp. In
addition, our findings demonstrate that lupin meal has a promising potential for
use in common carp and mirror carp feeds with importance for the aquaculture
industry.

IV
Table of Contents
Copyright Statement ................................................................................................ I
Title page ................................................................................................................ II
Abstract .................................................................................................................. III
List of Tables ......................................................................................................... IX
List of Figures ......................................................................................................... X
List of Plates .......................................................................................................... XI
List of Abbreviations.............................................................................................. XII
Acknowledgments .............................................................................................. XVII
Dedication ......................................................................................................... XVIII
Author’s Declaration............................................................................................ XIX
Chapter 1: General review and introduction ............................................................ 1
1.1 Global Fisheries and Aquaculture Status .......................................................... 2
1.2 Aquafeed production ......................................................................................... 5
1.3 Plant protein sources ........................................................................................ 7
1.4 Lupins in Aquafeeds: A review ........................................................................ 10
1.4.1 Introduction ........................................................................................... 10
1.4.2. Lupins composition .............................................................................. 11
1.4.3 Anti-nutritional Factors in Lupins (ANFs) ............................................... 17
1.4.4 Lupins in aquafeeds .............................................................................. 19
1.5 Enzyme Applications in Aquaculture ............................................................... 23
1.6 Synergen™ (Alltech Inc, .USA) ....................................................................... 25
1.7 Carp (Cyprinids) .............................................................................................. 27
1.7.1 Nutrient requirements ............................................................................ 28
1.7.1.1 Protein Requirement .......................................................................... 28
1.7.1.2 Amino acid requirement ..................................................................... 29
1.7.1.3 Energy ................................................................................................ 31
1.7.1.4 Lipid requirements .............................................................................. 31
1.7.1.5 Fatty acid requirements ...................................................................... 32
1.7.1.6 Carbohydrate requirements ................................................................ 32
1.7.1.7 Vitamin requirements ......................................................................... 33
1.7.1.8 Minerals ................................................................................................. 34
1.8 Aims and objectives of research program ....................................................... 36
V
Chapter 2 .............................................................................................................. 37
General Materials and Methods ............................................................................ 37
2.1 Feed formulation and Diet preparation ............................................................ 38
2.2 Fish and husbandry ........................................................................................ 39
2.3 Feed and Weighing ......................................................................................... 39
2.4 Water quality ................................................................................................... 39
2.5 Growth and feed utilization calculations .......................................................... 41
2.6 Chemical and proximate analysis ................................................................... 42
2.6.1 Moisture .................................................................................................... 42
2.6.2 Ash ........................................................................................................... 42
2.6.3 Lipid .......................................................................................................... 43
2.6.4 Protein ...................................................................................................... 44
2.6.6 Gross Energy ............................................................................................ 45
2.7 Histology ......................................................................................................... 47
2.7.1 Light microscopy ....................................................................................... 47
2.7.2 Electron microscopy ................................................................................. 49
2.8 Haematological parameters ............................................................................ 51
2.8.1 Haematocrit (Hct) ...................................................................................... 51
2.8.2 Haemoglobin (Hb) ..................................................................................... 52
2.8.3 Determination of differential leukocyte counts .......................................... 53
2.9 Microbiological investigations ......................................................................... 54
2.9.1 Fish dissection .......................................................................................... 54
2.9.2 Plating and colony counts ......................................................................... 54
2.10 Statistical analysis ......................................................................................... 55
Chapter 3:Evaluate the effect of white lupin (L. albus) inclusion and dietary
supplementation of Synergen™ on growth performance and feed utilization in diet
for common carp (Cyprinus carpio) ....................................................................... 56
3.2 Introduction ..................................................................................................... 56
3.3 Materials and methods .................................................................................... 61
3.3.1 Experimental dietary preparation .............................................................. 61
3.3.2 Fish rearing ............................................................................................... 62
3.3.3 Rearing conditions .................................................................................... 63
3.3.4 Sampling ................................................................................................... 63
3.3.5 Growth and feed utilization calculations ....................................................... 63
VI
3.3.6 Chemical analysis ..................................................................................... 65
3.3.7 Histology ................................................................................................... 65
3.3.8 Light microscopy ....................................................................................... 65
3.3.9 Statistical analysis .................................................................................... 65
3.4 Results ............................................................................................................ 65
3.4.1 Growth and feed utilization ....................................................................... 65
3.4.2 Carcass composition ................................................................................ 68
3.4.3 Histological examination ........................................................................... 71
3.4.3.1 Intestinal histology ................................................................................. 71
3.4.3.2 Liver histology…………………………………………………………………73
3.5 Discussion ...................................................................................................... 74
3.6 Conclusion ...................................................................................................... 80
Chapter 4: Partial replacement of soya protein concentrate (SPC) meal by white
lupin (L.albus) seed meal and Synergen™ supplementation in complete diets for
juvenile mirror carp (Cyprinus carpio) ................................................................... 81
4.2 Introduction ..................................................................................................... 81
4.3 Materials and Methods .................................................................................... 85
4.3.1 Diets ......................................................................................................... 85
4.3.2. Rearing condition ..................................................................................... 88
4.3.3 Fish ........................................................................................................... 88
4.3.4 Sampling ................................................................................................... 89
4.3.5 Growth and feed utilization calculations .................................................... 89
4.3.6 Chemical analysis ..................................................................................... 89
4.3.7 Histology ................................................................................................... 89
4.3.8 Light microscopy ....................................................................................... 89
4.3.9 Electron Microscopy ................................................................................. 90
4.3.10 Haematological parameters .................................................................... 90
4.3.12 Microbiology ............................................................................................ 91
4.3.13 Statistical analysis .................................................................................. 91
4.4 Results ............................................................................................................ 91
4.4.1 Growth and feed utilization ....................................................................... 91
4.4.2 Carcass composition ................................................................................ 96
4.4.3 Histological examination ........................................................................... 98
4.4.3.1 Gut histology .......................................................................................... 98
VII
4.4.3.2 Liver histology ...................................................................................... 108
4.4.4 Blood parameters ................................................................................... 109
4.4.5 Culture-based analysis ........................................................................... 112
4.5 Discussion .................................................................................................... 113
4.6 Conclusion .................................................................................................... 120
Chapter 5 ............................................................................................................ 121
General discussion and conclusion .................................................................... 121
References ......................................................................................................... 131
Appendix II .......................................................................................................... 145
Diploma supplement ........................................................................................... 145

VIII
List of Tables
Table 1.1 The state of fisheries and aquaculture production in the world
(Excluding aquatic plants)……………………... …….…………………………..…….. 4
Table1.2 Chemical composition of major lupin species (%) .................................. 13
Table 1.3 Essential amino acid profile for major lupin species .............................. 14
Table 1.4 Fatty acid profile of major lupin species (%).......................................... 15
Table 1.5 Mineral content of major species of lupin .............................................. 16
Table 1.6 Essential amino acid requirement for common carp as percentage . …..30
Table 1.7 Summary of the vitamin requirement of common carp mg/kg dry diet.33
Table 1.8 Recommended macro and micro mineral requirements for carp. ......... 35
Table 3.1 Proximate analysis of the diet components .......................................... 62
Table 3.2: Formulation and proximate analysis of the experimental diets (dry
weight) .................................................................................................................. 64
Table 3.3 Growth performance and feed utilization of common carp fed the
experimental diets for 10 weeks. (n=2) ................................................................. 67
Table 3.4 Proximate composition of initial fish carcasses and fish after 10 weeks
feeding on the experimental diets (n = 4) .............................................................. 69
Table 3.5 Posterior Intestinal morphology of fish fed on the experimental diets for
10 weeks (n = 4) ................................................................................................... 70
Table 3.6 Liver histological analyses of fish fed the experimental diets for 10
weeks. (n = 4) ....................................................................................................... 72
Table 4.1 Dietary formulations and proximate composition and energy content of
the experimental diets ........................................................................................... 87
Table 4.2 Estimated essential amino acid profile in the experimental diets g amino
acid /kgˉ¹ diet……………………………………………………………………………88
Table 4.3 Growth performance and feed utilization of carp fed the experimental
diets. (n=2) ............................................................................................................ 95
Table 4.4: Carcass composition of initial and final carp fed the experimental diets
(expressed as percentage dry weight). (n = 4) ...................................................... 97
Table 4.5 Intestinal morphology of fish fed on the experimental diets for 10 weeks.
(n = 6) ................................................................................................................. 100
Table 4.6 Liver histological analyses of fish fed the experimental diets for 10
weeks.(n = 6) ...................................................................................................... 107
Table 4.7 Haematological parameters of common carp after 10 weeks of feeding
on experimental diets. (n=6) ............................................................................... 110
Table 4.8 Log viable counts (CFU gˉ¹) from the digesta mirror carp. (n=4)……112
IX
List of Figures
Figure 1.1 The state of capture fisheries and aquaculture production in the world.. 4
Figure 1.2 Aquaculture productions by region: quantity and percentage of world
total production (excluding aquatic plants and non-food products) in 2010 ............ 5
Figure 1:3: Global consumption of fishmeal by major aquaculture species group in
2010 ...................................................................................................................... 10
Figure 1.4 Global productions of lupins by country in 2009 .................................. 11
Figure 3.1 Posterior Intestinal bulbs of the common carp stained by haematoxylin
and eosin (Scale bar 100 μm). .............................................................................. 71
Figure 3.2 Haematoxylin and eosin stained sections of livers of mirror carp. ....... 73
Figure 4.1 Haematoxylin and eosin stained section of the mid gut of mirror carp
(Scale bar 50μm). ............................................................................................... 101
Figure 4.2 Haematoxylin and eosin stained section of the posterior of mirror carp
(Scale bar 50μm). ............................................................................................... 102
Figure 4.3 Alcian blue and PAS stained section of the mid gut of mirror carp (x10)
(Scale bar 50μm) ................................................................................................ 103
Figure 4.4 Alcian blue and PAS stained section of the hind gut of mirror carp (x
10). (Scale bar 50μm) ......................................................................................... 104
Figure 4.5 Comparative SEM micrographs of posterior intestine of mirror carp fed.105
Figure 4.6 Comparative TEM micrographs of the posterior intestine ......... ……..106
Figure 4.7 Haematoxylin and eosin stained sections of livers of mirror carp. ..... 108
Figure 4.8 Blood cells of mirror (Cyprinus carpio L) ............................................ 111

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