EFFECTS OF PARENTAL SIZE AND AGE ON LARVAL GROWTH AND
DEVELOPMENT: IMPLICATIONS FOR IMPROVED INTENSIVE LARVAL
YELLOW PERCH (Percaflavescens) CULTURE TECHNIQUES
By

Michael Alan Oetker

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1998

ABSTRACT
EFFECTS OF PARENTAL SIZE AND AGE ON LARVAL GROWTH AND
DEVELOPMENT: IMPLICATIONS FOR IMPROVED INTENSIVE LARVAL
YELLOW PERCH (Percaflavescens) CULTURE TECHNIQUES
By

Michael Alan Oetker

Yellow perch aquaculture has been constrained by inadequate larval culture
techniques due, in part, to the small size of firSt feeding larvae. This study was designed
to: 1) identify mouth gapes for first feeding larvae, 2) estimate heritabilities, and 3)
correlate Artemia cyst diameter to nauplius hatching size. Mouth gapes for larval yellow
perch less than 10 mm total length (TL) were described by linear regression models:
width = 0.081599 + 0.006664 (TL)2 + e and height # 0.060535 + 0.007155 (TL)2 + e.
Heritabilities (hz), based on spawner TL and age, were estimated for larval yellow perch
TL (h2 = 0.1393), mouth gape width (h2 = 0),-and mouth gape height (h2 = 0.2289).
Separating Artemia cysts into discrete size categories facilitated the significant, positive
correlation of cyst diameter to nauplius size. These results can be used to help design

breeding programs and develop intensive larval yellow perch culture feeding strategies.

ACKNOWLEDGMENTS

I want to thank my major professor, Dr. Don Garling, for his support, guidance,
and friendship during my development as a researcher and through the completion of this
study. I also thank the rest of my graduate committee, Dr. Robert Batie and Dr. Pat
Muzzall, for their time and for their assistance provided in this study. Special recognition
goes to the Bay Port Aquaculture researchers who provided information, support, and
needed materials throughout the study. My appreciation goes to past and present
members of Michigan State University, Department of Fisheries and Wildlife for their
support and friendship during my graduate program. Finally, I send my love and thanks
to family and friends.

This research was supported by the North Central Regional Aquaculture Center
under grant number 95-38500-1410 from the United States Department of Agriculture
Cooperative State Research, Education, and Extension Service. The US. Government is
authorized to produce and distribute reprints for governmental purposes notwithstanding

any copyright notation appearing hereon.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ........................................................................................................... xi
INTRODUCTION ............................................................................................................... 1
Objectives ................................................................................................................ 3
Hypothesis statements .............................................................................................. 5
CHAPTER 1
EFFECTS OF ARTEMIA CYST DIAMETER ON NAUPLII SIZE
Introduction .............................................................................................................. 9
Objectives ............................................................................................ .....10
Literature Review ................................................................................................... 11
Methods .................................................................................................................. 16
Statistical Analysis ................................................................................................. 20
Results .................................................................................................................... 21
Discussion .............................................................................................................. 32
Summary and Conclusions .................................................................................... 36
CHAPTER 2

EFFECTS OF PARENTAL SIZE AND AGE ON DIFFERENCES OBSERVED IN EGG
SIZE, MOUTH GAPE SIZE, AND TOTAL LENGTH OF LARVAL YELLOW PERCH
AT HATCH

Introduction ............................................................................................................ 38

Objectives ................................................................................................. 39
Literature Review ................................................................................................... 40
Methods

Maternal Influence .................................................................................... 46
Statistical Analysis ................................................................................................. 52

Paternal Influence ..................................................................................... 57
Statistical Analysis ................................................................................................. 61
Results .................................................................................................................... 64
Discussion ............................................................................................................ 103
Summary and Conclusions .................................................................................. 119

iv

CHAPTER 3
EXPERIMENTAL TANK CULTURE DESIGN AND DIET STRATEGY
EVALUATION FOR FEEDING LARVAL YELLOW PERCH

Introduction .......................................................................................................... 122
Objectives ............................................................................................... 123
Literature Review ................................................................................................. 124
Methods
Culture System Design ........................................................................... 130
Egg and Larval Collection Procedures .................................................... 131
Feeding Procedures ................................................................................. 133
Changes for Trial Two ............................................................................ 136
Statistical Analysis ............................................................................................... 140
Results
Trial One ................................................................................................. 143
Trial Two ................................................................................................ 150
Discussion ............................................................................................................ 154
Summary and Conclusions .................................................................................. 164
APPENDICES
Appendix A. Artemia hatching meditun using artificial seawater ...................... 165
Appendix B. Analytical methods used for determining hatching percent .......... 166
Appendix C. Results of the statistical analysis conducted for objectives 1 and 2
................................................................................................................. 1 68
Appendix D. Calculation of heritability (hz) estimates ....................................... 169
Appendix E. Calculation of dominance (d2) estimates ....................................... 170

Appendix F. Results of the statistical analysis conducted for female spawner total
length, weight, age, egg ribbon weight, total fecundity, and number of
eggs per gram of eggs (objectives 3, 4 and 5) ......................................... 171

Appendix G. Results of Two factor Analysis of Variance (ANOVA) testing for
female spawner total length, weight, egg ribbon weight, total fecundity,
and number of eggs per gram of eggs (objective 3) ................................ 172

Appendix H. Results of simple linear regression analysis testing of female
spawner size to egg ribbon weight, total fecundity, and number of eggs
per gram of eggs (objective 3) ................................................................ 173

Appendix I. Results of factorial analysis testing for the determination of
significant differences between areas of the egg ribbon for number of eggs
per gram of eggs, chorion shell diameter, and yolk diameter ................. 174

Appendix J. Results of simple linear regression analysis of female spawner TL
and weight on chorion shell and egg yolk diameters .............................. 175

Appendix K. Results of One-way Analysis of Variance (AN OVA) testing for
larval total length, larval mouth gape width, and larval mouth gape height
for each day sampled post-fertilization ................................................... 176

Appendix L. Results of simple linear regression analysis of female spawner total
length and weight on larval total length, mouth gape width, and mouth
gape height .............................................................................................. 177

Appendix M. Results of simple linear regression analysis of larval total length on
mouth gape width and height across all female spawner size groups ..... 178

Appendix N. Results of linear regression analysis of larval total length on mouth
gape width and height for each female spawner size groups (SG) ......... 179

Appendix 0. Results of Analysis of Covariance (AN COVA) testing larval total
length on mouth gape width and height between each spawner size
group ....................................................................................................... 180

LIST OF REFERENCES ................................................................................................. 181

vi

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

LIST OF TABLES

Label designation for each micro-sieve based on the mesh size ......................... 17

Averages and percentages of San Francisco Bay (SFB) and Great Salt Lake

(GSL) Anemia cyst dry weight for each micro-sieve ......................................... 22
Averages of San Francisco Bay (SFB) and Great Salt Lake (GSL) Artemia cyst
diameters for each micro-sieve ........................................................................... 23

Averages of San Francisco Bay (SFB) and Great Salt Lake (GSL) Artemia
nauplii length, width, and appendage length for the M8200 and M8280 micro-

sieves ................................................................................................................... 27
Hatching percentage of San Francisco Bay (SFB) and Great Salt Lake (GSL)
Artemia strains in micro-sieves M8200 and M8280 .......................................... 31
Separation of female yellow perch broodstock into six female spawner size
groups (8G) based on total length ....................................................................... 48

Averages for spawner total length (TL), weight, and age for each female
spawner size group (8G) ..................................................................................... 65

Averages for spawner total length (TL) and weight for each female spawner
completed age ..................................................................................................... 66

One-way Analysis of Variance (AN OVA) contrast statements for spawner total
length (TL), weight, and age between female spawners groups (8G) ................ 68

Table 10. Averages for number of eggs per gram in each position of the egg ribbon

(anterior, median, and posterior) for female spawner size groups (8G), SG 226
and SG 301 ........................................................................................................ 69

Table 11. Averages for egg yolk diameter in each position of the egg ribbon (anterior,

median, and posterior) for female spawner size groups (SG), SG 226 and SG
301 ..................................................................................................................... 69

vii

Table 12.

Table 13.

Table 14.

Table 15.

Table 16.

Table 17.

Table 18.

Table 19.

Table 20.

Table 21.

Table 22.

Table 23.

Table 24.

Table 25.

Averages for chorion shell diameter in each position of the egg ribbon
(anterior, median, and posterior) for female spawner size groups (SG), SG 226
and SG 301 ........................................................................................................ 70

One-way Analysis of Variance (AN OVA) contrast statements for chorion shell
and egg yolk diameters between positions of the egg ribbon (anterior, median,
and posterior) for female spawner size groups (SG), SG 226 and SG 301 ....... 72

Averages for egg ribbon weight, number of eggs per gram, and total fecundity
for each female spawner size group (SG) ......................................................... 73

Averages for number of egg per gram, egg ribbon weight, and total fecundity
parameters for each completed age of female spawners ................................... 74

One-way Analysis of Variance (ANOVA) contrast statements for egg ribbon
weight between female spawner size groups (SG) ........................................... 76

Contrast statements for total fecundity based on the Kruskal-Wallis non-
parametric test between female spawner size groups (8G) ............................... 78

Average for egg yolk and chorion shell diameters in each female spawner size
group (8G) ........................................................................................................ 79

One-way Analysis of Variance (AN OVA) contrast statements for chorion shell
diameter between female spawner size groups (SG) ........................................ 81

Average for larval TL in each female spawner size group (SG) sampled
through days 15 to 19 post-fertilization ............................................................ 82

Average for larval mouth gape width in each female spawner size group (8G)
sampled through days 15 to 19 post-fertilization .............................................. 83

Average for larval mouth gape height in each female spawner size group (8G)
sampled through days 15 to 19 post—fertilization .............................................. 84

One-way Analysis of Variance (AN OVA) contrast statements for larval TL on
day 18 post-fertilization between female spawner size groups (SG) ................ 86

One-way Analysis of Variance (AN OVA) contrast statements for larval TL on
day 19 post-fertilization between female spawner size groups (SG) ................ 87

One-way Analysis of Variance (AN OVA) contrast statements for larval mouth
gape width on day 19 post-fertilization between female spawner size groups
(8G) ................................................................................................................... 88

viii

Table 26.

Table 27.

Table 28.

Table 29.

Table 30.

Table 31.

Table 32.

Table 33.

Table 34.

Table 35.

Table 36.

Table 37.

Analysis of Covariance (ANCOVA) contrast statements for larval mouth gape
width regression coefficients between female spawner size groups (8G) ........ 90

Analysis of Covariance (AN COVA) contrast statements for larval mouth gape
width regression intercepts between female spawner size groups (SG) ........... 91

Analysis of Covariance (AN COVA) contrast statements for larval mouth gape
height regression coefficients between female spawner size groups (8G) ....... 92

Analysis of Covariance (ANCOVA) contrast statements for larval mouth gape
height intercepts between female spawner size groups (8G) ............................ 93

Estimation of maternally and paternally derived sourcesiof variation from the
average additive genetic strain effects, including the specific combining ability
for larval total length (TL) on days 13 to 18 post-fertilization and larval mouth
gape width and height on day 14 post-fertilization ........................................... 95

Factorial contrast statements for average additive genetic strain differences
between fast growth and slow growth for larval total length (TL) on days 13 to
18 post-fertilization and larval mouth gape width and height on day 14 post-

fertilization ........................................................................................................ 96
Averages for larval total length (TL) by genotype combinations for days 13 to
18 post-fertilization ...................... g ..................................................................... 97
Average for larval mouth gape width and larval mouth gape height by
genotype combinations on day 14 post-fertilization ......................................... 98

Estimates of heritability and dominance for larval total length (TL) on days 13
to 18 post-fertilization and larval mouth gape width and height on day 14 post-
fertilization ..................................................... . ................................................... 99

Estimates of average heterotic effects based on mating fast growth sires with
slow growth dams (hs) and mating slow growth sires with fast growth dams
(hd) for larval total length (TL) on days 13 to 18 post-fertilization and larval
mouth gape width and height on day 14 post-fertilization ............................. 101

Factorial contrast statements for differences between average non-additive
genetic strain effect of sire and dam for larval total length (TL) on days 13 to
18 post-fertilization and larval mouth gape width and height on day 14 post-
fertilization ...................................................................................................... 102

Larval yellow perch stocking levels for each tank during trial one ................ 134

ix

Table 38.

Table 39.

Table 40.

Table 41.

Table 42.

Table 43.

Table 44.

Table 45.

Table 46.

Description of feeding strategies for larval yellow perch during trial one ..... 135
Larval yellow perch stocking levels for each tank during trial two ................ 137
Description of feeding strategies for larval yellow perch during trial two ..... 139
Average larval yellow perch TL over time for each diet strategy during the first
feed trial .......................................................................................................... 143
Gas bladder inflation and feed acceptance rates over time for each diet strategy
during the first feed trial ................................................................................. 144
Average survival rates over time for each diet strategy during the first feed

trial ................................................................................................................. 146
Average mortality rates over time for each diet strategy during the first feed
trial .................................................................................................................. 146
Average survival rates over time for each diet strategy during the second feed
trial .................................................................................................................. 151
Average mortality rates over time for each diet strategy during the second feed

trral .................................................................................................................. 151

Figure 1.
Lake

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

LIST OF FIGURES

Average cyst diameter (micrometer) for commercially available Great Salt
Artemia strain from each micro-sieve ................................................................ 24

Average cyst diameter (micrometer) for commercially available San Francisco
Bay Artemia strain from each micro-sieve ........................................................ 25

Comparison of Great Salt Lake Artemia strain Instar I nauplii length, width,
and appendage length for cysts collected from M8200 and M8280
micro-sieves ....................................................................................................... 28

Comparison of San Francisco Bay Artemia strain Instar I nauplii length,
width, and appendage length for cysts collected from M8200 and M8280 '
micro-sieves ....................................................................................................... 29

Comparison of first Instar I nauplii for the commercially available Great Salt
Lake and San Francisco Bay Artemia strains fi'om micro-sieve M8200 and
M8280 ................................................................................................................ 30

Map showing sampling location of yellow perch broodstock in Lake Huron and
study site at Bay Port Aquaculture research facilities near Lake Michigan ...... 47

Experimental design for Objective 5, paternal influence on the larval TL and
mouth gape size of larval yellow perch .............................................................. 58

Experimental illustration of two-tailed t-based distribution for Objective 5,
selection of growth rates (slow vs. fast) based on age-length fi'equency
distributions ....................................................................................................... 60

Averages (1 standard deviations) of female spawner total length by each
completed age .................................................................................................. 104

Figure 10. Simple linear regression model for the natural log of female spawner total

length to weight relationship .......................................................................... 106

xi

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Simple linear regression analysis for female spawner total length to total
fecundity relationship ..................................................................................... 109

Simple linear regression analysis for female spawner weight to total fecundity
relationship ..................................................................................................... 1 10

Simple linear regression analysis for larval total length to mouth gape height
relationship ..................................................................................................... 113

Simple linear regression analysis for larval total length to mouth gape width
relationship ..................................................................................................... 115

Survivorship curve for larval yellow perch during the first feed trial age ..... 147

Instantaneous Growth Rates (IGR) of larval yellow perch during first feed
trial ................................................................................................................. 148

Instantaneous mortality rates (IMR) for larval yellow perch during first feed
trial ................................................................................................................. 149 ‘

Survivorship curve for larval yellow perch during the second feed trial ...... 152

Instantaneous mortality rates (IMR) for larval yellow perch during the second
feed trial ......................................................................................................... 153

xii

INTRODUCTION

In the North Central Region (NCR) of the United States (U .8.), yellow perch
(perch, Percaflavescens) is an excellent candidate for commercial aquaculture. It is
recognized for its value as a food fish and for certain biological characteristics that
benefit aquaculture (Calbert 1975). With the inception of the North Central Regional
Aquaculture Center (NCRAC) in 1988, perch became a high-priority species for
aquaculture production research and has been funded every year (Batterson, personal
communication, 1997). Throughout the NCR, researchers and fish-culturists believe that
perch is an important species for aquaculture development (Garling 1991; Starr 1991;
Hushak et al. 1992) based on market presence and buyer demand (Gleckler et al. 1991).

Perch is one of the more valuable sport, commercial, and forage fish species in the
U8. (Hokanson and Kleiner 1974; Jobes 1952; Ney and Smith 1975; Smith 1977;
Tharratt 1959; Thorpe 1977; Tsai and Gibson 1971; Whiteside et al. 1985) with
commercial harvests fi'om the Great Lakes comprising the major supply of perch fillets in
the US. However, recent declines in perch populations throughout Lake Michigan have
had significant impacts on the industry. In January 1995, the Perch Task Group (YPTG)
was asked to investigate the reasons for the decline in Lake Michigan perch populations

(Hess 1996). In all states surrounding Lake Michigan, commercial fishing for perch has

been closed for the 1998 fishing season. In addition, recreational fishing harvests have
been reduced.

Larval perch are small at the time of hatching and first feeding, and their size may
limit their ability to utilize a large portion of potential food supplies. Wong and Ward
(1972) have speculated that larval perch are restricted to limited amounts of prey due to
the maximum mouth gape width. Mouth gape limitations have occurred when the size of
the entire prey exceeded the size of the mouth gape (Hansen and Wahl 1981; Wong 1972)
or the diameter of the esophagus (Kestemont et al. 1996; Raisanen and Applegate 1983).
This appears to be the most limiting factor affecting the survival of larval perch.

Two approaches could be used to help increase survival rates of larval perch. The
first approach could concentrate on developing a method that would provide the smallest
possible Anemia nauplii at first feeding. Variations observed in cyst diameters and
nauplii length within a single strain may indicate a possible correlation between cyst
diameter and nauplii length. The smaller diameter cysts may potentially result in smaller
Anemia nauplii lengths. If a correlation exists between nauplii size and cyst diameter,
then a process could be developed to initially hatch the smallest diameter cysts. The
smallest nauplii could be fed first, since the small size of certain first feeding larval fish
and crustacean limits the ability to utilize a large portion of available Artemia nauplii.

A second approach that could be used to help increase survival rates of larval
perch would focus on the selection of broodstock. Identifying the effects of paternal size
and age on their offspring could help lead to selecting broodstock which might favor
increased survival rates in intensive culture conditions. Mansueti (1964) observed that

perch egg diameters varied in size, which suggests that eggs from the same female or

from different females varied in size. The size of eggs can influence development and
growth of larvae (Kamler 1992). However, no correlation has been reported between the
size of the female perch spawner and the average size of eggs and larvae. Larger sized
females could potentially produce larger eggs and larvae.

An alternative to this approach is genetic improvement through selective breeding
and genetic analysis. Perch broodstock has not been genetically improved through
selective breeding programs nor has any significant genetic analyses been completed. A
basic approach in comparing genetic differences was completed on perch. The study
compared survival and growth of larval perch, which were collected from various
geographical sources of wild broodstock and raised in intensive culture conditions
(NCRAC 1997). The results of the study indicated there was not a single source of perch
broodstock that produced superior larvae. However, genetic selection for larval perch
could help improve survival and other desirable characteristics.

This thesis is divided into three chapters and explores several strategies for
improving intensive culture of larval perch including, new uses of Artemia nauplii,
identifying the effects of parental size and age on offspring, and developing an intensive

culture system and feeding strategy.

OBJECTIVES

The goal of this research project was to develop and enhance current intensive
larval perch culture techniques. The specific objectives for this study were:

1) to evaluate strains of Artemia for variations in cyst diameter

2) to evaluate the effect of different cyst diameters on nauplii size (length and width) at
the Instar I stage. i

3) to determine the relationships of maternal size to total fecundity, egg size, larval total
length (TL) at the time of hatch, and mouth gape size at first feeding for larval
perch;

4) to define the range of mouth gape sizes for larval perch less than 10 mm TL;

5) to determine the relationships of parental size and age to mouth gape size and TL of
larval perch at the time of hatch; .

6) to determine the significance of gape size on growth, survival, and feed acceptance

during intensive larval culture conditions.

The first chapter includes Objective 1 and 2 and details the variation observed in
diameters of Artemia cysts, along with the potential use of size variation for larval perch
culture. The second chapter includes Objectives 3, 4, and 5, which attempt to identify the
reasons for size differences observed in larval perch at hatch based on parental influence.
The third chapter includes Objective 6, which describes the diet strategies and culture

methods used in this study.

HYPOTHESIS

The specific hypotheses tested for each objective in this study are:

Objective 1 :
Null Hypothesis l__l: All Arternia cysts are equal in size based on the diameter.
Prediction l_l: The cysts can be separated into groups using the diameter of the chorion
shell. If a large variation exists in the diameter of the cysts, then the groups will be
statistically different from each other based on One-way AN OVA testing.

Large variations in cyst diameters have been reported, but correlating the size of
newly hatched nauplii has not been studied. Objective 1 is designed to separate cysts into
size categories based on the diameter and test the significance between cyst size
categories. Two strains of brine shrimp, San Francisco Bay and Great Salt Lake, were
evaluated.

Objective 2:
Null Hypothesis 2_1: All newly hatched Arternia nauplii are the same size (length,
width, and appendage length).
Prediction 2_l: Nauplii size (length, width, and appendage length) can be correlated to
cyst diameter. If a significant, positive correlation exists, larger cyst diameters will result

in a statistically significant larger nauplii.

Large variations in nauplii length have been reported, but correlating the size of
newly hatched nauplii has not been studied previously. Objective 2 is designed to
correlate cyst diameter size categories to the length, width, and appendage length of
newly hatched nauplii.

Objective 3:
Null Hypothesis 3_1: All perch larvae are equal in total length (TL) at the time of
hatching.
Prediction 3_1: The predictor variable, larval TL, can be correlated to female spawner
TL. If a significant correlation exists, the female spawner size groups will be
significantly different from each other. Analysis of Variance (AN OVA) procedures can
test the differences between female spawner size groups.
Null Hypothesis 3_2: All spawned and fertilized perch eggs are of equal diameter. V
Prediction 3_2: The predictor variable, egg yolk diameter and chorion shell diameter,
can be correlated to female spawner TL. If a significant correlation exists, egg diameters
from different sized female spawner groups will be significantly different. AN OVA
procedures can test egg diameter differences between female spawner size groups.
Null Hypothesis 3_3: Total fecundity, total number of eggs, will be equal for all female
spawners.
Prediction 3_3: The predictor variable, total fecundity, can be correlated to female
spawner TL. If a significant correlation exists, the female spawner size groups will be
significantly different from each other. AN OVA procedures can test the differences

between female spawner size groups.

The study for objective 3 was designed to describe and predict the effects of
female spawner TL on total fecundity, egg yolk diameter, egg chorion shell diameter, and
larval TL. The design allowed for testing the variation thatimight exist in larvae and eggs
from the same female spawner as well as between different female spawners. The study
for objective 3 was also designed to describe and predict the effects of female spawner
TL on various egg and larval parameters.

Objective 4:
Null Hypothesis 4_1: Larval mouth gape size, width and height, increases with an
increase in larval TL.
Prediction 4_1: The predictor variable, mouth gape width and height, can be correlated
to the larval TL. If a significant correlation exists, the slope of the line will be
significantly different from zero.

Objective 4 was designed to describe and predict the effects of larval TL on
mouth gape width and height. The design allowed for testing the variation that might
exist in larvae from the same female spawner as well as between different spawners.

Objective 5:
Null Hypothesis 5_l: All perch larvae have an equal TL at the time of hatching.
Prediction 5_l: The predictor variable, larval TL , can be correlated to female and male
TL and age. If a significant correlation exists, the slope of the line will be significantly
different from zero.

Objective 5 was designed to test the significance of the paternal contribution
observed in Objective 3 and 4. The study was also designed to estimate heritability for

larval TL, mouth gape width and height, based on the sire and darn components of the

broodstock used. A true estimation of heritability was not calculated, since the design of
this study includes a fixed assignment of parental stock. However, the estimation of non-
additive genetic variance, dominance, was calculated.

Objective 6:
Null Hypothesis 6_1: All perch larvae have an equal survival rate.
Prediction 6_1: If all perch larvae have an equal chance to survive, then other factors can
be tested. The type of diet fed to first feeding larval perch can be correlated to survival
over time. If a significant correlation exists, the slope of the regression line will be
significantly different from zero. ANCOVA procedures can test the difference between
regression lines for each diet.
Null Hypothesis 6_2: All perch larvae have an equal growth rate.
Prediction 6_2: If all perch larvae have an equal chance to grow at the same rate, then
other factors can be tested. The type of diet fed to first feeding larval perch can be
correlated to growth over time. If a correlation exists, the slope of the regression line will
be significantly different from zero. ANCOVA procedures can test the difference
between regression lines for each diet.

Objective 6 was designed using the results from Objective 3. The present study
failed to result in rejecting the null hypothesis 3_1 (review Chapter 2, this thesis). This
indicated that there were no significant differences between larvae from different sizes of
female spawners. Hence, all larvae were pooled for further studies. Objective 6 was

designed to test the effects of different diets on the survival and growth of larvae.

CHAPTER 1

EFFECTS OF ART EMIA CYST DIAMETER ON NAUPLII SIZE

INTRODUCTION

Since 1933, many species of larval fish and crustacea have been intensively
cultured using Arternia nauplii as first food. In some cases, Artemia nauplii were the only
available source of live food for the youngest stages of most cultured species of larval
fish and crustacea (Bardach et al. 1972; Kinne and Rosenthal 1977). High survival rates
are associated with feeding Arternia nauplii, which has resulted in a large aquaculture
demand.

In the commercial Arternia industry, Arternia cysts are graded and sold based on
various parameters such as nutritional value, hatching rate, cysts per gram, and naupliar
size. Depending on the quality of the Arternia cysts being sold, the variation of reported
values for these parameters could be quite large within each graded batch. The variations
for each of these parameters have been investigated and compared. However, the

correlation between cyst diameter and nauplii length has not been studied previously.

10

When a large variation in cyst diameter occurs, another grading could be used to
separate cysts into more uniform diameter size categories. This would facilitate an
evaluation of the effect of cyst size on nauplii size at hatch. Smaller diameter cysts may
potentially result in smaller Artemia nauplii lengths. The smaller nauplii could be fed to
first feeding larval fish and crustaceans, which are limited by mouth gape size. Larger
diameter cysts could be used when the larvae have grown large enough to eat larger
nauplii.

By making more Artemia nauplii available to larvae limited by mouth gape, less
Arternia would have to be cultured and fed. Less waste from uneaten Arternia nauplii
would collect in the tanks and reduce the amount of Arternia needed. This process could
potentially make the feeding process more efficient and could reduce feed costs.

This study was designed to explore the variation in cyst diameters, the impact of
cyst diameter on nauplii size, and the potential application of cyst grading to larval fish
and crustacean culture. San Francisco Bay (SF B) and Great Salt Lake (GSL) strains of

Arternia cysts were compared.

OBJECTIVES

l) to evaluate strains of Arternia for variations in cyst diameter
2) to evaluate the effect of different cyst diameters on nauplii size (length and width) at

the Instar I stage.

ll

LITERATURE REVIEW

Many species of larval fish and crustacea have been successfully raised on newly hatched
Arternia nauplii, including: Eurasian perch (V lavon‘ou et al. 1995), perch (Hinshaw 1985;
Starr, personal communication, 1996; DabrowSki 1998), striped bass (Webster and Lovell
1990a; Lemm and Lemarie 1991), bluegill (Mischke 1995), smallrnouth bass (Ehrlich et
al. 1989), shrimp and prawn (Sorgeloos et al. 1983), as well as many others. Arternia
comprises approximately 85% of the total live foodused for aquaculture worldwide
(Lovell 1990).

The aquaculture demand for Arternia cysts was met by the harvest of natural areas
until the 1960’s, when a severe shortage of cysts stimulated new research on the optimal
use of Arternia (review Sorgeloos et al. 1986). Methods for processing cysts and
increased hatching success were two examples of the results from the research. From
these advances, uses of Arternia for aquaculture operations were made more efficient. In
1993, another shortage in the harvest of Arternia cysts occurred. After Arternia cyst
reserves were depleted in 1995, prices soared to 500% above 1994 costs (Sawtell,
personal communication, 1996). Problems continue to plague the supply of Arternia
cysts. During the 1997 collection season, the Utah Division of Wildlife Resources

(Perschon, personal communication, 1997) enacted an emergency closure of the Great

12

Salt Lake Arternia strain harvest on October 27, when the season quota of 4.5 million
pounds of Arternia cysts had been collected.

The Great Salt Lake is an important source of Arternia cysts and has represented
up to 90% of the world’s market supply (Sawtell, personal communication, 1996), but
approximately 90% of the harvest is exported to prawn farms in Asia (Jensen, personal
communication, 1997). The high price of Arternia and the uncertainties of supply have
renewed interests in the development of more efficient uses of Arternia in aquaculture
(Starr, personal communication, 1996).

The demand for Arternia cysts has grown with the growth of intensive larval fish
and crustacean culture. Contributing to the increased demand for Arternia has been the
low survival of larvae fed formulated feeds and the high survival of larvae fed Arternia
nauplii. Many larvae are unable to survive on formulated feeds alone, and in some cases,
newly hatched Arternia nauplii were the only available source of live food for the
youngest stages of most cultured species of larval fish and crustacea (Bardach et al. 1972;
Kinne and Rosenthal 1977). This is partly due to a rudirnentarily developed alimentary
tract of larval fish that is ofien unable to effectively use formulated diets at first feeding
(Lovell 1990). Short, poorly developed alimentary tracts, rapid evacuation rates, and low
production of digestive enzymes are considered to be some of the constraints of larval
food digestion (Kamler 1992). Enzymes derived from live food have been identified as
providing a substantial contribution to the total enzymatic activity in the alimentary tract
(Lauff and Hofer 1984) which Dabrowski (1979) suggested were needed for digestion of
food by larvae. However, survival of certain larvae raised in intensive culture conditions

and fed Arternia nauplii is still low. To help increase survival rates, researchers and

13

culturists have been searching for the smallest possible prey or formulated diets that still
provide the necessary nutritional requirements (Brown et al. 1996).

While Artemia nauplii have been used to successfully raise many types of larval
fish and crustacea, a search continues to identify smaller strains of Arternia. Feeding
smaller Artemia could help to increase the survival of intensively cultured larvae.
However, a different approach could be explored to help reduce production 'costs and
increase larval survival rates. Instead of searching for smaller Arternia strains, developing
ways to reduce the variation in cyst diameter and nauplii length could be explored.

Several authors have reported large statistical variations for various Arternia
parameters (V anhaecke and Sorgeloos 1980; Beck and Bengtson 1982; Quynh et al.
1988; Webster and Lovell 1990b; Webster and Lovell 1991). In addition, different strains
from various geographical locations were compared and the values reported. Vanhaecke
and Sorgeloos (1980) described variations in cyst diameters fiom different geographical
locations. Mean cyst diameter values reported ranged from 224 to 285 um and standard
deviations ranged from 9.7 to 19.8. The authors also reported nauplii length for strains
from different geographical locations. Mean nauplii length values ranged from 429 to
517 pm with standard deviations that ranged from 16.5 to 37.2. The cysts/gram ratio for
the GSL brand was reported to be 280,000 dehydrated cysts/ gram, while one Argent
brand reported 330,000 dehydrated cysts/ gram. Other parameters have also varied
depending on grades and brands. Some strains of Arternia provided high levels of
essential highly unsaturated fatty acids (20:5033 and 18x03), while other strains did not
have significant amounts (Sorgeloos et al. 1983; Quynh et al. 1988; Webster and Lovell

1990a). In the commercial Artemia industry, cysts are graded and sold based on various

14

parameters such as nutritional value, hatching rate, cysts/gram ratio, and naupliar size.
Higher values of these parameters result in higher prices (Sawtell, personal
communication, 1996). Depending on the quality of the Artemia cysts being sold, the
variation of reported values for these parameters could be quite large within each graded
batch.

The variations observed in cyst diameters and nauplii length within a single strain
may indicate a possible correlation between cyst diameter and nauplii length. A more
detailed analysis of these variations might lead to specific applications in aquaculture.
For example, if the cysts can be separated into different size categories according to
diameter, then it might be possible to evaluate nauplii size at hatch for each category.
Another grading could be used to separate cysts into more uniform diameter size
categories. By grading cysts into more uniform diameter size categories, the effect of cyst
size on nauplii size at hatch would be facilitated for evaluation. Smaller diameter cysts
may potentially result in smaller Arternia nauplii lengths.

If a significant, positive correlation exists between nauplii size and cyst diameter,
then a process could be developed to initially hatch the smallest diameter cysts. The
smallest nauplii could be fed first, since the small size of certain first feeding larval fish
and crustacea lirrrits the ability to utilize a large portion of available Arternia nauplii. To
be potential food, an organism must be small enough for larvae to ingest (Siefert 1972).
Mouth gape limitations of first feeding larvae occurs when the size of the prey exceeds
the size of the mouth gape (Hansen and Wahl 1981; Wong 1972) or the diameter of the
esophagus (Kestemont et al. 1996; Raisanen and Applegate 1983). Mouth gape size has

been observed to limit the optimal utilization of Artemia nauplii for frrst feeding Eurasian

15

perch (Percafluviatilis) larvae (Kestemont et al. 1996). Hyatt (1979) observed that
stickleback larvae could consume invertebrates with a maximum body width almost equal
to the mouth size. By providing the smallest Arternia nauplii, a greater percentage of
nauplii could be available as food, since the larvae are not restricted by the size of nauplii.

As larval size increases, the size of Artemia nauplii could be increased by
culturing larger diameter cysts. The morphological capability to consume larger prey
increases with growth (Wong and Ward 1972; Werner 1974). Larger diameter cysts
could be saved for later uses when the larvae have grown large enough to eat larger
Arternia nauplii. Larger nauplii could also be saved for later in the feeding process when
nauplii “packing” could be used. Packing Arternia refers to a method of increasing fatty
acid profiles, which are ofien absent in nauplii (see review in Sorgeloos et al. 1986).
After approximately 12 hours, newly hatched nauplii molt and begin feeding. Typically,
a food source that is high in essential fatty acids is provided. The nauplii retain the newly
acquired fatty acids and are fed to larval fish and crustacean as a complete diet.

This strategy could potentially make the feeding process more efficient. By
making more Arternia nauplii available to larvae limited by mouth gape, less Artemia
would have to be cultured and fed. Less waste from uneaten Arternia nauplii would
collect in the tanks and reduce the amount of Arternia needed. Feed costs would be

reduced and water quality improved.

l6

METHODS

San Francisco Bay' and Great Salt Lake2 Arternia cysts were compared for the
variations in cyst diameter and effects on nauplii size. Cyst diameters were separated into
groups using micro-sieves constructed from nitex plankton screen stretched over PVC
pipe. The nitex mesh sizes used were labeled with the prefix MS (Table 1) and were
separated into 200, 220, 236, 243, 253, 265, 280 and 300 um sizes. The pipe diameter
was 4 inches and made of schedule 40 PVC. Nitex plankton screen was held in place
using 4-inch slip to slip couplings made of schedule 40 PVC. The screens were placed in
descending order beginning with the 300 um screen.

Arternia cysts were separated by diameter using the micro-sieves. Six grams of
cysts were weighed and added to tap water at room temperature for hydration. Afier one
hour, the cysts were placed into the micro-sieves and washed through with tap water.
Triplicate trials of each strain were run with samples of 100 collected from each screen.
Filter containers and micro-sieves were washed thoroughly between sample trials.

Cysts from each screen were rinsed with tap water into 250 ml Millipore plastic
filter funnels. Excess .water was filtered off using a six-place kit Millipore filter system

with 47 mm, #1 glass filters. After removing excess water, the concentrated cysts were

 

' San Francisco Bay (Platinum grade Argentemia, Argent Chemical Laboratories, Inc., Seattle,

WA; lot BP0103K)
2 Great Salt Lake (Bonneville Arternia lntemational, Inc., Salt Lake City, UT; no lot number)

17

 

 

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18

placed under a dissecting microscope. The dissecting microscope was connected to a
camera, which digitized the video image. Diameters were measured using digital analysis
software, OPTIMAS Imaging System, BioScanTM.

‘ After measuring the diameter of the cysts, the percent of cysts collected on each
micro-sieve was calculated. Cysts were rinsed from the filter into aluminum pans and
allowed to dry in an oven at 38 °C for 48 hours (modified from Sorgeloos et al. 1986).
The dry cysts were weighed and calculated as a percentage of the total.

Nauplii size at hatch for the 200 and 280 um size categories were evaluated. The
separated cysts were hatched separately and samples collected to measure nauplii length
according to diameter. Three Arternia cyst samples. weighing 2.5 grams each were placed
into room temperature tap water. After one hour, the hydrated cysts were placed into the
top of the stacked micro-sieves and rinsed through with tap water. The cysts in the 200
and 280 um micro-sieves were collected and placed into a 1.5 L beaker with l L of
hatching medium. Procedures for hatching Artemia and making the hatching medium
(Appendix A) follow the guidelines reviewed by Sorgeloos et al. (1986). After
approximately 21 hours of incubation at 28 °C, first Instar I nauplii were randomly
collected fiom the triplicate samples. Procedures for random nauplii collection are
outlined in Sorgeloos et al. (1986). The nauplii were preserved in Lugol’s solution
(Edmondson 1959). Sample sizes of 20 were selected after conducting power analysis
tests (Snedecor and Cochran 1989). Arternia nauplii size was determined by measuring
the 20 Instar I nauplii along the long axis, at the widest point, and the length of the

appendages. The nauplii were placed under a dissecting microscope and measured using

19

digital analysis software, OPTIMAS Imaging System, BioScanTM. Methods for

determining hatching percentage (Appendix B) are outlined in Sorgeloos et al. (1986).

20

STATISTICAL ANALYSIS

Once separated into the different size categories, cyst diameters were measured
and statistically analyzed. The success of separating the cysts into the different size
categories was determined by calculating the mean cyst diameter for each micro-sieve and
comparing the means. Significance was based on an alpha level of 0.05%.

The means were tested first for normal distribution and equal variances. If the
sample size was greater than 2,000, the test for normal distribution was based on the
Kolmogorov D statistic (Neter et al. 1996). If the sample size was less than 2,000, the test ‘
for normal distribution was based on the Shapiro- Wilk statistic W (N eter et al. 1996). The
assumption of equal variances was tested using Hartley’s F-maximum test (Sokal and
Rohlf 1995). If the assumption of equal variances was violated, the nonparametric
Kruskal-Wallis test was used (Miller 1985). One-way Analysis of Variance (AN OVA)
was used to test differences between means from each micro-sieve. If the AN OVA test
indicated an overall significance, contrast statements were used to determine which
micro—sieves were significantly different from each other.

The mean nauplii size for the 200 and 280 um micro-sieves were tested for
normal distribution and equal variances. Two sample t-tests were used to test significant

differences between means from each screen and significant differences between strains.

21

RESULTS

The first objective of this study was to evaluate the variation in cyst diameters for
from each sample used. Using the stacked micro-sieves, Arternia cysts were separated
into different diameter size categories. The means and percentage of the total cyst
biomass used were calculated (Table 2). The majority of cysts were collected in micro-
sieves M8236, M8265, and M8280. These micro-sieves represented 67.9% of the total
for GSL and 67.3% for SFB. Very few cysts were collected from the micro-sieves
M8200 and M8220. These micro-sieves represented 6.0% of the total for GSL and 9.2%
of the total for SFB. Many of these cysts in these two micro-sieves were not hydrated and
were not used for measurements. The reasons for the failure to hydrate were not
investigated.

A sample of cyst diameters from each micro-sieve was measured, and the means
and standard deviations were calculated (Table 3). A general increase in variance was
observed as screen size increased. The data were not normally distributed (p = 0.0001)
over all treatments, and the assumption of equal variances across treatments was violated
(p > 0.05; Appendix C). The non-parametric test indicated that were significant

differences (Appendix C) which are summed in Figures 1 and 2.

 

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The second objective of this study was to determine if the Instar I nauplii size was
correlated to cyst diameter. Cysts from micro-sieves M8200 and M8280 were chosen
because the cyst diameters were significantly different (Figure 1; Figure 2) and were at
the opposite ends of the stacked micro-sieve (Table 1). The means for nauplii length,
widths, and appendage length were calculated and compared (Table 4). The data were
normally distributed for all parameters, and the variances were homogeneous across
treatments (Appendix C). One-way AN OVA indicated that there were statistically
significant differences between nauplii from the different micro-sieves for both strains
(Appendix C). Contrast statements indicated significant differences between micro-
sieves (Figure 3; Figure 4).

The parameters measured for both strains of Arternia were compared (Table 4;
Figure 5). The t-tests indicated that there were significant differences between strains for
nauplii width and appendage length in micro-sieve M8200. The SFB strain was
significantly larger for both parameters. The t-tests also indicated that there were
significant differences between strains for nauplii length and appendage length in micro-
sieve M8280. The GSL strain had a longer length than the SFB strain, but the SFB strain
had a larger appendage length than the GSL strain. Hatching percentage for both sizes

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32

DISCUSSION

Variations in cyst diameters from different-geographical locations have been
described by Vanhaecke and Sorgeloos (1980). Their study revealed large statistical
variation in Arternia cyst diameters for the SFB and GSL strains (ism = 224.7um, 0'2 =
153.76; You: 252.5 pm, 02 = 169). Similar values were found in the current study (ism
= 241.8um, 02 = 0.589; RGSL = 246.1 mm, 0'2 e 0.736). The large variation observed
indicated that the cysts could be separated into size categories.

Once the Arternia cysts were separated into different size categories according to
diameter, a more detailed analysis of these variationsjrevealed the distribution of cyst
diameters. For both strains, the majority of Arternia cysts were found in micro-sieves
M8236 (GSL — 15.4%; SFB - 17.9%), M8265 (GSL — 23.6%; SFB — 23.5%), and
M8280 (GSL — 28.9%; SFB — 25.9%). Less Arternia cysts were found in the remaining
micro-sieves. Although the number of Arternia cysts collected in each micro-sieve was
significantly different, the distribution helped reduce the number of micro-sieves needed.
By reducing the number of micro-sieves, the process of separating Anemia cysts was
improved.

The stacked micro-sieves were further improved by analyzing the cysts collected
in each micro-sieve. The cyst diameters were measured and statistically analyzed. For

the SF B Arternia strain, there were no significant differences between micro-sieves

33

M8236 vs. M8243 and M8243 vs. M8256, and the M8243 micro-sieve was removed.
For the GSL Artemia strain, there were no significant differences between micro-sieves
M8220 vs. M8236 and M8280 vs. M8300, and the micro-sieves, M8236 and M8300
were removed. The inability to achieve significant differences between some of the
micro-sieve groups may have been the result of three possible factors. First, the mesh
sizes may have been too similar. For example, some of the micro-sieves had a maximum
difference of 20 um, while the minimum difference was 7 pm (Table 1). The difference
between micro-sieves M8236 and M8243 was 7 pm. The mesh sizes represented the
Nitex screen availability in the commercial industry. The second reason for the inability
to achieve significant differences may have resulted from contamination. In all trials, it
was noted that there were leaks between several of the micro-sieves. The Nitex screen,
which was stretched over the PVC pipe, bunched up and prevented the PVC coupling
from fitting completely. As a result, some water and Arternia cysts escaped through the
side of the stacked micro-sieves and may have contaminated other micro-sieves. A third
reason for the lack of significant differences between some micro-sieves was the large
percentage of cysts collected in micro-sieves M8265 and M8280. The cysts clogged the
Nitex screen and made it difficult to process all cysts through the stacked micro-sieves.
Dehydrated cysts could not be used for this experiment, since the cysts have a convex
shape.

Statistically significant differences between micro-sieves and a more uniform
diameter size made it possible to evaluate nauplii size at hatch. Vanhaecke and Sorgeloos
(1980) reported variations in newly hatched Arternia nauplii length from the GSL and

SFB strains (25m = 429.5 pm, 02 = 689; RGSL = 487.5 pm, 0'2 = 894). Similar nauplii

34

length values were found in the current study (251:3 = 447nm, (:2 = 961; 52051. = 452 pm,
62 = 1225).

Nauplii size at hatch was evaluated by taking Arternia cysts from the micro-sieves
M8200 and M8280. Cysts from these two micro-sieves were chosen because theicyst
diameters were significantly different (Figure 1; Figure 2) and were at the opposite ends
of the stacked micro-sieves (Table 1). For both strains in the current study, nauplii had
statistically significant differences in length, width, and appendage length. This indicated
that there was an increase in nauplii size as Arternia cysts increased in size. Smaller
diameter cysts produced smaller Arternia nauplii sizes. If cysts were separated into more
uniform sizes, the smaller cysts could be cultured first, and the nauplii could be fed to
larval fish and crustaceans with the appropriate mouth gape and esophagus. As larvae
increase in size, the size of Arternia nauplii could be increased by culturing progressively
larger diameter cysts.

In the final part of this study, the differences between these two Arternia strains
were compared. It is generally accepted that the SFB strain is one of the smallest nauplii
overall (Sorgeloos et al. 1986). However, comparisons of the same size cyst diameter
from both strains indicated the opposite. GSL cysts collected from the micro-sieve
M8200 resulted in smaller newly hatched nauplii than SF B. This indicates that GSL
nauplii hatched from the smallest cysts can be fed to larval fish and crustaceans. This
trend was not repeated for larger diameter cysts.

Reducing size variation in cysts and, consequently nauplii size, has the potential
to make the feeding process more efficient when using newly hatched Arternia nauplii.

This process would help to provide a greater percentage of small Arternia nauplii to first

35

feeding larvae that are limited by mouth gape size. Less Arternia would have to be
cultured and fed, which reduces the amount of Arternia needed. Less waste from uneaten

Arternia nauplii would collect in the tanks and feed costs could potentially be reduced.

1)

2)

3)

4)

5)

36

SUMMARY AND CONCLUSIONS

The high prices and decreased supply of Arternia have renewed interest in developing
more efficient use of Arternia for aquaculture.

Newly hatched Arternia nauplii from the GSL strain were as small or smaller than
nauplii from the SFB strain. Either strain could be fed to first feeding larval fish and
crustaceans. However, the SF B strain is marketed as the smallest nauplii and should
be fed to small larvae. The demand and price for the SF B strain is higher, reaching as
much as $100 per lb, while the GSL strain is priced less than $35 per 1b.

Large statistical variations in cyst diameters were observed for the SFB and GSL
Arternia strains.

Nauplii hatched from cysts collected in the micro-sieves M8200 and M8280 had
statistically significant differences in length, width, and appendage length for both
strains, SF B and GSL. These results indicate that there was a significant, positive
correlation between Arternia cyst diameter and nauplii length, width, and appendage
length.

Although separating Arternia cysts by size would enhance their use, the process used
in this study would not be efficient for small-scale aquaculture operations. After the
hydration and separation processes, the Arternia cysts would have to be dehydrated for

storage (review Sorgeloos et al. 1986).

37

6) After harvesting, separating cysts into smaller size categories could be done during
cyst processing, but prior to dehydrating the cysts. The cysts could be graded,

dehydrated, and sold according to diameter.

CHAPTER 2

EFFECTS OF PARENTAL SIZE AND AGE ON DIFFERENCES OBSERVED IN EGG
SIZE, MOUTH GAPE SIZE, AND TOTAL LENGTH OF LARVAL YELLOW PERCH
AT HATCH

INTRODUCTION

Successful intensive larval culture of perch is limited by high mortality rates. The
small size of larval perch limits their ability to search and feed on certain prey. In
addition, Wong and Ward (1972) suggested that limited prey selection was due to a
restriction of gape width.

Variations in the size of eggs and larval fish fi'om the same female or among
females could influence development and growth. Larger sized females and males could
potentially produce larger larvae with larger mouths. Several species of larval fish with
larger mouths grew faster than larvae with smaller mouths (Shirota 1970).

This study was designed to identify the effects of parental size and age on
differences in eggs and larvae of perch. In the first year of this study, the relationships
between maternal size and age to total fecundity, egg size, larval TL at the time of hatch,
and mouth gape size at first feeding were identified. The range of mouth gape sizes for
larval perch under 10 mm TL were defined. In the second year, the relationships of

parental (both male and female) size and age to larval perch mouth gape size and TL at

38

39

the time of hatch were identified. Estimates of heritability were calculated for potential

selection of desirable characteristics.
OBJECTIVES

3) to determine the relationships of maternal size to total fecundity, egg size, larval TL at
the time of batch, and mouth gape size at first feeding for larval perch;

4) to define the range of mouth gape sizes for larval perch under 10 mm TL;

5) to determine the relationships of paternal size and age on larval mouth gape size and

TL of perch at the time of hatch.

4O

LITERATURE REVIEW

Intensive culture of larval perch must be refined to achieve commercial
production success (NCRAC 1997). However, larvae of perch are difficult to rear
intensively due to their small size (Mansueti 1964; Nickum 1978; Best 1981). Perch
larvae have been observed to hatch within the range of 4.7 to 6.6 mm TL (Mansueti 1964;
Hokanson and Kleiner 1974; Ney 1978). After hatching, the larvae begin absorbing the
yolk sac and developing a more complete and functional mouth (for review, see Mansueti
1964). While larvae have not been observed feeding immediately after hatching,
exogenous feeding has been observed to begin before the yolk sac has been fully
absorbed. First feeding larvae were as small as 6.0 mm TL (Siefert 1972). By the time
the larvae had reached approximately 7.0 mm TL, the yolk sac had been completely
absorbed (Mansueti 1964; Siefert 1972).

The transition period from yolk absorption or endogenous feeding to exogenous
feeding characterizes the transition from prolarvae to postlarvae (Hubbs 1943). This
transition period has also been described as the “critical period” for larval fish because of
the increased mortality associated with the metamorphosis of larvae (Hjort 1914; 1926).
In general, failure to start exogenous feeding when the yolk sac is completely absorbed

results in mortality due to starvation (Wang and Eckrnann 1994). This period of high

41

mortality appears to be the most limiting factor for developing larval perch intensive
culture at commercial levels.

Since larval perch are small at the time of hatch and first feeding, their size may
limit their ability to utilize a large portion of potential food supply. To be potential food,
an organism must be small enough to be ingested and slow enough to be captured by the
larvae (Siefert 1972). Wong and Ward (1972) have speculated that larval perch are
restricted to limited amounts of prey due to the maximum mouth gape width. Mouth
gape limitations have occurred when the size of the prey exceeded the size of the mouth
gape (Hansen and Wahl 1981; Wong 1972) or the diameter of the esophagus (Kestemont
et al. 1996; Raisanen and Applegate 1983). Month gape size has limited the prey
selection for other planktivorous fish (Wankowski 1979) and has limited the optimal
utilization of Arternia nauplii for first feeding Eurasian perch (Percafluviatilis) larvae
(Kestemont et al. 1996).

Researchers and culturists have been searching for the smallest possible prey or
formulated diet that provides their nutritional requirements to help increase survival of
larval perch raised in intensive culture conditions (Brown et al. 1996). Identifying the
mouth gape size of first feeding perch larvae could help researchers and culturists identify
the appropriate sized prey or formulated diet.

Attempts have been previously made to determine the maximum mouth gape size
for various larval fish. Using a modified equation developed by Shirota (1970), Guma’a

(1978) determined the maximum mouth gape height for larval Eurasian perch using the

following equation:

(2.1) maximum mouth gape height = \l U 2 + L 2

42

where U and L are the lengths of the upper and lower jaws respectively, when opened at
90°. The calculated mouth gape height was used with larval TL to develop a linear
regression model. By using the linear regression model, the maximum mouth gape height
for natural stocks of Eurasian perch could be determined for first feeding larvae. The
maximum size of prey or formulated food for first feeding Eurasian perch larvae was
estimated to be 684 um.

However, it is not clear from the available data or methods, if Guma’a (1978)
measured mouth gapes for larvae under 10 mm TL. If mouth gape heights were not
measured for larvae smaller than 10 mm TL, statistical predictions on mouth gape sizes
cannot be made for smaller lengths (T empelrnan, personal communication, 1997). The
relationship between the mouth gape size and length of larvae less than 10 mm TL might
not be linear. The rate of increase may be much higher or lower than predicted by a
straight-line regression. For example, Henderson and Ward (197 8) concluded that the
body weight of perch increased exponentially after hatch. If the rate of growth or
increase in mouth gape sizes is exponential for Eurasian perch or perch larvae under 10
mm TL, then the linear regression model cannot be applied.

Wong and Ward (1972) measured the mouth gape widths of larval perch that were
greater than 10 mm TL. Their results indicated that the mouth size of perch larvae
initially increased more rapidly than body length. The measurements were made using a
calibrated, tapered brass cone with increments of 0.2 mm. Arts and Evans (1987)
measured the mouth gape width of larval perch using a modification of the brass cone

technique. Larval fish smaller than 10 mm TL could not be measured effectively using

43

either technique. The maximum mouth gape size for larval perch less than 10 mm TL,
first feeding larvae, has not been identified.

While some researchers have studied the theoretical maximum prey size that
larval fish can consume, other researchers have studied the size preference that larval fish
exhibit. Stickleback larvae consumed invertebrates with a maximum body width almost
equal to the mouth gape size (Hyatt 1979). However, Dabrowski and Bardega (1984)
concluded that cyprinid larvae consumed zooplankton prey that were only 40 - 6O % of
the total mouth gape.

Various approaches can be taken to help increase the survival of larval perch
raised in intensive culture conditions. Researchers have concentrated on developing
feeding strategies using small prey, such as small zooplankters and newly hatched
Arternia nauplii, or by providing a small, nutritionally complete diet. Some researchers
have attempted to raise larval perch on a commercial scale using both types of live food
(NCRAC 1995; Starr, personal communication, 1996; Dabrowski 1998) with limited
success. Other researchers that have attempted to use formulated diets exclusively to
raise larval perch have not been successful (Brown et al. 1996).

Melard and Kestemont (1994: in Kestemont et al. 1996) have illustrated the
importance of mouth size. While approximately 60-70 % of newly hatched Eurasian
perch larvae consumed a small strain Arternia nauplii, the remaining 3040 % disappeared
due to starvation or lack of vigor throughout the first week of experiments. The authors
speculated that the inability to consume Arternia nauplii was potentially due to

differences in mouth size between those that survived and those that died.

44

Another method for increasing the survival of larval perch raised in intensive
culture conditions could concentrate on selecting larvae with desirable characteristics or
selecting broodstock that produce larval offspring with desirable characteristics. Shirota
(1970) concluded that several species of larval fish with larger mouths grew faster
compared to larvae with smaller mouths. Selection for larger larvae or larvae with larger
mouths could potentially increase the survival of larval fish past the critical period.
Positive correlations between larval TL and mouth gape size have been established for
perch larvae greater than 10 mm TL (Wong and Ward 1972; Art and Evans 1987).
Longer larvae would have larger mouth gapes. By selecting for larvae with longer TL,
the potential for survival increases. However, researchers have not developed methods
for selecting longer larvae.

Wang (1994), Wang and Eckrnann (1994), and Kestemont et a1. (1996) have
suggested that eggs from the Eurasian perch have varied in size from the same female or
from different females, which could influence development and growth of larvae.
Similarly, Mansueti (1964) observed that perch egg diameters varied in size. The egg
diameters ranged from 1.6 to 2.1 mm before water hardening and expanded to 1.7 to 4.5
after water hardening. The variations observed in egg diameter might account for
variations observed in larval TL. Perch larvae hatch at variable lengths. Researchers
have reported TL values ranging 4.7 to 6.6 mm TL (Mansueti 1964; Hokanson and
Kleiner 1974; Ney 1978). However, no correlation has been reported between the size of
the female perch spawner and the average size of eggs and larvae. The size of eggs and

larvae from some marine fish increase with the size of female spawners within the sarrre

strain of fish (Kamler 1992).

45

Genetic improvement through selective breeding and genetic analysis can
improve desirable characteristics. European carp and freshwater salrnonids were some of
the first fish to be genetically improved through carefully controlled experiments (Moav
1979). As a result, fish have been observed with genetically increased fecundity rates,
egg size, hatching percent, growth, and height/length ratio. Perch broodstocks have not
been genetically improved through selective breeding programs nor has any significant
genetic analyses been completed. A basic approach in comparing genetic differences
among perch compared survival and growth of intensively cultured larval perch, which
were collected from various geographical sources (NCRAC 1997). The results indicated
that no single source of perch broodstock produced superior larval offspring.

Genetic analysis of desirable characters is required to design efficient selection
programs and predict expected gains (Moav 1979). In particular, the additive genetic
variance and its proportionate amount for each phenotype can be used to predict whether
selection will be effective (Tave 1993). Estimates of heritability are calculated using the
additive genetic variance, which ranges from 0 to 1 (for review, see Falconer 1989). In
cases where heritabilities are closer to 0, selection for that particular trait is not expected
to improve the existing mean genetic value (Purdom 1993). By establishing a breeding
program with carefully designed experiments, the gains observed in European carp and
fieshwater salrnonids potentially could be observed for perch. Genetic selection could

help improve survival and other desirable characteristics in larval perch.

46%

METHODS

Year 1: Using maternal size and age as indicators for egg and larval size of
perch ,

Perch broodstock was collected from the outer Saginaw Bay, Lake Huron for a
two week period beginning on April 19, 1996 and transported to Bay Port Aquaculture in
West Olive, Michigan (Figure 6). Eggs were collected over a two week period from
April 22 to May 4. All experiments were conducted at Bay Port Aquaculture’s hatchery

facilities.
Female Spawner and Egg Analysis

The broodstock was held in a flow through system supplied with 11 °C aerated
well water. Females were separated into six female spawner size groups by length (Table
6). Attempts were made to replicate each spawner size group five times. Data collected
for the female spawners included TL, weight and age. The TL to the nearest 1 mm of the
spawners (fi'om the tip of the head to the tip of the tail, with lobes compressed) was
determined using a measuring board. Weights were recorded to the nearest 1 g using a

top loading, electronic balance. Scale samples were taken from below the lateral line on

47

'5 "s5 . ..
a. ' Lake ' ‘-
o‘ (I Huron
° 1
Bay Port, MI
(sampling site)
Bay Port Aquaculture,
West Olive, MI
Lake, (study site)
chrgan

 

Figure 6. Map showing sampling location of yellow perch broodstock in Lake Huron and
study site at Bay Port Aquaculture research facilities near Lake Michigan.

48

 

 

cmmémm own Om

mNmAOm Sm Om

oomtchm Eh Om

mm; mm EN Om

cmmémm cum Om

ES mmutgm 8m Om
fiwefl .88. 9.8m 3% .885an Ema—om

 

dawne— ES no woman AOmV museum 3% 3:26am in 83 6.03385 neon 26:9» 2an~ mo cowauaom .0 033.

49

the left side (DeVries and F rie 1996). Age was determined after the spawning season by
counting the scale annuli and reporting completed years of life. Age analysis was
checked and verified by a second independent analysis of every third scale.

Egg ribbons were collected from each of the female spawner size groups (Table
6). Data collected for each egg ribbon included weight, number of eggs per gram of eggs,
total fecundity, chorion shell diameter, and yolk diameter. For all measurements, a video
recorder connected to an Olympus dissecting microscope was used to record all egg and
larval images. The video images were converted into a digital image using the
OPTIMAS Imaging System, BioScanTM. For each female, the eggs were manually
stripped into a dry bowl. Two egg samples (approximately 5 g and 1 g) were collected
from the posterior (i.e. situated nearest to the urogenital vent) pertion of the egg ribbon,
weighed, and recorded to the nearest 0.1 g using a top loading, electronic balance. The
remaining egg ribbon was weighed for total fecundity rate calculations.

After weighing the lg egg sample, the eggs were mixed with well water and
allowed to water harden in a separate container. After one hour, the egg sub-samples
were fixed in Stockard’s solution (Galat 1972). After 48 hours, the Stockard’s solution
was replaced with a 10% formalin solution. The eggs were counted in August 1996, and
the number of eggs per gram of eggs was determined and the diameters of the chorion
shell and yolk were measured.

After weighing the 5 g sample, the eggs were fertilized using rrrilt from one or
more males. The dry method was used because it enhances fertilization rates for eggs
(Piper et a1. 1982). In a dry container, milt was mixed with the eggs before water was

added. The fertilized eggs were allowed to water harden for 20 min before being placed

50

into an incubating unit. After the egg samples were fertilized and water hardened, they
were randomly placed in specially designed incubator chambers (Westrnaas 1992). The
incubator chambers were randomly placed in an eight tray vertical incubator unit and
supplied with 11.5 °C +/- 0.5 °C well water with a flow rate of 12 L/min (Leitritz and
Lewis 1980).

In addition to the samples collected from the six female spawner size groups, egg
samples were collected from a sub-sample of the female spawner size groups. The sub-
samples were collected from three fish in SG 226 and three fish in SG 301 (Table 6).
These two female spawner size groups were chosen because adequate numbers of gravid
females were available and provided a large enough size difference for contrasts. Two
egg samples (approximately 5 g and 1 g) were collected fiom the anterior, median, and
posterior positions of each egg ribbon. The posterior portion of the egg ribbon was the
region of the egg ribbon that was closest to the urogenital vent. The median portion was
the region of the egg ribbon that was the mid-point between the two ends. The 5 g and 1 g
egg samples were weighed, fertilized, and fixed using the same methods described above.
The remaining egg ribbon was weighed to calculate the total fecundity rate. The
parameters for number of eggs per gram of eggs, diameters of the chorion shell and yolk

were determined using the OPTIMAS Imaging System, BioScanTM.

51

Larval Ofifspring Analysis

Beginning with the first day of the hatch, five prolarvae were randomly selected
from each incubator chamber. The larvae were mildly agitated first and then selected
with a Pasteur pipette. The opening of the pipette was large enough to aspirate larvae
without causing damage. Larvae were anesthetized using CO2 saturated tonic water.
After the larvae were anesthetized, they were placed in a 90 mm diameter Petri dish
containing 10 ml of distilled water. The larvae were placed on the lefi ventral side for TL
measurements. Methods for measuring the mouth gape were modified from Arts and
Evans (1987). While on the ventral side, a needle was inserted into the mouth to separate
the jaws. Care was taken to insure that the mouth was not stretched or the larvae
damaged. The distance from the upper to the lower jaw was measured as the maximum
gape height. The larvae were placed on the dorsal side to measure the mouth gape width.
The needle was carefully inserted without stretching the mouth or causing damage to the
larvae. The width of the jaw was measured at the point where the two jaws connected.
Sampling continued daily in the same manner until all larvae were removed from the

chambers.

52

STATISTICAL ANALYSIS

Variations within groups of female spawners and differences between groups of
female spawners were tested using One-way analysis of variance (AN OVA) (Snedecor
and Cochran 1989). Variation was also tested for eggs taken at different areas of the
same egg ribbon and compared to another spawner size group using Two factor ANOVA
(Neter et a1. 1996). Relationships were analyzed using simple linear regression analysis.
All values, unless otherwise stated, were based on a significant alpha level = 0.05%. The
test for normal distribution was based on the Shapiro- Wilk statistic W (N eter et al. 1996).
If the data were not normally distributed, they were transformed to create a normal
distribution. The appropriate transformation was determined using the Box-Cox
procedure (Tempelrnan, personal communication, 1997). The assumption of equal
variances was tested using a Modified Levene 19 test (Neter et al. 1996). If the assumption
of equal variances were violated, differences between the means were determined using
the non-parametric Kuskal- Wallis test instead of One-way AN OVA (Miller 1985). If the
variances were not homogeneous for the two factor AN OVA, the data were ranked
transformed (Conover and Iman 1981). All linear regression models were based on the

following basic model (Neter et al. 1996):
(22) y = 130 + 131(x)+ e

where [30 was the regression coefficient for the intercept;

53

Br was the regression coefficient for the slope of the line;

x was the explanatory variable;

y was the response variable;

e was the residual error term.
Analysis of covariance (AN COVA) was used to test for statistically significant
differences between regression coefficients in thelinear regression models (Neter et al.
1996). In cases where a single explanatory variable did not explain the relationship well,
multiple linear regression models were explored using a stepwise regression selection

procedure (N eter et al. 1996).
Female Spawner Analysis

Data collected on the female spawner groups included TL, weight, and age.
Means for each of the female spawner group parameters were calculated and tested for
normal distribution and homogeneity of variances. One-Way Analysis of Variance
(ANOVA) tests were used to determine overall statistical significance between groups of
females. AN OVA contrast statements were used to determine statistical significance
between group comparisons. Simple linear regression analysis was used to describe and
predict the relationship between female spawner TL and weight. The linear regression

model was based on the power function:
(2.3) W= a L”,
where W was weight;

L was length;

54

a and b were the regression parameters (Carlander 1969; Anderson and
Neumann 1996).

The simple linear regression model can also be written as:
(2.4) 1°810(W) = a’ + b * log... (L) + e,
where a’ was logm (a) and the y-intercept parameter;

b was the slope regression parameter;

e was the residual error term.

Egg Analysis

Data collected on the egg ribbons for two spawner groups included number of
eggs per gram of eggs, chorion shell diameter, and yolk diameter, sampled from three
different areas of the egg ribbon: anterior, median, and posterior. A 2 x 3 factorial design
was used to test significance between 10cations of the egg ribbon. Means and standard
deviations for each female spawner group parameter were calculated and tested for
normal distribution and homogeneity of variances. Factorial analysis was used to
determine overall statistically significant differences between sampling locations in the
egg ribbon. Contrast statements were used to estimate statistically significant differences
between the locations in the ribbon within the two spawner size groups.

Data collected on the egg ribbons for each spawner group included total
fecundity, egg ribbon weight, number of eggs per gram of eggs, chorion shell diameter,
and yolk diameter. Means and standard deviations for each female spawner group

parameter were calculated and tested for normal distribution and homogeneity of

55

variances. One-way ANOVA tests were used to determine overall statistical significance
between groups of females. AN OVA contrast statements were used to determine
statistical significance between group comparisons. Simple linear regression analysis
was used to describe and predict the relationship between female spawner TL or weight
with total fecundity, egg ribbon weight, number of eggs per gram of eggs, chorion shell
diameter, and yolk diameter. The linear regression models were based on the basic model

(2.1).

Larval Ofifspring Analysis

Data collected on the larval offspring for each spawner group included total
length, mouth gape width and height. Larvae were separated into six groups based on the
female spawner size classification.

Means and standard deviations for each larval group parameter were calculated
and tested for normal distribution and homogeneity of variances. One-way AN OVA tests
were used to determine overall statistical significance between the six different groups of
perch larvae. AN OVA contrast statements were used to determine statistical significance
between group comparisons. Simple linear regression analysis was used to describe and
predict the relationship between female spawner TL or spawner weight to larval TL,
larval mouth gape width, and larval mouth gape height. The linear regression models
were based on the basic model (2.1). Simple linear regression models were developed
that described and predicted the relationship between larval TL and larval mouth gape

width and height. Individual linear regression models were developed for each larval

56

group. ANCOVA was used to test for statistically significant differences between the
regression coefficients in the different larval group linear relationships. The initial mouth
gape size and rate of mouth gape increase were tested. To standardize the hatching
period of larval perch, the time of hatching was standardized from day 0, the day when

the eggs were fertilized.

57

METHODS

Year 2: Effects of perch parental size and age on larval total length and mouth gape
width and height

The additive genetic variance and non-additive genetic variance (dominance) for
TL, mouth gape width and height were estimated using a cross-classification design
(Dickerson 1969; Falconer 1989; Tave 1993). Two slow growing and two fast growing
male perch were crossed with each of the four females, two slow growing and two fast
growing (Figure 7). This mating produced eight families sired by slow growing males
and eight families sired by fast growing males.

Male and female spawners were selected from the pool of spawning stock
collected from the outer Saginaw Bay, Lake Huron during a two week period starting
April 15, 1997 and were transported to Bay Port Aquaculture. The spawning stock was
held in a flow through system supplied with 11 °C well water.

Age of the spawners was determined before selection and analyzed at a later time
for verification. Spawners were selected based on age-length frequencies for native perch
in Lake Huron based on data provided by the Michigan Department of Natural Resources,
Fisheries Division (Fielder, personal communication, 1997). The TL’s were compared to
age and a sampling frequency was calculated. Based on a normal distribution (Central

Limit Theorem) for the age selected, an 80% two tailed t-based distribution was

Fast Growth Female; 11 = 2

Slow Growth Male; n = 2 A Fast Growth Male; 11 = 2

 

Figure 7. Experimental design for Objective 5, paternal influence on the larval TL and
mouth gape size of larval yellow perch.

59

calculated for TL (Snedecor and Cochran 1989). The test was used to select slow and fast
grth rates based on TL for the age (Figure 8). TL’s in the upper limit of the 10% tail
represented a fast grth rate. TL’s in the lower limit of the 10% tail represented a slow
growth rate.

For each female, the eggs were manually stripped into a dry bowl. Eight samples
of 5 g were initially cut from the egg ribbon and weighed. The egg samples were
randomly placed into separate, dry containers. The milt from each of the four male
spawners randomly fertilized two of the egg samples from each female spawner before
water was added. Each cross was replicated twice. The fertilized eggs were allowed to
water harden for 30 rrrin before being placed in a 55 gallon cooler. The eggs were
transported back to the MSU aquaculture laboratory within three hours of fertilization.

After arriving at the lab, the fertilized egg samples were placed in 11.5 °C 1 0.5 °C
well water bath. After the eggs had tempered, they were placed in specially designed
incubator chambers (Westrnaas 1992). The incubator chambers were randomly placed in
an eight tray vertical incubator unit and supplied with 11.5 °C : 0.5 °C well water with a
flow rate of 12 L/min (Leitritz and Lewis 1980).

Beginning on the second day of the hatch, the larvae were randomly selected in
each incubator chamber for measurements of TL. On day 5 of the hatch, 10 larvae were
randomly selected for measurements of mouth gape width and height. While still in the
incubating chamber, the larvae were randomly sampled by mildly agitating and selecting
with a Pasteur pipette. The opening of the pipette was large enough to aspirate larvae
without causing damage. Larvae were anesthetized using CO2 saturated tonic water.

Sampling continued daily in the same manner until all larvae were sampled.

6O

  
 

 

 

 

Age x

F

r

e

q

‘1 Lower 10% Limit

e

n . .
c Upper 10% ert
y

k
Length (mm TL)_
Slow growth ' Fast growth

Figure 8. Experimental illustration of two-tailed t-based distribution for selection of
growth rates (slow vs. fast) based on age-length frequency distributions in Saginaw Bay,
Lake Huron (data provided by the MI DNR; Dave Fielder 1997).

61

STATISTICAL ANALYSIS

The average additive and non-additive genetic strain effects for perch growth
rates, fast and slow, were estimated. The average non-additive strain effect was separated
into the two reciprocal crosses and estimated. The variations around the average additive
and non-additive genetic strain effects were estimated as the additive and non-additive
genetic individual effects. The non-additive genetic strain effect was partitioned into two
heterotic effects based on mating fast growth dams with slow grth sires (hd) and slow
growth dams with fast growth sires (hs).

Detection of significant treatment and interaction (specific combining ability)
effects were performed on coded data. Variance components were estimated and used to
determine the relative contributions of maternally and paternally derived sources of
variation to the total variation. The specific combining ability was also partitioned out
(Falconer 1989). Contrast statements were used to determine significance between fast
and slow genotypes and between the average non-additive genetic strain effects of sire

and dam (ha and hd).

62

The ANOVA model used to test for significance was based on a partially nested

factorial design (Winer 1971). The model was:

(2-5) Yijklm = 11 + Cr (af)ik + cs (aslik + Sirej (sire groupi) + dam] (dam groupk) + '

sirej’“ darn, (sire group, * dam groupk) + hs(Recip)12 + hd(Recip)2l + eijklm

where, p. was the overall mean;

of was the covariate for fast growth;
of = 1, when siref x damf cross;
= 0.5, when siref x dams cross or sires x damf cross;
= 0, when sires x darns cross;
cs was the covariate for slow growth;
c5 = 0, when siref x damf cross;
= 0.5, when siref x darns cross or sires x damf cross;
= 1, when sires x darns cross;
(arm was the additive effect of genotype for fast growth;
(ors )ik was the additive effect of genotype for slow growth;

sirej (sire groupi) was the effect of the jth male within the ith male group;
i = 1, when sire was genotype fast growth;
= 2, when sire was genotype slow growth;

daml (dam groupk) was the effect of the 1th female within the kth female

63

group;
j = 1, when dam was genotype slow growth; 1
= 2, when dam was genotype fast growth;
sire,- * darn, (sire group, * dam groupk) was the effect of the jth male by the
lth female within the ith male and kth female group;
hs was the covariate for male fast growth (i=1) by female slow growth
(k=2) heterotic component;
hs = 1, when sire, x darns cross;
= 0, otherwise;
hd was the covariate for male slow grth (i=2) by female fast growth
(k=l) heterotic component; ‘
hd = 1, when sires x damf cross;
= 0, otherwise;
(Recip),2 was the heterotic effect of male fast growth (i=1) and female
slow grth (k=2), and;
(Recip)2, was the heterotic effect of male slow growth (i=2) and female

fast growth (k=1).

For the determination of heritability and dominance, the variance component was
partitioned for the sire and dam combinations based on Becker (1975). Models described
in Falconer (1989) were used to estimate heritability and dominance for TL, mouth gape

width, and mouth gape height for each day (Appendix D and B, respectively).

64

RESULTS

Female Spawner Analysis

Weight and TL for each female was measured, and the age determined from the
scales. The means and standard deviations for each female spawner size group were
calculated (Table 7). The means for spawner TL, weight, and age increased from the
smallest to the largest female spawner size group. Means and standard deviations for
spawner TL and weight at each completed age were also calculated (Table 8). The means
for spawner TL and weight increased fi'om the smallest to the largest female spawner age
group.

The data were normally distributed for TL (p = 0.0980) and age (p = 0.0509) but
were not normally distributed for weight (p = 0.0074, Appendix F). The weight data
were log transformed to bring the data back to a normal distribution (p = 0.0874). The
test for the assumption of equal variances across female spawner size groups indicated a
violation (p < 0.05) of equal variances for all three parameters. Since hypothesis testing
was not conducted on these parameters, nonparametric tests were not used. Two factor
AN OVA tests indicated an estimated overall statistical significance (p = 0.0001) for the
age-group effect in spawner TL and weight (Appendix G). However, the age effect was

not significant. The age effect was eliminated and One-way AN OVA tests were

65

Table 7. Average spawner total length (TL), weight, and age for each female spawner
size group (SG). Values are reported as means 1 standard deviations (sd).

 

 

 

 

Parameter Group N Mean : sd Minimum Maximum
Female SG 201 6 221.50 : 3.50 mm 216 mm 225 mm
Spawner SG 226 5 242.60 1 5.46 237 250

Total SG 251 7 259.14 1 7.40 252 274
Length 86 276 7 280.71 1: 4.92 276 287
SG 301 6 312.83 1 8.06 301 322
SG 326 4 340.50 : 11.24 328 350
Female 86 201 6 119.17 : 7.28 g 109 g 129 g

Spawner SG 226 5 150.60 : 13.13 139 171

Weight SG 251 7 212.71 1 24.63 181 260
86 276 7 269.86 3: 20.03 248 301

SG 301 6 395.33 j; 26.61 365 428

SG 326 4 497.75 : 39.87 460 541

Female SG 201 6 4.5 i 0.84 4 6
Spawner SG 226 5 5.4 j; 0.55 5 6
Age 86251 7 6.3 i 1.13 5 8
SG 276 7 6.6 7: 1.27 5 8

SG 301 6 7.3 _t 1.03 6 9

SG 326 4 8.5 i 0.58 8 9

 

66

Table 8. Averages for spawner total length (TL) and weight for each female spawner
completed age. Values are reported as means : standard deviations (sd).

 

 

 

Parameter Age N Mean : sd Minimum Maximum
Female 4 4 220.0 i 3.37mm 216 224
Spawner 5 8 254.9 : 21.15 224 287

Total length 6 7 259.7 : 25.95 225 301

7 7 288.3 : 30.18 252 322
8 6 298.0 : 29.83 258 334
9 3 339.0 : 19.05 317 350
Female 4 4 118.0 f: 8.87g 109 129
Spawner 5 8 191.6 : 62.28 119 301
Weight 6 7 213.7 : 89.31 124 380
7 7 3121 : 96.73 200 428
8 6 343.8 : 104.37 223 468
9 3 486.7 3: 78.23 397 541

 

67

conducted on group effects, which were statistically significant (Appendix F). The tests
indicated an estimated overall statistical significance (p = 0.0001) for both parameters,
female spawner TL and weight (Appendix F). Spawner TL and weight contrast
statements indicated statistical significance between all group means (Table 9). Age
contrast statements indicated statistical significance between the following groups: SG
201 vs. so 251, so 201 vs. so 276, so 201 vs. so 301, so 201 vs. so 326, so 226 vs.
SG 301, SG 226 vs. SG 326, SG 256 vs. SG 326,. and SG 301 vs. SC 326 (Table 9).
Linear regression analysis was used to describe the relationship between female
spawner weight and TL. The response variable, log(weight) parameter, was highly
correlated to the predictor variable, log(spawner .T L) parameter (r2 = 0.9820) and the

slope of the line was statistically significant from zero (p = 0.0001, Appendix H).

Factorial Analysis of positions within the egg ribbon

The means and standard deviations for number of eggs per gram of eggs (Table
10), egg yolk diameter (Table 11), and chorion shell diameter (Table 12) were calculated
for each position of the egg ribbon. The mean values demonstrated an increase from the
anterior position to the median position of the egg ribbon. The mean values for number
of eggs per gram of eggs decreased fi'om the median position to the posterior position for
both female spawner size groups (Table 10). This trend was also observed for egg yolk
diameter (Table 11) and chorion shell diameter (Table 12), in SG 226. However, the
mean values for both of these parameters in SG 301 continued to increase fiom the

median position to the posterior position.

68

 

 

 

 

 

 

 

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Table 10. Averages for number of eggs per gram in each position of the egg ribbon
(anterior, median, and posterior) for female spawner size groups (SG), SG 226 and SG

301. Values are reported as means 1 standard deviations (sd).

 

 

 

Group Position N Mean 1' sd Minimum Maximum
SG 226 Anterior 3 634.61 : 181.84 um 477.18 833.65
SG 226 Median 3 662.60 1 166.89 530.00 850.00
SG 226 Posterior 3 653.75 i 143.20 516.13 801.95
SG 301 Anterior 3 635.15 1 32.54 600.00 664.23
SG 301 Median 3 669.78 1 15.58 653.04 683.87
SG 301 Posterior 3 659.35 1: 37.78 618.60 693.22

 

Table 11. Averages for egg yolk diameter in each position of the egg ribbon (anterior,

median, and posterior) for female spawner size groups (SG), 86 226 and SG 301. Values

are reported as means 1 standard deviations (sd).

 

 

 

Group Position N Mean : sd Minimum Maximum
SG 226 Anterior 75 0.3160 : 0.0468 um 0.1954 0.3946
SG 226 Median 75 0.3355 : 0.0489 0.2391 0.4435
SG 226 Posterior 75 0.3274 : 0.0809 0.1323 0.4548
SG 301 Anterior 75 0.2879 j; 0.0330 0.2097 0.3405
86 301 Median 75 0.3514 1 0.0451 0.2463 0.4493
SG 301 Posterior 75 0.3926 j; 0.0423 0.3091 0.5025

 

 

70

Table 12. Averages for chorion shell diameter in each position of the egg ribbon
(anterior, median, and posterior) for female spawner size groups (SG), SG 226 and SG
301. Values are reported as means 1 standard deviations (sd).

 

 

 

Group Position N Mean : sd Minimum Maximum
SG 226 Anterior 75 1.3731 1 0.0632 um 1.2158 1.4838
SG 226 Median 75 1.4003 1 0.0685 1.2702 1.5581
SG 226 Posterior 75 1.3918 : 0.1107 1.1415 1.5758
SG 301 Anterior 75 1.3343 1; 0.0436 1.2333 1.4057
SG 301 Median 75 1.4225 1 0.0636 1.2793 1.5672
SG 301 Posterior 75 1.4822 _+_ 0.0630 1.3622 1.6528

 

71

The data were normally distributed for all of the parameters (Appendix I). The
assumption of homogeneous variances across positions for the chorion shell and egg yolk
diameters was violated (p < 0.05) but was not violated (p > 0.05) for the parameter
number of eggs per gram of eggs (Appendix I).

Since the assumption of homogeneous variances was violated for the chorion shell
and egg yolk diameter, the data were ranked transformed. Multi-factorial analysis was
completed using the ranked transformed data which demonstrated an overall statistically
significant difference (p = 0.0001, Appendix 1). Contrast statements for both parameters
indicated statistically significant differences (p < 0.05) between the three positions of the
egg ribbon in SG 301 (Table 13). There were no statistically significant differences (p >
0.05) between positions of the egg ribbon in SG 226 (Table 13).

The number of eggs per gram of eggs data were not ranked transformed, and
multi-factorial analysis was completed which demonstrated that there were no statistically
significant differences (p = 0.9984) between positions of the egg ribbon (Appendix I).
The female spawner size group, SG 226, was more variable than SG 301, but there were

no significant differences between the positions within the two groups.

Egg Analysis

Data for egg ribbon weight, total fecundity, and number of eggs per gram of eggs
were collected. The means and standard deviations were calculated for the parameters by
female spawner size groups (Table 14) and by completed age (TablelS). The means

calculated for egg ribbon weight and total fecundity data demonstrated an

72

 

 

 

 

 

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73

Table 14. Averages for egg ribbon weight, number of eggs per gram, and total fecundity
for each female spawner size group (SG). Values are reported as means 1 standard
deviations (sd).

 

 

 

 

Parameter Group N Mean : sd Minimum Maximum
Egg ribbon SG 201 6 37.24 i 5.75 g 31.78 g 47.64 g
Weight SG 226 5 53.23 i 5.04 49.29 62.02

SG 251 7 65.21 : 12.63 49.39 83.76
SG 276 7 80.19 i 8.62 72.83 97.13
SG 301 6 121.94 1 18.14 101.14 143.15
SG 326 4 176.79 : 19.36 154.07 200.38
Number of SG 201 6 689.80 1 63.42 573.04 753.75
eggs perg SG 226 14 671.50 : 126.84 479.18 850.00
SG 251 7 662.41 : 72.67 572.78 784.21
SG 276 7 644.80 i 114.75 492.38 784.36
SG 301 15 632.00 : 39.68 583.33 674.86
SG 326 4 591.11 : 66.27 524.10 671.43
Total SG 201 6 25910.34 _t 5965.33 18211.32 35908.65
Fecundity SG 226 5 37934.64 : 7291.07 30597.69 49089.03
SG 251 7 42934.97 3: 7991.43 32073.01 56178.42
SG 276 8 52416.78 _+_ 6263.50 46586.38 62487.27
SG 301 5 79823.62 1 12659.95 62154.17 94436.27
SG 326 4 107802.80 : 17953.27 81004.90 119173.40

 

74

Table 15. Averages for number of egg per gram, egg ribbon weight, and total fecundity
parameters for each completed age of female spawners. Values are reported as means :
standard deviations (sd).

 

 

 

 

Parameter Age N Mean : sd Minimum Maximum
Number of 4 4 672.50 3: 70.23 573.04 733.33
Eggs per gram 5 8 738.67 3; 46.98 670.71 796.55
6 7 648.53 : 89.38 547.75 784.21
7 7 632.03 3: 67.29 579.45 771.95
8 6 582.93 : 60.72 492.38 649.38
9 3 661.94 3: 19.48 ‘ 639.53 674.86
Egg ribbon 4 4 35.90 i 2.85 g 31.78 38.36
Weight 5 8 60.47 : 16.36 32.20 83.76
6 7 71.28 : 32.11 47.64 137.99
7 7 96.99 : 31.37 59.94 143.15
8 6 113.08 :54.96 49.39 200.38
9 3 154.18 _+_ 38.75 109.82 181.44
Total 4 4 24293 i 4283 18211 28131
Fecundity 5 8 44629 : 11771 22381 57125
6 7 45553 : 19952 30598 88915
7 7 61057 t 19955 36982 94436
8 6 64953 t 31288 32073 119173
9 3 101715 : 23910 74113 116037

 

75

increase fiom the smallest to largest female spawner size groups (Table 14). These two
parameters also increased from age 4 to age 9 (Table 15). The means calculated for
number of eggs per gram of eggs data demonstrated a decrease from the smallest to
largest female spawner size groups (Table 14). The mean number of eggs per gram of
eggs increased from age 4 to age 5, decreased from age 5 to age 8, and increased from age
8 to age 9 (Table 15).

The data for number of eggs per gram of eggs were normally distributed (p =
0.4726) but were not normally distributed for egg ribbon weight (p = 0.0013) and total
fecundity (p = 0.0028, Appendix H). The egg ribbon and total fecundity data were log
transformed which converted the data into a normal distribution (p = 0.3603 and p =
0.73 72, respectively).

The assumption for homogeneous variances across female spawner size groups
was tested and did not indicate a violation (p > 0.05) for egg ribbon weight and number
of eggs per gram of eggs (Appendix F). Two factor ANOVA tests indicated an estimated
overall statistical significance (p = 0.0001) for the age-group effect in log(egg ribbon
weight) and log(total fecundity), but was not statistically significant overall (p = 0.0834)
for number of eggs per gram of eggs (Appendix G). The age effect was not statistically
significant for log(egg ribbon weight) and log(total fecundity) parameters. The age effect
was eliminated and One-way AN OVA tests were conducted on size group effects, which
were statistically significant. The tests indicated an overall statistical significance (p =
0.0001) for the log(egg ribbon weight) data (Appendix F). Contrast statements
demonstrated statistical significance between all groups except for the group contrast SG

226 vs. SG 251 (p = 0.1091, Table 16). Statistical significance was found

76

Table 16. ANOVA contrast statements for egg ribbon weight. Mean values for egg
ribbon weight are compared between female spawner size groups (SG). Significance
determined on the 0.05 % alpha level.

 

 

 

 

 

 

Contrast df Estimate p-value
SG 201 vs SG 226 1 4.55 0.0414
SG 201 vs SG 251 1 16.51 0.0003
SG 201 vs SG 276 1 38.92 0.0001
SG 201 vs SG 301 1 140.58 0.0001
SG 201 vs SG 326 1 305.28 0.0001
SG 226 vs SG 251 1 2.73 0.1091
SG 226 vs SG 276 l 13.84 0.0008
SG 226 vs SG 301 1 84.10 0.0001
SG 226 vs SG 326 1 221.60 0.0001
SG 251 vs SG 276 1 5.13 0.0312
SG 251 vs 86 301 1 67.92 0.0001
86 251 vs SG 326 1 207.01 0.0001
so 276 vs so 301 1 36.79 0.0001
SG 276 vs SG 326 1 155.16 0.0001
SG 301 vs SG 326 1 47.16 0.0001

 

77

between groups SG 201 vs. SG 226 and SG 251 vs. SG 276, although the p-values were
marginal (p = 0.0414 and p = 0.0312, respectively). One-way AN OVA test of the
number of eggs per gram of eggs data demonstrated that there were no overall statistical
significance (p = 0.4816, Appendix F). No contrasts were conducted.

The test for homogeneity of variances indicated a violation for the total fecundity
data. Nonparametric testing indicated overall statistically significant differences between
groups (overall chi-square value, p = 0.0001, Appendix F). The group contrasts that were
statistically significant were 86 201 vs. SC 301 and SG 201 vs. SG 326 (Table 17).

Linear regression analysis was used to describe the relationship between female
spawner TL or spawner weight to log(egg ribbon), log(total fecundity), and number of
eggs per gram of eggs. The response variables, log(egg ribbon)'and log(total fecundity),
were highly correlated to the predictor variables, spawner TL and spawner weight
(Appendix H). The analysis indicated that the 1‘2 values ranged fiom 0.8567 to 0.9089
and that the slope of each line was statistically significant from zero (p = 0.0001). The
response variable, number of eggs per gram of eggs, was not highly correlated to the
predictor variables, spawner TL and spawner weight (Appendix H). The analysis
indicated that the 1'2 values were 0.0983 to 0.1000, respectively, and that the slope of both
lines were not statistically significant from zero (p = 0.0667 and p = 0.0643,
respectively).

Data for the chorion shell and egg yolk diameters were collected. The means and
standard deviations calculated for the two parameters did not indicate a trend across
female spawner size groups (Table 18). For both parameters, the data were normally

distributed (p = 0.8979 and p = 0.9915, respectively, Appendix F). The test for

78

Table 17. Contrast statements for total fecundity based on the Kruskal-Wallis non-
parametric test. Mean values for total fecundity are compared between female spawner
size groups (SG). Significance determined if D is greater than R.

 

 

 

 

 

 

Contrast D R D>R
SG 201 vs SG 226 7.23 21.25 no
SG 201 vs SG 251 10.40 19.53 no
SG 201 vs SG 276 16.83 18.96 no
86 201 vs SG 301 25.43 21.25 yes
SG 201 vs SG 326 29.03 21.25 yes
SG 226 vs SG 251 -3.17 20.55 no
SG 226 vs SG 276 -9.60 20.01 no
86 226 vs SG 301 ~18.20 22.20 no
SG 226 vs SG 326 -21.80 22.20 no
SG 251 vs 86 276 -6.43 18.17 no
86 251 vs 86 301 -15.03 20.55 no
86 251 vs SG 326 -18.63 20.55 no
SG 276 vs SG 301 -8.60 20.01 no
SG 276 vs SG 326 -12.20 20.01 no
SG 301 vs 86 326 -3.60 22.20 no

 

79

 

 

 

 

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80

homogeneity of variances across female spawner size groups did not indicate a violation
for either parameter. Although statistically significant, the One-way AN OVA tests
indicated overall statistical significant differences between groups for the chorion shell
diameter (p = 0.0381, Appendix F). Group contrasts for the chorion shell diameter
demonstrated statistical significance between SG 226 vs. SG 251, SG 226 vs. SG 276,
and SG 226 vs. SC 301 (Table 19). One-way AN OVA tests did not indicate overall
statistically significant differences between groups for the egg yolk diameter (p = 0.0661,
Appendix F).

Simple linear regression analysis was used to describe the relationship between
female spawner TL or spawner weight to chorion shell and egg yolk diameters. The
response variables, chorion shell diameter and egg yolk diameter, were not correlated to
the predictor variables, TL and weight (Appendix J). The analysis indicated that the r2
values ranged fiom 0.0003 to 0.0005 and that the slope of each line was not statistically

significant from zero (p > 0.05).

Larval Offspring Analysis - Maternal Contribution

In general, all means increased over time with the exception of the means for
larval TL, which indicated a decreasing trend over time for SG 301 (Table 20). Larval
mouth gape width and height increased over time (Table 21 and 22, respectively). The
data were normally distributed (p = 0.3876 and p = 0.0942, respectively) for the larval
mouth gape width and height parameters but were not normally distributed (p = 0.0345)

for larval TL (Appendix F). The larval TL data were squared which transformed the data

81

Table 19. ANOVA contrast statements for chorion shell diameter. Mean values for
chorion shell diameter are compared between female spawner size groups (SG).
Significance determined on the 0.05 % alpha level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 l 2.99 0.0839
SG 201 vs SG 251 1 0.67 0.4147
SG 201 vs SG 276 l 0.03 0.8721
86 201 vs SG 301 1 0.44 0.5090
86 201 vs SG 326 1 0.16 0.6928
SG 226 vs SG 251 1 7.86 _ 0.0051
SG 226 vs SG 276 1 6.49 0.0110
86 226 vs 86 301 l 5.70 0.0171
SG 226 vs 86 326 1 1.35 0.2459
86 251 vs 86 276 1 0.70 0.4013
SG 251 vs SG 301 1 ' 0.01 0.9266
86 251 vs SG 326 l . 1.40 0.2367
SG 276 vs SG 301 1 0.40 0.5253
SG 276 vs 86 326 l 0.38 0.5352
SG 301 vs 86 326 1 1.02 0.3123

 

82

Table 20. Average for larval TL in each female spawner size group (SG) sampled
through days 15 to 19 post-fertilization. Values are reported as means : standard
deviations (sd).

 

 

 

 

 

 

 

Day N Mean : sd Minimum Maximum
SG 201 15 6 5.5267 1 0.1903 mm 5.3695 5.8819
16 11 5.3173 :1; 0.3617 4.7749 5.7604
17 14 5.2473 1 0.5155 4.5120 6.0746
l8 17 5.4784 1: 0.3945 4.8205 6.2897
19 11 5.5822 : 0.5948 4.3713 6.3593
SG 226 15 3 5.3813 1 0.6013 4.9773 6.0723
16 13 5.4232 : 0.4138 4.6354 6.0025
17 13 5.4091 : 0.5611 4.5888 6.2455
18 28 5.4560 1; 0.5427 4.3724 6.3445
19 33 5.0480 1 0.5672 4.1058 6.1159
SG 251 15 2 5.0358 1 0.0826 4.9773 5.0942
16 11 5.2085 : 0.3324 4.7168 5.8087
17 12 5.3289 3: 0.5793 4.1597 6.5572
18 9 5.3478 1 0.5019 4.3811 6.0457
19 10 5.5124 1 0.5246 4.7065 6.4660
SG 276 15 6 5.2323 : 0.1632 4.9544 5.3973
16 13 5.3462 1 0.2631 4.8837 5.8384
17 14 5.5768 1 0.2925 5.0599 6.1604
l8 14 5.2107 1 0.5443 4.5067 6.0326
19 13 5.6695 7: 0.3796 5.1452 6.3152
SG 301 15 14 5.4054 1 0.3611 4.7272 5.9896
16 28 5.3018 :5 0.5079 4.4626 6.2793
17 32 5.1814 1 0.5486 4.1672 5.9049
18 39 5.0413 1 0.5280 4.3002 6.1363
19 8 4.9037 1 0.2768 4.5084 5.1884
SG 326 15 2 5.1778 1 0.2883 4.9740 5.3816
16 4 5.1235 _+_- 0.3198 4.7366 5.5074
17 2 5.3795 : 0.0890 5.3165 5.4424
18 4 5.5479 : 0.2232 5.3509 5.8357
19 3 5.0420 1 0.2362 4.7702 5.1972

 

83

Table 21. Average for larval mouth gape width in each female spawner size group (SG)
sampled through days 15 to 19 post-fertilization. Values are reported as means :1;
standard deviations (sd).

 

 

 

 

 

 

 

Day N Mean : sd Minimum Maximum
SG 201 15 6 0.2435 : 0.0586mm 0.1545 0.3232
16 11 0.2772 : 0.0469 0.1878 0.3292
17 14 0.2892 :5 0.0508 0.1844 0.3782
18 17 0.2881 : 0.0583 0.1815 0.4444
19 11 0.3103 : 0.0406 0.2537 0.3909
SG 226 15 3 0.2388 1: 0.0663 0.1920 0.3147
16 13 0.2663 : 0.0348 0.2180 0.3549
17 13 0.2395 : 0.0669 0.1373 0.3190
18 28 0.2922 i 0.0477 0.1915 0.3751
19 33 0.2632 : 0.0502 0.1601 0.3623
80 251 15 2 0.2009 : 0.0126 0.1920 0.2098
16 11 0.2278 : 0.0378 0.1801 0.3102
17 12 0.2635 1: 0.0445 0.1836 0.3248
18 9 0.3101 i 0.0954 0.1949 0.4727
19 10 0.2863 : 0.0551 0.1955 0.3450
SG 276 15 6 0.2785 : 0.0419 0.2142 0.3327
l6 13 0.2622 : 0.0545 0.1754 0.3597
17 14 0.2909 : 0.0502 0.2120 0.3519
18 14 0.2686 : 0.0524 0.2087 0.3741
19 13 0.2972 :1; 0.0299 0.2435 0.3479
86 301 15 14 0.2696 : 0.0332 0.2068 0.3229
16 28 0.2632 1 0.0519 0.1857 0.3558
17 32 0.2497 1 0.0572 0.1308 0.3443
18 39 0.2590 : 0.0526 0.1564 0.3664
19 8 0.2674 1 0.0485 0.1902 0.3398
SG 326 15 2 0.2962 i 0.0228 0.2801 0.3124
16 4 0.2636 1 0.0616 0.1719 0.3023
17 2 0.2599 1 0.0486 0.2255 0.2942
18 4 0.3046 : 0.0122 0.2934 0.3179
19 3 0.2840 1 0.0296 0.2572 0.3157

 

84

Table 22. Average for larval mouth gape height in each female spawner size group (SG)
sampled through days 15 to 19 post-fertilization. Values are reported as means 1
standard deviations (sd).

 

 

 

 

 

 

 

Day N Mean : sd Minimum Maximum
SG 201 15 2 0.2593 : 0.0163mm 0.2478 0.2708
16 11 0.2745 : 0.0602 0.1778 0.3626
17 14 0.2668 1 0.0493 0.1851 0.3597
18 17 0.2979 j; 0.0667 0.1886 0.4185
19 11 0.3125 : 0.0680 0.2338 0.4167
SG 226 15 3 0.2310 3: 0.0332 0.1981 0.2644
16 10 0.2705 : 0.0326 0.2125 0.3333
17 13 0.2395 i 0.0661 0.1434 0.3371
18 28 0.2730 1 0.0617 0.1477 0.3774
19 33 0.2561 1 0.0571 0.1145 0.3583
SG 251 15 2 0.2143 : 0.0230 0.1981 0.2305
16 6 0.2186 : 0.0234 0.1959 0.2598
17 12 0.2454 : 0.0392 0.1739 0.2961
18 9 0.3101 : 0.0985 0.2145 0.4601
19 10 0.2744 1 0.0595 0.1542 0.3513
SG 276 15 6 0.2559 1 0.0261 0.2125 0.2867
16 13 0.2549 1 0.0645 0.1586 0.3729
17 14 0.2763 -_1—_ 0.0548 0.1667 0.4021
18 14 0.2671 : 0.0594 0.1848 0.3825
l9 13 0.2880 i 0.0381 0.2452 0.3860
SG 301 15 14 0.2569 1 0.0367 0.1717 0.3143
16 28 0.2427 i 0.0479 0.1697 0.3664
17 32 0.2450 1; 0.0568 0.1342 0.3474
18 39 0.2540 : 0.0494 0.1624 0.3638
19 8 0.2645 : 0.0417 0.2028 0.3225
86 326 15 2 0.2797 1 0.0176 0.2673 0.2921
16 4 0.2570 3: 0.0297 0.2139 0.2803
17 2 0.2964 3: 0.0409 0.2675 0.3254
18 4 0.3033 1: 0.0349 0.2608 0.3385
19 3 0.2648 : 0.0386 0.2213 0.2948

 

85

to a normal distribution (p = 0.0679). The assumption of homogeneous variances across
female spawner size groups was tested and demonstrated no violations of this
assumption.

In general, all means increased over time with the exception of the means for
larval TL, which indicated a decreasing trend over time for SG 301. One-way AN OVA
tests were used to demonstrate statistically significant differences between offspring
within female spawner size groups across all days sampled. The data for larval TL2 were
statistically significant overall (p = 0.0127 and p = 0.0004) for days 18 and 19 post-
fertilization, respectively (Appendix K). Contrast statements for larval TL2 on day 18
indicated statistically significant differences between groups SG 201 vs. SG 301 and SG
226 vs. SG 301 (Table 23). Contrast statements for. larval TL2 on day 19 indicated
statistically significant differences between groups SG 201 vs. SG 226, SG 201 vs. SG
301, SG 226 vs. SG 251, SG 226 vs. SG 276, 86 251 vs. SG 301 and SG 276 vs. SG 301
(Table 24). The data for larval mouth gape width were statistically significant overall (p
= 0.0497) on day 19 post-fertilization (Appendix K). Contrast statements for larval
mouth gape width on day 19 indicated statistically significant differences between groups
SG 201 vs. SG 226, SG 201 vs. SG 301, and SG 226 vs. SG 276 (Table 25). The data for
larval mouth gape height were not statistically significant for any days post-fertilization.

Since there were no significant differences between egg size from the different
spawner size groups, chorion shell diameter and egg yolk diameter were not used as
predictor variables to describe any relationship between these maternal characteristics and
any offspring characteristic. Instead, spawner TL and spawner weight were used to

describe the linear relationships with the response variables, larval TL, larval mouth gape

86

Table 23. AN OVA contrast statements for larval TL on day 18 post-fertilization. Mean
values for larval TL on day 18 post-fertilization are compared between female spawner
size groups (SG). Significance determined on the 0.05 % alpha level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 l 0 0.9510
SG 201 vs SC 251 1 0.36 0.5496
86 201 vs SG 276 1 1.97 0.1638
86 201 vs SG 301 1 8.12 0.0053
86 201 vs SG 326 1 0.05 0.8271
SG 226 vs SG 251 1 0.36 0.5523
86 226 vs 86 276 1 2.21 0.1398
86 226 vs 86 301 1 10.67 0.0015
86 226 vs SG 326 1 0.07 0.7930
SG 251 vs SG 276 1 0.37 0.5464
SG 251 vs SG 301 1 2.47 0.1193
SG 251 vs SG 326 l 0.38 0.5404
SG 276 vs 86 301 l 1.07 0.3035
SG 276 vs 80 326 1 1.23 0.2708
SG 301 vs SG 326 1 3.27 0.0733

 

87

Table 24. AN OVA contrast statements for larval TL on day 19 post-fertilization. Mean
values for larval TL on day 19 post-fertilization are compared between female spawner
size groups (SG). Significance determined on the 0.05 % alpha level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 l 9.35 0.0031
SG 201 vs SG 251 1 0.13 0.7176
SG 201 vs SG 276 1 0.13 0.7179
86 201 vs SG 301 l 8.81 0.0041
86 201 vs SG 326 1 3.00 0.0878
SG 226 vs SG 251 1 6.30 0.0143
SG 226 vs SG 276 l 13.72 0.0004
SG 226 vs SG 301 1 0.64 0.4274
86 226 vs 86 326 1 0.01 0.9174
SG 251 vs SG 276 l 0.53 0.4676
86 251 vs SG 301 1 6.62 0.0121
86 251 vs 86 326 1 2.17 0.1455
SG 276 vs SG 301 l 11.56 0.0011
SG 276 vs 86 326 1 3.97 0.0502
SG 301 vs 86 326 1 0.14 0.7110

 

88

Table 25. AN OVA contrast statements for larval mouth gape width on day 19 post-
fertilization. Mean values for larval mouth gape width on day 19 post-fertilization are
compared between female spawner size groups (SG). Significance determined on the
0.05 % alpha level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 1 8.60 0.0045
SG 201 vs SG 251 l 1.42 0.2373
86 201 vs SG 276 1 0.48 0.4895
SG 201 vs SG 301 1 4.00 0.0492
SG 201 vs SG 326 1 0.77 0.3837
86 226 vs SG 251 1 1.92 0.1699
86 226 vs SG 276 l 5.06 0.0275
86 226 vs 86 301 1 0.05 0.8172
SG 226 vs SG 326 1 0.56 0.4576
SG 251 vs SG 276 1 0.32 0.5764
86 251 vs SG 301 1 0.74 0.3913
86 251 vs SG 326 l 0.01 0.9395
86 276 vs SG 301 1 2.06 0.1554
SG 276 vs 86 326 1 0.20 0.6563
86 301 vs SG 326 1 0.28 0.5976

 

89

width, and larval mouth gape height. The simple linear regression analysis indicated that
the maternal characteristics were not highly correlated to the offspring characteristics.
While the slopes for all of the lines were statistically significant from zero (p values
ranged from 0.0170 to 0.0294), the r2 values ranged from 0.0007 to 0.0100 (Appendix L).

Simple linear regression analysis was used to describe the relationship between
larval TL and larval mouth gape width and height. The response variables, larval mouth
gape width and height, were linear related to the predictor variable, larval TL (r2 = 0.4330
and r2 = 0.4577, respectively, Appendix M). The slopes for both lines were statistically
significantly different from zero (p = 0.0001). These relationships were reduced into the
six different spawner size groups and analyzed. The analysis indicated linear
relationships between the response and predictor variables. The r‘2 values ranged from
0.3487 to 0.6100 and the p values ranged from 0.0001 to 0.0100 (Appendix N).

AN COVA was used to determine if the rate of increase for larval mouth gapes
was different for the six spawner size groups. The analysis indicated that there were
significant differences between spawner size groups (p = 0.0001, Appendix 0). Contrast
statements indicated that there were no significant differences overall between the rates of
growth of the mouth gape width (Table 26) and between initial mouth gape widths (Table
27). The contrast statements indicated that there were significant differences between the
rates of growth of the mouth gape height (Table 28) and between initial mouth gape
heights (Table 29). The contrast statements that were significant for the rate of grth
are SG 201 vs. SG 226, SG 201 vs. SG 301, SG 226 vs. SG 276, and SG 276 vs. SG 301.

The contrast statements that were significant between intercepts are SG 201 vs. SG 226,

90

Table 26. ANCOVA contrast statements for larval mouth gape width regression
coefficients. Mean values for larval mouth gape width regression coefficients are
compared between female spawner size groups (SG). Significance detemrined on the
0. 05 % alpha level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 1 0.58 0.4470
86 201 vs SG 251 1 0.18 0.6758
SG 201 vs 86 276 1 0.41 0.5231
SG 201 vs SG 301 1 0.96 0.3274
SG 201 vs SG 326 l 0.06 0.8103
SG 226 vs SG 251 l 1.44 0.2315
SG 226 vs SG 276 l 2.10 0.1483
SG 226 vs SG 301 1 0.07 0.7906
86 226 vs 86 326 1 0.28 0.5975
86 251 vs SG 276 l 0.05 . 0.8313
SG 251 vs 86 301 1 1.99 0.1595
86 251 vs SG 326 1 0.00 0.9570
SG 276 vs SG 301 1 2.76 0.0973
86 276 vs SG 326 1 0.00 0.9647
SG 301 vs 86 326 1 0.37 0.5452

 

91

Table 27. AN COVA contrast statements for larval mouth gape width regression
intercepts. Mean values for larval mouth gape width regression intercepts are compared
between female spawner size groups (SG). Significance determined on the 0.05 % alpha
level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 1 1.93 0.1656
SG 201 vs 86 251 1 2.86 0.0917
SG 201 vs SG 276 1 0.84 0.3609
86 201 vs SG 301 1 2.32 0.1283
SG 201 vs 86 326 1 0.51 0.4769
SG 226 vs 86 251 1 0.32 0.5727
SG 226 vs SG 276 1 0.15 0.6951
SG 226 vs 80 301 1 0.01 0.9331
SG 226 vs SG 326 l 2.48 0.1159
SG 251 vs SG 276 1 0.73 0.3943
SG 251 vs 86 301 1 0.27 0.6025
86 251 vs SG 326 1 3.30 0.0700
SG 276 vs 86 301 1 0.23 0.6291
SG 276 vs SG 326 1 1.67 0.1968
SG 301 vs SG 326 1 2.72 0.1002

 

92

Table 28. AN COVA contrast statements for larval mouth gape height regression
coefficients. Mean values for larval mouth gape height regression coefficients are
compared between female spawner size groups (SG). Significance determined on the
0.05 % alpha level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 1 5.57 0.0188
SG 201 vs SG 251 1 1.52 0.2190
SG 201 vs SG 276 l 0.03 0.8711
SG 201 vs SG 301 1 7.86 0.0053
SG 201 vs SG 326 l 0.29 0.5894
86 226 vs SG 251 1 0.56 0.4561
SG 226 vs SG 276 1 4.08 0.0440
86 226 vs SG 301 l 0.27 0.6041
80 226 vs 86 326 1 0.10 0.7478
86 251 vs 86 276 1 1.05 0.3054
86 251 vs 86 301 1 1.27 0.2608
86 251 vs SG 326 1 0.00 0.9854
86 276 vs 86 301 1 5.90 0.0157
SG 276 vs 86 326 l 0.21 0.6433
SG 301 vs SG 326 l 0.22 0.6379

 

93

Table 29. AN COVA contrast statements for larval mouth gape height intercepts. Mean
values for larval mouth gape height regression intercepts are compared between female
spawner size groups (SG). Significance determined on the 0.05 % alpha level.

 

 

 

 

 

 

Contrast DF Estimate p-value
SG 201 vs SG 226 1 7.16 0.0078
SG 201 vs SG 251 1 5.45 0.0201
86 201 vs SG 276 1 5.17 0.0235
86 201 vs 86 301 l 7.93 0.0051
SG 201 vs SG 326 1 0.16 0.6908
86 226 vs SG 251 1 0.02 0.8883
86 226 vs SG 276 1 0.05 0.8251
SG 226 vs SG 301 1 0.00 0.9917
86 226 vs SG 326 1 4.27 0.0394
SG 251 vs SG 276 1 0.10 0.7549
SG 251 vs 86 301 1 0.02 0.8899
SG 251 vs 86 326 1 3.96 0.0473
SG 276 vs SG 301 1 0.06 0.8091
86 276 vs 86 326 1 3.50 0.0623
86 301 vs 86 326 1 4.48 0.0350

 

94

SG 201 vs. SG 251, SG 201 vs. SG 276, SG 201 vs. SG 301, SG 251 vs. 36 326, and SG

301 vs. SG 326.
Larval Offspring Analysis - Parental Contribution

Analysis was completed over a period of six days (days 13 to 18 post-fertilization)
for the measurement of larval TL. Measurements'of larval mouth gape width were
completed on day 14 post-fertilization. From the AN OVA model, the maternally and
paternally derived sources of variation were estimated for larval TL, mouth gape width,
and mouth gape height (Table 30). Larval TL estimates ranged from 0 to 0.1278
(maternal) and 0 to 0.0427 (paternal) over the Six sampling days. The maternal and
paternal variation estimates for mouth gape width and height were 0.00005 and 0.0001,
respectively. The specific combining ability of the, two strains (fast and slow growth
rates) was estimated for larval TL, mouth gape width, and mouth gape height (Table 30).
The estimates for larval TL ranged from 0 to 0.1751. The estimates for mouth gape width
and height were 0.00007 and 0.0002, respectively. Differences between the average
additive genetic strain effects (fast and slow growth rates) were compared. A statistically
significant difference was observed on day 13 post-fertilization (p = 0.0072, Table 31).

Means and standard deviations for larval TL (Table 32) and mouth gape width
and height (Table 33) were calculated for each genotype combination over all days
sampled. Estimates of heritability and dominance were calculated for larval TL, mouth
gape width, and mouth gape height (Table 34). Heritability estimates for larval TL

ranged from 0 to 0.5863. The heritability estimates for month gape width was 0 and was

95

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97

Table 32. Averages for larval total length (TL) by genotype combinations for days 13 to
18 post-fertilization. Values are reported as means : standard deviations (sd).

 

 

 

 

 

Sire fast growth x Dam fast growth
Days post-fertilization N Mean :1; sd
13 40 5.3632 : 0.2093 mm
14 80 4.9212 : 0.4294
15 40 5.2210 1 0.3451
16 30 5.4021 1 0.4146
17 24 5.5929 1 0.5852
18 19 5.6430 3: 0.5654
Sire fast growth x Dam slow growth
Days post-fertilization N Mean _t sd
13 40 4.9878 : 0.2826 mm
14 80 4.9092 1 0.4690
15 40 5.1148 : 0.3964
16 36 5.4853 : 0.4269
17 38 5.5545 : 0.5565
18 23 5.7696 1 0.5334
Sire slow growth x Dam fast growth
Days post-fertilization N Mean : sd
13 40 5.1336 : 0.1896 mm
14 80 5.2428 3: 0.3036
15 40 5.3103 1 0.3168
16 38 5.4159 : 0.5838
17 16 5.3437 : 0.7601
18 10 6.0379 : 0.4016
Sire slow growth x Dam slow growth
Days post-fertilization N Mean : sd
13 40 5.0483 i 0.3034 mm
14 80 4.8997 j; 0.3179
15 40 5.2365 : 0.3825
16 39 5.5892 : 0.4474
17 40 5.5590 1 0.3996

18 30 5.8534 _+_: 0.4627

 

98

Table 33. Average for larval mouth gape width and larval mouth gape height by
genotype combinations on day 14 post-fertilization. Values are reported as means 1
standard deviations (sd).

 

 

 

 

 

Genotype combination N Mean 1 sd

Sire fast growth x Dam fast growth

Mouth gape width 40 0.2744 1 0.0395 pm

Mouth gape height 40 0.2521 3: 0.0331
Sire fast growth x Dam slow growth

Mouth gape width 40 0.2566 : 0.0339

Mouth gape height 40 0.2481 : 0.0383
Sire slow growth x Dam fast growth

Mouth gape width 30 0.2569 : 0.0371

Mouth gape height 30 0.2470 j; 0.0355
Sire slow growth x Darn slow growth

Mouth gape width 30 0.3023 : 0.0275

Mouth gape height 30 0.2738 1 0.0361

 

99

Table 34. Estimates of heritability and dominance. Estimates include larval total length
(TL) for days 13 to 18 post-fertilization and larval mouth gape width and height on day
14 post-fertilization.

 

 

 

Parameter Days Heritability Dominance
Post-fertilization
Larval TL
13 0 1.1006
14 0.1393 1.5668
15 0.0544 0.3816
16 0 1.4284
17 0.5269 1.5201
18 0.5863 0
Mouth gape width 14 0 0.7303
Mouth gape height 14 0.2289 0.2087

 

100

0.2289 for mouth gape height. The dominance estimates for larval TL ranged from 0 to
1.5668. The dominance estimates for mouth gape width was 0.7303 and was 0.2087 for
mouth gape height. The dominance estimates were broken down into two heterotic
effects and estimated (Table 35). Only days 13 and 14 post-fertilization showed a
statistically significant average dominance effect. On day 13 post-fertilization, a
significant heterotic effect for male fast growth and female slow growth was observed
(hs, p = 0.0188). On day 14 post-fertilization, another heterotic effect, male slow growth
and female fast growth, was significant (hd, p = 0.0484). However, the contrast
statement comparing these two heterotic effects were not significantly different from each

other (p = 0.1058 and p = 0.1055, respectively, Table 36).

Table 35. Estimates of average heterotic effects based on mating fast growth sires with

101

slow growth dams (hs) and mating slow growth sires with fast growth dams (hd).

Estimates include larval total length (TL) for days 13 to 18 post-fertilization and larval

mouth gape width and height on day 14 post-fertilization.

 

 

 

Parameter Days hs hd
Post-fertilization
Larval TL
13 0.0188 0.5701
14 0.9938 0.0484
15 0.2593 0.4195
16 ' 0.9681 0.7499
17 0.8622 0.6067
18 0.5231 0.2694
Mouth gape width 14 0.0072 0.0154
Mouth gape height 14 . 0.1434 0.0742

 

102

Table 36. Factorial contrast statements for differences between average non-additive
genetic strain effect of sire and dam. Estimates include larval total length (TL) for days
13 to 18 post-fertilization and larval mouth gape width and height on day 14 post-
fertilization. Significance determined on the 0.05 % alpha level.

 

 

 

Parameter Days DF p-value
Post-fertilization
Larval TL
13 1 0.1058
14 1 0.1055
15 1 0.1213
16 1 0.8051
17 1 0.6001
18 1 0.7128
Mouth gape width 14 1 0.7468
Mouth gape height 14 1 0.9816

 

103

DISCUSSION

Female Spawner Analysis

Caution needs to be taken when interpreting the values calculated for the female
spawning data. Although, the females were randomly selected, the spawning stock was
not entirely reflective of the perch population in Saginaw Bay, Lake Huron. The sample
size was too small to make specific population comparisons, however, general
comparisons can be made.

The AN OVA contrast statements indicated that the TL means for each female
spawner size group were significantly different from the other spawner size groups. This
complete separation of the size groups allowed for the comparison of maternal and larval
offspring characteristics in this study. The mean spawner TL values calculated for each
completed age (Table 8; Figure 9) were similar to the values calculated by Hile and Jobes
(1941)3 and were comparable to the unweighted means reported for the strain of perch in
Saginaw Bay, Lake Huron (review Carlander 1969). Values calculated by Diana and Salz
(1990)3 were less than the values reported in this study.

In the simple linear regression analysis, spawner weight was log transformed

because it was not normally distributed. Although spawner TL was normally distributed,

 

3 Values were estimated from figures.

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105

it was log transformed to describe the weight-length relationship (Anderson and
Neumann 1996). The transformations of the data provided a linear relationship and

resulted in the following weight-length linear regression equation (Figure 10):
(2.6) loglo (W) = -13.675 + 3.417 * loglo (L) + e.

The y-intercept estimate was lower than the natural log values reported by Parker (1958,
in Carlander 1969) for perch in Flora Lake, WI. The value of the y-intercept afier
transforming from the natural log is -2.6156. This value compares to —2.3982 reported
for perch in Saginaw Bay, Lake Huron (El-Zarka 1959). The slope regression estimate
falls within the range (2.65 to 3.54) of reported values (review Carlander 1969). While
the sample size of this study was considerably less than the studies reviewed by Carlander
(1969), the estimates were comparable. The sample of females did not produce any

females that could be considered “out of the ordinary.”

Factorial Analysis of positions within the egg ribbon

Number of eggs per gram of eggs, chorion shell diameter, and egg yolk diameter
for each region of the egg ribbon were compared. There were no significant differences
between the positions within the egg ribbons for SG 226. There were, however,
significant differences between the positions within the egg ribbons for SG 301. The
difference observed in the different positions of the egg ribbon is potentially due to the
location within the ovary. Toetz (1966; in Kamler 1996) compared differences between

eggs from the anterior and posterior positions within the ovary of bluegill

106

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107

(Lepomis macrochirus). The dry weight, energy content, nitrogen content, and
fertilizability were not significantly different between the two positions.

The diameters of the chorion shell and egg yolk for the eggs in SG 301 were
smallest in the anterior portion and largest in the posterior portion of the egg ribbon. The
ratio between the smallest and largest chorion shell diameter was 1.34 and 2.40 for the
egg yolk diameter. The chorion shell diameter ratio is lower than the ratio reported for
herring (Clupea harengus, Blaxter and Hempel 1963), which indicates that the variability
in egg size observed in this study was comparable to reported values in other fish species.

The variability observed within the egg ribbon could be explained by food
availability. Mejen (1940; in Kamler 1996) described the mechanism of regulation for
this process. When food is adequate, all oocytes near large or small blood vessels receive
sufficient nutrients for development. If there is a food shortage during egg development,
the nutrient flow is restricted in the oocytes near small blood vessels. This can result in
an increase in variability and a decrease in average egg size. In the cases of extreme food
shortages, female spawners can utilize lipids present in the gonads for metabolic purposes
(Nikolskij 1974; in Kamler 1996). While egg size can be influenced by the amount of
food available, Springate et al. (1985) did not find any differences in egg size for rainbow
trout (Onchorhynchus mykiss) eggs developed during an induced period of starvation.
The researchers also did not find significant differences in protein, lipid, and ash
percentages of the egg dry matter.

While there were significant differences observed between the positions of the egg
ribbon, these differences have been observed in other studies. However, the effect of

food availability may not have a large effect on egg size, chemical composition, and

108,

offspring viability. Any effect of food limitation on egg quality can be compensated by
the reduction of total fecundity (Kamler 1996). The analysis continued with the

assumption that the posterior portion was representative of the entire egg ribbon.

Egg Analysis

Mean fecundity values were higher than the values reported in Carlander (1969).
However, all sample sizes, including this study, were small. The total fecundity
regression models developed in this study were comparable to reported values in
Carlander (1969). The total fecundity-spawner TL relationship resulted in the following
regression model (Figure 11): I

(2.7) log (y) = -5.939 + 3.417 log (x) + e.

The y—intercept estimate is lower, but the regression slope estimate almost identical to the
values reported for Lake Michigan (Brazo et al. 1975) and Lake Erie (Hartman et a1.
1980). The total fecundity-spawner weight relationship resulted in the following
regression model (Figure 12):

(2.8) log (y) = 2.550 + 0.900 log (x) + e.

The y-intercept estimate is higher and regression slope estimate is lower than the values
reported for Lake Erie (Sztramo and Teleki 1977). The observed differences may be the
result of differences in sample sizes or geographic locations. Sample sizes from earlier
studies were much larger than the sample size of this study.

In addition to total fecundity, egg ribbon weight or egg biomass per female can be

used to express reproductive effort (Calow 1979). However, the relationship between egg

109

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ribbon weight or number of eggs per gram of eggs to spawner TL or weight have not
been previously reported for perch. No comparisons could be made between the
relationships. Since the total fecundity follows the same trends reported by other
researchers, it might be assumed that egg ribbon weight would follow similar trends.
While this study indicated that there was a linear association for the total
fecundity-spawner size and egg ribbon weight-spawner size relationships, the number of
eggs per gram of eggs-spawner size relationship was not linear. The trend of the
calculated means indicated that this parameter decreased as spawner size increased. This
trend is possibly related to egg size. However, the results of this study indicated that
there were no significant differences found between chorion shell diameter or egg yolk
diameter for the different spawner size groups. The linear regression models constructed
poorly represented the egg-spawner size relationship. Schneider (1972) was unable to
establish any relationship between perch spawners (97 — 343 mm TL) and egg size, egg

yolk size, fry length, or hatching success.

Larval Offspring Analysis - Maternal Contribution

Linear regression models were constructed to describe the larval offspring
characteristics-spawner size relationship. Like Schneider (1972), a relationship could not
be maintained between the larval offspring characteristics and spawner size. The
resulting r-square values in this study were less than 0.0300.

A different approach was attempted to establish a relationship between larval

offspring and spawner size. First, linear regression models were constructed to describe

112

the mouth gape width and height-larval TL relationships. Guma’a (1978) constructed a
linear regression model for the mouth gape height-larval TL relationship. Although the
data were derived differently, the linear model described the relationship well (r = 0.99).

The following linear regression model was constructed by Guma’a (1978):

(2.9) Gmx = 0.2152 + 0.0781 (L) + e,
where Gmax is the maximum gape height;

L is the total length of the larvae;

e is the residual error term.
This linear regression model compares to the following model constructed in this study
(Figure 13):

(2.10) y = 0.060535 + 0.007155 (x) + e,

where y is the mouth gape height;

x is the larval TLZ;

e is the residual error term.
Although the model (2.8) was constructed using data collected from the Eurasian perch,
the two species are closely related and are considered biologically similar (Thorpe 1977).
A comparison of models (2.8) and (2.9), results in an observed difference for maximum
mouth gape height. For example, 6.0 mm TL was the smallest TL that the larvae were
observed to begin exogenous feeding (Siefert 1972). Using this length, model (2.8)
predicted a maximum mouth gape height of 684 um and model (2.9) predicted in a
maximum mouth gape height of 318 pm. The difference observed between these two
models may be due to several factors. First, the difference in data collection techniques

may have resulted in the difference observed. Guma’a (1978) measured the jaws at a 90°

113

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114

angle. In this study, care was taken to minimize any damage to the larvae. The mouth
was only opened less than 45°. Secondly, it is not clear from the available data or
methods, if Guma’a (1978) measured the mouth gape height of Eurasian perch larvae less
than 10 mm TL. If larvae less than 10 mm TL were not used, this could account for the
observed differences. Statistical predictions on the mouth gape cannot be made for
values outside of the range investigated (Tempehnan, personal communication, 1997).
Third, the difference could potentially be due to different broodstock used. Researchers
in the NCR have observed differences in larval offspring from different sources of
broodstock (NCRAC 1997). While this study was unable to establish a relationship
between the size of the broodstock and the larval offspring, differences were observed in
larval offspring from different sources of spawning stock. All stocks showed the greatest
amount of growth at 22 °C. For temperatures higher than 22 °C, growth rates were
higher in stocks originating fi'om the southern US, but growth rates were higher for
stocks originating from the northern U.S. when temperatures were lower than 22 °C.

A second linear regression model was constructed for the larval TL-mouth gape
relationship. Data collected for the mouth gape width were used to construct the
following model (Figure 14):

(2.11) y = 0.0585 + 0.0624 (x) + e,

where y is the mouth gape width;
x is the larval TL’;
2 is the residual error term.
Using the example of 6.0 mm TL for first feeding larval perch, the mouth gape width can

be estimated to be approximately 322 um. Since the mouth gape width of larval perch

115

y = - 0.0357 +10.0576(x)2 + e'
r2 = o 3174

’ __ ”—1

 

 

 

0.45 —

T
V.
O

I 1
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O

(19191110101111) {{1le odes qmour [BAJ‘B’I

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20 25 30 35 40 45
Larval (total length)2 (mm)

15

10

Figure 14. Simple linear regression analysis for larval (total length)2 to mouth gape width relationship.

116

less than 10 mm TL has never been reported in the literature, comparisons cannot be
made.

Linear regression models were constructed for larval TL-mouth gape relationships
in each female spawner size group (Appendix N). This division of models allowed the
comparisons between female spawner size groups for initial larval mouth gape size and
rate of increase. In this experiment, no statistically significant differences for mouth gape
width were observed between female spawner size groups (Table 26 and 27).

Statistically significant differences for larval mouth gape height were observed between
female spawner size groups. The rate of increase for mouth gape height was significantly
different for SG 201 vs. SG 226, SG 226 vs. SG 276, and SG 276 vs. SG 301 (Table 28).
The rate of increase for the contrast 86 201 and SG 276 was 0.009 and 0.006 for SG 226
and SG 301 (Appendix N). The rates of increase for the other female spawner size
groups fell in between 0.006 and 0.009. This may have indicated that the rates of
increase fell over a range of values, and the two opposite extremes of the range were
significantly different. Overall, the rates of increase may not be too different. Statistical
differences for initial mouth gape height were observed between a range of female
spawner size groups. A consistent pattern did not emerge from the statistical analysis and
may be a result of large variations.

The variations observed in this study might be the result of the fertilization
methods used. For this study, eggs from a single female spawner were fertilized with one
or more males. In addition, egg samples were taken over a period of two weeks. During
this time, researchers were unable to keep male spawners used in this study separate from

new broodstock. Male spawners were chosen at random. Consequently, the same male

ll7

spawners were not used to fertilize all of the eggs in this study and several males were
often used at one time to ensure that enough milt was available for fertilization. The
results of using several males may have contributed to the large variation observed in this

portion of the study.

Larval Offspring Analysis - Parental contribution

The estimates of heritability were very low for larval TL, mouth gape width, and
mouth gape height. All estimated values were less than 0.5. The value of heritability can ’
range from 0 to 1, but values less than 0.5 indicate that selective improvement for these
values is unlikely. Moav and Wohlfarth (1976) estimated the heritability for increased
growth rate in European carp to be approximately 0. While an estimate for heritability is
only a prediction on the effectiveness of selection (Tave 1993), Moav and Wohlfarth
(1976) investigated the selection for improved growth rates over five generations. The
result was negative and the conclusion was that the additive genetic variance for this trait
had reached a plateau due to natural selection and past, uncontrolled, artificial selection.
Purdom (1993) offered another explanation for the lack of response to increased growth
rates. This trait may depend heavily on environmental conditions. Examples of variables
that can result in environmental variation are food availability, food quality, water
temperature, and predation. Variance due to environmental conditions, especially if not
controlled, can confound with the additive genetic variance, and eliminate any
possibilities to quantify improvement due to breeding by interacting with the genome

(Tave 1993).

118

The low heritability values in this study are a reflection of the estimates for
additive genetic variance (review Falconer 1989; Appendix D). The additive genetic
variance was partitioned into maternally and paternally derived sources of variation.
Estimates for both sources of variation were low, which suggests large non-additive
genetic and environmental effects. Non-additive genetic variations result from the
interaction between alleles at each locus. In this study, two breeding combinations
resulted in significantly larger larvae (larval TL). On day 14 post-fertilization, a non-
additive genetic or dominance effect was observed for sire fast grth and dam slow
growth, but on day 15 post-fertilization, the non—additive genetic effect was observed for
sire slow growth and dam fast growth. However, this is not significant since non-additive
genetic variance cannot be inherited (Tave 1993). This form of genetic variance depends
on interactions and is disrupted during meiosis, which eliminates the possibility of
transfer from parent to offspring.

In this study, variation due to the environment was not partitioned out and was
included in the error term. This was the result of unknown factors influencing the
broodstock. Otherwise, the environmental condition was similar for all offspring.

The results of this study indicate that the low heritability for larval TL, mouth
gape width, and mouth gape height do not currently warrant a selection or breeding
program with current available broodstock. As Moav and Wohlfarth (1976) suggested,
these traits may have reached a plateau due to natural selection. However, if a captive
broodstock is developed, then another heritability study should be completed. There is
still potential for improvement if environmental conditions for the broodstock are

controlled.

1)

2)

3)

119

SUMMARY AND CONCLUSIONS

The width and height of the mouth gapes for first feeding larval perch larvae were
identified. For a 6.0 mm TL larvae, the mouth gape width was approximately 322
um, and the mouth gape height was approximately 318 um. With the mouth gape
identified, researchers can focus on providing suitable diets that are small enough for
the larvae to consume.

Positive linear relationships were significant between female spawner size (TL and
weight) and egg ribbon weight and total fecundity. However, linear relationships
were not significant between female spawner size and number of eggs per gram of
eggs, chorion shell diameter, and egg yolk diameter. The inability to establish a
correlation between female spawner size and these egg characteristics may be due to
large environmental variation. Examples of variables that can result in environmental
variation are food availability, food quality, water temperature, and predation.
Linear relationships could not be established between female spawner size and larval
TL and size of the mouth gape, which could have been the result of large paternal
influences or environmental variation. The genetics study attempted to identify the
amount of influence fiom both maternal and paternal sources and from the

environment.

4)

5)

6)

7)

120

The genetics study was designed to partition out the influence of the maternal and
paternal sources of variation on larval TL, mouth gape width and height. Overall, the
paternal contribution to the total variation was small (0 — 0.1278). The overall
residual term, including any environmental variance, was larger (0.0484 - 0.2104),
which indicates the large environmental influence. Another genetics experiment
should be conducted, but only after some of the environmental variables can be
controlled (i.e. — a captive broodstock).

Estimates of heritability were calculated for larval TL, mouth gape width, and mouth
gape height. All values of heritability were less than 0.5, which indicates that
selection for improvement of these characteristics will be unlikely. Since this study
included a fixed assignment of the parental stock, a true estimation of heritability
could not be calculated. However, the estimates of heritability provide a valuable
insight to the possibility of starting breeding programs, which could select for other
desirable characteristics.

The number of broodstock used and the number of larvae sampled were large enough
to estimate the genetic variance components (review Falconer 1989). However, by
increasing the number of broodstock used, the amount of variation in the population
would be better represented.

The large dominance values (0.2087 - 1.5668) indicate that the variations in larval TL
and mouth gape sizes occurred by chance. Larger values for larval TL and the size of
the mouth gape resulting from a breeding program could not be duplicated, since this

is a result of cell division.

121

8) The results of this study indicate that, through natural selection, larval TL and the size
of the mouth gape for perch may have reached a plateau and cannot be increased
through selective improvement. If artificial selection for these traits operates in the
same direction as natural selection (i.e. - larger mouths and longer lengths), then it
may be difficult to improve on natural selection. However, other traits that are

important for culture, such as survival could be investigated

CHAPTER 3

EXPERIMENTAL TANK CULTURE DESIGN AND DIET STRATEGY
EVALUATION FOR FEEDING LARVAL YELLOW PERCH

INTRODUCTION

Researchers and fish culttuists have been trying to develop successful feeding
strategies and culture systems for larval perch.‘ However, current intensive larval culture
techniques remain inadequate and cannot supply enough advanced fry and fingerlings for
research, stocking, and food production requirements. With the development of out-of-
season spawning techniques, successful intensive culture techniques become essential.

One reason for the slow development of intensive culture techniques has been the
small size of perch larvae. This morphological icharacteristic makes it difficult to develop
culture systems and to provide a suitable diet or feeding strategy for the small larvae.

This study was designed to develop feeding strategies and culture methods for
intensive culture systems. Diet strategies were based on methods developed in other
larval perch and walleye culture research (Barrows et al. 1993; Moore et al. 1994; Starr,
personal communication, 1996; Summerfelt 1996). Culture methods were based on a
combination of successful larval culture methods used for walleye and mahi mahi (Kim et

al. 1993; Peterson et al. 1997).

122

123

Findings from the first two chapters of this thesis were incorporated into the
design of this study. Based on the results of chapters 1 and 2, all larvae were pooled and

different diet strategies were compared for growth and survival.

OBJECTIVE

6) to determine the significance of mouth gape size on growth, survival, and feed

acceptance during intensive larval culture conditions.

124

LITERATURE REVIEW

Researchers and fish culturists in the North Central Region have been trying to
develop successful feeding strategies and culture systems for larval perch using both,
pond and tank rearing approaches to produce advanced perch larvae. However, intensive
larval culture techniques remain inadequate and fail to meet commercial production
demands.

In the pond rearing approach, eggs have been collected from wild or captive
broodstock, which have been stocked as fertilized eggs or hatched larvae into intensively I
managed, fertilized ponds (Manci et al. 1983; Malison and Held 1992). The fertilized
ponds provided a natural forage base for the larvae to survive and grow. At a later time,
the larvae could be harvested and habituated to formulated feeds and intensive culture
conditions. However, pond production of larval perch has been associated with highly
variable survival, growth, and condition of the fish (NCRAC 1995). Results can vary
annually as well as by location. In addition, perch stocked into ponds can be subjected to
stress from zooplankton forage base depletion and excessive handling during harvest
(Malison and Held 1995). These types of stress have been observed to result in higher
rates of disease and to increase the amount of time to habituate to feed, resulting in

significant mortality rates (Hussain and Summerfelt 1991).

125

In tank rearing of larval perch, larvae could be monitored during all stages of
development, and the environmental and nutritional variables could be controlled by
manipulation for the long-term goal of maximum growth and survival (Calbert and Huh
1976; Hale and Carlson 1972; Hinshaw 1985). This culture method is essential if out-of-
season spawning for perch is to be developed (Dabrowski 1998). Together, these
methods could potentially supply perch year round for research, stocking, and food
production requirements.

Despite advancements, development of intensive culture techniques has been slow
(NCRAC 1997). Larval perch are difficult to rear intensively due to their small size
(Mansueti 1964; Nickum 1978; Best 1981), which has been reported to range from 4.7 -
7.0 mm total length (TL) at hatch (Mansueti 1964; Ney 1978; Whiteside et al. 1985;
Heidinger and Kayes 1986). In addition to this morphological limitation, perch larvae
have been observed to have small mouth gapes, which suggests that the larvae are
restricted to certain prey due to the restriction of mouth gape width (Wong and Ward
1972). Larval perch are limited by the mouth gape when the size of their prey exceeds
their mouth gape (Hansen and Wahl 1981; Wong 1972) and possibly the diameter of the
esophagus (Kestemont et al. 1996; Raisanen and Applegate 1983). The size of the mouth
gape, width and height, has been identified for larval perch less than 10 mm TL (review
Chapter 2, this thesis). By identifying the mouth gape size for first feeding larval perch,
the appropriate sized prey or pellet can be provided. For example, if the mouth width of
first feeding larval perch is approximately 322 um, then the larvae are capable of

consuming prey or pellets that are less than this size. Care should be taken to avoid

126

providing prey or formulated diets that exceed the mouth gape or diameter of the
esophagus.

Several researchers have been exploring the use of formulated diets as the initial
food for first feeding larval perch (Starr, personal communication, 1996; NCRAC 1997).
Formulated diets would be ideal for intensive culture of perch larvae if the diets were
accepted by the larvae and were nutritionally complete. However, first feeding perch
larvae do not accept and survive readily on formulated diets (Best 1981; Starr, personal
communication, 1996). The development of intensive larval walleye culture also went
through the similar problems of providing an appropriately sized and nutritionally
balanced diet (Barrows et al. 1988).

Currently, larval perch intensive culture feeding strategies have employed
captured or cultured species of zooplankton and phytoplankton (Hale and Carlson 1972;
Noble 1973; Raisanen and Applegate 1983; Confer and Lake 1987; Ansari and Qadri
1989; NCRAC 1995) or feeding newly hatched Arternia nauplii (Hinshaw 1985;
Mansueti 1964; McCormick 1976). The use of captured zooplankton presents several
problems for any early season or out-of-season spawning and is often an unreliable food
source. Problems may arise if zooplankton blooms do not occur at the same time as the
first feeding larval stage. For example, the necessary food requirements of larval fish are
sometimes not met due to a difference in timing between first feeding and zooplankton
availability. It can also be difficult to find appropriate sizes or abundance of zooplankton.

At different times of the year, certain zooplankton species might not be able to
supply the necessary nutritional requirements of the larval fish. Zooplankton population

structure and size varies considerably during a growing season and between years due to

127

differentenvironmental conditions (Houde 1967; Mills et al. 1986). In addition to the
size of the zooplankton biomass, the caloric content of zooplankton may vary among
species and seasonally within a species (Schindler et al. 1971).

Since larval fish depended on the chemical properties of their food (Watanabe et
al. 1983), large numbers of zooplankton are needed to fulfill the fishes nutritional
requirements. The National Research Council (1993) reported docosahexaenoic acid
(DHA, 22:6w3), eicosapentaenoic acid (EPA, 20:5(03), and linolenic acid (18:0)3) are
essential fatty acids for several species of fish. Levels of DHA and EPA were higher in
young perch than in their major prey item, Daphnia (review Dabrowski et al. 1993). In
addition, a single species of zooplankton is often incapable of supplying the long-chain
fatty acids needed for growth and survival of larval fish. The lipid content of Daphnia
varied throughout egg development and the molt cycle (Tessier at al. 1983).

Researchers that have focused on collecting zooplankton from rivers and lakes for
feeding larval fish have discovered that this has not been a reliable source of food for
commercial scale operations (Kamler 1992). The collection process can be labor
' intensive and problems can be magnified during periods of reduced zooplankton
abundance. In addition, zooplankton have been identified as potential carriers of
parasites and feeding wild zooplankton has been observed to increase the risk of pathogen
introduction (Spotte 1970).

The problems associated with capturing zooplankton can be avoided by culturing
zooplankton. Mass production of live feeds has been extensively used throughout Japan
with success (Kamler 1992). However, zooplankton culture has been associated with an

inability to provide adequate varieties of species as well as an adequate abundance for

128

commercial operations. Plankton and zooplankton monocultures require large amounts
of space and skilled labor.

Binkowski (see review in NCRAC 1995) has developed a “green tank water”
(GTW) method for culturing several zooplankton species in single, indoor tanks. The
GTW culture is added regularly as a food source for first feeding larval perch. After four
to six days, the perch larvae can be fed with newly hatched Artemia nauplii. While the
need for monoculture has been eliminated, this method has gained limited acceptance for
commercial applications, in part due to an inability to duplicate the research results.

An alternative to zooplankton culture could be Arternia culture. While the cost of
Arternia cysts can be high (825-100 lb'l), the newly hatched Arternia nauplii provide a
readily available diet with high nutritional quality in the form of fatty acids (Lovell 1990;
Webster and Lovell 1990b). In addition, Arternia can be “packed” which can increase
levels of highly unsaturated and essential fatty acids (Lemm and Lemarie 1991).
Approximately 12 hours after hatching, Artemia nauplii molt into the Instar 11 stage when
feeding begins (Sorgeloos et al. 1986). Typically, the nauplii can be fed a food source
that has concentrated levels of fatty acids which helps to increase the nutritional value.

Feeding Arternia nauplii is a viable diet strategy for intensive commercial culture
for first feeding larval perch. Survival rates have been reported as high as 85.3% after 14
days for European perch fed Arternia nauplii at first feeding (V lavonou et al. 1995).
Perch larvae can also be raised on Arternia nauplii as first food (Mansueti 1964; Hale and
Carlson 1972; Hinshaw 1985; Starr, personal communication, 1996).

Although newly hatched Arternia nauplii are an excellent source of food for first

feeding larval fish, there are two drawbacks. First, Artemia cysts can be expensive,

129

especially for certain characteristic traits such as a high hatching rate. Second, the daily
production of Artemia nauplii can be labor intensive and usually requires additional
equipment and space. One alternative to the use of Arternia nauplii is decapsulated
Arternia cysts. The cysts have a non-digestible chorion shell, which can be removed
using a chemical method (Bruggeman et al. 1979). The decapsulated cysts have a
biochemical composition comparable to newly hatched nauplii and have a 30 to 40 %
higher energy content (Schauer et al. 1980; Seidal et a1. 1982). Verreth et al. (1987) and
Vanhaecke et a1. (1990) have reported using decapsulated Artemia' cysts as a food source
for first feeding larvae of carp (Cyprinus carpio) and African catfish (Clarias
gariepinus).

Intensive culture techniques need to be. improved, if a continuous supply of perch
can be provided to meet the needs of reSearch, food. production, and stocking. By
developing intensive larval perch culture techniques; the options available to the hatchery
manager increases (West and Leonard 1978).1When'out-of-season spawning techniques
are developed, intensive larval culture techniques will be necessary to help supply perch

year round for research, stocking, and food production requirements.

130

METHODS

Larval feeding strategy experiments were designed to compare survival and
growth rates during the first 45 days using different larval diets. During the first
experimental trial (year 1) for feeding strategies, there was no attempt to separate female
spawners into separate size groups. Data analyzed from year one were used to determine
strategies for feed trials in year two. Based on the results from year one (review Chapter
2 in this thesis), all larvae from the different adult female spawners were pooled in the

second experimental trial (year 2).

Culture System Design

Cylindrical, 60 L polyethylene black plastic tanks with conical bottoms were used
to culture the perch larvae. The conical bottom design was similar to culture tanks
designed by Kim et al. (1993). The culture tanks had a 42 cm diameter with a 76 cm
depth. In the center of each culture tank, an erect tubular screen was used to keep the fish
larvae from entering the drain, which was located at the bottom of each tank. Since a
large amount of diet was provided, a screen was constructed, which provided a large
surface area and reduced the amount of clogging. The screen was constructed out of a 3.8

cm inside diameter (ID) PVC pipe with approximately 75% removed and covered with

131

150 um Nitex mesh. The screen ran the length of the pipe, which was 78 cm long and
extended 6.5 cm above the water surface.

The culture tanks were initially supplied with 11 °C aerated well water.
Gradually, the well water supply was mixed with heated water from the Bay Port facility.
The temperature of the water was increased over the next seven days and held constant at
17 i 3 °C. The water depth was controlled using an external standpipe with the water
volume set to 50 L. Water was supplied to the culture tanks using a similar design that
has been used for intensive culture tank systems for walleye larvae (Barrows et al. 1993;
Moore et al. 1994). The water was supplied below the surface, along the tank wall
through a vertical manifold. The manifold was constructed using a 13 mm 11) PVC pipe
with 10 - 1.6 mm diameter holes. The manifold was pointed in the direction to deliver a
clockwise flow. A water spray was used on the surface and pointed in the direction of the I
flow to minimize any disruption of flow patterns. The flow rate was regulated to 0.5
maximum exchanges per hour (0.42 liters per min) for the prolarvae stage and increased
to 0.75 exchanges per hour (0.625 liters per min) at the onset of exogenous feeding
(Summerfelt 1996). The culture tanks were illuminated using overhead, incandescent
lights with less than an estimated 100 lux. The culture tank walls and screens were

cleaned every two days using a siphon.

Egg and Larval Collection Procedures

Perch spawning stock was collected from the outer Saginaw Bay, Lake Huron

during a two week period beginning on April 19, 1996 and on April 15, 1997. The

132

spawning stock was transported to Bay Port Aquaculture in West Olive, Michigan
(Figure 6) and held in a flow through system supplied with 11 °C aerated well water.

The egg ribbon was manually stripped from each ripe female into a dry bowl to
enhance fertilization rates (Piper et. al. 1985). Milt was mixed with the eggs before water
was added. The fertilized eggs were allowed to water harden for 10 min before being
placed into an upwelling incubation unit. Eggs were incubated using techniques
developed by Bay Port Aquaculture researchers (Starr, personal communication, 1996).

After hatching, approximately 11-12 days post fertilization, larvae from several
incubating units were pooled into one collection tank. The water within the collection
tank was mildly agitated to achieve an even mixture of larvae. All culture tanks were
randomly stocked, on day 0, with perch larvae at a density of 160 larvae per liter (8,000
larvae per tank) using a Jensorter Electronic Fry Counter, Model FC (Bend, Oregon,
US).

Sub-samples of five larvae were collected every three days post stocking to
determine percent gas bladder inflation, verify diet consumption and measure TL. Larvae
were euthanized using CO2 saturated tonic water. After the larvae were euthanized, they
were placed in a 90 mm diameter Petri dish containing 10 ml of distilled water. Larvae
were observed under a dissecting microscope with light provided from the bottom to
determine gas bladder inflation (GBI). Inflated gas bladders were readily detected by
observation (Barrows et al. 1993). After the initial two week sampling period, larvae
were sampled once per week until completion of the study. At each cleaning, moribund

larvae were collected and counted to estimate mortality and survival.

133

Feeding Procedures

Four different larval diet strategies were compared (3 replicates per treatment) in
12 culture tanks (Table 37). Two Arternia strains, Great Salt Lake4 (GSL) and San
Francisco Bay‘ (SF B), were compared. Arternia cysts and nauplii from these two strains
were used as initial food for first feeding larval perch. Incubation of cysts followed
standard methods (Hoff and Snell 1993) and decapsulation of cysts followed procedures
discussed by Sorgeloos et al. (1977) and Bruggeman et al. (1979).

The control group was initially fed newly hatched Arternia nauplii from the GSL
strain. This control group designation was based on previous successful research
conducted by researchers at the Bay Port research facility (Starr, personal
communication, 1996). For the other three feeding strategies, decapsulated Arternia cysts
from both strains were used as the initial food for first feeding larval perch. The feeding
strategies were randomly assigned to culture tanks (SAS, 1988).

Feeding began on day 2 with an initial Artemia cyst or nauplii density of 3 per ml
for each feeding. The Arternia density was increased to 5 per ml for each feeding
beginning on day 4 and was increased to 7.5 per ml on day 9. On day 13, the first diet
transition began. The initial diet amount was reduced by 25% each day and replaced with
the first transition diet (Table 38). This continued until the initial diet was entirely

replaced. On day 27, the second diet transition began. The transition occurred in the

 

‘ Great Salt Lake (Bonneville Arternia lntemational, Inc., Salt Lake City, UT; no lot number)
5 San Francisco Bay (Platinum grade Argentemia, Argent Chemical Laboratories, Inc., Seattle,
WA; lot BP0103K)

 

134

 

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135

 

 

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136

same manner as described above. The larval starter diet consisted of BioKyowa A and B

with a pellet size of 50 — 200 um.
Changes to Equipment and Feeding Strategies for Trial Two

Perch larvae were pooled and collected using the same methods as described for
year one. All tanks were stocked volumetrically. with perch larvae at a density of
approximately 160 larvae per liter (8,000 larvae per tank). For transportation, larvae were
placed into 12 - 40 L bags with 10 L of 11 °C aerated well water. The bags were filled
with pure oxygen and sealed. The larvae were transported to Michigan State University’s
Aquaculture Laboratory in East Lansing, Michigan. where larval feeding experiments
were conducted. The larvae were tempered to 16 °C before being randomly stocked into
the culture tanks. The culture tanks were supplied. with heated well water. The
temperature of the water was increased to 20°C over the next seven days and held
constant to i 1 °C. The culture tanks were illuminated using overhead, fluorescent lights
with an estimated 400 lux. The culture tanks were cleaned every two days.

Tank design was modified based on findings from the first trial. Initially, a false,
flat bottom was provided to increase the bottom surface area. A similar modification in
the tank design of Kim et al. (1993) had been used for benthic marine fish larvae at the
Oceanic Institute, University of Hawaii (Garling, personal communication, 1996).

Four different larval diet strategies were compared (3 replicates per treatment) in
twelve culture tanks (Table 39). The diets consisted of newly hatched Arternia nauplii

from two different strains, SFB and GSL. Feeding began on day 2 with an initial Arternia

137

 

 

 

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138

nauplii density of 3 per ml for each feeding. The Arternia density was increased to 5 per
ml for each feeding beginning on day 4 and was increased to 7.5 per ml on day 9. On day
13, the first diet transition began. The initial diet amount was reduced by 25% each day
and replaced with the first transition diet (Table 40). This continued until the initial diet
was replaced entirely. On day 27, the second diet transition began. The transition

occurred in the same manner as described above.

139

 

 

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140

STATISTICAL ANALYSIS

Variation in larvae TL means for each diet strategy were calculated and tested for
normal distribution and homogeneity of variances. The test for normal distribution was
based on the Shapiro- Wilk statistic W (Neter et al. 1996). The assumption of equal
variances was tested using a Modified Levene ’5 test (N eter et al. 1996). One-way
Analysis of Variance (ANOVA) tests were used to determine overall statistical
significance between TL from the different diet strategies. Variation in mean TL was
compared over time for the different diet strategies. The percentage of G31 and feed
acceptance were calculated and compared over time.

Growth and mortality were represented as instantaneous growth rates (IGR) and
instantaneous mortality rates (IMR). IGR and IMR were calculated using methods fiom

Ricker (1975). Means for larval TL were used to calculate IGR:

 

(3.1) u= mum—ML.)
tx + i
where, p. is the IGR;

Lx, LN are the lengths at time x and x + i;
tx H is the time at x + i represented as days.

In addition, a survivorship curve was used to show the corresponding larval survival to

any point in time (Deevey 1947):

(3.2)

(3.3)

141

5=N

X _L

No
where, fix is the change in population size;
N0 is the initial population size;

Nx is the population size at any point in the future.

Larval mortalities were used to calculate IMR:

Z= ln(Nx)-1n(N...)

l

 

where, Z is the IMR;
Nx is the initial population size;
NM is the population size time at x + i;

i is the time represented as days.

142

RESULTS
Trial One

Since each tank was treated as an experimental unit, the observations from each
replicate were combined into means for each of the four different feeding strategies. The
means and standard deviations for larval TL were calculated every three days during the
first feeding trial (Table 41). The data were normally distributed (p = 0.7839), and the
test for the assumption of equal variances across feeding strategies was not violated (p >
0.05). One-way ANOVA tests did not indicate any statistical differences between the
mean larval TL for each feeding strategy or for each day measured.

Feed acceptance and GBI were measured at the same time. The first observation
of GBI occurred on day 6 in groups DG 2 and DS 1 (Table 42). By day 9, GBI rates for
the experiment groups ranged from O % to 87 % (Table 42). GBI was not observed in the
control group before day 10. The group DS 1 indicated 100 % GBI by day 12 (Table 42).
Feed acceptance was low throughout the study. The first observation of feed acceptance
occurred on day 9 (Table 42). Approximately 7 % of the larvae in the DS 1 feeding
strategy had accepted the diet. In contrast, none of the larvae sampled in this feeding

strategy had accepted the diet on day 12. However, 20 % of the larvae from the DG 2

143

 

 

 

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144

 

 

 

 

 

 

 

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145

feeding strategy had accepted the diet by day 12. By day 15, 13 % to 20 % of larvae
remaining in the three feeding strategies had accepted the diet.

Survival for all diet strategies was above 99 % for the initial six days after
stocking (Table 43; Figure 15). Beginning on day 8, survival rates decreased to 65 % for
the control group. The survival rates for the rest of the diet strategies were lower than
reported for the previous six days but ranged from 89 % to 91 % (Table 43). By day 10,

0.5 % of the 8,000 larvae stocked in each of the control group culture tanks were

remaining. The survival rates for the rest of the diet strategies ranged from 71 % to 74 %.

By day 10, almost 100 % mortality was observed in the control group, and all sampling
was discontinued. By day 12, the experiment for the control group was terminated. For
the remaining culture tanks, survival rates ranged from 0.52 % to 1.65 %. By day 16,
there were no larvae surviving in the culture tanks, except for 0.15 % in the DS 1 culture
tanks.

The growth of perch larvae was measured as larval TL and calculated as IGR.
While the TL of the larvae increased over time (Table 41), the IGR initially increased
until day 6 and began to decrease (Figure 16).

The number of mortalities in the control group occurred sooner than the other
feeding strategies. The number of mortalities peaked by day 10 (Table 44). For the other
three feeding strategies, the number of mortalities peaked on day 12. These observations
are reflected in the IMR calculations. The IMR peaked at 2.42 on day 10 for the control
group (Figure 17). The IMR for the other three feeding strategies peaked on day 14 but

was less, ranging fiom 1.22 to 1.77 (Figure 17).

C'

146

 

 

 

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150

Trial Two

After the larvae were stocked and allowed one day to adapt to the new
surroundings, a period of high mortality was observed. By day 3 and 4, mortality rates
approached 96 % to 99 % (Table 45). Between sampling periods 2 and 3, the number of
larvae mortalites ranged from 7831 to 8065 (Table 46) and survival dropped to
approximately zero (Figure 18). Measurements of TL, GBI, and feed acceptance were
not recorded. During the period of high mortality on days 3 and 4, _IMR values ranged
from 1.8 to 2.9 (Figure 19). By day 6, the IMR values were less than 0.5, but increased to

a range of0.85 to 1.63 by day 10 (Figure 19).

151

 

 

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154

DISCUSSION

Although considerable knowledge is known about intensive culture methods for
several species of larval fish, including walleye (review Moore et al. 1994a; Moore et al.
1994b; Summerfelt 1996; Moore 1996), less is known about perch and Eurasian perch.
Different culture systems have been used to investigate optimal conditions for culturing
perch; however, a single, universal intensive culture method has not been accepted.
Comparing prey requirements can be hampered by the different experimental designs and
rearing conditions used (Buckley et al. 1987). Some optimal conditions of environmental
factors, such as; temperature, flow rate, and light intensity have been identified (Houde
1969; Hokanson and Kleiner 1974; Calbert and Huh 1976; Hokanson 1977), but optimal
diets and feeding strategies are unknown (Brown et al. 1996).

This study attempted to investigate the growth and survival of larval perch using
different diets and strategies. Feed acceptance, GBI, and survival of perch larvae raised
on Arternia in the first 14 d of life was lower than reported for other previous studies
(Hinshaw 1985; Starr, personal communication, 1996). The TL for larval perch stocked
into the culture tanks on day 0 were within the range reported by Heidinger and Kayes
(1986). By day 9, the yolk sac was almost completely absorbed with only the oil droplet
remaining. At this stage, the larvae were approximately 6.2 mm TL. Similarly, Siefert

(1972) had observed that larval perch had accepted feed at 6.0 mm TL with complete

155

yolk sac absorption at 7.1 mm TL under natural conditions. However, egg size (Blaxter
and Hempel 1963) and temperature (Ware 1975) can affect the length of time that larvae
remain in the yolk sac stage. In this study, the first observation of feed acceptance
occurred on day 9 when the larvae were approximately 6.3 mm TL (mean temperature =
13.8 °C).

Larval mortalities were observed in this study beginning on day 8. Hokanson and
Kleiner (1974) had observed mortality in non-feeding perch larvae by day 9 at 19.8 °C.
The pattern of larval mortality observed was similar to larval walleye mortalities
described by Li and Mathias (1982). Although, the larvae had not begun to feed,
mortality rates remained low despite complete yolk absorption, which occurred on day 8.
The remaining oil globule might have provided a nutritional “buffer” between the onset
of exogenous feeding or starvation (Li and Mathias 1982). However, by day 12, the
mortality rates ranged from 81 to 100 % in this study. The IGR values calculated for this
study support the timing of the endogenous to exogenous feeding transition. The IGR
values peaked for all feeding strategies on day 6, but decreased afier this point (Figure
14). This may have indicated that the larvae consumed most of the endogenous feeding
reserves. Without supplementation fi'om exogenous feed sources, growth decreased and
mortality increased. Significant mortality rates were observed for the first time on day 6
(Figure 13). If the larvae had accepted the diet, the IGR values probably would have
increased. The trend would have appeared to be similar to the observations for larval lake
Whitefish (Coregonus clupeaformis) IGR values (Brown and Taylor 1992).

The low feed acceptance rate observed in this study may have resulted from an

inability by the larval perch to consume the newly hatched Arternia nauplii at the onset of

156

first feeding. This factor alone may indicate the reason for the inability of the larvae to
consume the diet. By day 6, the larvae had reached an average TL of 5.94 mm which was
close to the TL of first feeding larval perch (Siefert 1972). The linear relationship
established between larval TL and mouth gape width indicated that the mouth gape width
should have been approximately 310 to 317 pm (review Chapter 2, this thesis).
Researchers have reported that the critical dimension for ingestion of oblong prey is the
maximum width including all appendages (Blaxter 1965; Arthur 1976). The Arternia
nauplii used in this study had a maximum appendage width that ranged from 484 to 764
pm (review Chapter 1, this thesis). This indicated that the larvae were mouth gape
restricted at the beginning of exogenous feeding. Using this strain of Artemia, only
larvae longer than 7.69 mm TL at this critical point could have survived (review Chapter
2, this thesis).

Previous studies have reported using Arternia nauplii as food for first feeding
larval perch with limited success (Mansueti 1965; Hale and Carlson 1972; Hinshaw 1985;
Starr, personal communication, 1996). However, differences in nauplii size may have
accounted for the different success rates. Differences in nauplii appendage widths have
been observed for different Arternia strains (review Chapter 1, this thesis). Maximum
appendage widths for the SFB strain ranged from 285 to 418 pm. This range of
maximum appendage widths would be small enough for 5.52 mm TL, or greater, first
feeding larval perch.

Although decapsulated Arternia cysts have not been used previously for larval
perch culture, the cysts can provide an excellent alternative to newly hatched Artemia

nauplii. The decapsulated cysts can be handled as an inert diet and have comparable

157,

nutritional qualities (Schauer et al. 1980; Seidel et al. 1982). In addition, the diameters of
decapsulated cysts are small enough to be consumed by first feeding larval perch.
Vanaecke and Sorgeloos (1980) compared different sources of hydrated, decapsulated

cysts. The diameters of the GSL strain averaged between 207.7 to 210.5 pm for the SF B

strain and 234.8 to 241.6 um. Decapsulated Arternia cysts have been used for larval
culture of carp and Afiican catfish (V erreth et al. 1987; Vanhaecke et al. 1990). Unlike
free-swimming Arternia nauplii, hydrated decapsulated cysts sink to the bottom. To help
alleviate this problem, aeration was used within the tank to keep the cysts suspended in
the water column. However, the aeration was discontinued after day 1, because the larvae
became disoriented. The air pressure from the aeration system was too high and could
not be reduced significantly without affecting theefficiency of the air stones. Although
the cysts sank to the tank bottom, 6.7 % of thelarvae were feeding by day 9 in the DS 1
diet strategy and 20 % by day 12 in the DG 2 diet strategy (Table 41). A better method
might be used to deliver the decapsulated Arternia cysts over the entire day rather than
during four short feeding periods.

Colesante (1996) discussed a similar method used to deliver Arternia nauplii to
larval walleye. Afier hatching, the nauplii were placed into a feeder that dispensed every
8 min for a 3-sec interval, 24 hrs/day. At each feeding, the feeder dispensed
approximately 500-600 ml of the nauplii solution into feeder tubes. The feeder tubes
directed the solution to different parts of the tank. The amount of nauplii released during
each feeding was not reported, but the target feeding rate was 800-1000 nauplii/fish/day.
By using this method, the decapsulated cysts would not lose significant nutritional

quality, as is the case with nauplii. Newly hatched nauplii continue to grow and use the

158

energy reserves, which significantly reduces the nutritional content (Sorgeloos et a1.
1986)

A second method could have employed the use of dry, decapsulated Arternia
cysts. Unlike freecswimming nauplii and hydrated cysts, the dry, decapsulated cysts will
float on the water surface and sink afier hydrating. Vanhaecke et al. (1990) successfully
used the dry, decapsulated cysts to raise larval carp. The growth rates of the larvae were
comparable to the larvae fed newly hatched nauplii.

Another reason for the differences in survival rates for first feeding larval perch
fed Arternia nauplii, may be due to differences in broodstocks used in previous studies.
Blaxter (1965) observed that differences in mouth gape existed between different races of
herring and concluded that these differences could be significant in early survival. The
same may be true for perch. Researchers have not utilized a single broodstock source in
the various experiments. Some sources of broodstock may be better suited for intensive
culture conditions or may have offspring that are larger, have larger mouth gapes, or both.
Researchers in the NCR have conducted evaluations of offspring performance taken from
different wild stocks as an initial step for a broodstock development program (N CRAC
1997). The research indicated that variations occurred between sources of broodstock
and identified geographically disparate populations but did not identify a single source
that performed better than all others (NCRAC 1997). Variations within strains of
broodstock have been identified, including fast and slow growth rates (Moav 1979;
Kamler 1992; review Chapter 2, this thesis). When the gametes from a slow growing

female perch and a fast growing male perch were crossed, the combination resulted in

159

larger larvae (review Chapter 2, this thesis). All of these variables may contribute to
differences observed when comparing results between studies.

Two other factors that might have contributed to the high mortality rates observed
in the study were the behavior of the larvae and the design of the culture tanks. These
two factors combined together may have caused higher mortalities than might be
expected. The culture tank included a conical bottom design to collect excess feed and
fecal material. This design was expected to help facilitate waste removal. However,
when the larvae were not swimming, they sank to the bottom and displayed a resting
behavior. Approximately 50 % of the larvae remained on the bottom of the culture tanks.
With the design of this culture system, the larvae were concentrated into a small area and
were covered with uneaten Arternia nauplii or decapsulated cysts. This sinking motion
has previously been observed in larval walleyes (Li and Mathias 1982) and perch (Ross et I
al. 1977) during intensive culture conditions. The reasons for remaining on the bottom of
the culture tanks are unclear. This behavior may have been the result of non-inflated gas
bladders. GBI averaged approximately 13 % in two of the diet strategy culture tanks and
0 % in the other two by day 6 (Table 6). Although, the GBI rates increased over time, the
combination of a resting behavior and uneaten feed collection may have led to early
mortalities.

While non-inflated gas bladders may help to explain the swimming behavior
exhibited by the perch larvae, there was no explanation of larvae remaining on the bottom
for extended periods of time while being covered by uneaten food. The water
temperature may have had an effect on the swimming ability of the larvae. Temperature

is an important factor. It directly affects organogenesis and metabolism. Teleost larvae

160

are unable to compensate for temperature induced metabolic alterations (Wang et al.
1987). Lower water temperatures may have reduced the ability to swim. The average

temperature was 13.8 °C through the first nine days of the experiment. This average

temperature was higher than the 13 °C used by Houde (1969) to observe swimming
abilities of larval perch and walleye. The water‘temperatures in this study (12.0 to 15.0
°C) did not reach the optimal temperature range for larval culture (20 to 23.9 °C)
determined by Hokanson (1977). An additional factor that might have had an affect on
the swimming behavior was the flow rate. Summerfelt (1996) determined the flow rate
used in this study as the optimal flow rate for intensive culture conditions of larval
walleye. Although both perch and walleye are poor swimmers as newly hatched larvae,
perch have a better relative swimming ability than larval walleye as the yolk is absorbed
(Houde 1969). The flow rate used in these experiments should have been within an
optimal flow rate range.

The perch larvae exhibited another behavior that may have contributed to the high
mortality rates observed in this study. Many of the remaining larvae that did not exhibit
the sinking or resting behavior had a tendency to stay very near to the sides of the culture
tanks. Often, the larvae appeared to rest with their heads touching the side of the tank.
This behavior has also been observed in larval walleyes during intensive culture
conditions (Bristow and Summerfelt 1994; Summerfelt 1996). Researchers have
suggested that this behavior (also known as clinging behavior) may be a result of stress
(Kise and Meade 1986) or may be an attraction to light reflected from the sides of the
tanks (Summerfelt 1996). To help reduce this behavior, researchers have investigated the

use of turbid water (Bristow and Summerfelt 1994; Bristow et al. 1996) and higher light

161

intensities that ranged from 100 — 200 lux (Colesante 1996). For the second feeding trial,
a higher light intensity was used (estimated to be 400 lux). A previous study used a high
light level that supported a mean survival rate of 44 % for perch larvae fed Arternia
nauplii as the first food (Hinshaw 1985). The higher light levels (95 lux) used in the
second feeding trial appeared to eliminate the clinging behavior.

After analyzing the results of the first feeding trial, the design of the culture
system and feeding strategy changed. A false, flat bottom was added to the culture tanks
(similar to Kim et al. 1993 ). This change offered a larger surface area for any larvae
resting on the bottom. Based on research conducted by Bay Port researchers (review
Chapter 1, this thesis), the decapsulated Anemia cysts were replaced with newly hatched
Arternia nauplii as the first food. The GSL strain was offered again as the control, but the
newly hatched nauplii from the SF B strain was introduced. While the maximum
appendage width of the SFB strain was small enough for first feeding perch larvae to eat,
observations were unable to be made since the majority of the larvae did not survive past
day 3.

The reasons for the sudden mortalities remain unclear. Afier stocking the larvae
into the culture tanks, the larvae were observed to be actively swimming and possibly
inflating the gas bladders. Many of the larvae were swimming at the surface, which
caused little ripples to form on the surface. It appeared that the larvae might have been
trying to inflate the gas bladders. By day 3, approximately 90 % of larvae were inactive
on the tank bottom and by day 4, mortality rates were as high as 99 %. It might be
possible that water temperature may have played a significant role. In the first

experiment, the initial water temperature was 11 °C and was warmed gradually over a

162

period of 15 days. In the second experiment, the initial water temperature was 16 °C.
Since the larvae were incubated in 11 °C well water, the higher culture temperature may
have been too high for the larvae to survive. Higher water temperature could have
increased larval metabolism to the point where the endogenous energy reserves were
depleted at a faster rate. At higher temperatures, larvae must be fed shortly after the
swim-up stage (Hokanson and Kleiner 1974). However, Hokanson and Kleiner (1974)
reported a median period of 9 days between swim-up and mortality of unfed larvae at
19.8 °C, and larvae hatched at lower temperatures had an increased chance of survival
when transferred to higher temperatures. It remains unclear if temperature played a
significant role in the mortalities observed in this study.

The sudden mortality rates observed in this study may have been the result of
handling stress induced during transportation. The trip fiom the Bay Port facility to the
MSU aquaculture facility was approximately 2 hours. Steps were taken to minimize
stress and damage to the larvae. These steps included packing larvae into coolers and
five-gallon containers to minimize shifiing and covering the containers to block direct
sunlight. Handling stress also seems unlikely since Bay Port researchers transport larvae
at higher densities and over longer distances with high success rates.

While this study was unable to culture a significant number of larvae to an
advanced fingerling stage, several lessons were learned. First, perch larvae can be raised
successfully on Arternia as the initial food (Mansueti 1965; Hale and Carlson 1972;
Hinshaw 1985; Starr, personal communication, 1996). The small size of the mouth gape
and the large size of the diet provided restricted larval perch in this study. A small

nauplii strain of Arternia is necessary for acceptance. Second, perch larvae can be raised

163

on decapsulated cysts. While this study produced limited results, refining a feed
dispensing system might increase the acceptance of decapsulated cysts. Finally, the
selection of broodstock is an important step for successful culture of larval perch.
Through genetic selection, important culture characteristics can be enhanced. Freshwater
salrnonids have gone through the process of selection, which has resulted in higher
fecundity rates larger egg sizes, higher hatching percentages and higher growth rates
(Moav 1979). Controlled selection experiments should be designed to begin the process

of developing perch stocks with traits favorable to intensive culture.

1)

2)

3)

4)

5)

164

SUMMARY AND CONCLUSIONS

Small strain Arternia nauplii, such as the SFB strain, should be used for first feeding
larval perch.

Decapsulated Arternia cysts were used with some success as the first food for larval
perch. Hydrated, decapsulated cysts can be used with limited aeration, but
dehydrated cysts would be better, since they .tend to float on the surface and sink over
a longer period of time. i i

The flat bottom added to the culture tanks. helped to spread out the larvae when they
exhibited the resting behavior.

Developing intensive culture techniques for perch larvae increases the number of
options for culturists. With out-of-season spawning techniques and intensive culture
techniques, a supply of perch could be made available year round. Intensive culture
of larval perch would reduce the amount of variablity observed in pond culture by
controlling the environment and food.

A selection program, which identifies perch that grow well in intensive culture
conditions, should be started to aid intensive larval culture techniques. This could be
started by using perch raised entirely in intensive culture conditions as the broodstock

for future cultures.

APPENDICES

APPENDIX A: Arternia hatching medium using artificial seawater

Table VII

Artificial seawater used in Arternia hatching and culturing

Hatching mediuma Culturing mediuma

 

Evaporated sea salt (N aCl) 5.0 31.08
MgSO4b 1.3 7.74
MgCl2 1.0 6.09
CaClz 0.3 1.53
KCI" 0.2 ' 0.97
Ncho3" 2.0 2.00

 

 

" In gram salts (technical grade) per liter tap water.
5 Should be dissolved separately in warm tap water before addition to the solution of other salts.

165

 

APPENDIX B: Analytical methods used for determining hatching percent

APPENDIX 1

Analytical methodology for hatching characterization of Artemia cysts.

HATCHING PERCENTAGE — Method A

A.l.

A2.

A3.

A.4.

A.5.

A.6.

A.7.

Incubate approx. 250 mg of cysts in approx. 100 ml of 25 ppt seawater, under
continuous illumination (1,000 lux) at 25 °C in a conical tube (preferentially) or in a
graduated cylinder (see schematic drawings under the hatching efficiency section,
same appendix); provide aeration from bottom to keep all cysts in suspension
(aeration should be low enough to prevent foaming).

After 1 hour, take 10 subsamples (i=10) of approx. 100 cysts each using an
automatic micropipet (preferentially) or a 0.5 ml glass pipette with the tip cut away
if the opening is too small for the cysts/nauplii.

Pipet cysts for each subsample onto a separate paper filter.

Count cysts on each paper filter using a dissection microscope (ci); calculate the
mean value (c).

Rinse cysts from paper filters into individual glass petridishes or small plastic tubes
(5 ml); add 35 ppt pre-aerated seawater and incubate under continuous illumination
(> 1000 lux) at 25 °C.

After 48 hours, fixate nauplii by adding a few drops of lugol’s solution.
Preparation of lugol’s solution:

- dissolve 10g K1 and 5 g 12 in 20 ml distilled water;

- dissolve 5g Na-acetate in 50 ml distilled water;

- mix the 2 solutions and store in a dark bottle in refrigerator.
The pharmaceutical disinfective iodine tincture can be used as an alternative for
the lugol’s solution.

Count nauplii under dissection microscope (ni) and calculate the mean value (n).
Practical method for nauplii counting:

- construct small filter nets made of PVC-tube (8 cm diameter, 1-2 cm height)
which 100um nylon filter are glued;

- drain contents of hatching tube or petridish over filter; rinse tube or petridish
and empty contents over filter;

- place filter in petridish filled with water; shaking and moving filter in and out
of water will ensure more even distribution of the nauplii over the filter as to
facilitate counting;

- remove filter fi'om petridish, dry filter from underneath using a towel or filter

paper;

166

 

167

- place filter under microscope for counting; eventually place filter in plastic
petridish which has been graduated as to facilitate counting

A.8. Hatching percentage H% = n x 100

c
A.9. Practical example

Number of cysts counted on

 

 

Sample filter (Ci) Number of nauplii (ni) . Hatching %
l l 12 76 67.9
2 89 72 80.9
3 102 75 73.5
4 110 79 71.5
5 99 69 69.7
6 108 70 64.8
7 103 76 73.8
8 97 69 71.1
9 98 65 66.3
10 115 80 69.6
n 70.9
Standard deviation (s.d.) 4.5
A.10. Remarks

- this method can only be used when the cysts have been properly processed
(i.e. not containing empty cyst shells).

- correct sampling and counting is indicated by a standard deviation of less than
5%.

APPENDIX C: Results of the statistical analysis conducted for objectives 1 and 2

 

 

 

 

168

 

 

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APPENDIX D: Calculation of heritability (hz) estimates

Appendix D. Calculation of heritability (hz) estimates using the following model
(Falconer 1989).

h2=VG/Vp

where, VG was the genotypic variance component;
V}: was the phenotypic variance component;
VG = 2 * (VSIRE + VDAM);
VP = VSIRE + VDAM + VERROR;
where, Vsmfi was the male contribution to the additive variance
component;
VDAM was the female contribution to the additive variance component;
VERROR was the remaining unexplained contribution to the additive variance

component.

169

APPENDIX E: Calculation of dominance (d2) estimates

Appendix E. Calculation of dominance (d2) estimates using the following model
(Falconer 1989).

d2=VI/Vp

where, V; was the interaction variance component between sire and dam;
V1: VSIRE‘DAM " 4;
VP = VSIRE + VDAM + VSIRE‘DAM + VERROR;
where, VsmgoDAM was the sire by dam interaction of the non-additive
variance component;
V3135 was the male contribution to the non-additive variance component;
VDAM was the female contribution to the non-additive variance
component;
VERROR was the remaining unexplained contribution to the non-additive

variance component.

170

APPENDIX F: Results of the statistical analysis conducted for female spawner total
length, weight, age, egg ribbon weight, total fecundity, and number of eggs per gram
of eggs (objectives 3, 4 and 5)

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171

APPENDIX G: Results of Two factor Analysis of Variance (AN OVA) testing for
female spawner total length, weight, egg ribbon weight, total fecundity, and
number of eggs per gram of eggs (objective 3)

 

 

172

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APPENDIX H: Results of simple linear regression analysis testing of female
spawner size to egg ribbon weight, total fecundity, and number of eggs per
gram of eggs (objective 3)

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Osm.03 Egag... n x 0.023

 

 

 

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173

APPENDIX 1: Results of factorial analysis testing for the determination of
significant differences between areas of the egg ribbon for number of eggs
per gram of eggs, chorion shell diameter, and yolk diameter

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174

APPENDIX J: Results of simple linear regression analysis of female spawner TL
and weight on chorion shell and egg yolk diameters

 

O..W.03 B2302.|Vwo. n x 0.3.3

 

 

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175

APPENDIX K: Results of One-way Analysis of Variance (ANOVA) testing for
larval total length, larval mouth gape width, and larval mouth gape height for
each day sampled post-fertilization

 

 

 

 

 

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176

APPENDIX L: Results of simple linear regression analysis of female spawner total
length and weight on larval total length, mouth gape width, and mouth gape height

 

 

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177

APPENDIX M: Results of simple linear regression analysis of larval total length on
month gape width and height across all female spawner size groups

 

 

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Go Cu 0%. "x95 3

one... 8...... o + ... 38...... + 32...... u . o ....%....W.. ".50....588 “20.

0.0.6... 03.0.... 02.02%. .002).

 

098% 0... 8:30...
0.080.. ..0 $0.00 23.0.. ....0 523 3% 5.5... ..o 59.0. .0.0. .0>..0. ..o 0.3.0.... 8.0.0.3. .00.... 0.9.... ..o 3.30M. .3. 5000......

178

Appendix N. Results of linear regression analysis of larval total length on mouth
gape width and height for each female spawner size groups (SG)

 

 

.A...m=0. .0.0. .0>.0.. u x 0.2.3

 

 

 

 

 

 

 

.8... . 3...... o + .... .28.... + ... .8... u . o + .... .. + ... n .....2. 5.52 ... o.
8...... ....8... o + ... .88.... + 8.8.... n . ......Mowfiwwmafiuflwwofioz .... c.
8...... 8...... o + .... .88.... + .....8... n . ....me “wowmmamw “H.592 .... o.
8.... 8...... o + .... .88.... + 8.8... n . ..ummmwflow .mfiwuwhflh .... o.
8...... 8...... o + .... .28.... + .....8... - u .....ww “wannahm wwmwofioz ... o.
...... 8...... o + .... .....8... + 88...... u . .....flfiflm%fl.%fioz .... 0.
8...... 8...... o + .... .88.... + 38...... n . ......wm. “weafltmw .ugwmmfioz ... c.
8.... 8...... o + .... 8.8.... + «8.8... n . ..fimdfiwfifiwwnfiwWfiwoz ... o.
...... 8...... o + .... ......o... + .388... u . ....me flammamw www.mfioz .... o.
3...... 8...... o + .... .. 8...... + 8.3.... u . ..fim°w.mww%.fi%ufl%.fiwoz ... o.
....8... 8...... o + .... .38.... + .....8... u .. ......mw. flownattmfiu manage... .... o.
8...... 8...... o + .... 8.8.... + 888.. u . ..fiwowfiwwmufiuufihwmawoz .... 0.
0.0.6.-. 020?... 0050......— .0..o.>. 9.0.0
.69 .....oE

0... .0030... 0.080.. ..000 .0.. 23.0.. ....0 50.3 2.0m 5.5... ..o 5mg. .0.... .0>.0. ..o ......000 so...0.w0. .00.... ..o ......3. .Z 5000......

179

Appendix 0. Results of Analysis of Covariance (AN COVA) testing larval total
length on mouth gape width and height between each spawner size group

 

 

.A5m00. .0.0. .0>.0d n x 0.0..3

 

...... 8...... o + 0.. .. + ... u 0...... 5.52
A5w00. .0.0. .0>.0.. u 0. 0.2.3
. . .
0.3 o .80 o 0 + A... .0 + on n 50.3 5.5.).
0.00.... 00.0>-.. .003).

 

.....o.m 0... .0030...
..000 0003.0. 03.0.. 000 50.3 000m 5000. 0.. 5w00. .0.0. .0>.0. $5.0. 3.50023 0000.350 .0 ......05. .0 3.0.3. .0 0.00003.

180

 

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