INDUCED SPAWNING OF SPOTTED GAR Lepisosteus oculatus, THE EFFECTS
OF THYROID HORMONES ON SPOTTED GAR EGGS AND LARVAE, AND
THE EFFECTS OF LIVE FEEDS ON CANNIBALISM RATES IN ALLIGATOR
GAR Atractosteus spatula LARVAE
A Thesis
Submitted to the Graduate Faculty
of Nicholls State University
in Partial Fulfillment
of the requirements for the Degree
Master of Science in Marine and Environmental Biology
By
Kent Tyler Bollfrass
B.S. Fisheries and Water Resources, University of Wisconsin- Stevens Point, 2007
Fall 2012
CERTIFICATE
This is to certify that the thesis entitled “Induced spawning of spotted gar
Lepisostues oculatus, the effects of thyroid hormones on spotted gar eggs and larvae, and
the effects of live feeds on cannibalism rates in alligator gar Atractosteus spatula larvae”
submitted for the award of Master of Science to Nicholls State University is a record of
authentic, original research conducted by Kent Bollfrass under our supervision and
guidance and that no part of this thesis has been submitted for the award of any other
degree, diploma, fellowship, or other similar titles.
APPROVED:
SIGNATURE:
DATE:
Quenton Fontenot Ph.D.
Associate Professor of
Biological Sciences
Committee Chair
a
a
Allyse Ferrara Ph.D.
Associate Professor of
Biological Sciences
a
a
Gary Lafleur Ph.D.
Associate Professor of
Biological Sciences
a
a
Christopher Green Ph.D.
Assistant Professor of
Aquaculture
Louisiana State University
a
a
i
ABSTRACT
Larval rearing is a limiting factor in gar production due to cannibalism during the
larval and early juvenile life stages. Providing adequate live feeds and increasing early
growth rates to limit the duration of cannibalism have been a priority to improve gar
aquaculture. Gar production can be used for current and future gar conservation
programs, food fish production, and scientific research. This thesis contains two
experiments focusing on increasing larval and juvenile growth and developmental rates
and increase survival. The first experiment demonstrated the use of an intramuscular
injection of thyroid hormones, thyroid stimulating hormone (TSH), thyroxine (T4),
triiodothyronine (T3), in spotted gar Lepisosteus oculatus broodstock to increase egg and
larval thyroid hormone levels potentially increasing growth and development rates and
improve survival. The first experiment also documented the success of the spotted gar
spawning protocol previously developed in the Bayousphere Research Laboratory at
Nicholls State University. Eggs were removed from each tank within 72 hours of
spawning, measured (mm) and thyroid hormones levels were quantified (N=3). Embryos
were measured (N=3) for egg (mm) and yolk size (mm) and yolk volume (µL) was
calculated using yolk diameter measurements. Larvae were tested (N=3) for hatch rate
(%) and survival (%). Larvae were measured for total length (mm), snout length (mm),
and snout proportion to body length (%). T4 levels (ng/individual ± standard error) were
significantly higher in the fertilized embryos of the T4 (6.0 ± 1.78) and T3 (5.1 ± 0.35)
treatments compared to the control (0.9 ± 0.24) treatment. T3 levels (ng/ individual ±
standard error) were significantly higher in the fertilized embryos of the T3 (4.2 ± 1.54)
treatment compared to the control (0.5 ± 0.01) treatment. T3 levels (ng/ individual ±
ii
standard error) were significantly higher in the 0 DPH larvae of the T4 (5.0 ± 1.54)
treatment compared to the control (0.8 ± 0.12) treatment. Salmon gonadotropin releasing
hormone analog 20 µg/mL and domperidone 10 mg/mL (Ovaprim®) consistently
induced spawning of wild caught spotted gar. Although thyroid hormones injected into
the broodstock were absorbed by the developing embryos, there was no significant effect
on the performance of eggs or larvae. The second experiment used an industry standard
live feed, enriched Artemia salina, and an innovative live feed, newly hatched gulf
killifish Fundulus grandis, to improve larval alligator gar Atractosteus spatula growth
and survival. Alligator gar total length (mm) and weight (mg) were measured on 5, 12,
and 20 DPH and survival (%), natural mortality (%), and cannibalism (%) were
calculated on 20 DPH. Growth was not different among all treatments at 20 DPH.
Survival was higher (F= 10.57 2,6 ; P= 0.0108) in the killifish treatment (67.3 ± 3.62%)
than the control (31.4 ± 7.85%). Because natural mortality was similar among all
treatments, higher survival was due to less cannibalism (F=9.21 2,6 ; P=0.0148) in the
killifish treatment (21.7 ± 1.65%) than the control (52.7 ± 7.48%). Although alligator gar
cannibalism was reduced using killifish as a live feed source, producing enough killifish
will likely be the limiting factor for this rearing method. Because thyroid hormones did
not increase spotted gar egg and larval performance, I do not recommend using
intramuscular injection of thyroid hormones during the production of spotted gar.
Because of higher alligator gar larval survival rates, I do recommend using newly hatched
gulf killifish when rearing larval and early juvenile alligator gar.
iii
ACKNOWLEDGEMENTS
To my family, Karl, Sharon, and Olivia Bollfrass, I would like to say thank you
for their unending support of what I choose to do in life. I could not have even considered
starting this venture without your help. To my committee members, Quenton, Allyse,
Gary, and Chris, I would like to say thank you for your guidance and unwavering
commitment to me and my project. The knowledge and skills you have taught me are
much appreciated and will be put to good use. I would like to extend a special thanks to
Gary for introducing me to the cultural landscape of Southern Louisiana, and likewise to
Quenton and Allyse for showing me the beauty of one of the world’s great cities, New
Orleans. To my fellow graduate students that were, for better or worse, forced to be in the
same social circle as me I would like to thank you for providing me with friendship and
help with my project when I needed it. Without your help in the field, lab, and downtown
Thibodaux my project and time at Nicholls State University would not have been a
success. A special thank you to Ricky Campbell at the Private John Allen Fish Hatchery,
Tupelo, MS for providing me with alligator gar larvae when my other attempts to
produce them had failed. Finally, I would like to thank Nicholls State University for
funding me and my project during my time as a student.
iv
TABLE OF CONTENTS
Certificate…………………………………………………………………………………..i
Abstract……………………………………………………………………………………ii
Acknowledgements……………………………………………………………………… iv
Table of Contents………………………………………………………………………….v
List of Figures………………………………………………...………………………….vii
List of Tables……………………………………………………………………………..ix
List of Abbreviations...........................................................................................................x
Introduction………………………………………………………………………………..1
Chapter One……………………………………………………………………………….5
Abstract……………………………………………………………………………5
Introduction………………………………………………………………………..7
Methods………………………………………………………………………......11
Results……………………………………………………………………………18
Discussion………………………………………………………………………..30
Chapter Two……………………………………………………………………………...34
Abstract…………………………………………………………………………..34
Introduction………………………………………………………………………36
Methods…………………………………………………………………………..40
Results……………………………………………………………………………47
Discussion………………………………………………………………………..57
Conclusion……………………………………………………………………………….62
v
Recommendations………………………………………………………………………..64
Works Cited……………………………………………………………………………...61
Appendices……………………………………………………………………………….82
Biographical Sketch……………………………………………………………………...99
Curriculum Vitae……………………………………………………………………….100
vi
Figure 1:
LIST OF FIGURES
Daily mean cumulative survival (± SE) of larvae from control (black line),
TSH (dotted line), T4 (dashed line), and T3 (dotted and dashed line)
treatments spawned from wild caught spotted gar injected with DMSO
(control), TSH, T4, or T3 and Ovaprim®…………….…………..……...23
Figure 2:
Mean total length (mm ± SE) of larvae from control (black line), TSH
(dotted line), T4 (dashed line), and T3 (dotted and dashed line) treatments
spawned from wild caught spotted gar injected with DMSO (control),
TSH, T4, or T3 and Ovaprim®……………………..……………………24
Figure 3:
Mean snout length (mm ± SE) of larvae from control (black line), TSH
(dotted line), T4 (dashed line), and T3 (dotted and dashed line) treatments
spawned from wild caught spotted gar injected with DMSO (control),
TSH, T4, or T3 and Ovaprim®…………………………………………..25
Figure 4:
Mean snout length proportion of body length (%; ± SE)of larvae from
control (black line), TSH (dotted line), T4 (dashed line), and T3 (dotted
and dashed line) treatments spawned from wild caught spotted gar injected
with DMSO (control), TSH, T4, or T3 and Ovaprim®.............................26
Figure 5:
Mean (± SE) T4 levels (ng/ individual)of eggs and larvae from control
(black line), TSH (dotted line), T4 (dashed line), and T3 (dotted and
dashed line) treatments spawned from wild caught spotted gar injected
with DMSO (control), TSH, T4, or T3 and Ovaprim®...……….……….28
Figure 6:
Mean (± SE) T3 levels (ng/ individual) of eggs and larvae from control
(black line), TSH (dotted line), T4 (dashed line), and T3 (dotted and
dashed line) treatments spawned from wild caught spotted gar injected
with DMSO (control), TSH, T4, or T3 and Ovaprim®……………..…...29
Figure 7:
Mean (± SE) total length (mm) of alligator gar Atractosteus spatula larvae
fed a dry diet supplemented with either 1st instar Artemia salina (black
line), enriched 2nd instar enriched Artemia salina (dashed black line), or
Fundulus grandis larvae (gray line) until 12 days post hatch (DPH). All
treatments were fed only dry diet between 12 and 20 DPH. ...……..…...48
Figure 8:
Mean (± SE) coefficient of variation for log total length of alligator gar
Atractosteus spatula larvae at 12 days post hatch (DPH) and 20 DPH fed a
dry diet supplemented with either 1st instar Artemia salina (black bars),
enriched 2nd instar enriched Artemia salina (white bars), or Fundulus
grandis larvae (gray bars) until 12 DPH…………………….……...……49
vii
Figure 9:
Mean (± SE) wet weight (mg) of alligator gar Atractosteus spatula larvae
fed a dry diet supplemented with either 1st instar Artemia salina (black
line), enriched 2nd instar enriched Artemia salina (dashed black line), or
Fundulus grandis larvae (gray line) until 12 days post hatch (DPH)……50
Figure 10:
Mean (± SE) coefficient of variation for log wet weight of alligator gar
Atractosteus spatula larvae at 12 days post hatch (DPH) and 20 DPH fed a
dry diet supplemented with either 1st instar Artemia salina (black bars),
enriched 2nd instar enriched Artemia salina (white bars), or Fundulus
grandis larvae (gray bars) until 12 DPH…………………….…………...51
Figure 11:
Mean (± SE) total biomass (g) of alligator gar Atractosteus spatula larvae
at 20 DPH fed a dry diet supplemented with either 1st instar Artemia
salina, enriched 2nd instar enriched Artemia salina, or Fundulus grandis
larvae until 12 days post hatch (DPH). All treatments were fed only dry
diet between 12 and 20 DPH…………………………………………….52
Figure 12:
Mean (± SE) total survival (%) of alligator gar Atractosteus spatula larvae
at 20 days post hatch (DPH) fed a dry diet supplemented with either 1st
instar Artemia salina, enriched 2nd instar enriched Artemia salina , or
Fundulus grandis larvae until 12 DPH. All treatments were fed only dry
diet between 12 and 20 DPH…………………..…..…………………….53
Figure 13:
Mean (± SE) natural mortality (%) of alligator gar Atractosteus spatula
larvae at 20 days post hatch (DPH) fed a dry diet supplemented with either
1st instar Artemia salina, enriched 2nd instar enriched Artemia salina, or
Fundulus grandis larvae until 12 DPH………………….………...……..55
Figure 14:
Mean (± SE) cannibalism (%) of alligator gar Atractosteus spatula larvae
at 20 days post hatch (DPH) fed a dry diet supplemented with either 1st
instar Artemia salina, enriched 2nd instar enriched Artemia salina , or
Fundulus grandis larvae until 12 DPH…...………..………………….....56
viii
Table 1:
LIST OF TABLES
Mean (±SE) temperature (°C) and range and photoperiod (hours of
daylight; ± SE) of spawning tanks for five spotted gar spawning trials in
2011.……………………………………………………………………...13
Table 2:
Spawning attempts, number of successful spawns, and % success of wild
caught spotted gar broodstock injected with Ovaprim® to induce
spawning....................................................................................................19
Table 3:
Mean (± SE) total length (mm), weight (kg), and prepelvic girth (mm) for
wild caught spotted gar broodstock from spawning trials in 2011………20
Table 4:
Combined mean (± SE) egg and yolk diameter (mm), yolk volume (µL),
hatch rate and survival to first feeding (%), days to yolk sac absorption,
days to 50% mortality, days to 100% mortality, total length at hatch (mm),
wet and dry weight (mg), and condition factor of larvae spawned from of
wild caught spotted gar injected with DMSO (control), TSH, T4, or T3
and Ovaprim®………..….………………………………………………21
Table 5:
Particle size (mm) and protein and lipid (%) content for each type of feed
given to larval alligator gar for each period of days post hatch………….43
Table 6:
Mean (±SE) for each water quality variable for each recirculating system
used to rear alligator gar larvae fed a dry diet supplemented with either 1st
instar Artemia salina, enriched 2nd instar enriched Artemia salina , or
Fundulus grandis larvae until 12 DPH.………………………...…..……45
ix
LIST OF ABBREVIATIONS
DMSO
Dimethyl Sulfoxide
DPH
Days post hatch
DPS
Days post spawn
GnRHa
Gonadotropin releasing hormone analog
Nicholls
Nicholls State University
T3
Triiodothyronine
T4
Thyroxine
TH
Thyroid hormones
TSH
Thyroid stimulating hormone
x
INTRODUCTION
Global aquaculture production has reached 60 billion tons of seafood worth $119
billion (US) and now most commercial fisheries are either fully exploited (57%) or over
exploited (29.9%) and cannot support expanded harvest (Food and Agriculture
Organization 2012). Considering that wild harvest is nearly maximized and the demand
for seafood will increase as the global human population increases, aquaculture
production may need to fill the gap in demand (Diana 2009; Food and Agriculture
Organization 2012). Aquaculture may also have positive impacts on wild fish populations
when used as a management tool decrease harvest pressure or to reintroduce or replenish
depleted stocks (Diana 2009). Replenishment and stock enhancement programs can
increase population size by directly placing reared individuals into habitats where
populations have declined (Bell et al. 2008). Aquaculture is a valuable tool that can be
used to increase wild fish populations (Bartley and Bell 2008; Okuzawa et al. 2008).
Fish populations are declining worldwide and nearly one out of four fisheries
collapsed between 1950–2000 (Mullon et al. 2005) because of habitat loss (Aarts et al.
2003; Hughes 2002), habitat degradation (Whitfield and Cowley 2010), or pressure from
recreational and commercial fisheries (Food and Agriculture Organization 2012). Many
spawning habitats have been degraded rendering spawning grounds unsuitable to support
eggs and larvae, which limits recruitment (Seaman 2007). Alligator gar Atractosteus
spatula spawning habitat, well vegetated shallow water (Suttkus 1963), has been
destroyed, modified, or made inaccessible by hydrologic modifications. Alligator gar
have faced extermination efforts by regulatory agencies that perceived all gar species as
nuisances that consumed game species and damaged fishing equipment (Burr 1931;
1
Scarnecchia 1992). Gar species are no longer exterminated by agencies and eradication
efforts have ceased (Scarnecchia 1992). are now a sought-after species by recreational
and commercial anglers for sport and consumption (Garcia de Leon 2001). Habitat loss,
habitat degradation, and fishing pressure have caused alligator gar populations to parallel
the worldwide fisheries trend of decline in abundance and species range (O’Connell et al.
2007).
To be considered suitable for aquaculture production, fish species should be able
to be reared during every stage of the life cycle, produce viable embryos and larvae in
culture systems, tolerate high densities and poor water quality conditions, achieve desired
size quickly, and be in demand by markets or conservation agencies to be considered
suitable for aquaculture production (Webber and Riordan 1976). Rearing techniques must
be specifically adapted to individual species based on life history traits and other special
requirements to maximize production efficiency. Gar species are potential candidates for
aquaculture because they can complete their life cycle in captivity (Aguilera et al. 2002;
Mendoza et al. 2008a), can spawn or be induced to spawn in culture systems (Aguilera et
al. 2002; Mendoza et al. 2008a; Mendoza et al.2008b), have fast growth rates up to 5.06
mm/day (Aguilera et al. 2002), and tolerate poor water quality (Boudreaux et al. 2007).
However, there are still limits to gar culture including high cannibalism rates during the
early juvenile period (Mendoza et al. 2002, Mendoza et al. 2008a) and limited live feeds
availability, which exacerbates cannibalism (Mendoza et al. 2008a; Mendoza et al..
2008b). Aquaculture techniques used to produce gar to replenish wild stocks or to satisfy
a commercial market can be improved. Cannibalistic behavior is common during the
early stages of development in many cultured species, but cannibalism rates declines as
2
individuals increase in size (Hecht and Pienaar 1993). Decreasing cannibalism in gar
aquaculture is necessary for conservation programs and for developing food fish
production facilities.
Cannibalism limits larval and juvenile alligator gar production and conservation
efforts, recent research has focused on developing techniques that can decrease
cannibalism and improve early rearing (Mendoza et al. 2008a). Among the key areas of
study, the use of live feeds and thyroid hormones (TH) have been identified as potential
ways to improve larval and juvenile alligator gar rearing (Mendoza et al. 2002; Mendoza
et al. 2008a; Mendoza et al. 2008b, Clay et al. 2011). The use of live feeds can reduce
cannibalism by keeping cultured fish satiated (Hecht and Pienaar 1993; Chiu and Chang
2002). The use of thyroid hormones can increase the speed of larval development rates
(Tagawa and Hirano 1987; Ayson and Lam 1993; Brown 1997; Mendoza et al. 2002;
Kang and Chang 2004), which may shorten the duration of cannibalistic activity.
Alligator gar cannibalism ceases at total lengths greater than 104 mm (Clay 2009) and
increased growth rates may lead to a decrease in overall cannibalism rates. Spotted gar
Lepisosteus oculatus were used as a surrogate species for alligator gar in maternal TH
injection trials because spotted gar are smaller than alligator gar (Mendoza et al. 2008a),
and are easier to handle, transport, and hold in tanks for use in experiments, are relatively
more abundant than alligator gar, and are genetically similar to other gar species (Wiley
1976; Nelson 1994).
The first part of this project used spotted gar as a model species to determine if
TH injected into broodstock caused a difference in hatch rates, larval growth, larval
development, and survival. My hypothesis was that increased TH levels would lead to
3
increased growth rates, thus reducing the duration of cannibalism during the larval and
early juvenile stages.
The second part of the project used enriched 2nd instar Artemia salina and newly
hatched Gulf killifish Fundulus grandis larvae to determine if live feeds increase growth
and decrease cannibalism of larval and early juvenile alligator gar. My hypothesis was
that live feeds would increase alligator gar growth rates and improve survival by reducing
cannibalism.
4
TESTING THE EFFICACY OF INDUCED SPAWNING WITH AND WITHOUT
THYROID HORMONES IN THE SPOTTED GAR Lepisosteus oculatus
CHAPTER ONE
ABSRACT
Induced spawning of fish in the laboratory can provide larvae for experimental
studies, wild stock augmentation, or production of food fish. I am reporting on a protocol
for spawning wild-caught spotted gar Lepisosteus oculatus in the laboratory and have
used that protocol to determine if thyroid hormones injected into the broodstock result in
more robust larvae. Injection of 0.5 mL/kg of salmon gonadotropin releasing hormone
analog 20 µg/mL and domperidone 10 mg/mL (Ovaprim®) usually results in spawning
activity within 48 hours. Broodstock were also injected with thyrotropin (TSH),
thyroxine (T4), or triiodothyronine (T3) with dimethyl sulfoxide (DMSO) as a vehicle. A
control group of broodstock received only the Ovaprim® and DMSO. Trials were
conducted in 2-m diameter aerated static systems containing artificial spawning substrate.
Eggs were removed from each tank within 72 hours after spawning, measured (mm), and
thyroid hormones levels quantified. T4 levels (ng/individual ± standard error) were:
Control = 0.91 ± 0.235, TSH= 1.09 ± 0.165, T4= 5.97 ± 1.78, and T3= 5.06 ± 0.345 in
the eggs and Control= 0.73 ± 0.075, TSH= 0.97 ± 0.095, T4= 1.12 ± 0.289, and T3= 0.71
± 0.055 in the 0 days post hatch (DPH) larvae. T3 levels (ng/individual ± standard error)
were: Control= 0.51 ± 0.039, TSH= 0.80 ± 0.131, T4= 1.08 ± 0.480, and T3= 4.18 ±
1.541 in the eggs and Control= 0.82 ± 0.119, TSH= 1.02 ± 0.411, T4= 4.97 ± 1.542, and
T3= 1.14 ± 0.321 in the 0 DPH larvae. Embryos were measured (N=3) for egg (mm) and
5
yolk size (mm) and yolk volume (µL) was calculated using yolk diameter measurements.
Larvae were tested (N=3) for hatch rate (%) and survival (%). Larvae were measured for
total length (mm), snout length (mm), and snout proportion to body length (%).
Ovaprim® consistently induced spawning of wild caught spotted gar. Although thyroid
hormones injected into the broodstock was detected in the developing embryos, there was
no measureable effect on egg size, hatch rate, larval size, growth, or survival.
6
INTRODUCTION
Gars are apex predators that serve important functions in riverine and estuarine
ecosystems (O’connell et al. 2007; Mendoza et al. 2008a), are popular sport fish (Quinn
2010; Mendoza et al. 2008a), and are important food fish in some regions (Garcia de
Leon et al. 2001). Gar species have declined in abundance and range because of habitat
loss and alteration from anthropogenic hydrologic modifications (Mendoza et al. 2008a)
and aquaculture based production may be necessary for short term management of some
species (Aguilera et al. 2002). There is potential for commercialization of gar aquaculture
because they have high growth rates (Aguilera et al. 2002; Mendoza et al. 2008b) and can
tolerate poor water quality conditions by breathing atmospheric oxygen (Boudreaux et al.
2007). High rates of cannibalism limit production of juvenile gar in captivity, and
conservation efforts typically release larval alligator gar before they become cannibalistic
(Mendoza et al. 2008a; 2008b).
Producing viable embryos from captive fish can be important for species
conservation, food fish production, and scientific research. Spawning can occur in
culture settings without manipulation by the culturist (Sink et al. 2010) but induced
spawning is sometimes required to surmount the absence of natural spawning events.
Many methods are often used to induce spawning including manipulation of photoperiod
and temperature (Garcia-Lopez et al. 2009; Phelps et al. 2009; Sarkar et al. 2010),
injection of hormones (Cerqueira and Tsuzuki 2009; Phelps et al. 2009; Sink et al. 2010;
Aranda et al. 2011), strip spawning (Myers and Hershberger 1991), or adding spawning
substrate to culture tanks or pools (Clement and Stone 2010; Horne et al. 2010). The use
of salmon gonadotropin releasing hormone analog (Ovaprim®) for several gar species
7
was reviewed in Mendoza et al. (2008a). In this study, I used a spawning protocol slightly
modified from Aguillera et al (2002) and Mondoza et al (2002) that has been used at
Nicholls State University since 2003, but until now remained unpublished.
Thyrotropin releasing hormone is secreted from the hypothalamus and stimulates
the anterior pituitary gland to secrete thyrotropin (TSH), which then stimulates the
thyroid gland to secrete the thyroid hormones, thyroxine (T4) and triiodothyronine (T3;
Blanton and Specker 2007). Both T4 and T3 are biologically active in fish, but T4 has
been found to be converted into T3 by the liver due to increased biological activity (Eales
et al. 1999). Thyroid hormones are important for fish embryonic development (Tagawa et
al. 1990; Walpita et al. 2007), larval development (Tagawa and Hirano 1987; Brown
1997), metamorphosis (Power et al. 2001; Einarsdottir et al. 2006; Klaren et al. 2008),
and growth (Tagawa and Hirano 1987; Ayson and Lam 1993; Kang and Chang 2004),
with artificially increased TH levels leading to higher survival rates in cultured fish
(Brown et al. 1989; Kang and Chang 2004). The initial source of TH within the yolk sac
of larvae is maternally derived and is used until the individual is capable of producing
endogenous hormones (Kobuke et al. 1987). Thyroid hormones (TH) injected
intramuscularly into maternal broodstock have been shown to effectivly increase TH
levels in eggs and larvae of fish (Yamano 2005) and are a potential tool to produce more
larvae that have higher growth and developmental rates (Lam 1994).
Alligator gar larvae have shown a response to TH in past research. Alligator gar
larvae fed a continuous dose of TH had increased snout developmental rates compared to
larvae not exposed to TH (Mendoza et al. 2002). Alligator gar given a continuous dose of
thiourea, which inhibits thyroid function, had decreased snout development rates
8
compared to larvae not exposed to thiourea (Mendoza et al. 2002). To date, no study of
gar broodstock being injected with TH prior to spawning to test the effects on larval
growth, development, and survival have been reported. An advantage to maternal
injection of TH into broodstock is decreasing the potential safety hazard of exposing the
culturists to TH compared to continuous dosing through feeding. Treating gar with TH
can increase snout development, which may lead to higher rates of cannibalism from
larger fish with a cohort (Mendoza et al. 2008a). Cannibalism rates have reportedly
ceased when gar juveniles grow >104 mm (Clay 2009) or > 200 mm (Mendoza et al.
2008a) and limiting the duration alligator gar are cannibalistic could be done with TH,
which can influence growth (Tagawa and Hirano 1987; Ayson and Lam 1993; Kang and
Chang 2004). Spotted gar are a good species to use in maternal TH injection trials
because individuals are a smaller (Mendoza et al. 2008a) and easier to handle for use in
experiments, are relatively more abundant, easier to handle, and are genetically similar to
other gar species (Wiley 1976; Nelson 1994; Amores et al 2010).
The current chapter describes a spawning protocol using Ovaprim®, and spawning
substrate for spotted gar Lepisosteus oculatus that has been used by Nicholls State
University (Nicholls) investigators to produce viable spotted gar embryos. To take
advantage of the large number of spawns used to report on the spawning protocol, a
thyroid hormone (TH) injection experiment was conducted simultaneously using the
same fish. If an increase in TH levels increases growth and development rates so that
juveniles grow out of the cannibalistic stage more quickly, cannibalism during the culture
period may be reduced. The objectives of this study were to describe a reliable spawning
protocol to produce viable embryos from wild-caught spotted gar in a laboratory setting
9
and to determine the effects of broodstock injection of TH on eggs and larvae of spotted
gar.
10
METHODS
Broodstock collection and maintenance
Mature spotted gar (broodstock) were collected from Bayou Chevreuil
(N29°55.885’ W090°46.138’) in February through April 2011 and the Atchafalaya River
Basin (N30°19.394’ W091°32.441’) in March 2011 in southern Louisiana using
monofilament gill nets (2.5, 3.5, or 5.1 cm bar mesh). Broodstock from Bayou Chevreuil
and the Atchafalaya River Basin were transferred in 60-L containers containing 30 L of
water to two 3.6-m diameter, 9,600-L recirculating round tanks (holding tanks) at the
Nicholls State University (Nicholls) farm. Holding tanks were kept outdoors underneath
a pole barn receiving no direct sunlight but exposed to seasonal temperature and
photoperiod. Broodstock were not fed for the duration of the experiement.
Spawning
To induce spawning, broodstock were treated with an intramuscular injection of
0.5 mL of Ovaprim® (salmon gonadotropin releasing hormone analog (20 µg/mL) and
domperidone (10 mg/mL)) per kg body weight on 12 March, 19 March, 2 April, 9 April,
and 23 April, 2011. To determine the effect of injected thyroid hormones on egg size,
larval size, growth, and survival, broodstock were also intramuscularly injected with of
one of three hormones and transferred to spawning tanks. Treatment hormones were TSH
(4 IU/ kg of fish body weight), T4 (20 mg/kg body weight), or T3 (20 mg/kg body
weight) using Dimethyl Sulfoxide (DMSO; 5 mL/kg body weight) as a vehicle. Control
broodstock were injected with 0.5 mL/ kg body weight of Ovaprim® and DMSO (5 mL/
kg body weight) only.
11
Spawning tanks were 2-m diameter, 1,600 L circular tanks filled with 1,200 L of
dechlorinated municipal water. Tanks were static with mild aeration and were kept
outdoors under a pole barn receiving no direct sunlight but were exposed to seasonal
temperature and photoperiod (Table 1). Green pompoms attached to weighted 2.54 cm
diameter PVC pipes 0.5 m long were submerged as spawning substrate to imitate
submerged aquatic vegetation and covered half of the basal area of the spawning tanks.
For each treatment replicate, 12 mature spotted gar were used with a target ratio
of 3 male per female based on sexually dipmorphic snout morphology (Love 2002). Sex
of each individual was confirmed by post spawn by inspection of gonads (Ferrara and
Irwin 2001).
Egg Collection and Distribution
Eggs were collected two days after the initiation of spawning activity or when
fertilized eggs were first observed in the tanks. Pompoms with attached eggs were
removed and placed in 60-L containers with enough static water to cover all substrate and
eggs. Eggs that were not attached to substrate were removed with a fine-mesh net and
placed in the containers. Eggs from each treatment were then randomly distributed to
measurement and hatch rate test tank. Dead and unfertilized eggs were removed to limit
fungal growth and to more easily distribute fertilized eggs.
Egg measurements
To measure egg and yolk diameter (mm) and determine yolk volume for each
treatment replicate, 25 fertilized eggs were photographed next to a stage micrometer
using a digital camera and a Nikon SMZ800 stereomicroscope. Egg and yolk diameter
were measured from images of eggs using ImageTool 3.0 (University of Texas Health
12
Table 1. Mean (±SE) temperature (°C) and range and photoperiod (hours of daylight:
sunlight) of spawning tanks for five spotted gar spawning trials in 2011.
Injection date
Temp
Temp Range
Photoperiod
12 March
16.0 ± 0.51
12.0 – 19.0
11.9: 12.1
19 March
19.5 ± 0.49
17.1 – 21.6
12.1: 11.9
13
2 April
19.6 ± 1.13
15.4 – 22.6
12.6: 11.4
9 April
22.2 ± 1.03
17.9 – 27.1
12.8: 11.2
23 April
23.2 ± 0.42
21.8 – 24.1
13.2: 10.8
Science Center, San Antonio, Texas) software after calibration with the stage micrometer
and measuring the. Yolk volume was calculated using the formula for sphere volume as
Yolk Volume = (4/3) x π x (yolk diameter/2)3
Eggs were then stored in 2 mL microcentrifuge tubes and frozen at -80°C for analysis of
hormone levels.
Hatch Rate
To determine hatch rates (%) for each treatment replicate, 100 fertilized eggs
were placed in each of three 7.6-L buckets filled with 5.0 L of dechlorinated municipal
water. Buckets were placed in circulating raceways fitted with a thermostatically
controlled heater to maintain temperature at 21.7 ± 0.03 °C (mean ± SE). The number of
individually hatched eggs were recorded daily until all eggs hatched or died.
All remaining fertilized eggs from each spawn were placed in separate 90-L
rearing tanks (23.6 ± 0.36 °C) with a 270-L sump with biofiltration and allowed to hatch
for use in larval measurements, mortality test, and hormone analyses. Larvae were fed
Zeigler® AP100 (250-450µm; Ziegler Brothers Inc, Gardners, PA) floating larval fish
feed from 5 DPH (day of first feeding; Mendoza et al. 2008b) until 9 DPH, Silver Cup
Trout® (590-840µm; Skretting USA, Tooele, UT) fry artificial granulated crumble from 8
DPH until 12 DPH, and Silver Cup Trout® (830-1380µm; Skretting USA, Tooele, UT)
fry artificial granulated crumble from 11 DPH until 15 DPH.
Larval Measurements
At time of hatch, 25 larvae from each treatment replicate were removed from
rearing tanks and total length (TL; mm), snout length (SnL; mm), wet weight (WW; g),
14
and dry weight (DW; g) were measured. SnL proportion of total length (SnLP; %) was
calculates as
SnLP= (SL/TL) x 100
and condition factor (K) were calculated as
K = 10 x [wet weight / (TL)3]
TL and SL were measured for each larva by photographing larvae and a stage
micrometer using a digital camera and a Nikon SMZ800 (Nikon Instruments, Inc,
Melville, NY) stereomicroscope. Images of larvae were measured using ImageTool 3.0
software by calibrating the program with the stage micrometer and measuring larvae.
WW was measured by placing individual larvae on previously tared and labeled
20 mL disposable aluminum weighing dishes, dried with a KimWipe®, and were weighed
on an Ohaus Adventurer Pro Series (Ohaus Corporation, Parsippany, New Jersey)
balance to the nearest 0.1 mg. DW was measured by placing weighing dishes with larvae
in a 70 °C Fisher Isotemp 500 (Fisher Scientific Inc) series oven and reweighed until a
constant weight was obtained.
Mortality Test
To determine if mortality rate differed among treatments, 100 individual 0 DPH
larvae were placed in each of three 7.6-L buckets with 5.0 L of water for each treatment
replicate. Larvae were not fed and dead larvae were removed daily to determine
mortality rates at 5 DPH and at yolk sac absorption. Also, days to 50% mortality and days
to 100% mortality were determined for each replicate. Buckets were maintained in
recirculating raceways fitted with a thermostatically controlled heater to maintain a
temperature of 21.8 ± 0.05°C. From the 19 March spawn, the T4 treatment yielded only
15
enough larvae to have two replicates for the mortality trials and the 23 April spawn
yielded only enough larvae from the control treatment for one replicate for the mortality
trial. Larvae were observed daily to determine yolk sac absorption, which was
considered to be when half of remaining larvae were free swimming.
Hormone Analysis
To measure T3 and T4 concentration in larvae, thirty larvae per treatment
replicate were removed from the rearing tanks on 0, 3, 6, 9, 12, and 15 DPH. Samples
were maintained at -80°C until being freeze dried. Samples were sent to Universidad
Autonoma de Nuevo Leon in Monterrey, Mexico for thyroid hormone extraction and
analyses. Thyroid hormone extraction was done by homogenizing the caudal peduncles
of 30 larvae in 1.0 mL 95% ethanol with 6–N–propyl -2–thiouracil (extraction solution;
1mM) with a mortar and pestle. The extraction solution did not allow conversion of T4 to
T3 within the samples. Homogenate for each sample was centrifuged at 1700 G for 10
min at 4°C. Supernatant was removed from each sample and placed in a 1.5 mL
microcentrifuge tube and placed in 37°C oven overnight. Each sample was individually
reconstituted in 0.9 mL barbital buffer 0.11 M (pH 8.6) and 0.1 ml 95% ethanol. A
radioimmunoassay was done according to Evered et al (1976) on each sample to quantify
thyroid hormone (ng/individual) levels in each sample.
Statistical Analysis
All analyses were done using Statistical Analysis System (SAS), with significance
determined at the α level of 0.05. Egg diameter, yolk diameter, yolk volume, dry weight
at hatch, wet weight at hatch, condition factor at hatch, days to first feeding, days to yolk
absorption, and days to 50% survival were compared using analysis of variance
16
(ANOVA). Hatch rate was arcsine transformed and compared using ANOVA. Total
length, snout proportion, and snout proportion to body length were compared using
repeated measures ANOVA.
17
RESULTS
Spawning
Spawning success rate appeared to be lower for the control treatment than all
other treatments (Table 2). Although 14 out of 20 (70%) spawning attempts produced
viable embryos (i.e. were successful), one T4 and one T3 treatment did not produce
sufficient numbers of viable embryos for further analyses. There were at least three
successful spawns per treatment that produced adequate viable embryos for study. For
male broodstock, total length (524.0 ± 2.76; mm ± SE), weight (0.6 ±0.01; kg ± SE), and
prepelvic girth (162.2 ± 1.56; mm ± SE) did not differ significantly among treatments
(Table 3). For female broodstock, total length (604.5 ± 10.01; mm ± SE), weight (0.9
±0.46; kg ± SE), and prepelvic girth (198.6 ± 3.80; mm ± SE) did not differ significantly
among treatments.
Egg Measurements and Hatch Rates
Mean egg diameter (4.0 ± 0.02; mm ± SE) ranged from 3.9 to 4.1 mm and was
not significantly different among treatments (Table 4). Mean yolk diameter (2.6 ± 0.01;
mm ± SE) ranged from 2.6 to 2.7 mm and was not significantly different among
treatments (Table 4). Mean yolk volume (9.6 ± 0.13; µL ± SE) ranged from 9.2 to 10.0
µL and was not significantly different among treatments (Table 4). Hatch rate (78.1 ±
4.78; % ± SE) ranged from 64.8 to 84.2% and was not significantly different among
treatments (Table 4).
18
Table 2. Spawning attempts, number of successful spawns, and % success of wild caught
spotted gar broodstock injected with Ovaprim® to induce spawning. Broodstock were
also injected and DMSO (control), TSH, T4, or T3.
Attempts
Success*
% Success
Control
7
3
42.8
TSH
4
3
75
T4
4
4
100
T3
5
4
80
*One T4 and one T3 successful spawn did not produce sufficient viable eggs for further
analyses.
19
Table 3. Mean (± SE) total length (mm), weight (kg), and prepelvic girth (mm) for wild
caught spotted gar broodstock from spawning trials in 2011.
Treatment
Sex
Control
M
F
531.5 ± 1.62
603.7 ± 20.22
M
F
0.55 ± 0.027
.94 ± 0.094
M
F
163.8 ± 0.26
203.1 ± 5.95
TSH
T4
Length
515.3 ± 3.19
524.6 ± 3.12
591.6 ± 9.71 621.6 ± 38.93
Weight
0.56 ± 0.024
0.54 ± 0.018
0.88 ± 0.044
0.98 ± 0.169
Girth
158.9 ± 3.79
158.2 ± 5.96
193.3 ± 5.43 201.5 ± 15.29
20
T3
526.5 ± 14.81
583.8 ± 13.37
0.58 ± 0.019
0.83 ± 0.046
164.5 ± 5.77
193.3 ± 8.81
Table 4. Mean (± SE) egg and yolk diameter (mm), yolk volume (µL), hatch rate and
survival to first feeding (%), days to yolk sac absorption, days to 50% survival, total
length at hatch (mm), wet and dry weight (mg), and condition factor of larvae spawned
from of wild caught spotted gar injected with DMSO (control), TSH, T4, or T3 and
Ovaprim®. There were no significant differences among treatments for any variable
Variable
Egg Diameter
Yolk Diameter
Yolk Volume
Hatch Rate
Survival to 1st Feeding
Yolk Absorption
Days to 50% Survival
Wet Weight at Hatch
Dry Weight at Hatch
Condition (K) at Hatch
Control
TSH
T4
T3
4.1 ± 0.13
2.6 ± 0.03
9.3 ± 0.34
64.8 ± 6.17
100.0 ± 0
8.8 ± 0.12
19.8 ± 0.83
8.7 ± 0.56
3.8 ± 0.31
0.18 ± 0.009
3.9 ± 0.02
2.6 ± 0.09
9.8 ± 0.94
79.3 ± 7.76
97.3 ± 0.27
8.6 ± 0.36
18.8 ± 1.83
9.5 ± 0.24
3.8 ± 0.21
0.16 ± 0.015
3.9 ± 0.07
2.6 ± 0.13
9.2 ± 1.34
79.9 ± 13.73
98.3 ± 0.98
8.8 ± 0.18
19.6 ± 0.44
8.5 ± 0.80
3.7 ± 0.66
0.17 ± 0.022
4.1 ± 0.21
2.7 ± 0.07
10.0 ± 0.70
84.2 ± 10.94
99.0 ± 0
8.7 ± 0.27
17.0 ± 0.51
9.5 ± 0.64
3.7 ± 0.26
0.18 ± 0.016
21
Mortality Test
Percent survival to first feeding (98.5 ± 0.62; ± SE) ranged from 97.3 to 100 %
and was not significantly different among treatments (Table 4). Mean days to yolk sac
absorption (8.7 ± 0.14; days ± SE) ranged from 8.6 to 8.8 days and was not significantly
different among treatments (Table 4). Mean days to 50% survival (18.1 ± 0.65; days ±
SE) ranged from 17.0 to 19.8 days and was not significantly different among treatments
(Table 4). Most mortality occurred between 12 and 24 DPH and was likely due to
starvation (Figure 1).
Larval Measurements
Larval wet weight at hatch (9.1 ± 0.28; mg ± SE ) ranged from 8.5 to 9.5 mg, and
mean dry weight at hatch (3.8 ± 0.16; mg ± SE ) ranged from 3.7 to 3.8 mg and neither
was significantly different among treatments (Table 4). Mean condition factor at hatch
(0.17 ± 0.01; ± SE) ranged from 0.16 to 0.18 and was not significantly different among
treatments (Table 4). Mean total length for all treatments increased from 8.1 ± 0.09 mm
on 0 DPH to 21.9 ± 0.49 mm on 15 DPH and was not significantly different among
treatments at any sample day (Figure 2). Growth was asymptotic with minimal growth
after 9 DPH and growth rate was 1.3 ± 0.03 mm/day from 0 - 9 DPH and 0.9 ± 0.04 mm/
day from 0 - 15 DPH (Figure 2). Snout length for all treatments increased from 0.4 ±
0.01 mm to 2.78 ± 0.25 mm and was not significantly different among treatments (Figure
3). Mean snout length proportion of total body length for all treatments increased from
5.1 ± 0.11% to 12.6 ± 0.70% and was not significantly different among treatments
(Figure 4).
22
Figure 1. Daily mean cumulative survival (± SE) of larvae from control (black line),
TSH (dotted line), T4 (dashed line), and T3 (dotted and dashed line) treatments spawned
from wild caught spotted gar injected with DMSO (control), TSH, T4, or T3 and
Ovaprim®. Larvae were unfed during this trial.
23
Figure 2. Mean total length (mm ± SE) of larvae from control (black line), TSH (dotted
line), T4 (dashed line), and T3 (dotted and dashed line) treatments spawned from wild
caught spotted gar injected with DMSO (control), TSH, T4, or T3 and Ovaprim®.
24
Figure 3. Mean snout length (mm ± SE) of larvae from control (black line), TSH (dotted
line), T4 (dashed line), and T3 (dotted and dashed line) treatments spawned from wild
caught spotted gar injected with DMSO (control), TSH, T4, or T3 and Ovaprim®.
25
Figure 4. Mean snout length proportion of body length (%; ± SE)of larvae from control
(black line), TSH (dotted line), T4 (dashed line), and T3 (dotted and dashed line)
treatments spawned from wild caught spotted gar injected with DMSO (control), TSH,
T4, or T3 and Ovaprim®.
26
Hormone Analysis
Levels of T4 were higher (F= 9.4
7,14 ;
P= 0.0165) in newly spawned eggs in the
T4 and T3 treatments but were not significantly different among treatments after 3 DPH
(Figure 5). Levels of T3 were higher (F= 3.77 7,14 ; P=0.0003) in 0 DPH larvae in the T4
treatment and in spawned eggs of the T3 treatment but were not significantly different
among treatments after 3 DPH (Figure 6).
27
*
*
Figure 5. Mean (± SE) T4 levels (ng/individual)of eggs and larvae for each day post
hatch (from control (black line), TSH (dotted line), T4 (dashed line), and T3 (dotted and
dashed line) treatments spawned from wild caught spotted gar injected with DMSO
(control), TSH, T4, or T3 and Ovaprim®. Asterisks indicate T4 levels that were different
from controls.
28
*
*
Figure 6. Mean (± SE) T3 levels (ng/individual) of eggs and larvae for each day post
hatch from control (black line), TSH (dotted line), T4 (dashed line), and T3 (dotted and
dashed line) treatments spawned from wild caught spotted gar injected with DMSO
(control), TSH, T4, or T3 and Ovaprim®. Asterisks indicate different T4 levels that were
different from controls.
29
DISCUSSION
Reliable production of fish larvae is required for conservation and food
production efforts. In this study I tested whether the injection of Ovaprim® with and
without TSH, T4, and T3 led to different spawning, hatching, and growth rates. Based on
the 70% spawning success of this study, it appears that spotted gar can be reliably
produced by injecting the broodstock with GnRHa. After spawning, egg collection for
different species of fish can be accomplished by using inline egg traps in broodstock
culture systems (Phelps et al. 2009), spawning substrate (Clement and Stone 2011; Horne
et al. 2011), or strip spawning and is an important component to developing a reliable
spawning program. I did not test if the spawning substrate influenced spawning success,
but the pompoms made it easy to collect the adhesive eggs. The technique described in
this paper resulted in a relatively high hatch rate as compared to channel catfish Ictalurus
punctatus (64 %; Mitchell et al. 2009), red snapper Lutjanus campechanus (42 %; Phelps
et al. 2009) and California yellowtail Seriola lalandi (68 %; Stuart et al. 2010), was
similar to paddlefish Polyodon spathula hatch rate (79 %; Horvath et al. 2006), but not as
high as reported for California halibut Paralichthys californicus and white seabass
Atractoscion nobilis (85 % and 90 % respectively; Stuart et al. 2010). Regardless, spotted
gar can be reliably produced in an aquaculture setting.
Providing the proper feed for first feeding larvae is problematic for many fish
species and early starvation is a major source of mortality for many aquaculture species
(Rao 2003). Spotted gar can survive for up to 24 days without feeding and readily
consume artificial feed and newly hatched Artemia nauplii. I did not determine the point
of no return. After the onset of first feeding, fast growth rate is important for larval fish as
30
the number of potential predators decreases (Pepin et al. 1992; Letcher et al. 1996) and
potential prey items increases with growth (Rao 2003). Alligator gar larval growth rates
are reported to be higher (1.55 – 5.06 mm/day; Aguilera et al. 2002) than larval spotted
gar growth rates, spotted gar have potential for high growth rates and approached 1
mm/day through 15 DPH in this study. Fast growth rates contribute to spotted gar being a
good aquaculture species as mortality rates tend to decrease with increased fish size after
the larval stage (Pepin 1991; Mendoza et al. 2008a).
Artificially increased TH levels increased growth rates in larval rabbitfish Siganus
guttatus, rockfish Sebasts schlegeli, and matrinxã Brycon cephales (Ayson and Lam
1993; Kang and Chang 2004; Urbinati et al. 2008), larval development rates of many
species including zebrafish Danio rerio, Atlantic halibut Hippoglossus hippoglossus, and
Senegalese sole Solea senegalensis (Tagawa and Hirano 1987; Brown 1997; Power et al.
2001; Einarsdottir et al. 2006; Klaren et al. 2008), and larval survival in striped bass
Morone saxatilis, Japanese parrotfish Oplegnathus fasciatus, and rockfish Sebasts
schlegeli (Brown et al. 1989; El Zibdeh et al. 1996; Kang and Chang 2004). Initial TH
levels in mature oocytes are from maternal input, and are used by larvae until they are
physiologically capable of producing their own TH (Kobuke et al. 1987). Thus, TH levels
within an individual larva tend to increase with age. Injection of TH into the broodstock
resulted in elevated levels in spawned eggs and newly hatched larvae as expected
(Yomano 2005). I could not detect an effect on hatch rate, size, growth rate, or
development rate of spotted gar. TH injection was found to be inconsistent at increasing
larval performance with striped bass Morone saxatilis, goldenstriped amberjack Seriola
lalandi, and Japanese parrotfish Oplegnathus fasciatus showing increased survival and
31
red sea bream Pagrus major, rabbitfish Siganus guttatus, and Japanese whiting Sillago
japonica showing no increase in performance (Yomano 2005). Conversely, alligator gar
larvae given a continuous dose of T3 had increased snout development (Mendoza et al.
2002) indicating that a continuous dose of TH would have increased development in
spotted gar larvae.
Spotted gar embryos from the control treatment had higher levels of T3/egg than
did alligator gar embryos (0.00117 ng; Aguilera 1999). To compare TH levels in eggs of
spotted gar from the control treatment to embryos of other species reviewed in Yomano
(2005) the TH levels were standardized to ng/g of egg. Spotted gar eggs from the control
treatment had T4 levels of 27.9 ± 5.13 (ng/g ± SE) and T3 levels of 15.7 ± 0.84 (ng/g ±
SE). Levels of T4 in control spotted gar embryos were as high as any other reviewed
species (Power et al. 2001; Yomano 2005) and levels of T3 were higher than any other
reviewed species except brown trout Salmo trutta (52 ng/g; Power et al. 2001) and
chinook salmon Oncorhynchus tschawytscha (20 ng/g; Yomano 2005). High levels of
both T4 and T3 may indicate that thyroid hormones are not a limiting factor for
embryonic and larval growth and development in spotted gar resulting in no difference
among treatments. A negative feedback loop in the hypothalamic-pituitary-thyroid axis
most likely stopped production of TRH in the hypothalamus and TSH in the anterior
pituitary and was the reason increased TH levels did not persist in spotted gar larvae
(Costa-e-sousa and Hollenberg 2012). Increased levels of TH in spotted gar embryos and
were back to baseline levels between 0 and 3 DPH, which may explain why there was no
difference among treatments.
32
Temperature is the most influential variable in fish growth (Bakanov et al 1987)
and in this study may have limited the spotted gar larvae from responding to the TH.
Maximum metabolic rate decreases with decreasing temperature, which limits metabolic
scope (Claireaux et al 2000; Mallekh and Lagardere 2002). In this study spotted gar
larvae were reared at 23.6 ± 0.36°C but it is unknown what temperature spotted gar
larvae growth rates are maximized or growth is optimized. Alligator gar achieve
maximum growth rates at 31°C (Aguilera et al 2002) indicating that spotted gar may need
to be reared at higher temperatures for maximum growth rates or highest potential for
growth. A response to the TH treatments may not have been detected because the
temperature was too low, limiting metabolic scope and a potential difference among
treatments may have been nullified.
Despite increased levels of T4 and T3 in spotted gar larvae after injecting
broodstock with TH, no measured effect on egg and larval performance was found.
Therefore, injecting spotted gar broodstock with TH may not be a useful tool for
improving spotted gar larvae production in the future. There is potential for injecting
spotted gar broodstock with TH to improve spawning success in a laboratory setting.
Also, because other methods of treating larvae with TH have produced positive growth,
development, and survival results in other species, and use of these methods on spotted
gar should not be ruled out.
33
CANNIBALISM CAN BE DECREASED IN ALLIGATOR GAR Atractosteus spatula
IN AQUACULTURE SYSTEMS WHEN FED WITH LIVE FEEDS
CHAPTER TWO
ABSTRACT
Alligator gar Atractosteus spatula are apex predators, a popular sport fish, and an
important food fish species in localized markets. Unfortunately, alligator gar abundance
and range has decreased because of anthropogenic hydrologic modifications to riverine
systems, which limits recruitment. Because of the reduction in abundance and range,
many states have management plans that include stocking aquaculture produced juvenile
alligator gar. Production of alligator gar is limited by cannibalism during the larval and
early juvenile developmental stages in aquaculture systems. Therefore, reducing alligator
gar cannibalism rates has been identified as a priority to improve survival and yield of
this species in aquaculture. Alligator gar can be reared using only formulated diets, but
the greatest reduction in cannibalism rates has been accomplished when co-feeding
Artemia salina with formulated diets. The effects of co-feeding formulated diets with
three live feeds, 1st instar Artemia salina (control), enriched 2nd instar Artemia salina, and
newly hatched gulf killifish Fundulus grandis larvae were examined for alligator gar
larvae. Total length (mm) and weight (mg) were measured on 5, 12, and 20 DPH and
survival (%), natural mortality (%), and cannibalism (%) were calculated on 20 DPH.
Growth was not different among treatments at 20 DPH. Survival was greater (F=
10.57 2,6 ; P= 0.0108) in the killifish treatment (67.3 ± 3.62 %) than the control (31.4 ±
7.85 %). Natural mortality was similar among all treatments, greater survival can be
34
explained by reduced cannibalism (F=9.21 2,6 ; P=0.0148) in the killifish treatment (21.7 ±
1.65 %) as compared to the control (52.7 ± 7.48 %). Alligator gar cannibalism was
reduced using killifish as a live feed source; producing sufficient numbers of killifish to
suppress cannibalism will likely be the limiting factor for using this rearing method.
Although recent interest in killifish production as baitfish may result in partnerships
between alligator gar and killifish producers that could provide sufficient numbers of
killifish larvae for alligator gar production.
35
INTRODUCTION
Alligator gar Atractosteus spatula serve important ecological functions in riverine
and estuarine ecosystems (O’connell et al. 2007; Mendoza et al. 2008a), are popular sport
fish (Mendoza et al. 2008a), and are important food fish in some markets (Garcia de Leon
et al. 2001). Alligator gar abundance has decreased due to habitat loss and alteration due
to anthropogenic hydrologic modifications (Mendoza et al. 2002; Mendoza et al. 2008a).
To address alligator gar declines within the United States, management plans have been
developed for Alabama (Armstrong, Jr. 2007), Arkansas (Barnett et al. 2011), Illinois
(Thomas and Hilsabeck 2008), Kentucky (Oster 2010), Mississippi (Deviney and Polles
2012), Tennessee (Todd 2005), and Texas (Texas Parks and Wildlife 2012). Federal
efforts at the Warm Springs National Fish Hatchery in Warm Springs, GA, and the
Private John Allen National Fish Hatchery in Tupelo, MS produce alligator gar via
aquaculture techniques for stocking depleted habitats as a short term management
strategy (Aguilera et al. 2002). Alligator gar have also been stocked by the Mexican
Ministry of Agriculture’s Tancol Aquaculture Center in Tamaulipas Mexico since 1982
(Mendoza et al 2008b). High growth rates (Aguilera et al. 2002; Mendoza et al. 2008b)
and tolerance of water quality conditions considered poor for many species (Boudreaux et
al. 2007) make alligator gar suitable for aquaculture.
Cannibalism occurs in most fish culture systems but the rate increases as the size
distribution of larvae and young juveniles increases in heterogeneity (Hecht and Pienaar,
1993; Kestemont et al. 2003; Correa and Cerqueira, 2007) or if the larvae are not are not
fed to satiation (Hecht and Pienaar, 1993; Chiu Liao and Chang, 2002). High rates of
cannibalism limit production of larval and juvenile alligator gar in captivity, and reducing
36
cannibalism is an important step for improving alligator gar larval and juvenile survival
(Mendoza et al. 2008a; Mendoza et al. 2008b; Clay et al. 2011). Light duration and
intensity, water clarity, stocking density, and feeding frequency can also affect the rate of
cannibalism (Hecht and Pienaar 1993) but usually as a result of decreasing size
heterogeneity within the culture tanks (Baras and Jobling 2002; Correa and Cerqueira
2007; Arockiaraj and Appelbaum 2011). Reducing cannibalism has been accomplished
for some species by providing artificial refuge in culture tanks (Abdel-Tawwab et al.
2006), reducing light intensity (Qin et al. 2004), reducing daylight hours (Arockiaraj and
Appelbaum 2011), size grading (Hseu et al. 2004), feeding to satiation (Chiu and Chang
2002), improved nutrition (Abdel-Tawwab et al. 2006), and increased growth rates
reducing the duration of cannibalism (Baras et al. 2011). Although it is not possible to
completely eliminate cannibalism in culture systems, a reduction in cannibalism
improves economic efficiency and enhances the ability to meet production goals (Baras
and Jobling 2002).
Larval alligator gar growth rates are related to feed rate and may reach 5.06
mm/day TL (Mendoza et al. 2002; Mendoza et al. 2008b). Achieving maximum growth
rates may reduce cannibalism for the entire culture period (Baras et al. 2011) because as
the fish increase in size, cannibalism decreases due to a decrease in the mouth size to
body size proportion (Hecht and Pienaar 1993; Baras 1998). Alligator gar larvae and
juveniles are highly cannibalistic in culture tanks (Mendoza et al. 2008a; Mendoza et al.
2008b; Clay et al. 2011), but are non-cannibalistic once reaching 104 mm TL (Clay
2009). Therefore, maximizing growth rates has the potential to reduce overall
cannibalism during early production. Rearing alligator gar from larva to adult can be
37
accomplished by feeding only formulated dry diets (Mendoza et al. 2002; Mendoza et al.
2008a; Mendoza et al. 2008b), however co-feeding dry diets with a live feed source
improves larval alligator gar growth and reduces cannibalism (Clay et al. 2011).
Providing a larger, more nutrient rich live feed may stimulate the early feeding response
(Rao 2003) in larval alligator gar thus improving early growth rates and reducing
cannibalism. The presence of larger, more nutritious live feeds may also reduce larval
alligator gar cannibalism by providing a search image of live feeds rather than other
alligator gar (Ishii and Shimada 2010; van Leeuwen and Jansen 2010).
Artemia spp. are a common live feed for fish in the aquaculture industry
(Sorgoloos et al. 2001). Although larval alligator gar can be reared solely on dry feed
(Mendoza et al. 2008a; Mendoza et al. 2008b), growth and survival of larval alligator gar
are improved when dry feed is supplemented with newly hatched Artemia spp. for the
first seven days of feeding (Clay et al.. 2011). While newly hatched Artemia spp. may
stimulate larval alligator gar to feed, enriching Artemia spp. may provide a more nutrient
rich diet (Tuncer et al. 1993; Biswas et al. 2006; Hamre and Harboe 2008). Artemia spp.
develop through a series of stages and form mouthparts and begin filter feeding during
the 2nd instar stage (McEvoy et al. 1996; Makridis and Vadstein 1999; Gelabert
Hernandez 2001). Nutrients placed in Artemia spp. culture tanks are consumed by the
feeding Artemia spp. and are then transferred to fish that consume the enriched 2nd instar
Artemia spp. (Sargent et al. 1999a; 1999b; Tonheim et al. 2000). Also, 2nd instar Artemia
spp. are approximately 50% larger (Sorgeloos et al. 2001) than 1st instar Artemia spp.
(<500 μm total length; Vanhaecke and Sorgeloos 1980) and nutrient enrichment is a
common practice in larviculture (Dhont and Van Stappen 2003). Gulf killifish Fundulus
38
grandis are native to Louisiana estuaries and are abundant, easily collected, adapt to
culture systems well, and spawn readily in culture systems (Hsiao and Meier 1986;
Perschbacher et al. 1995; Green et al. 2010; Coulon et al. 2012). After collection,
fertilized eggs can be quantified volumetrically and air incubated so that hatching desired
numbers of individuals can be coordinated to a specific time between 8 and 24 days post
spawn (Perschbacher et al. 1995). Killifish larvae are a suitable prey size (<7.0 mm total
length (TL), <0.7 mm at vent depth; Coulon et al. 2012) for alligator gar larvae, which
are >14.2 mm long and have a mouth gape of 1.2 ± 0.12 mm at time of first feeding at 5
DPH (Aguilera et al. 2002). Gulf killifish eggs can be easily obtained and quantified,
embryos can be hatched at a desired time, and larvae are a suitable prey size, gulf killifish
may be a viable cultured live feed for larval alligator gar.
The goal of this study was to determine if supplementing a dry feed diet with
nutrient enriched 2nd instar Artemia salina or live Gulf killifish larvae increases growth
and survival of larval alligator gar compared to supplementing a dry feed diet with 1st
instar Artemia salina. Because alligator gar begin exogenous feeding 5 DPH, feeding
treatments began on 5 DPH and continued through 12 DPH and all treatments received
only dry feed from 13 DPH through 20 DPH (Clay et al 2011). The objective of this
study was to compare the size and survival of larval alligator gar among the 1st instar, 2nd
instar, and killifish treatments at 12 DPH and at 20 DPH.
39
METHODS
Live Feeds Production
Production of 1st instar Great Salt Lake Artemia Cysts Artemia salina (Inve; Salt
Lake City, Utah) was performed in cone tanks with 17.0 L of water (17.4 ± 0.43ppt, 8.2 ±
0.07 pH, 27.8 ± 0.87°C). Artemia salina cysts (2.5 g/L of water) were incubated for 24
hours with moderate aeration to promote hatching. Hatched 1st instar Artemia salina were
harvested by siphoning the cone tanks and filtering the discharge water with a fine mesh
net and rinsed with freshwater. Artemia salina were filtered out of the freshwater with a
fine mesh net and fed ad libitum to alligator gar larvae in experimental tanks.
Production of enriched 2nd instar Artemia salina was performed in cone-bottom
cylinder tanks with 72.0 L of water (16.8 ± 0.35ppt, 8.2 ± 0.09 pH, 21.1 ± 0.42°C).
Hatched 1st instar Artemia salina from 37.5g of cysts were incubated in the 72.0-L conebottom cylinder tank for an additional 24 hours for enrichment with Easy Selco Artemia
nauplii enrichment (Inve; Salt Lake City, Utah; 0.6 g/L of water). The enriched 2nd instar
Artemia salina were then harvested with a fine mesh net and rinsed with freshwater.
Artemia salina were filtered out of the freshwater with a fine mesh net and fed ad libitum
to alligator gar larvae in experimental tanks.
Adult Gulf killifish were collected with four Gee minnow traps (22.9 x 44.5 cm
with 2.5 cm openings and 0.6 cm mesh) in Grand Isle, LA (N 29° 14’ 6.02” W 090° 0’
8.97) from February 2011 to January 2012. Killifish were then transported to the
Bayousphere Research Laboratory at Nicholls State University and held in two indoor
recirculating systems outfitted with bead filters for filtration. Each circular tank held
40
3,000 L of 8.0 ± 0.57ppt saltwater maintained at ambient room temperature (20.1 ±
0.47°C) and pH 6.9 ± 0.26. Killifish were fed Silver Cup Trout 1.0mm, 2.0mm, and
3.0mm (Skretting; Tooele, Utah) at 4 % body weight per day, determined by batch
weight prior to stocking in tanks. To collect eggs, Spawntex spawning substrate (Aquatic
Eco-systems, Inc; Apopka, Florida) was suspended in the rearing tanks approximately 15
cm below the water surface (Gothreaux and Green 2012). Eggs were collected every
morning from spawning substrate and enumerated volumetrically. Eggs were placed
between moist poly foam and transferred to containers for incubation. Incubation took
place for 15 days at ambient room temperature (21.5 ± 0.08°C). Eggs were hatched in
water from the alligator gar treatment systems at 15 days after collection and were fed to
alligator gar larvae (5 killifish larvae per gar larva per day).
Experimental Larval Alligator Gar Production
Alligator gar larvae were obtained at 2 DPH from the Private John Allen Fish
Hatchery in Tupelo, MS and transported to Nicholls State Universityon 24 April, 2012.
Alligator gar larvae were reared in two 95-L circular tanks containing 60 L of water in an
eight tank recirculating system in a greenhouse (Clay et al 2012; 4.3 ± 0.01 ppt, 8.3 ±
0.02 pH, 23.5 ± 1.18 °C). The recirculating system included a 265-L sump for
biofiltration and thermostat regulated heater to maintain minimum temperature (21°C).
Experimental Design
Two recirculating systems with water running through eight 95-L circular tanks
containing 30 L of 4.1 ± 0.01 ppt saltwater were used from 24 April, 2012 through 13
May, 2012. Four tanks in one system and five tanks in the other system were stocked
with 100-5 DPH alligator gar larvae. Each system included a 265 L sump with
41
biofiltration and a thermostat regulated heater to maintain minimum temperature (21°C).
Each tank had a top draining standpipe and flow to each tank was maintained at 0.5
L/minute. Tanks were cleaned daily by siphoning excess food and wastes. Dead
individuals were removed from each tank at time of cleaning and counted as natural
mortalities.
Alligator gar larvae were fed one of three feeding regimes; 1st instar Artemia
salina ad libitum and floating dry diet ad libitum (1st instar hereafter) at 7:00 am, 12:00
am and 5:00 pm daily, enriched 2nd instar Artemia salina ad libitum and floating dry diet
ad libitum (2nd instar hereafter) at 7:00 am, 12:00 am and 5:00 pm daily, or larval Gulf
killifish at a rate of 5 killifish/gar at 7:00 am daily and floating dry diet ad libitum
(killifish hereafter) at 7:00 am, 12:00 am and 5:00 pm daily. All live feeds were fed to
alligator gar larvae from 5 DPH to 12 DPH. Floating dry diets were fed three times per
day from 5 DPH to 20 DPH (Table 5). Feeding behavior (i.e. cannibalistic activity and
successful capture of live feed) was observed and recorded multiple times daily in each
experimental tank.
Water Quality
Temperature (°C), dissolved oxygen (mg/L), pH, and salinity (ppt) were measured
once at 7 am and once at 5 pm using a handheld temperature-oxygen-conductivitysalinity meter (YSI model 556 MPS; Yellow Springs, Ohio). Nitrite-N (mg/L) was
measured once at 7 am and once at 5 pm using Nitrver 3 Nitrite Reagent Powder Pillows
(Hach; Loveland, Colorado).Total ammonia nitrogen (TAN; mg/L) was measured once at
7 am and once at 5 pm using Nitrogen-Ammonia Reagent Powder Pillows (Hach).
Unionized ammonia was calculated using TAN, temperature, and pH measurements
42
Table 5. Particle size (µm) and protein and lipid (%) content for each type of feed given
to larval alligator gar for each period of days post hatch. Adapted from Clay et al. 2011.
Feed
Zeigler AP100 floating larval fish feed
SilverCup Trout fry artificial starter
SilverCup Trout fry artificial #1 crumble
43
Size
Protein/ Lipid
DPH Fed
250-450
440-590
590-840
50/12
48/14
48/14
5-12
9-15
12-20
(Emerson et al. 1975). All water quality parameters were not significantly different for
both systems (Table 6).
Length, Weight, and Total Biomass
Total length (± 0.1 mm) of alligator gar larvae was measured for three subsamples
of ten larvae were measured from both rearing tanks at the beginning of the experiment
on 5 DPH, ten larvae from each treatment replicate tank were measured on 12 DPH, and
all remaining larvae were measured from each treatment replicate tank on 20 DPH. All
larvae were photographed and measured with a stage micrometer using ImageTool 3.0
software(University of Texas Health Science Center, San Antonio, Texas). Mean
coefficient of variation was calculated for total length on 12 and 20 DPH.
Wet weight (± 0.1 mg) was determined for three subsamples of ten larvae from
both rearing tanks at the beginning of the experiment on 5 DPH, ten larvae from each
treatment replicate tank on 12 DPH, and all remaining larvae from each treatment
replicate tank on 20 DPH. Individual larvae were placed on previously weighed and
labeled 20 mL disposable aluminum weighing dishes and were weighed on an electronic
balance (Ohaus Adventurer Pro Series; Ohaus Corporation, Parsippany, New Jersey) to
the nearest 0.1 mg. Dry weight (mg) was measured by placing weighing dishes with
larvae in a Fisher Isotemp 500 series oven at 70°C and reweighed until a constant weight
was obtained. Wet weight from all fish on 20 DPH was used to calculate total biomass
for each treatment replicate.
Survival, Natural Mortality, and Cannibalism
44
Natural mortality was counted daily for each treatment replicate to determine total
mortality counts. All remaining larvae in each treatment replicate were counted to
determine final survival Using final survival and daily mortality counts, cannibalism was
Table 6. Mean (±SE) for each water quality variable for each recirculating system used
to rear alligator gar larvae fed a dry diet supplemented with either 1st instar Artemia
salina, enriched 2nd instar enriched Artemia salina , or Fundulus grandis larvae until 12
DPH. The last column represents the mean for both systems.
Variable
Temperature °C
Dissolved Oxygen (mg/L)
pH
Salinity (ppt)
Nitrite (mg/L)
Total Ammonia Nitrogen (mg/L)
Unionized Ammonia (mg/L)
System 1
System 2
Mean
24.1 ± 0.54 23.8 ± 0.48
24.0 ± 0.12
9.0 ± 0.22
9.1 ± 0.21
9.1 ± 0.05
8.2 ± 0.03
8.2 ± 0.04
8.2 ± 0.01
4.1 ± 0.06
4.1 ± 0.06
4.1 ± 0.01
0.3 ± 0.06
0.2 ± 0.03
0.3 ± 0.02
0.2 ± 0.07
0.1 ± 0.06
0.1 ± 0.03
0.01 ± 0.004 0.01 ± 0.002 0.01 ± 0.0002
45
calculated as
Total Cannibalism = (# larvae initially stocked – # lost to natural mortality) - Survivors
Statistical Analysis
All analyses were done using Statistical Analysis System (SAS), with significance
determined at the α level of 0.05. Total length, weight, total biomass, and all water
quality parameters were compared using analysis of variance (ANOVA). Total %
survival, % cannibalism, and % natural mortality were arcsine transformed and compared
using ANOVA. Coefficient of variation of total length and weight was log transformed
and compared using ANOVA.
46
RESULTS
Total Length
At 12 DPH alligator gar larvae in the 2nd instar treatment had a greater mean total
length (36.3 ± 0.31 mm) than larvae in the other two treatments (F= 22.99 2,6 ; P= 0.0015;
Figure 7) but was not significantly different among treatments at 20 DPH. Mean growth
rates of alligator gar larvae for all treatments combined was 2.8 ± 0.11 mm/day from 5
DPH to 12 DPH and 3.3 ± 0.19 mm/day from 12 DPH to 20 DPH. Total length was least
variable (F= 8.50 5,12 ; P= 0.0012) for alligator gar larvae in the 2nd instar treatment at 12
DPH (Figure 8).
Wet Weight and Total Biomass
Mean wet weight of alligator gar larvae from all treatments increased from 28.6 ±
0.62 mg at 5 DPH to 1,063.1 ± 27.56 mg at 20 DPH and was not significantly different
among treatments at any sample day (Figure 9). Wet weight was least variable (F= 13.39
5,12 ;
P= 0.0001) in the 2nd instar treatment at 12 DPH (Figure 10) but was not
significantly different among treatments by 20 DPH. Mean total biomass of alligator gar
larvae for all treatments at 20 DPH was 42.2 ± 7.75 g and was higher in the killifish
treatment (F= 20.29 2,6 ; P= 0.0021) than in the other treatments (Figure 11).
Survival, Mortality, and Cannibalism
Mean survival of alligator gar larvae at 20 DPH for all treatments was 47.5 ±
8.58% and was higher (F= 10.57 2,6 ; P= 0.0108) for the killifish treatment than the 1st
instar treatment (Figure 12). Mean natural mortality of alligator gar larvae for all
treatments was 29.8 ± 0.84% and was not significantly different among treatments
47
A
B
C
A
A
A
Figure 7. Mean (± SE) total length (mm) of alligator gar Atractosteus spatula larvae fed
a dry diet supplemented with either 1st instar Artemia salina (black line), enriched 2nd
instar Artemia salina (dashed black line), or Fundulus grandis larvae (dotted line) until
12 days post hatch (DPH). All treatments were fed only dry diet between 12 and 20 DPH.
Tukey groupings indicate differences among treatments for each day.
48
Figure 8. Mean (± SE) coefficient of variation for log total length of alligator gar
Atractosteus spatula larvae at 12 days post hatch (DPH) and 20 DPH fed a dry diet
supplemented with either 1st instar Artemia salina (black bars), enriched 2nd instar
Artemia salina (white bars), or Fundulus grandis larvae (gray bars) until 12 DPH. All
treatments were fed only dry diet between 12 and 20 DPH. Means with a common letter
are not different.
49
Figure 9. Mean (± SE) wet weight (mg) of alligator gar Atractosteus spatula larvae fed a
dry diet supplemented with either 1st instar Artemia salina (black line), enriched 2nd instar
Artemia salina (dashed black line), or Fundulus grandis larvae (dotted line) until 12 days
post hatch (DPH). All treatments were fed only dry diet between 12 and 20 DPH.
50
A
AB
A
A
A
B
Figure 10. Mean (± SE) coefficient of variation for log wet weight of alligator gar
Atractosteus spatula larvae at 12 days post hatch (DPH) and 20 DPH fed a dry diet
supplemented with either 1st instar Artemia salina (black bars), enriched 2nd instar
Artemia salina (white bars), or Fundulus grandis larvae (gray bars) until 12 DPH. All
treatments were fed only dry diet between 12 and 20 DPH. Means with a common letter
are not different.
51
A
B
B
Figure 11. Mean (± SE) total biomass (g) of alligator gar Atractosteus spatula larvae at
20 DPH fed a dry diet supplemented with either 1st instar Artemia salina (1st), enriched
2nd instar Artemia salina (2nd), or Fundulus grandis larvae (Fg) until 12 days post hatch
(DPH). All treatments were fed only dry diet between 12 and 20 DPH. Means with a
common letter are not different.
52
A
AB
B
Figure 12. Mean (± SE) total survival (%) of alligator gar Atractosteus spatula larvae at
20 days post hatch (DPH) fed a dry diet supplemented with either 1st instar Artemia
salina (1st), enriched 2nd instar Artemia salina (2nd), or Fundulus grandis larvae (Fg) until
12 DPH. All treatments were fed only dry diet between 12 and 20 DPH. Means with a
common letter are not different.
53
(Figure 13). Mean cannibalism was 38.0 ± 7.34% and was higher (F=9.21 2,6 ; P=0.0148)
in the 1st instar treatment than in the killifish treatment (Figure 14).
54
Figure 13. Mean (± SE) natural mortality (%) of alligator gar Atractosteus spatula larvae
at 20 days post hatch (DPH) fed a dry diet supplemented with either 1st instar Artemia
salina (1st), enriched 2nd instar Artemia salina (2nd), or Fundulus grandis larvae (Fg) until
12 DPH. All treatments were fed only dry diet between 12 and 20 DPH.
55
A
AB
B
Figure 14. Mean (± SE) cannibalism (%) of alligator gar Atractosteus spatula larvae at
20 days post hatch (DPH) fed a dry diet supplemented with either 1st instar Artemia
salina (1st), enriched 2nd instar Artemia salina (2nd), or Fundulus grandis larvae (Fg) until
12 DPH. All treatments were fed only dry diet between 12 and 20 DPH. Means with a
common letter are not different.
56
DISCUSSION
Aquaculture production can be used to augment wild fish populations in degraded
and depleted habitats (Neufeld et al. 2011; Wagner et al. 2012). Conservation aquaculture
programs produce fish to increase population abundance and range where populations
have decreased or have been extirpated (Bell et al. 2008). Desirable food species may be
reared in aquaculture facilities and sold to markets for consumption, thus reducing fishing
pressure of wild populations and allowing populations to possibly replenish via natural
reproduction. There are many bottlenecks in aquaculture production and limitations such
as cannibalism (Baras and Jobling 2002) and live feeds production (Bengston 2003;
Mendoza et al. 2008b). High production costs limit how many fish can be produced and
how many restocking programs can be funded. Despite bottlenecks in production,
aquaculture is a valuable tool that can increase wild fish populations in conjunction with
fishery regulations and habitat restoration (Bartley and Bell 2008; Okuzawa et al. 2008).
Alligator gar larvae and juveniles are currently produced at the Private John Allen
National Fish Hatchery in Tupelo, MS are distributed for conservation and management
purposes in Alabama (Armstrong, Jr. 2007), Arkansas (Barnett et al. 2011), Illinois
(Thomas and Hilsabeck 2008), Kentucky (Oster 2010), Mississippi (Deviney and Polles
2012), and Tennessee (Todd 2005). In addition to production for conservation programs,
alligator gar are a good candidate species for aquaculture production as a food fish in
localized markets (Garcia de Leon et al. 2001) because of high growth rates (Aguilera et
al. 2002; Mendoza et al. 2002; Mendoza et al. 2008b) and tolerance to poor water quality
(Boudreaux et al. 2007). However, a high rate of cannibalism during the larval and early
juvenile stages is the primary limitation to production and improving rearing methods to
57
reduce cannibalism should be a priority to increase production and efficiency (Mendoza
et al. 2008a). Rearing alligator gar by co feeding Artemia spp. with dry diets during larval
rearing reduced the rate of cannibalism when compared to rearing on dry diets only (Clay
et al. 2011). In this study, alligator gar larval cannibalism was reduced in the killifish
treatment as compared the 1st instar treatment. Natural mortality was similar among all
treatments so the reduction in cannibalism resulted in increased survival in the killifish
treatment as compared to the 1st instar treatment. Increased survival using larvae as live
feed during early rearing has also been documented in Pacific bluefin tuna Thunnus
orientalis when fed striped knifejaw Oplegnathus fasciatus larvae (Biswas et al. 2006).
Overall, the use of live feeds during larval alligator gar rearing reduced cannibalism and
improved production efficiency.
Larval fish typically become cannibalistic in culture tanks when available prey
items are no longer large enough for efficient feeding or are not in sufficient density
(Hecht and Pienaar 1993; Rao 2003). The reduction in alligator gar larval cannibalism in
the killifish treatment was due to size of the killifish being sufficient to keep the alligator
gar satiated longer than in the other treatments. Feeding behavior observations suggested
that alligator gar larvae in the 1st instar treatment decreased feeding frequency on 1st
instar Artemia salina at 9 DPH. At that time, behavior consistent with type I cannibalism
(tail first, partial digestion; Hecht and Applebaum 1988) began with the first evidence of
type II cannibalism (head first, complete digestion) starting on 12 DPH. No visual
evidence of cannibalist in the killifish treatment was seen until 16 DPH.
The rate of cannibalism can increase in culture systems as size variation among
individuals increases within tanks (Hecht and Applebaum 1988; Hecht and Pienaar
58
1993). Therefore, efficient alligator gar culture will require limiting size variation,
especially during the larval and young juvenile stages when cannibalism is most
prevalent (Mendoza et al. 2002; Mendoza et al. 2008b). Even though the alligator gar
larvae in the killifish treatment had the lowest cannibalism rates, the size variation was
greater at 12 DPH than in the other treatments (Figure 8; 10). Size variability of alligator
gar in the killifish treatment may have been due to differences in capture efficiency of
killifish larvae among individual alligator gar larvae before 10 DPH. Despite greater size
variation in the killifish treatment, there was no evidence of alligator gar larvae adopting
cannibalism as a feeding strategy when the killifish were present. Cannibalism in
aquaculture systems becomes an alternative feeding strategy when food is a limiting
factor (Hecht and Pienaar 1993). The presence of killifish larvae provided enough
predator-prey interaction for alligator gar larvae to not adopt a cannibalistic feeding
strategy.
Alligator gar are capable of digesting dry diets when feeding begins at 5 DPH
making rearing possible using only dry diets during all life stages (Mendoza et al. 2002;
Mendoza et al. 2008b). Clay et al. (2011) demonstrated that first-feeding alligator gar can
survive and grow when fed only a dry diet, but survival and growth was enhanced when
co-fed newly hatched Artemia nauplii. Alligator gar larvae in this study readily
consumed the Artemia salina and live killifish, but there was no growth difference among
the three feed treatments. Conservation programs need healthy, robust fish because reared
fish that are in poor condition perform poorly when stocked due to stress (Harmon 2009).
Alligator gar larvae in all treatments were similar in length and weight at 20 DPH but
because more alligator gar larvae were produced in the killifish treatment, total biomass
59
at 20 DPH was higher (60.4 ± 4.38 g) than the 1st instar treatment (28.4 ± 3.50 g) and the
2nd instar treatment (37.7 ± 2.92 g). This indicates that the killifish treatment could be
used to produce more alligator gar larvae for conservation aquaculture programs.
Alligator gar larvae in the 2nd instar treatment grew faster than alligator gar larvae
in the other two treatments, and were least variable in size through 12 DPH. Both initial
growth and homogeneity are potential advantages in future alligator gar production.
Feeding enriched Artemia has increased larval growth in beluga sturgeon Huso huso
(Jaladi et al. 2008), Pacific bluefin tuna Thunnus orientalis (Biswas et al. 2006), red sea
bream Pargus major (Andrade et al. 2012), and Japanese flounder Paralichthys olivaceus
(Sumule et al. 2003). Enriched Artemia fed to larvae provided other advantages such as
increased pigmentation in Atlantic halibut Hippoglossus hippoglossus (Gara et al. 1998)
and brown sole Pleuronectes herzensteini (Satoh et al. 2009) and improved salinity
tolerance in beluga sturgeon Huso huso (Jaladi et al. 2008). But alligator gar in the 2nd
instar treatment did not outperform the other treatments by 20 DPH indicating that from
12 to 20 DPH. Alligator gar larvae from the killifish treatment grew slower and were
more variable initially because of inefficient capture of prey items until 10 DPH.
Therefore a feeding regime for alligator gar larvae where they are weaned from enriched
2nd instar Artemia salina to killifish larvae may be effective for rearing alligator gar
larvae. It is common in aquaculture to offer progressively larger prey items to fish larvae
(Rao 2003). Advantages such as improved growth in larval mulloway Argyrosomus
japonicus (Ballagh et al. 2010), growth and survival of Pacific bluefin tuna Thunnus
orientalis (Biswas et al. 2006) and red sea bream Pargus major (Andrade et al. 2012),
improved rates of metamorphosis of Atlantic halibut Hippoglossus hippoglossus (Gara et
60
al. 1998) resulted after weaning larvae onto progressively larger prey items. Using a
progressive weaning regime, initial alligator gar larval growth should be fast and uniform
at least until 12 DPH when feeding 2nd instar Artemia salina and cannibalism would be
reduced through 20 DPH by providing killifish larvae.
Providing killifish larvae for alligator gar conservation programs may be a
limiting factor when using this feeding regime. On average, Gulf killifish produce viable
eggs at a maximum rate of 7.4 eggs per female per day (Green et al. 2010; Gothreaux and
Green 2010). At that rate of egg production and a feeding rate of 5 killifish larvae/gar,
alligator gar producers would need to rear at least 4 female and 1 male killifish
(Gothreaux and Green 2012) for every 6 larval alligator gar. However, because Gulf
killifish availability as a baitfish is limited by wild catch rates there has been recent
emphasis on producing gulf killifish from captive broodstock to provide the market with
a consistent, year-round supply (Green et al. 2010; Coulon et al. 2012). Therefore it may
be costly for a conservation program to rear enough adult killifish to meet killifish larvae
production demand. A partnership between separate alligator gar and killifish hatcheries
may be possible and killifish producers could sell viable eggs to the alligator gar
producers. Alligator gar producers would benefit from the purchase of killifish eggs by
producing more alligator gar larvae because of reduced cannibalism. Because of
increased alligator gar larval rearing efficiency due to reduced cannibalism, there is the
potential to reduce labor and production costs associated with larval rearing.
61
CONCLUSION
The spawning protocol for spotted gar developed in the Bayousphere Research
Laboratory at Nicholls State University can be used to produce spotted gar larvae for
conservation and research purposes. Conservation efforts may be needed in Ontario,
Canada, where the spotted gar has been listed as a threatened species (Species At Risk
Public Registry 2012). Although it is likely that there never was a large population of
spotted gar in Ontario, the small, current population faces challenges from habitat
degradation that may reduce future abundance (Bouvier and Mandrak 2010; Species At
Risk Public Registry 2012). Baseline growth, development, and survival data generated
by this study may be used by spotted gar producers for quality control purposes. Spotted
gar are genetically similar to other gar species (Wiley 1976; Nelson 1994) and are
possibly a suitable surrogate for future research on less abundant gar species. Because of
their relative small size, spotted gar are a good model species than can be cared for with
less space and equipment and broodstock are easier to handle as compared to larger gar
species such as alligator gar.
Intramuscular injection of TH into spotted gar broodstock increased TH levels of
spawned larvae, but there was no measureable size effect among treatments. However,
TH might be a valuable tool to increase the success rate of the current spawning protocol.
There is evidence to suggest that TH interacts with reproductive systems (Cyr and Eales
1996; Arcand-Hoy and Benson 1998). Increased TH levels have been found seasonally
during spawning but have yet to be directly linked to reproduction (Cyr and Eales 1996).
An alternative explaination for increased TH levels in adult fish during reproduction is
for enough TH to transfer into oocytes during oogenesis (Power et al. 2001). Embryos
62
and larvae need adequate levels of maternal TH to transfer into the yolk sac to develop
properly until they produce an endogenous source (Kobuke 1987).
Alligator gar cannibalism was lower in the killifish treatment than the 1st instar
treatment and directly influenced overall survival rate by 20 DPH. Therefore, a live feed
source like Gulf killifish larvae that are larger than Artemia salina should be used at some
point during alligator gar larval and early juvenile culture. As larval fish grow, weaning
onto progressively larger live feed during larval culture generally improves growth and
survival (Rao 2003). Overall alligator gar survival rates increased because cannibalism
rates were decreased. This increase in survival could be important to future alligator gar
production efforts because more gar could be produced more efficiently. The potential for
increased production and efficiency of alligator gar culture could also help conservation
efforts implement management plans.
The results of the live feeds study was done on a small scale. Producing enough
Gulf killifish larvae to conservation programs for alligator gar would be difficult.
However, the reduction in cannibalism during this study warrants testing of larval
alligator gar production using Gulf killifish larvae on a larger scale to determine financial
and production efficiency.
63
RECOMMENDATIONS
Because there were very few embryos produced from the 12 March spawns,
which occurred at lower temperatures, I believe that it is most efficient to use this
spawning protocol when mean water temperature is 19.5 °C or greater (Table 1). This is
consistent with natural spawning, which occurs in the wild when water temperature
reaches 20.0 °C (Eschelle and Riggs 1972).
Because there were no growth effects on spotted gar eggs and larvae, I do not
recommend using an intramuscular injection of TH into broodstock to enhance larval
performance. But, studies to determine if TH can increase the spawning success rate in
spotted gar and other fish should be done to improve the spawning protocol. Studying TH
for use improving the spotted gar spawning protocol should only be done if effects of TH
excreted by the broodstock on the environment are understood. Studies to fill the gap of
knowledge regarding the relationships between thyroidal and reproductive systems in fish
should also be done.
As is important in any aquaculture production, alligator gar rearing protocols
during the larval and juvenile stages can continue to be refined for maximum efficiency
and output. Studies should be done to determine efficacy of a feeding regime consisting
of Gulf killifish of increasing size as alligator gar larvae and juveniles increase in size. A
feeding regime of weaning alligator gar from enriched 2nd instar Artemia salina at first
feeding (5 DPH) to Gulf killifish around 10 DPH would be an effective early feeding
regime that would allow for maximum growth and survival. Rearing alligator gar larvae
on Gulf killifish larvae would be effective at reducing cannibalism past 20 DPH and that
64
Gulf killifish larvae presence in alligator gar culture tanks will limit cannibalism until gar
juveniles outgrow cannibalistic behaviors (104 mm; Clay 2009).
There are also future experiments I propose to reduce alligator gar cannibalism
rates during production. The use of anti-thyroids has been shown to reduce alligator gar
snout development (Mendoza et al. 2002), which can be theoretically linked to reduced
cannibalism but has never been tested. A study testing cannibalism rates after using antithyroids and TH to alter development of alligator gar should be done.
During observation of alligator gar larvae feeding, I noticed that gar predation on
killifish larvae was greatest during the morning. Alligator gar larvae are visual predators
and I believe they do not feed as much at night as during the day. Reducing photoperiod
has reduced cannibalism in juvenile barramundi Lates calcarifer culture (Arockiaraj and
Appelbaum 2011) and should be studied in alligator gar. Conversely, this study should
also investigate whether a longer photoperiod would allow for more feeding throughout
the day to increase amount of feed consumed per day. This would theoretically increase
growth rates and reduce cannibalism by shortening the duration that alligator gar are
cannibalistic.
65
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receptors. General and Comparative Endocrinology. 152: 206-214.
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Whitley, A.K. and P.D. Cowley. 2010. The status of fish conservation in South African
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Wiley, E.O. 1976. The phylogeny and biogeography of fossil and recent gars
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Aquaculture. Japan Aquaculture Research Quarterly. 3: 161-168.
81
APPENDICIES
Appendix I: Sex, total length (mm), weight (kg), and prepelvic girth (mm) for wild
caught spotted gar broodstock injected with TSH, T4, or T3 and spawning hormones,
Spring 2011.
Treatment
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Trial
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
Sex
f
f
f
m
m
m
m
m
m
m
m
m
f
f
f
f
f
f
f
m
m
m
m
m
m
m
m
m
m
m
m
f
f
Predicted Sex
f
m
f
f
m
m
m
m
m
m
m
m
f
f
f
f
f
f
f
f
m
m
m
m
m
m
m
m
m
m
m
f
f
82
Total Length
666
547
491
543
499
577
497
532
557
546
521
513
680
581
645
635
658
601
666
504
549
533
548
481
513
525
511
570
560
543
506
645
660
Weight
1.3
0.6
0.4
0.4
0.4
0.7
0.5
0.5
0.6
0.5
0.5
0.5
1.3
0.7
1.1
1.2
1.1
1.1
1.2
0.4
0.6
0.6
0.6
0.4
0.4
0.5
0.5
0.5
0.6
0.7
0.6
1.1
1.1
Girth
236
185
163
151
156
181
160
166
177
161
162
164
231
194
221
215
214
210
217
149
180
171
173
147
153
167
166
156
174
168
155
211
210
Control
Control
Treatment
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
3
3
Trial
3
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
f
f
Sex
f
m
m
m
m
m
m
m
m
m
m
f
f
f
f
m
m
m
m
m
m
m
m
m
m
m
m
f
f
f
f
f
f
m
m
m
m
m
m
m
f
Predicted Sex
f
m
f
m
m
m
m
m
m
m
m
f
m
f
f
m
m
f
f
m
m
m
m
m
m
m
m
f
f
f
m
f
f
m
m
m
m
m
m
83
534
575
Total Length
612
508
591
580
532
530
531
532
536
514
488
562
497
688
616
508
527
589
543
585
560
522
543
540
574
519
531
607
602
560
543
688
616
508
527
476
568
532
538
0.6
0.9
Weight
1.1
0.5
0.7
0.7
0.6
0.6
0.6
0.7
0.6
0.5
0.5
0.5
0.4
1.4
0.9
0.5
0.5
0.8
0.5
0.7
0.7
0.6
0.6
0.7
0.7
0.5
0.6
1.1
0.9
0.6
0.6
1.4
0.9
0.5
0.5
0.4
0.7
0.6
0.6
164
200
Girth
215
160
180
177
161
154
165
176
163
149
153
158
151
240
203
166
164
192
156
176
177
166
171
183
175
163
163
207
205
177
177
240
203
166
164
152
181
165
172
T3
T3
Treatment
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
2
2
Trial
2
2
3
3
3
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
3
m
m
Sex
m
m
f
f
f
f
f
f
f
m
m
m
m
m
f
m
m
m
m
m
m
m
m
m
m
m
f
f
f
f
f
m
m
m
m
m
m
m
f
m
m
Predicted Sex
m
m
f
f
m
m
m
m
m
f
m
m
m
m
m
f
f
m
m
m
m
m
m
m
m
m
f
f
m
m
m
f
m
m
m
m
m
m
f
84
586
573
Total Length
487
576
595
502
575
600
511
574
549
555
445
548
474
464
694
486
510
514
546
544
539
569
502
547
570
504
721
591
572
586
581
581
571
491
505
521
483
518
647
0.7
0.7
Weight
0.4
0.6
0.9
0.6
0.8
1
0.5
0.8
0.8
0.7
0.4
0.8
0.5
0.4
1.3
0.4
0.4
0.6
0.6
0.6
0.6
0.6
0.5
0.6
0.7
0.5
1.4
0.7
0.7
0.9
0.8
0.7
0.6
0.4
0.5
0.5
0.4
0.5
0.9
183
186
Girth
156
170
201
165
179
191
164
182
188
170
138
183
142
132
230
143
146
171
173
170
165
172
156
178
181
157
229
178
186
200
192
181
170
154
168
161
152
159
195
T4
T4
Treatment
T4
T4
T4
T4
T4
T4
T4
T4
T4
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
3
3
Trial
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
f
f
Sex
f
f
f
f
f
f
m
m
m
f
f
f
m
m
m
m
m
m
m
m
m
f
f
f
f
m
m
m
m
m
m
m
m
f
f
f
f
f
m
f
f
Predicted Sex
m
m
m
m
m
m
m
m
m
f
f
m
f
m
m
m
m
m
m
m
m
f
f
f
m
f
f
f
f
f
f
f
f
f
f
m
m
m
f
85
658
585
Total Length
520
532
555
518
495
535
531
495
532
681
559
537
545
562
483
492
565
490
571
517
479
731
545
541
615
499
518
492
487
562
541
513
523
640
620
536
563
513
526
1
0.8
Weight
0.6
0.7
0.9
0.6
0.4
0.7
0.6
0.5
0.6
1.4
0.7
0.6
0.7
0.7
0.4
0.5
0.7
0.6
0.7
0.6
0.5
1.7
0.7
0.7
0.7
0.5
0.5
0.4
0.4
0.7
0.6
0.5
0.5
1.1
0.9
0.7
0.7
0.6
0.6
200
193
Girth
161
165
196
167
137
184
157
137
145
234
180
174
176
177
148
146
181
174
176
166
151
251
188
184
181
156
154
151
148
178
168
165
162
214
195
174
177
154
158
TSH
TSH
Treatment
TSH
TSH
TSH
TSH
3
3
Trial
3
3
3
3
m
m
Sex
m
m
m
m
m
m
Predicted Sex
m
m
m
m
86
570
452
Total Length
521
428
511
573
0.8
0.4
Weight
0.6
0.3
0.6
0.7
171
125
Girth
158
126
157
174
Appendix II: Mean egg diameter (mm), mean yolk diameter (mm), and mean yolk
volume (µl) for each treatment replicate of eggs spawned from wild caught spotted gar
injected with TSH, T4, or T3 and spawning hormones, Spring 2011.
Treatment
Control
Control
Control
T3
T3
T3
T4
T4
T4
TSH
TSH
TSH
Rep
1
2
3
1
2
3
1
2
3
1
2
3
Mean Egg Dm
3.85
4.28
4.16
3.88
3.87
4.52
3.81
3.97
4.06
3.98
3.94
3.89
87
Mean Yolk Dm
2.55
2.65
2.63
2.74
2.72
2.54
2.54
2.82
2.39
2.60
2.81
2.48
Mean Yolk Vol
8.64
9.73
9.59
10.82
10.60
8.62
8.63
11.78
7.27
9.25
11.60
8.47
Appendix III: Hatch rate (%), time to yolk absorption (days), time to 100 % mortality
(days), and time to 50 % mortality (days) of each treatment replicate of eggs and larvae
spawned form wild caught spotted gar broodstock injected with TSH, T4, or T3 and
spawning hormones, Spring 2011.
Treatment
Control
Control
Control
Control
Control
Control
Control
Control
Control
T3
T3
T3
T3
T3
T3
T3
T3
T3
T4
T4
T4
T4
T4
T4
T4
T4
T4
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
Rep
1
1
1
2
2
2
3
3
3
1
1
1
2
2
2
3
3
3
1
1
1
2
2
2
3
3
3
1
1
1
2
2
2
3
3
3
Hatch %
68
74
71
10
5
23
58
57
61
90
97
99
49
67
71
86
99
100
49
59
50
94
83
94
93
99
98
86
47
62
80
83
81
96
86
93
Yolk Abs
9
9
8
9
.
.
9
.
.
9
9
9
9
9
9
9
9
9
9
9
.
9
9
9
8
8
9
9
9
9
9
9
9
9
9
9
88
100 % Mort
24
23
23
24
.
.
20
.
.
19
19
19
18
18
18
19
20
17
23
22
.
20
23
21
22
22
19
25
25
25
19
22
19
16
17
18
50 % Mort
20
21
21
19
.
.
19
.
.
18
18
18
17
17
16
17
16
16
20
20
.
19
21
20
19
19
18
22
22
22
18
20
18
15
15
17
Appendix IV: Wet weight at hatch (mg), dry weight at hatch (mg), total length at hatch
(TL; mm), and condition factor at hatch of each treatment replicate of larvae spawned
from wild caught spotted gar broodstock injected with TSH, T4, or T3 and spawning
hormones, Spring 2011.
Treatment
Control
Control
Control
T3
T3
T3
T4
T4
T4
TSH
TSH
TSH
Rep
1
2
3
1
2
3
1
2
3
1
2
3
Wet Weight
9.32
7.59
9.25
9.91
9.07
9.55
8.42
9.93
7.17
10.75
9.09
8.64
89
Dry Weight
3.97
3.19
4.23
4.12
3.93
3.4
3.77
4.83
2.55
3.64
4.13
3.24
Condition Factor
0.17
0.17
0.20
0.19
0.15
0.14
0.15
0.21
0.14
0.19
0.20
0.15
Appendix V: Mean total length (mm), mean snout length (mm), and mean snout
proportion of body length (%) of each treatment replicate of larvae up to 15 days post
hatch (DPH) spawned from wild caught spotted gar broodstock injected with TSH, T4, or
T3 and spawning hormones, Spring 2011.
Treatment
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
Control
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T4
T4
T4
T4
T4
Rep
1
2
3
1
2
1
2
1
2
1
2
1
2
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
DPH
0
0
0
3
3
6
6
9
9
12
12
15
15
0
0
0
3
3
3
6
6
6
9
9
9
12
12
12
15
15
15
0
0
0
3
3
Total Length
8.24
7.68
7.89
11.92
11.28
17.08
16.83
19.56
19.66
20.52
22.55
21.13
22.73
8.12
8.42
8.87
12.70
12.06
10.78
18.21
16.77
15.97
20.31
19.24
19.81
21.56
21.32
20.10
21.08
23.76
21.10
8.18
7.81
8.02
12.17
11.34
90
Snout Length
0.48
0.39
0.44
0.70
0.70
1.16
1.39
1.83
1.69
2.18
2.38
2.52
2.75
0.43
0.43
0.44
0.81
0.79
0.65
1.45
1.16
1.20
2.00
2.08
2.03
2.52
2.67
2.23
2.77
3.30
2.81
0.42
0.38
0.37
0.66
0.80
Snout Proportion
5.79
5.04
5.44
5.86
6.24
6.76
8.23
9.35
8.57
10.65
10.53
11.93
12.10
5.29
5.10
4.94
6.37
6.56
6.03
7.97
6.91
7.51
9.82
10.52
10.23
11.55
12.52
11.09
13.12
13.88
13.26
5.07
4.86
4.64
5.41
7.07
T4
Treatment
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
TSH
3
Rep
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
3
DPH
6
6
6
9
9
9
12
12
12
15
15
15
0
0
0
3
3
3
6
6
6
9
9
9
12
12
12
15
15
15
10.28
Total Length
15.90
16.33
15.15
18.98
20.76
19.04
19.54
23.45
19.41
20.55
24.83
20.75
8.31
7.24
8.32
12.07
12.11
10.68
17.50
17.57
15.43
19.14
20.83
18.87
19.72
22.55
19.70
19.79
24.28
20.53
91
0.74
Snout Length
1.07
1.25
1.05
1.67
2.38
1.94
1,83
2.67
2.27
2.28
3.36
2.70
0.39
0.39
0.39
0.67
0.75
0.76
1.28
1.59
1.17
1.66
2.05
1.89
1.99
2.73
2.27
2.15
3.38
2.53
7.22
Snout Proportion
6.75
7.65
6.96
8.79
11.45
10.17
9.37
11.39
11.77
11.11
13.55
13.03
4.65
5.08
4.65
5.55
6.19
7,09
7.34
9.03
7.59
8.68
9.86
10.04
10.10
12.12
11.52
10.84
13.92
13.33
Appendix VI: Thyroxine levels (T4; ng/ individual) and triiodothyronine levels (T3; ng/
individual) of eggs and larvae spawned from wild caught spotted gar broodstock injected
with TSH, T4, or T3 and spawning hormones, Spring 2011.
Treatment
Control
Control
T3
T3
T3
T4
T4
T4
TSH
TSH
TSH
Control
Control
T3
T3
T3
T4
T4
T4
TSH
TSH
TSH
Control
Control
T3
T3
T3
T4
T4
T4
TSH
TSH
TSH
Control
Control
Age
Egg
Egg
Egg
Egg
Egg
Egg
Egg
Egg
Egg
Egg
Egg
0
0
0
0
0
0
0
0
0
0
0
3
3
3
3
3
3
3
3
3
3
3
6
6
Rep
1
2
1
2
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
1
2
3
1
2
T4
1.14
0.67
5.50
5.30
4.38
9.03
6.00
2.88
0.79
1.36
1.12
0.65
0.80
0.76
0.77
0.60
0.63
1.11
1.63
1.13
0.80
0.97
1.80
1.56
1.26
1.30
1.20
1.55
1.70
1.68
2.02
1.72
1.14
2.20
2.03
92
T3
0.47
0.55
6.31
5.05
1.19
0.72
0.49
2.03
0.56
1.01
0.82
1.01
0.63
1.65
1.22
0.55
6.51
1.88
6.51
1.46
0.19
1.39
3.43
1.07
1.07
2.24
0.87
5.35
1.03
0.49
1.30
6.38
0.88
3.85
0.62
T3
Treatment
T3
T3
T4
T4
T4
TSH
TSH
TSH
Control
Control
T3
T3
T3
T4
T4
T4
TSH
TSH
TSH
Control
Control
T3
T3
T3
T4
T4
T4
TSH
TSH
TSH
Control
Control
T3
T3
T3
T4
T4
T4
6
Age
6
6
6
6
6
6
6
6
9
9
9
9
9
9
9
9
9
9
9
12
12
12
12
12
12
12
12
12
12
12
15
15
15
15
15
15
15
15
1
Rep
2
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
1
2
3
1
2
1
2
3
1
2
3
1.50
T4
2.58
2.00
2.38
1.55
2.00
1.83
2.48
1.43
4.03
2.63
2.43
2.70
2.57
2.07
2.90
2.60
2.07
2.33
3.03
3.43
2.53
2.77
3.63
2.53
2.77
2.83
2.67
3.10
2.67
2.83
4.05
4.30
4.30
3.95
4.05
3.45
3.65
5.40
93
1.48
T3
7.65
0.48
3.25
16.28
11.31
0.25
16.28
0.87
21.70
3.76
5.15
2.92
0.69
1.40
1.23
1.34
6.31
1.72
5.43
21.70
0.55
4.06
4.76
6.16
4.34
3.66
3.69
21.26
4.21
0.83
2.42
9.59
3.40
5.07
5.43
1.20
7.67
3.26
TSH
Treatment
TSH
TSH
15
Age
15
15
1
Rep
2
3
4.00
T4
6.40
4.00
94
12.06
T3
11.77
7.39
Appendix VII: Water quality parameters pH, salinity (ppt), dissolved oxygen (DO;
mg/L), temperature (°C) of two recalculating systems used to rear alligator gar larvae
during live feeds experiment.
Date
4/25/2012
4/26/2012
4/26/2012
4/27/2012
4/27/2012
4/28/2012
4/28/2012
4/29/2012
4/29/2012
4/30/2012
4/30/2012
5/1/2012
5/1/2012
5/2/2012
5/2/2012
5/3/2012
5/3/2012
5/4/2012
5/4/2012
5/5/2012
5/5/2012
5/6/2012
5/6/2012
5/7/2012
5/7/2012
5/8/2012
5/8/2012
5/9/2012
5/9/2012
5/10/2012
5/10/2012
5/11/2012
5/11/2012
5/12/2012
5/12/2012
System
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Time
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
pH
8.36
8.14
8.43
8.21
8.31
8.25
8.46
8.27
8.41
8.17
8.18
8.11
8.17
8.35
8.26
8.31
8.28
8.29
8.27
8.18
8.20
8.22
8.14
8.31
8.16
8.31
8.12
8.06
8.10
8.15
8.20
8.00
7.89
8.05
8.16
Salinity
4.43
4.45
4.40
4.10
4.48
4.51
4.53
4.58
4.60
4.65
3.56
3.98
4.08
4.11
4.12
4.15
3.78
4.04
4.05
4.09
4.11
4.14
3.88
3.91
3.91
3.95
3.94
3.97
3.99
4.18
4.08
4.12
4.06
4.08
4.08
95
DO
8.97
8.43
9.16
4.41
9.04
9.68
9.14
9.37
8.80
9.49
9.25
8.70
8.98
8.97
8.56
8.96
8.93
8.85
8.31
9.79
8.50
9.61
8.61
9.63
8.74
9.88
8.81
9.22
8.93
9.84
8.49
9.40
9.45
10.05
9.13
Temperature
24.01
21.26
26.74
23.40
25.22
22.43
25.97
22.07
25.95
22.05
27.32
21.99
26.60
23.05
25.39
22.34
26.71
23.02
28.67
23.42
26.58
22.64
27.75
22.82
25.52
21.23
27.18
23.08
26.98
21.43
26.55
21.93
21.96
21.35
22.79
5/13/2012
Date
4/25/2012
4/26/2012
4/26/2012
4/27/2012
4/27/2012
4/28/2012
4/28/2012
4/29/2012
4/29/2012
4/30/2012
4/30/2012
5/1/2012
5/1/2012
5/2/2012
5/2/2012
5/3/2012
5/3/2012
5/4/2012
5/4/2012
5/5/2012
5/5/2012
5/6/2012
5/6/2012
5/7/2012
5/7/2012
5/8/2012
5/8/2012
5/9/2012
5/9/2012
5/10/2012
5/10/2012
5/11/2012
5/11/2012
5/12/2012
5/12/2012
5/13/2012
1
System
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
AM
Time
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
8.02
pH
7.89
7.90
8.57
8.29
8.46
8.43
8.48
8.38
8.43
8.31
8.18
8.14
8.23
8.36
8.35
8.32
8.22
8.31
8.26
8.24
8.19
8.21
8.15
8.30
8.13
8.31
8.10
8.15
8.11
8.16
8.18
8.03
7.94
8.07
8.14
8.07
4.10
Salinity
4.95
4.76
4.03
4.10
4.07
4.29
4.31
4.35
4.50
4.55
4.05
4.07
4.06
4.09
4.10
4.13
3.51
3.78
4.00
4.04
4.05
4.08
3.94
3.97
3.97
4.00
4.00
4.03
4.04
3.92
3.95
3.99
4.10
4.12
4.06
4.00
96
9.78
DO
9.65
9.38
9.05
4.80
9.52
10.54
9.34
9.97
8.78
9.61
9.13
8.97
8.91
8.59
8.77
8.83
8.80
9.14
8.61
9.91
8.60
10.02
8.56
9.94
8.97
10.04
9.22
9.25
9.01
9.64
8.52
9.13
9.56
9.79
9.24
9.13
21.52
Temperature
23.67
21.38
26.14
23.01
23.86
21.66
25.23
21.95
25.52
21.85
26.62
21.82
25.95
22.98
25.02
22.10
25.90
22.83
27.76
23.38
26.26
22.58
26.88
22.69
25.45
21.28
26.57
23.16
26.89
21.80
25.70
21.73
21.54
21.34
22.68
22.03
Appendix XIII: Total length (mm) and weight (mg) of alligator gar larvae fed a dry diet
supplemented with either 1st instar Artemia salina, enriched 2nd instar Artemia salina, or
Fundulus grandis larvae until 12 days post hatch (DPH). All treatments were fed only dry
diet between 12 and 20 DPH.
Treatment
1st
1st
1st
1st
1st
1st
1st
1st
1st
1st
1st
1st
2nd
2nd
2nd
2nd
2nd
2nd
2nd
2nd
2nd
2nd
2nd
2nd
Killifish
Killifish
Killifish
Killifish
Killifish
Killifish
Killifish
Killifish
Killifish
Killifish
Killifish
Killifish
Rep
1
2
3
4
5
6
1
2
3
1
2
3
1
2
3
4
5
6
1
2
3
1
2
3
1
2
3
4
5
6
1
2
3
1
2
3
DPH
5
5
5
5
5
5
12
12
12
20
20
20
5
5
5
5
5
5
12
12
12
20
20
20
5
5
5
5
5
5
12
12
12
20
20
20
97
Total Length
14.46
15.22
15.07
15.82
15.90
15.49
32.82
33.47
32.40
62.25
69.38
57.33
14.46
15.22
15.07
15.82
15.90
15.49
36.93
35.66
36.25
61.44
58.45
59.44
14.46
15.22
15.07
15.82
15.90
15.49
35.09
35.23
33.95
62.69
59.75
61.59
Weight
26.11
28.82
27.93
29.20
30.59
29.17
190.41
208.10
173.78
1089.92
1432.70
819.17
26.11
28.82
27.93
29.20
30.59
29.17
210.76
177.89
187.25
1048.17
884.70
1064.77
26.11
28.82
27.93
29.20
30.59
29.17
226.38
212.68
192.97
1117.83
996.36
1114.40
Appendix IX: Total survival, cannibalism, natural mortality, and final biomass (g) at 20
days post hatch (DPH) of alligator gar larvae fed a dry diet supplemented with either 1st
instar Artemia salina, enriched 2nd instar Artemia salina, or Fundulus grandis larvae until
12 DPH. All treatments were fed only dry diet between 12 and 20 DPH.
Treat
Rep
Survival
Cannibalism Natural Mortality
Biomass
1st
1
30
47
23
32.70
1st
2
15
54
31
21.49
1st
3
38
34
28
31.11
2nd
1
31
42
27
32.49
2nd
2
43
32
25
38.04
2nd
3
40
30
30
42.59
FuGr
1
54
19
27
60.36
FuGr
2
53
22
25
52.81
FuGr
3
61
17
22
67.98
98
BIOGRAPHICAL SKETCH
Kent’s passion for fish began in Colon, MI in 1988 when he caught his first fish
while vacationing with his family. Since then, Kent has always been interested fish and
his hobbies and career have reflected that. Kent studied fisheries and water resources at
the University of Wisconsin- Stevens Point before pursuing his career in aquaculture in
San Diego, CA at the Hubbs- SeaWorld Research Institute. There Kent learned the skills
necessary to accept a graduate position in Thibodaux, LA and excel in the Marine and
Environmental Biology program rearing spotted and alligator gar and receiving his
master’ degree at Nicholls State University. The skills Kent has learned at Nicholls will
serve him well in whatever he decides to do in the future. Kent would like to continue his
career in aquaculture and someday run a research or production facility.
99
CIRRICULUM VITAE
KENT BOLLFRASS
204 Gloria St #1
Thibodaux, LA 70301
Phone: 952.807.4310
kbollfrass@its.nicholls.edu
EDUCATION
University of Wisconsin- Stevens Point, Stevens Point, WI
B.S. Fisheries and Water Resources, May, 2007
Nicholls State University, Thibodaux, LA
M.S. Marine and Environmental Biology, Dec, 2012
Thesis: Induced spawning of spotted gar Lepisostues oculatus, the effects of
thyroid hormones on spotted gar eggs and larvae, and the effects of innovative
live feeds on alligator gar Atractosteus spatula larvae cannibalism rates
EMPLOYMENT
Graduate Assistant, Nicholls State University, Thibodaux, LA, Fall 2010- present
Extensive use of aquaculture knowledge and field sampling techniques.
Responsible for creating, designing and implementing research project.
Successful with culture of Gulf Killfish Fundulus grandis, alligator gar
Atractosteus spatula, and spotted gar Lepisosteus oculatus, design and
construction of recirculating aquaculture systems, and excelled with lab and field
techniques. Operated state vehicles including trucks, trailers, and small
watercraft.. Responsible for instructing and teaching undergraduate biology lab
students.
Research Technician, Hubbs- SeaWorld Research Institute, San Diego, CA, March
2008- May 2010
Successful in larval, juvenile, and broodstock culture of California yellowtail
Seriola lalandi, white sea bass Atractoscion nobilis, and California halibut
Paralichthys californicus, as well as care for several species of broodstock and
juvenile rockfish and cabezon Scorpaenichthys marmoratus. Actively involved in
daily system maintenance and weekly water quality monitoring.
Intern, Lake and Pond Solutions, Elkhorn, WI, May-August 2006
100
Identified and implemented strategies for control of aquatic plants in private water
bodies. Installed and maintained aeration systems, pump systems, and fountains.
Responsible for driving company truck with trailer and boat.
MDU Field Supervisor, Prewire Specialists Inc, St. Paul, MN, Seasonal 2002-2008
Installed, repaired and upgraded video cable, and inspected completed work in
multifamily dwellings and commercial office buildings. Oversaw a crew of four
other technicians, trained new technicians, and provided customer service.
SKILS








Working knowledge of low voltage wiring and plumbing, skilled with hand and power
tools
Experience with water quality testing methods and equipment
Proficient with Microsoft Excel, Word, PowerPoint, Outlook, SAS and limited
experience with ArcGIS
Excellent written and oral communication skills
Proven work ethic, flexible, creative and innovative
Productive and effective in both solo and group assignments
Effective use of company and/ or state equipment and vehicles including trailers and
boats
Leadership, including training of new employees and management of small work group
SPECIAL PROJECTS
International Study, Universidad Autonoma de Neuvo Leon, Monterrey, Neuvo Leon,
MX, August, 2011
Alligator gar Atractosteus spatula and spotted gar Lepisosteus oculatus thyroid
hormone extraction and quantification as well as DNA and RNA extraction,
quantification, and analysis.
Spotted gar spawning project coordinator, Nicholls State University, Thibodaux, LA,
Spring 2011 and 2012.
Collected and maintained broodstock, administered spotted gar spawning
protocol, and shipped viable embryos for study to principle investigators
domestically and internationally. Coordinated and delegated work efforts with
other Nicholls State University students and staff.
Coastal Restoration Volunteer. Southeastern Louisiana, LA, August 2010- Spetember
2012
Maintained, collected, transported, and planted oyster grass Spartina alterniflora
and black mangrove Avicennia germinans for marsh restoration projects as well
as smooth cord grass Spartina patins and bitter panicum Panicum amirum for
barrier island restoration projects in Southeastern Louisiana.
PROFESSIONAL PRESENTATIONS
101
2012 World Aquaculture Society, Prague, Czech Republic
Title- Involvement of thyroid hormones in the reproduction and early
development of alligator gar Atractostues spatula and spotted gar Lepisostues
oculatus Roberto Mendoza, Sergio Castillo, Kent Bollfrass, Allyse Ferrara,
Quenton, Juan Pablo Lazo, Carlos Aguilera
2012 Nicholls State University, Calypseaux, Cocodrie, LA, Oral Presentation
Title- Effects of innovative live feeds on early rearing of alligator gar Atractosteus
spatula Kent Bollfrass, Quenton Fontenot, Allyse Ferrara, Chris Green
2012 Nicholls State University Research Week, Thibodaux, LA, Poster Presentation, 3rd
place poster competition, graduate student division
Title- Induced spawning of spotted gar Lepisosteus oculatus and effects of
injecting broodstock with thyroid hormones on egg and larval performance Kent
Bollfrass, Allyse Ferrara, Quenton Fontenot, Sergio Castillo, Roberto Mendoza
2012 Southern Division American Fisheries Society, Biloxi, MS, Oral Presentaion
Title- Induced Spawning of Wild-caught Spotted Gar (Lepisosteus oculatus) and
Effects of Injecting Broodstock with Thyroid Hormones on Egg Hatch Rate,
Larval Growth and Development, and Survival Kent Bollfrass, Allyse Ferrara,
Quenton Fontenot, Sergio Castillo, Roberto Mendoza
2011 Alligator Gar Technical Committee Meeting, Memphis, TN, Oral Presentation
Title- Thyroid Hormone Trials with Spotted Gar Surrogates and Larval Alligator
Gar Feeding Trials Kent Bollfrass, Allyse Ferrara, Quenton Fontenot, Sergio
Castillo, Roberto Mendoza, Carlos Aguilera, and Chris Green
2011 Lousiana Academy of Professional Biologists, Lafayette, LA, Oral Presentation
Title- Induced spawning of wild caught spotted gar Lepisosteus oculatus and
effects of injected thyroid hormones on eggs and larvae Kent Bollfrass, Allyse
Ferrara, Quenton Fontenot, Sergio Castillo, Roberto Mendoza
2011 Universidad Autonoma de Nuevo Leon, 2011 Monterrey, MX, Oral Presentation
Title- Induced spawning of wild caught spotted gar Lepisosteus oculatus and
effects of injected thyroid hormones on eggs and larvae Kent Bollfrass, Allyse
Ferrara, Quenton Fontenot, Sergio Castillo, Roberto Mendoza
2011 Nicholls State University, Calypseaux, Cocodrie, LA, Oral Presentation
102
Title- Improving larval alligator gar Atractosteus spatula rearing techniques Kent
Bollfrass, Allyse Ferrara, Quenton Fontenot, Sergio Castillo, Roberto Mendoza,
Chris Green
2010 World Aquaculture Society, San Diego, CA, Oral Presentation
Title- Egg disinfection of three marine finfish Kevin Stuart, Martha Keller, Kent
Bollfrass, and Mark Drawbridge
103