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Analysis of spawning behavior and gamete availability of the Florida pompano (Trachinotus carolinus) in a recirculating ...

Permanent Link: http://ufdc.ufl.edu/UFE0042163/00001

Material Information

Title: Analysis of spawning behavior and gamete availability of the Florida pompano (Trachinotus carolinus) in a recirculating aquaculture system.
Physical Description: 1 online resource (42 p.)
Language: english
Creator: Dippon, Elizabeth
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: aquaculture, behavior, gamete, mote, pompano, recirculating, reynolds, spawning
Biology -- Dissertations, Academic -- UF
Genre: Zoology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Marine finfish recirculating aquaculture systems are a viable way to produce environmentally-sustainable stock. However, one of the biggest challenges production managers face in recirculating systems is that fertilized egg yields are low or inconsistent. An economically valuable fish that is currently in population decline is the Florida pompano (Trachinotus carolinus). This is a preliminary study that focuses on the factors that contribute to the low fertilized egg yields of the Florida pompano in the recirculating aquaculture facility at Mote Aquaculture Research Park, a field station of the Mote Marine Laboratory. In this study, we used video analysis of spawns in combination with sampling and fertilized egg yield data to determine A) if pompano will exhibit natural spawning behavior in a recirculating system, B) does pompano gamete maturity remain consistent throughout the entire February-October spawning season, and C) what are the fertilization rates of pompano spawns in recirculating systems, and how does it compare to other captive pompano fertilization rates. From our data, we determined that pompano spawning behavior is not inhibited by recirculating aquaculture systems. In fact, both false (spawning behavior without the release of gametes) and true spawning behavior (with the release of gametes) occurred and were quantified. We also found that the production of mature gametes in males is highly variable and possibly the true cause of low fertilized egg yield. A relatively high proportion of males had mature gametes at the beginning (53%, February) and end (36%, September) of the spawning season, but in June, only 7% of males had mature gametes at the time of sampling. Female gamete maturity also peaked in February and September (70% and 60% of females had mature gametes in these months, respectively), but female gamete maturity did not plunge significantly like the male gamete maturity did. This pattern in gamete maturity could reflect a need for a 90-day break in the middle of the spawning season so adults can produce more mature gametes. Finally, fertilization rates of 0%-57% were much lower than other captive pompano studies and were also variable across the spawning season. Some causes for these low fertilization rates could be smaller egg sizes, small egg fecundity, and sperm limitation. Strong flow rate in the tanks during spawning could also cause fertilization dysfunction.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Elizabeth Dippon.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: St. Mary, Colette M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042163:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042163/00001

Material Information

Title: Analysis of spawning behavior and gamete availability of the Florida pompano (Trachinotus carolinus) in a recirculating aquaculture system.
Physical Description: 1 online resource (42 p.)
Language: english
Creator: Dippon, Elizabeth
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: aquaculture, behavior, gamete, mote, pompano, recirculating, reynolds, spawning
Biology -- Dissertations, Academic -- UF
Genre: Zoology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Marine finfish recirculating aquaculture systems are a viable way to produce environmentally-sustainable stock. However, one of the biggest challenges production managers face in recirculating systems is that fertilized egg yields are low or inconsistent. An economically valuable fish that is currently in population decline is the Florida pompano (Trachinotus carolinus). This is a preliminary study that focuses on the factors that contribute to the low fertilized egg yields of the Florida pompano in the recirculating aquaculture facility at Mote Aquaculture Research Park, a field station of the Mote Marine Laboratory. In this study, we used video analysis of spawns in combination with sampling and fertilized egg yield data to determine A) if pompano will exhibit natural spawning behavior in a recirculating system, B) does pompano gamete maturity remain consistent throughout the entire February-October spawning season, and C) what are the fertilization rates of pompano spawns in recirculating systems, and how does it compare to other captive pompano fertilization rates. From our data, we determined that pompano spawning behavior is not inhibited by recirculating aquaculture systems. In fact, both false (spawning behavior without the release of gametes) and true spawning behavior (with the release of gametes) occurred and were quantified. We also found that the production of mature gametes in males is highly variable and possibly the true cause of low fertilized egg yield. A relatively high proportion of males had mature gametes at the beginning (53%, February) and end (36%, September) of the spawning season, but in June, only 7% of males had mature gametes at the time of sampling. Female gamete maturity also peaked in February and September (70% and 60% of females had mature gametes in these months, respectively), but female gamete maturity did not plunge significantly like the male gamete maturity did. This pattern in gamete maturity could reflect a need for a 90-day break in the middle of the spawning season so adults can produce more mature gametes. Finally, fertilization rates of 0%-57% were much lower than other captive pompano studies and were also variable across the spawning season. Some causes for these low fertilization rates could be smaller egg sizes, small egg fecundity, and sperm limitation. Strong flow rate in the tanks during spawning could also cause fertilization dysfunction.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Elizabeth Dippon.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: St. Mary, Colette M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042163:00001


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ANALYSIS OF SPAWNING BEHAVIOR AND GAMETE AVAILABILITY OF THE
FLORIDA POMPANO (Trachinotus carolinus) IN A RECIRCULATING AQUACULTURE
SYSTEM.

















By

ELIZABETH A. REYNOLDS


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2010
































2010 Elizabeth A. Reynolds









ACKNOWLEDGMENTS

I would like to thank the chair and the members of my committee for their

mentoring and advice, the Mote Marine Laboratory for the use of their facilities and

collaboration, and my loving parents, grandparents, husband, and friends who

encouraged me and motivated me through this study.









TABLE OF CONTENTS

page

ACKNOW LEDGMENTS ........ ......... ......... ..... ............... .... ........... 3

LIST O F TA BLES ......... ...... ...................................................................... 5

LIST O F F IG U R E S .................................................................. 6

A B S T R A C T .............. ...... ........... ................. ....... .. ....................................... 7

CHAPTER

1 MARINE FINFISH AQUACULTURE SYSTEMS AND RELATED
REPRODUCTIVE CHALLENGES ..... .................. ................. 9

2 SPAWNING BEHAVIOR AND GAMETE AVAILABILITY OF FLORIDA
POMPANO ............... .............................. 15

Materials and Methods................... .... .. ..................... ......... 18
Study Facility and Housing Conditions ................... ............... 18
Experim mental Procedures .............. .... .. ................. ............... 19
Horm one Induction of Spaw ning .............. ................. .................................. 20
B ehaviora l F ilm ing E quipm ent............................... ................ ... .. ............... 20
Behavioral and Statistical Analysis........... ............................... .... ............ 21
R results ................ .............. ... .... ......... .. ............. .... 22
Male and Female Gamete Production ............ ................. .... .................. 22
Fertilized Egg Yield Across Spawning Cycles ............ .................................. 23
F a lse S paw n ing B e havio r........................................................ ... .. ............... 2 4
True Spawning Behavior .............. ......... .. ............. ........... .... ........... 25
Comparisons Among True Spawning Rushes...... ..... ................................. 26
Discussion ................................... ..... .. ............................ 27
Natural Spawning Behavior........................ .... ............... 27
Limited Gamete Quantity............................. .................... 28
Improving Fertilization Rates ................................ ................ ................... 31
C o n c lu s io n ......... ............................................. .................... ............... 3 3

LIST OF REFERENCES .................... ........ ......... ......... 37

BIOGRAPHICAL SKETCH ................................ .............................. 42









LIST OF TABLES

Table page

2-1 Proportion males and females with mature oocytes spawning season............ 35

2- 2 Fertilized egg yield of 2009 spawning season .............. ..... ............... 35

2-3 Egg diameter and female measurements.................................... 36









LIST OF FIGURES


Figure page

2-1 Spawning behavior video recording timeline ............. ...... .................. 34

2-2 Percent individuals with mature gametes ................................. ......... ........ 34









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ANALYSIS OF SPAWNING BEHAVIOR AND GAMETE AVAILABILITY OF THE
FLORIDA POMPANO (Trachinotus carolinus) IN A RECIRCULATING AQUACULTURE
SYSTEM.

By

Elizabeth A. Reynolds

August 2010

Chair: Colette M. St. Mary
Major: Zoology

Marine finfish recirculating aquaculture systems are a viable way to produce

environmentally-sustainable stock. However, one of the biggest challenges production

managers face in recirculating systems is that fertilized egg yields are low or

inconsistent. An economically valuable fish that is currently in population decline is the

Florida pompano (Trachinotus carolinus). This is a preliminary study that focuses on

the factors that contribute to the low fertilized egg yields of the Florida pompano in the

recirculating aquaculture facility at Mote Aquaculture Research Park, a field station of

the Mote Marine Laboratory. In this study, we used video analysis of spawns in

combination with sampling and fertilized egg yield data to determine A) if pompano will

exhibit natural spawning behavior in a recirculating system, B) does pompano gamete

maturity remain consistent throughout the entire February-October spawning season,

and C) what are the fertilization rates of pompano spawns in recirculating systems, and

how does it compare to other captive pompano fertilization rates. From our data, we

determined that pompano spawning behavior is not inhibited by recirculating

aquaculture systems. In fact, both false (spawning behavior without the release of









gametes) and true spawning behavior (with the release of gametes) occurred and were

quantified. We also found that the production of mature gametes in males is highly

variable and possibly the true cause of low fertilized egg yield. A relatively high

proportion of males had mature gametes at the beginning (53%, February) and end

(36%, September) of the spawning season, but in June, only 7% of males had mature

gametes at the time of sampling. Female gamete maturity also peaked in February and

September (70% and 60% of females had mature gametes in these months,

respectively), but female gamete maturity did not plunge significantly like the male

gamete maturity did. This pattern in gamete maturity could reflect a need for a 90-day

break in the middle of the spawning season so adults can produce more mature

gametes. Finally, fertilization rates of 0%-57% were much lower than other captive

pompano studies and were also variable across the spawning season. Some causes

for these low fertilization rates could be smaller egg sizes, small egg fecundity, and

sperm limitation. Strong flow rate in the tanks during spawning could also cause

fertilization dysfunction.









CHAPTER 1
MARINE FINFISH AQUACULTURE SYSTEMS AND RELATED REPRODUCTIVE
CHALLENGES

The struggle between the demand for commercial fishing and conservation has

proved to be a tumultuous battle. In response to this problem and the ever-increasing

demand for food fish, the development of recirculating aquaculture systems has been

proposed as an environmentally-friendly and sustainable solution to the demands for

commercial fish. Over the past century, commercial fishing for finfish has increased

exponentially (Watson and Pauly 2001). During the 1980s, overfishing was such a

threat to marine species that the government passed the Magnuson-Stevens Fishery

Conservation and Management Act in 1996 that required an end to over-exploitation of

fish populations and mandatory rebuilding of fish stocks (Rosenberg et al. 2006).

Unfortunately, even though there is evidence that biomass in stocks managed under

rebuilding plans has the potential to increase, overfishing still occurs in 45% of these

stocks (Rosenberg et al. 2006). Perhaps the most vulnerable species to

overexploitation are those that predictably spawn in the same area each season (de

Mitcheson et al. 2008). For example, seasonal spawning aggregations seen in many

shallow-water grouper such as Nassau grouper (Epinephelus striatus), Gag Grouper

(Mycteroperca microlepis), and Scamp Grouper (Epiniphelus morio) are often the target

of commercial fishing due to their predictability (Coleman et al. 1996; de Mitcheson et

al. 2008). The risk of rapid stock decline is increased as a result.

To combat the decline in fish populations due to overfishing and other causes of

stock decline (e.g. development, habitat degradation, and pollution), agencies have

attempted to increase marine fish yields through restocking natural populations

(increase spawning biomass), fisheries enhancement (overcome recruitment limitation),









and sea ranching (put, grow, and take method) (Bell et al. 2008). All of these

supplemental additions first require the production of juveniles (Bell et al. 2008). The

principle method of producing juveniles for release is through aquaculture.

Aquaculture is by no means a new system for producing fish. Historically,

aquaculture involved producing fish in inland ponds or coastal cages (Losordo et al.

1998; Holmer et al. 2003). Unfortunately, aquaculture in inland ponds relies on large

amounts of water (as much as 1 million gallons per acre to fill a pond, and an equal

amount every year to compensate for evaporation, and seepage; Losordo et al. 1998), a

large land area, and its productivity is limited by environmental factors. In addition, the

costs of nutrient effluent, shifts in biodiversity, and changes in water chemistry have

caused coastal cages to come under scrutiny (Holmer et al. 2003; Alongi et al. 2003;

Islam 2005). The necessity for a sustainable system for fish production is especially

important when aquaculture is expected to produce over half the total fish consumed by

the world population by 2030 (FAO 2000.) In the latter half of the 20th century,

aquaculture systems have changed toward environmentally sustainable facilities that

have little or no impact on the coastal ecosystems or waterways (Kupren et al. 2008).

For many years, scientists have been developing the vision of recirculating aquaculture

systems that require less land, have lower water impact, allow environmental control for

year-round production, and offer the flexibility to be located near prime markets (Masser

et al. 1992).

Recirculating aquaculture systems, or closed-system aquaculture, use a

recirculating water system instead of a flow-through design. In a study comparing the

energy use, water dependence, and environmental impact of a recirculating and flow-









through trout systems on the same waterway, it was shown that the water use of a

recirculating system was 93% less than that of a flow through design (D'Orbcastel et al.

2009). Also, the eutrophication potential of the recirculating system was 26-38% lower

than that of the flow-through system because of reduced waste release (D'Orbcastel et

al. 2009). Although d'Orbcastel et al. (2009) found that the recirculating systems used

24-40% more energy than traditional aquaculture systems due to aeration and water

treatment, it was acknowledged that early recirculating systems could be engineered

using biofilters and airlift designs that would reduce their energy use. Airlift is a cost-

effective design that provides water movement and aeration (Pfeiffer and Wills 2007).

Much work is underway on the exact design of the most effective and cost-efficient

biofilters, but in fact, many recirculating systems today already use biofiltration and airlift

technology as a means of water treatment (Pfieffer and Wills 2007). The technology of

recirculating aquaculture systems is rapidly advancing to limit environmental impact and

increase cost-effectiveness, but there are still many concerns about whether

recirculating systems can produce sufficient marine finfish to meet the present and

future demands.

One of the biggest challenges aquaculture managers are currently facing in

recirculating aquaculture systems is the inconsistency of reproductive success (the

production of fertilized eggs) (Holt et al. 2007; Cejko et al. 2008; Kucharczyk et al.

2008). The individual factors needed for reproductive success are predictable

spawning, the production of viable gametes, spawning participation, and stable

fertilization kinetics. Some causes for the reproductive breakdown specifically in

recirculating systems may be due to A) the lack of environmental stimuli, such as









temperature, photoperiod, and lunar phase, B) spatial constraints of depth and volume,

and C) a lack of spawning stimuli due to group size, unnatural spawning sex ratios, and

the absence of hormonal cues.

Substantial research has been done on photoperiod manipulation and spawning

success in marine finfish aquaculture. By advancing or delaying the annual photoperiod

in relation to the natural changes in the environment, researchers found that they could

control the spawning season of marine finfish (Roberts et al. 1978; Campos-Mendoza et

al. 2004; Van der Meeren and Ivannikov 2006). In some cases, Atlantic cod were

induced to have two spawning seasons each year, instead of only one, by truncating the

annual photoperiod (Van der Meeren and Ivannikov 2006). In many marine species,

specific changes in temperature can be an indicator of when to spawn and may actually

affect egg quality. For example, Atlantic halibut (Hippoglossus hippoglossus) that

experienced a temperature drop in late December were induced to spawn in the spring

and produced more eggs with a higher fertilization and hatch rate than fish that

experienced a temperature rise in late December (Brown et al. 2006). These important

environmental cues can be tightly controlled and manipulated with today's aquaculture

equipment, and thus fish can be induced to spawn via these techniques. Nonetheless,

there remain other problems that have not been adequately addressed in recirculating

aquaculture.

The problem of poor gamete quality goes deeper to the physiological level. Often

females will not ovulate in captivity as they do in their natural habitat. Previous studies

have hypothesized that this failure to ovulate is due to a lack of a luteinizing hormone

(LH) surge from the pituitary (Zohar and Mylonas 2001). Standard practices of









spawning fish in captivity include artificial injection of hormones that simulate LH-like

activity and induce the oocytes to undergo final oocyte maturation and ovulation

(Shirashi et al. 2005). In standard aquaculture spawning practices, females undergo

cannulation, or oocyte biopsy, to examine the stage of the oocytes. As described by

Shiraishi et al. (2008), only females with oocytes predominantly in the late stages of

vitellogenesis are selected for hormone injection (Ibarra-Castro and Duncan 2007). In

addition to female examination and injection, males are also examined for milt flow,

percent sperm motility, and duration of sperm motility. For example, in spotted rose

snapper (Lutianus guttatus), males were sampled with haematocrit tubes, the sperm

analyzed at 400X magnification, and the sperm was observed for motility (Ibarra-Castro

and Duncan 2007).

Two of the most widely used hormones to induce fish ovulation are human

chorionic gonadotropin (HCG) and gonadotropin releasing hormone analog (GnRHa)

(Shiraishi et al. 2008). Both hormones are injected into the fish intramuscularly. The

exact timing of the induced-spawn can be variable. In an experiment done with chub

mackerel (Scomberjaponicas), ovulation occurred about 33 hours post HCG-injection

and 36 hours post GnRH-injection (Shiraishi et al. 2008). However, in red snapper

(Lutjanus campechanus), most females ovulated between 24-32 hours post HCG-

injection and larval survival rate was highest when females spawned during this time

period (Bourque and Phelps 2007).

These hormone techniques are useful to induce mature fish to release gametes,

but little is known about the participation of individuals within the group during spawning.

Similarly, little is known about the actual fertilization kinetics in recirculating systems.









Historically, mixed-milt fertilization, or fertilization that includes combining eggs and milt

from many individuals in a single container (Wedekind et al. 2007), has been widely-

used to produce fertilized eggs, with varying success (Hoff et al. 1972; Kloth 1980).

However, this method of mixed-milt fertilization has been discouraged due to decreased

genetic variation from sperm competition, increased artificial selection, and

domestication of the stock population (Wedekind et al. 2007). The restocking of wild

fish populations is an important part of management, and both genetic variability and

quality of the stock must be maintained through natural spawning.

Due to the high water and land requirements of pond aquaculture and the effects

of effluent loading on the environment in coastal cages, the most environmentally

sustainable option for producing high quantities of fish remains recirculating aquaculture

systems. While research has been done on controlling environmental cues and

hormonal levels that facilitate spawning, much research is still needed to increase

annual fertilized egg yield. The specific questions for future progress in recirculating

systems will be can they provide a suitable environment for natural spawning behavior?

And can gamete production be optimized or at least controlled to produce consistent

yields of fertilized eggs? Once researchers have consistent production of fertilized

eggs, the next step, which has already been undertaken, is to develop cost-effective

systems to hatch eggs and rear juveniles (for a review of marine finfish larviculture

techniques see Lee and Ostrowski 2001).









CHAPTER 2
SPAWNING BEHAVIOR AND GAMETE AVAILABILITY OF FLORIDA POMPANO

Introduction

Florida pompano is one species that is not recovering as expected under its

overexploitation recovery plan. Economically, Florida pompano is one of the most

desirable marine fish with a price per pound (Retail market prices in 2005 were around

$8.00/Ib; Main et al. 2007) that is higher than most other US marine and freshwater fish

(Weirich et al. 2005). As a fish with such high market demand, it is not surprising that

the pompano stock condition was listed as decreasing due to overfishing for the years

1998-2007, based on catch-rate information (FWRI 2008). To limit the impact of

overexploitation and decline of natural populations as well as create an economically

lucrative industry, aquaculture techniques for the Florida pompano have been in

development for more than 40 years. Unfortunately, a recirculating system that

produces reliable yields of fertilized eggs has yet to be developed.

Both HCG and GnRHa can be used to induce spawning in Florida pompano, and

it has been documented that spawning occurs about 36 hours post-injection (Weirich et

al. 2005). In addition to hormone treatments, temperature shifts from 74 to 78F (24 to

26C) to 86 to 88F (30 to 31 C) can induce natural spawning when combined with a

photoperiod ranging from 11 hours (winter) to 13 hours (summer) (Hoff et al. 1972,

1978a, b; Kloth 1980; Weirich et al. 2005; Main et al. 2007; Weirich and Riley 2007).

Unfortunately, even while using these spawning methods, fertilization rates are highly

variable (0% to 91%, Main et al. 2007) and remain a large problem when producing

pompano on a commercial scale. While pompano can be conditioned to spawn under

artificially controlled conditions of photoperiod, temperature and hormone









injection/implantation, new aquaculture techniques must be developed to improve

fertilized egg yield.

One possible explanation for low fertilized egg yield in many marine fish,

including the Family Carangidae of which pompano are a member of (Graham and

Castellanos 2005), is that they naturally spawn in large groups (involving 100s of

individuals) and in captivity the group size is small and the quarters too confined. These

factors may inhibit natural spawning behavior and hence the production of fertilized

eggs. Few observations of pompano group-spawning in the field have been made, but

group spawning behavior is often stereotyped across taxa. Graham and Castellanos

(2005) described the spawning behavior of two members of the family Carangidae (the

permit, Trachinotus falcatus, and the yellow jack, Carangoides bartholomaei), of which

pompano is also a member. In permit and yellow jack, Graham and Castellanos (2005)

reported schools of at least 300 individuals aggregating about an hour before sunset.

As spawning began, subgroups of the large school broke off as a female swam rapidly

toward the surface followed by multiple males and followers. The female would stop

approximately 5 m from the surface and vibrated rapidly, releasing a cloud of eggs, the

males released their sperm essentially simultaneously and in very close proximity to

the female (Graham and Castellanos 2005). These spawning subgroups are very

typical in group-spawning species, however data have not been collected to determine if

females spawn once and then leave the aggregation, or if they spawn multiple times in

the same event. In wrasses, for example, females come to the aggregation, and spawn

a single clutch of eggs, and then leave (Colin 2010). Replicating this dramatic spawning









behavior in a tank setting may be an important limitation to high fertilized egg yield in

aquaculture.

Past attempts to spawn pompano have involved selecting individuals with mature

gametes, injecting them with a hormone, and either strip-spawning or allowing the

individuals to spawn in a separate tank (Hoff et al. 1978; Kloth 1980; Weirich and Riley

2007). While these techniques may produce a larger proportion of fertilized eggs,

pompano are especially delicate fish, and susceptible to the detrimental effects of

excessive handling. To maintain the effects of sexual selection and to limit handling of

the fish, natural group spawning behavior was the focus of our study (Forsberg 2008).

In this study, the entire group was allowed to participate in the spawn.

In this study, I examined whether pompano exhibit natural spawning behavior in

a commercial recirculating aquaculture system. This was done using video analysis of

spawning and by comparing the number of participants, spawning rush duration, and

spawning behavior to the limited descriptions of natural group spawning in carangids.

Video analysis of pompano group spawning behavior has never been conducted, and

the characterization of pompano spawning behavior in a recirculating system should

give an indication of whether pompano are receiving the necessary cues to promote

normal spawning behavior. In addition, the proportions of males and females producing

mature gametes vs. those adults that are found without mature gametes was assessed,

and from that analysis, a refined spawning schedule is proposed with the focus on

developing a schedule that provides consistent yields of fertilized eggs each season.









Materials and Methods

Study Facility and Housing Conditions

The work reported herein was conducted at the Mote Aquaculture Research Park

(MAP) in Sarasota, FL, a field station of the Mote Marine Laboratory. This private

marine research organization is dedicated to increasing both marine knowledge and

education in the community. Although Mote Marine Laboratory was built in 1955, the

Mote Aquaculture Park was established in 2001 and primarily serves as a facility to

further the development of sustainable marine and freshwater aquaculture technology.

In 2009, Mote Aquaculture Research Park housed 20-30 adult pompano in each

of two cylindrical indoor recirculating tanks (Groupl and Group2). Our study focuses

only on Groupl because a more complete data set from Groupl was collected. The

tanks are each 4.57m (15 ft) diameter 1.52m (5 ft) deep with a volume of about

28,000L. To maintain tank clarity and water quality there was a 0.085 m3 drop filter

(Aquaculture Systems Technologies, L.L.C, New Orleans, La) for solids separation, a

900-L moving bed for biofiltration, and 2 150-W High Output SMART HO UV units

(Emperor Aquatics, Inc, Pottstown, PA). The tanks were maintained at a temperature

of 28 1.00C during the spawning season. Salinity was held at 35 0.5ppt, D.O. at a

range of 24 and 59ppm and pH maintained between the ranges of 7.5 and 8.5. Lighting

was controlled in each tank using Solar 1000 series dimmer (BlueLine Aquatics, San

Antonio, TX), a system designed to simulate solar and lunar events. These events

closely followed natural solar and lunar patterns of the pompano spawning season of

February-October. Sex ratios began at 19M:10F during the first sampling and changed

to 14M:10F in April.









Experimental Procedures

Beginning February 2009 and continuing until October 2009, the pompano were

artificially induced to spawn every other month. Handling was restricted to one day

before the spawn. The purpose of this handling was to sample the gametes of the fish

over the course of the spawning season. Statistical tests (Fisher's exact tests, a=0.05

and unpaired t-tests) were performed to analyze any changes in gamete maturity

throughout the season. During fish handling, the water level of the tanks was dropped

by 60%, lab technicians corralled fish and one-by-one anesthetized the fish using a

separate tank filled with water and tricaine methanesulfonate (MS-222) at a

concentration of 200mg/L. Once the fish were anesthetized, weight, length and the

presence/absence of mature gametes were assessed. The presence of mature oocytes

was assessed using a cannulation biopsy. Oocytes recovered from the biopsy were

measured and staged. When oocytes were in vitellogenic or post-vitellogenic stages, at

least 450 pm in diameter, and the macronucleus had migrated to one side, the oocytes

were considered mature. Females with oocytes that were less than 450 pm in diameter

were considered to contain no mature gametes. All mature females were injected with

a maturation and ovulation-advancing hormone during each handling period. Females

were either injected with human chorionic gonadotropin (HCG), Ovaplant (active

ingredient Salmon gonadotropin sGnRHa), or Ovaprim (active ingredients Salmon

gonadotropin sGnRHa and Domperidone, a dopamine inhibitor), see below for more

detail.

The assessment of males involved determining the amount of milt that could be

manually expressed as well as measuring sperm motility using a compound

microscope. Percent motility as well as motility duration was recorded when a milt









sample could be obtained. Males were considered to have mature gametes when milt

was free-flowing when expressed and when sperm was active for at least 15 sec.

Males in which a sample was difficult or impossible to obtain, or whose sperm was not

motile under a microscope were categorized as having no mature gametes. Males

were injected with Ovaprim twice during the season to increase milt production, as

detailed below. After handling, the fish were revived by gill ventilation in the original

tank.

Hormone Induction of Spawning

Various hormones were used over the course of the breeding season in an

attempt to evaluate the different hormones' effects. During the course of the breeding

season, mature females were injected intramuscularly at each handling period with

HCG, Ovaplant, or Ovaprim. HCG (10001U/kg body weight) was injected in mature

females February, April, and September. In June, all females were injected with

Ovaprim (0.5ml/kg body weight for females with mature oocytes at the time of sampling,

0.25ml/kg body weight for females found without mature oocytes at the time of

sampling). In August and October, females were injected with Ovaplant based on size

(<1500g body weight, 75ug; >1500g body weight, 150ug). In August and October, all

males were injected with 0.5ml/kg body weight Ovaprim.

Behavioral Filming Equipment

Three underwater Neuros OSD (Neuros Technology International, LLC.,

Chicago, IL) cameras were used to record spawning behavior of the pompano during

February and April 2009. The cameras were installed near the bottom of the tanks with

a slight upward angle during the handling period. They were mounted on pvc pipe and

secured to the tank so water flow or fish interference would not dislodge the position









during filming. In February, data were captured on VHS tapes, but in April filming was

recorded directly to digital thumb drives. Three cameras were set to record from 1500-

1900 on February 12, 2009, and from 1330-1700 on April 8, 2009.

Behavioral and Statistical Analysis

The tapes were analyzed first by identifying possible spawning rushes. During

the tape analysis, the duration of the rush, number of participants, relative vertical

location at which the rush took place, whether eggs were found shortly after the rush,

and period between individual rushes were recorded. A Mann-Whitney U statistical test

(a=0.05) was performed to determine if there was a difference between the rush

intervals, rush durations, and number of participants between the February and April

spawns. Observed spawning rush behavior was categorized into false spawning

behavior (that which did not result in the release of eggs) and true spawning behavior

(that which did result in the release of eggs.) Differences between false spawning

behavior and true spawning behavior were determined by the measurable aspects of

the spawning rushes. During each spawning cycle, both fertilized and unfertilized eggs

were collected at the surface of the tank using a skimmer bar and deposited into an

external egg basket. It is important to note that only fertilized eggs are buoyant (Main et

al. 2007; Weirich and Riley 2007), however due to the efficient aeration of the

recirculating tank system, unfertilized eggs were also collected using the skimmer bar.

The total number of eggs was counted by volumetric estimates post-spawn, and the

percent fertilized eggs calculated. While not all fertilized eggs were able to be collected

via the skimmer bar due to the filtration system in the tank, the calculated percent

fertilized eggs is the maximum fertilization rate possible. Some egg samples were aged

under the microscope to estimate the spawning time that produced viable eggs.









Results


Male and Female Gamete Production

Handling data were recorded from February 2009 through October 2009 to

characterize variance in gamete production over the 2009 spawning season (Table 2-1).

On the first handling date in February 2009, the spawning group had the largest

proportion of males with mature gametes across all sampling dates (Figure 2-2). Ten of

19 males had mature gametes at the time of sampling (52%). By the next handling in

April 2009, the proportion of males with mature gametes significantly decreased

(Fisher's exact test; p=0.0191, a=0.05, df=1). Only 3 males out of 19 were found with

mature gametes at the time of sampling (16%). In June 2009, the proportion of males

with mature gametes was the lowest of the entire season, with only 1 male out of 14

total males (7%). The number of males with mature gametes increased slightly at the

next handling date, August 2009, to 2 males out of 14 total males (14%). By the next

sampling date, the proportion of males with mature gametes increased to the second

highest value of the season. September 2009, 5 males of 14 were found with mature

gametes (36%). The proportion of males with mature gametes on the last sampling

date, October 2009, was a moderate 3 males of 14 total males (21%). Thus the

proportion of males with mature gametes in the spawning group was highly variable,

with two peaks of male gamete maturity in February and September of the spawning

season. Although males were injected with Ovaprim in August and October, there was

no measurable difference in the proportion of males found with mature gametes in

February-June compared to August-October (unpaired t-test: t=0.09, p=0.9326, df=4,

se=0.153).









Female gamete maturity and fecundity was also recorded for the 2009 spawning

season (Table 2-1). Egg maturity was determined in part by egg diameter (Table 2-3).

Similar to the males, the highest proportion of females with mature oocytes was found in

February (70% females with mature gametes) and September (60% of females). For all

months other than February and September, the proportion of females with mature

oocytes was below 50% (April, 30%; June, 40%; August, 40%; October, 20%) (Figure 2-

2). Fecundity was also estimated by taking the total number of eggs collected and

dividing by the number of females with mature oocytes (Table 2-2). The largest number

of eggs collected in one spawning cycle was 324,000 eggs in September, 2009. During

this cycle, 6 females were found to have mature oocytes, and thus, the average number

of eggs per female was 54,000 eggs. The average number of eggs per female across

the 2009 season ranged from 32,500 eggs (August 2009) to 74,666 eggs (April 2009)

per female with mature oocytes.

Fertilized Egg Yield Across Spawning Cycles

In addition to gamete maturity and hormone injections, the total number of

fertilized eggs collected after each spawning cycle was recorded (Table 2-2). The two

peak time periods in fertilized egg collection corresponded to the months of peak male

gamete maturity. The largest numbers of fertilized eggs were collected in February

(112,128 fertilized eggs, 44% total eggs), April (127,904, 57%), and September

(139,639, 43%). From February to April, the number of fertilized eggs collected

increased slightly (112,128, 44%; 127,904, 57%), even though the numbers of females

with mature oocytes dropped by more than half; there were no fertilized eggs collected

in June. In August, only 47,970 (37%) fertilized eggs were collected, leading up to the









largest yield in September (139,639 fertilized eggs, 43%). In the final cycle, collected in

October, the number of fertilized eggs was very low at 20,893 fertilized eggs (29%).

False Spawning Behavior

Prior to gamete release, the majority of the school swam clockwise against the

current. However, rushes, where two to five fish broke out of the school and chased,

swam against the current, and stopped mid-swim occurred frequently. False spawning

rushes often included a sudden speeding up of a few fish to position themselves with

their flanks touching. The fish remained closely positioned for a few seconds, and then

the group broke up and the individual fish swam away. The closely swimming group

swam either up or down in the water column and often swam in the opposite direction to

the rest of the school. Their erratic swimming could be easily seen by flank flashing.

None of these rushes included vibrations, often associated with egg release, in any

participants.

False spawning rushes were captured April 2009 when recording began several

hours prior to the first collection of eggs at 13:30 (Figure 2-1). These false spawning

rushes began when one fish paused in the water column with a group of other

individuals closely surrounding it, and ended when it began swimming again. During

the false spawning rushes, those individuals not directly involved either ignored the rush

and kept swimming or followed the false spawning group in the direction of the current.

By 14:42, the school still swam against the current, but more often they either stopped

swimming or swam in the other direction. The school looked like a disorganized group

and individuals milling about in any direction.

The false spawning rushes in April 2009 began at 14:42 and continued until about

14:57. There were at least 6 false spawning rushes that lasted on average 3.6 sec









(SD=1.397 sec) with an average of 3.4 participants (SD=0.976 sec). The average time

interval between false spawning rushes was 2 min 59 sec (SD=2.018 min). These

events were undoubtedly false spawning rushes because there were still no eggs found

at 15:08. The true spawning rushes in April 2009, characterized by the female vibrating

during release of gametes, began at 16:07 and lasted until 16:37. No false spawning

rushes were observed in February 2009 (Figure 2-1).

True Spawning Behavior

A characteristic true spawning rush involved five or more individuals, with one

female in the center of a close group of 4-10 other fish, pausing in the water column.

The female then vibrated her whole body for 5-12 sec as she released eggs into the

water column. This extended period of female vibration was the most obvious

difference between true spawning and false spawning rushes. The group of fish

surrounded her and swam slightly behind her while remaining in close contact with her,

and the males presumably released their sperm as she released eggs (a milt cloud was

difficult to see due to the aeration of the tank). The whole group was either stationary in

the water column or swimming very slowly. All true and false spawning rushes occurred

between the midlevel and the upper half of the tank (20.76m from the tank floor).

The focal breeding group was injected between 10:15 and 12:00 on February 11,

2009, and eggs were found approximately 30 hours later, at 17:06 on February 12,

2009, in the 2-4 egg cell stages (eggs in this stage of cell division are 0-15 min post

fertilization). Fourteen true spawning rushes were observed between the time frame of

16:41 and 17:32 (Figure 2-1) despite the fact that only 8 females were found with

mature oocytes during handling on February 11,2009. The average number of

participants in each true spawning rush was 8.0 individuals (SD=2.5 fish), and the









average duration of the true spawning rushes was 7.7 sec (SD=2.79 sec). The

average time period between true spawning rushes was 3 min 54 sec (SD=2.70 min).

From this spawn cycle, a volumetric estimate of 112,128 fertilized eggs was found with

an estimated maximum fertilization rate of 44%.

When the focal breeding group was next induced to spawn on April 8, 2009, the

fish were injected between 10:15 and 12:00 on April 7, 2009. Eggs were found on April

8, 2009, approximately 30 hours later, at 16:30. Three cameras were set to record from

13:30-17:00 (Figure 2-1). Setting the camera to record earlier provided the opportunity

to view false spawning behavior. Beginning 16:07 and lasting until 16:37, while only 4

females were found with mature oocytes during handling on April 7, 2009, there were 8

recorded true spawning rushes that lasted on average 6.3 sec (SD=1.83 sec) with an

average of 5.5 participants (SD=1.5 fish). The average time interval between true

spawning rushes was 4 min 53 sec (SD=2.49 min). From this spawn cycle, an estimate

of 127,904 fertilized eggs was collected with an estimated maximum fertilization rate of

57%.

Comparisons Among True Spawning Rushes.

Mann-Whitney U tests were conducted to compare the February and April spawn

cycles. No differences were found between the true spawning rush intervals (Mann-

Whitney U test: U=38, p=0.336, a=0.05, ni=8, n2=13) or the true spawning rush

durations (Mann-Whitney U test: U=91.5, p=0.072, a=0.05, n1=9, n2=14). However,

significantly more fish participated in the true spawning rushes in February than in April

(February mean participants=8, April mean participants=5.5; Mann-Whitney U test:

U=102, p=0.0064, a=0.05, n1=9, n2=14) which correlates to the larger proportion of

adults found with mature gametes, and in particular males with mature gametes, in









February relative to April. On average, spawns included nearly as many males as were

sperm producing, suggesting either that males with mature gametes spawned

repeatedly or that males without mature gametes were participating in spawning rushes.

Discussion

Natural Spawning Behavior

From this study, we can conclude that natural pompano spawning behavior is not

inhibited by closed-system aquaculture. Clear distinctions between false spawning

rushes and true spawning rushes were quantified in the video analysis, although no

false spawning rushes were recorded for the February spawning event (Figure 2-1).

This may have been due to the later start time for recording spawning behavior at 15:00

in February and false spawning rushes were recorded in April at 14:42. The number of

participants for each spawning rush, was as expected for group spawning carangids

(Graham and Castellanos 2005). Natural observations of spawning groups of a close

relative to pompano, the permit, have been observed in which 5 to 9 individuals broke

off from the main group and swam toward the surface of the water, stopped about 15m

from the surface, and vibrated while releasing a puff of gametes (Graham and

Castellanos, 2005). While the average number of participants was different for the

February and April spawns, this was not surprising given the differing numbers of males

with mature gametes. Also, the group size and sex ratio used in our study appeared to

be acceptable for natural pompano spawning behavior.

Interestingly, there is strong evidence that females are participating in multiple

spawning rushes during each spawning cycle. In the February spawning cycle, 14

individual spawning events were observed, and out of 10 females in the group, only 7

were found with mature oocytes. Likewise, in the April spawning cycle, 8 spawning









rushes were observed and only 3 females with mature oocytes were identified. Thus it

would appear that on average 2 spawning rushes can be expected per fertile female. In

addition, the number of participants in each spawn also leads to the conclusion that

males or females that are not producing mature gametes are participating in the

spawning rushes. For instance, in April, only 3 males and 3 females were producing

mature gametes, but the spawning rushes consisted of 4-8 individuals. From this

information, it is possible that males with mature gametes are participating in multiple

spawning rushes, or that adults without mature gametes are also participating in

spawning behavior.

Limited Gamete Quantity

An obvious problem to consider further is the low percentage of individuals with

mature gametes. The percentage of males with mature gametes dropped significantly

over the 2009 spawning season. Low female fecundity of 0-74,600 eggs/female is also

a major consideration in our study. This fecundity is extremely low compared to early

natural estimates of 630,000 eggs (Finucane 1969), more conservative recent

estimations of 133,400- 205,500 eggs (Muller et al. 2002), and even captive stock

estimates of 123,000-772,000 eggs (Weirich and Riley 2007). The cause of this drop of

individuals with mature gametes could be the frequency of injection and spawning

throughout the season. From natural observations of juveniles, pompano appear to

have an extended spawning season that stretches from April to October, with a main

spawning peak found in spring (April-June) and another spawning peak in late summer

or early fall (July-October) (Finucane 1969). These peaks have been identified based

on the documented size of juveniles caught within that time period (Finucane 1969). A

supporting study reports young-of-year (YOY) juvenile pompano were collected off the









coast of Florida between April and November, with the majority of YOY collected in May

(Solomon and Tremain 2009). In addition, as many as four size categories of juveniles

have been collected at one time (Finucane 1969), indicating that pompano spawn over

a protracted period and may spawn multiple times during each spawning season.

Therefore, it is entirely possible that while natural populations are able to spawn the

entire season (April to October), with peaks of spawning in spring and late

summer/early fall, that individual fish are spawning less frequently and with some

recovery period between spawning activity.

Applying this hypothesis to the aquaculture setting, it may be more effective to

induce spawning once or serially in quick succession within the natural spawning peaks,

but with an extended resting period between peaks. For instance, instead of inducing

pompano to spawn every 60 days from February through October, an experiment where

pompano are induced to spawn once a month in March, April, and May, are permitted to

recover June and July, and then induced to spawn once a month in August, September,

and October may yield higher total eggs as well as a higher fertilization success rate.

By scheduling serial spawns within the two natural spawning peaks and a 90-day

recovery period between the two peaks, both males and females may produce a larger

quantity of sperm and eggs available to be induced with hormone implants. There is

evidence that even with serial spawning in a shortened period of time, fecundity,

fertilization rates, and spawning activity are not severely decreased when fish are

induced with GnRHa. By GnRHa implantation, pompano were induced to spawn six

times from July-October, and each spawning cycle lasted 2-6 days with average yields









of 150,000 to 772,000 eggs/female and average percent fertilization (floating eggs) of

81.8 to 96.9% (Weirich and Riley 2007.)

Sperm quantity could also be a limiting factor for egg fertilization when fish are

repeatedly induced to spawn over a protracted season. This sperm limitation

hypothesis is supported by the dual peaks of males gamete maturity found in our study.

The dual peaks of males with mature gametes found in February and September are

slightly further apart than the supporting data reflects, but both the data in our study and

natural observations indicate that the first spawning cycle of the year is the largest

(Finucane 1969). Hence the maximum proportion of males with mature gametes found

in February as the first spawn of the season corresponds to the natural first spawn in

April. In September, the number of males with mature gametes again spiked,

correlating to the natural second spike recorded by Finucane (1969). Between these

two peaks, the number of males with mature gametes drops to such low proportions

that the sperm produced is insufficient to give an appreciable yield of fertilized eggs.

Another possible explanation for poor gamete maturation in both males and

females could be nitrate concentrations in the broodstock tanks. Recirculating

aquaculture systems often have a higher nitrate level than natural water systems, and it

has been show that elevated levels of nitrate (57 1.52 mg/L) can disrupt endocrine

function in female Siberian sturgeon (Acipenser baeri) (Hamlin et al. 2008). Measured

NO3 levels in the pompano tanks from August-December 2009 averaged from 55.83

mg/L (Nov. 2, 2009) to 103.64 mg/L (Aug. 13, 2009) with a median of 84.62 mg/L.

These high nitrate levels could have contributed not only to the low female fecundity but

also to the low proportion of males with mature gametes. Nitrate concentrations are









now being measured and adjusted more frequently to prevent high levels of nitrate in

the system.

Hormone treatments have been used to increase gamete production over a

spawning season. GnRHa and HCG implants have been shown to increase

spermiation in fish such as the European Sea Bass (Dicentrarchus labrax) and

Japanese eels (Anguilla japonica) as well as in a variety of freshwater and marine fish

(Rainis et al. 2003; Dou et al. 2007). In Pompano, the use of a GnRHa pellet produces

greater egg production, higher fertilization rates, and a spawning period of up to 6 days

when compared to HCG (Weirich and Riley 2007). While GnRHa compounds (Ovaplant

and Ovaprim) were used in our study, on both males and females, use was limited and

there were no indications of increased spermiation or fecundity during the time which it

was used. The most likely reason that the proportion of fish with mature gametes did

not increase after the treatments is because the product used was not a slow-releasing

hormone, and it is sustained release of GnRHa over a period of days to weeks in male

fish that results in the most consistent elevation of spermiation (Zohar and Mylonas

2001). To increase gamete maturation, a GnRHa product that is slow-release, such as

a pellet, microsphere, or monolithic implant (Zohar and Mylonas 2001), and that is

applied consistently over the spawning season should be attempted.

Improving Fertilization Rates

While fertilization rates of field spawning data for pompano and related jacks are

unavailable, some studies have focused on another species of group spawners, e.g.,

the blueheaded wrasse (Thalassoma bifasciatum). The mean fertilization success

(defined as percent of total eggs that were successfully fertilized) for 304 field collected

spawns of T. bifasciatum was 76.5% (Petersen et al. 1992). In a captive pompano









spawning event in which one female was induced to spawn with four males, was taken

out of the tank, and another female was induced to spawn with the same four males, an

estimated total egg yield of 125,000 for each female was collected, with a fertilization

rate of 52% (Kloth 1980). More recently, a study by Weirich and Riley (2007) revealed

high female fecundity (on average 234,000 eggs per female) and high fertilization rates

of over 70% when pompano were injected with a GnRHa slow-release pellet and

allowed to spawn in a recirculating system. Maximum fertilization rates in our study

were less than 50% in most spawning cycles. One factor that may contribute to low

fertilization success is that in our study, is that we considered eggs 2450 pm in diameter

mature. Studies with higher fertilization success categorize eggs 2500 pm in diameter

as mature, and thus only induce spawning in those females with eggs 2500 pm in

diameter (Weirich and Riley 2007). While egg diameter is less reliable than

macronucleus placement in the determination of egg maturity, some of the eggs

released by our females may not be sufficiently mature to be fertilized.

Another issue that could be hindering fertilization success is the flow rate in the

tank. Analysis of natural spawns of T. bifasciatum revealed that fertilization success

(percent eggs fertilized) is higher in calmer water currents than in rough, turbulent

currents (Petersen et al. 1992). The flow rate of the recirculating system may be a

problem especially with smaller egg sizes. In sea urchins, larger eggs were found to

have significantly higher fertilization rates than smaller eggs (Levitan 1993), primarily

because they are easier to find by sperm in a turbulent or fast-moving environment.

Recirculating systems may have low fertilization success due to the strong current in the

tank which potentially disperses sperm and eggs too quickly. Especially with the









smaller eggs induced in our study, if the flow of the tank was disruptive enough, it could

prevent the sperm from fertilizing the eggs in the brief window of the spawning event.

To test the effects of water flow on fertilization rates, pompano should be induced to

spawn in the tanks with the aeration turned off, and the fertilization rates should be

compared to those achieved when fish are spawned with normal tank aeration and

pump systems.

Conclusion

The factors that hamper the successful and consistent spawning of pompano in

recirculating aquaculture systems are still largely unknown. However, from our study, it

is clear that the limiting factors of space and external cues in the recirculating system

are not hindering natural group spawning behavior in the pompano. In fact, strong

evidence for distinctions between false spawning and true spawning behavior have

been found, as well as evidence for multiple spawning in both sexes. While pompano

exhibit normal spawning behavior in a recirculating aquaculture system, further study

must explore the factors of inconsistent gamete production and fertilization rates found

in this species. One method that may improve both these factors is inducing serial

spawns within the two natural peaks in the spawning season. In addition, further testing

with GnRHa hormones should be done to improve gamete production. Finally, the

effects of water movement during spawning should be examined.











Group 1--February 12, 2009


I I I I I I I I I I
1200 1300 1400 1500 1600 1700 1800 1900 200C
Group 2--April 8, 2009

Key:
Total Recorded Period
False Spawning Rushes
True Spawning Rushes
Eggs Collected



Figure 2-1. Spawning behavior video recording timeline


80'',. -

70%

60%

50%

40% -ercent Males with

30% M- ature Gametes

20% percent Females with
Mature Gametes
10%

0%

P, &3 !( RI
115\ t-II q\k IN\ lb\' K,"


Figure 2-2. Percent individuals with mature gametes









Table 2-1. Proportion males and females with mature oocytes spawning season
% Females
Ripe % Males with Ripe with Mature
Males Mature Gametes Females Gametes Sex Ratio Injection
2/11/2009 10 52.6% 7 70.0% 19M:10F HCG F
4/7/2009 3 15.8% 3 30.0% 19M:10F HCG F
6/12/2009 1 7.1% 4 40.0% 14M:10F Ovaprim F
8/11/2009 2 14.3% 4 40.0% 14M:10F Ovaplant F, Ovaprim M
9/14/2009 5 35.7% 6 60.0% 14M:10F HCG-F
10/13/2009 3 21.4% 2 20.0% 14M:10F Ovaplant F, Ovaprim M


Table 2-2. Fertilized egg yield of 2009 spawning season
Spawning Date Total Number of Eggs Fertilized Eggs Mature Female Fertilization Rate
2/11/2009 256,000 36,571 43.80%
4/7/2009 224,000 74,666 57.10%
6/12/2009 no eggs none no eggs
8/11/2009 130,000 32,500 36.90%
9/14/2009 323,988 53,998 43.10%
10/13/2009 72,800 36,400 28.70%










Table 2-3. Egg diameter and female measurements
Sampling
Date 2/11/2009 4/7/2009 6/9/2009
Egg Egg
Weight Length Diameter Weight Length Diameter Length
Female (g) (cm) (pm) (g) (cm) (pm) Weight (g) (cm) Egg Diameter (pm)
1 1020 34.5 424-530 1115 35.1 424-530 1240 37.0 239-318
No
2 745 31.5 318-398 845 32.5 Sample 995 33.9 398-477
3 2000 45.6 371-477 2195 45.9 504-557 2280 46.1 450-530
4 850 31.6 504-557 925 32.2 318-398 1070 33.8 292-371
5 975 34.2 504-583 1005 34.4 398-504 1035 34.9 504-557
6 670 31.3 398-477 755 32.5 80-133 860 33.0 424-504
No
7 830 33.2 80-133 920 34.2 Sample 1070 35.3 80-133
8 900 34.1 424-530 1040 35.4 80-133 1215 37.1 80-133
9 705 31.5 398-504 755 32.1 80-133 770 32.2 185-265
10 685 30.7 53-80 780 31.8 80-133 860 32.8 106-159
8/11/2009 9/14/2009 10/13/2009
1 1510 39.5 53-80 1700 40.1 477-557 1755 40.8 159-212
2 955 34.2 477-530 1365 37.2 504-583 1430 38.2 80-133
3 2515 47.0 530-583 2535 47.2 530-610 2520 47.4 133-212
4 1300 36.3 80-133 1445 37.0 318-398 1560 37.0 371-424
5 1160 36.3 504-557 1195 36.5 239-292 1240 36.9 345-398
6 985 34.1 477-530 1000 34.2 504-610 1055 32.0 504-583
7 1270 37.4 80-133 1350 37.8 159-292 1460 38.6 212-292
8 1515 39.7 80-133 1620 40.0 318-371 1745 41.1 239-318
9 860 33.5 53-80 895 33.5 371-451 970 35.0 106-159
10 1075 35.0 106-159 1190 35.7 424-504 1280 36.5 504-583









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BIOGRAPHICAL SKETCH

Elizabeth Ann Dippon was born in Gainesville, FL in 1986. When she was three,

she and her parents moved to Oregon for her father to pursue a career with the Bureau

of Land Management. Elizabeth was raised in Portland and has loved animals and

music from a very young age. She has enjoyed playing the piano since age 6 and

learned the marimba in high school. Elizabeth enjoyed much success with music during

high school, earning three 2nd Place Awards at the State Music Festival for Marimba.

She graduated as Valedictorian from Westview High School in 2004. She earned a

B.A. in music performance as well as a B.S. in zoology from the University of Florida in

2008. Elizabeth pursued an M.S. in zoology in 2010 from the University of Florida.

Elizabeth was married to Jesse J. Reynolds in May 2008. Elizabeth and her

husband Jesse are very active in health and fitness. They own a youth performance

center and personal training studio, Accel Sports, Inc. in Jacksonville, FL as well as a

nutritional business, Advocare. Elizabeth loves helping her husband coach a nationally-

ranked Olympic Weightlifting team and helping people achieve their physical and

financial goals. Elizabeth is committed to helping others in health and personal

development and attributes all her accomplishment to her Lord and Savior Jesus Christ.

She is also especially thankful for all the support from her family, her husband, her

friends, and Henry.





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1 ANALYSIS OF SPAWNING BEHAVIOR AND GAMETE AVAILABILITY OF THE FLORIDA POMPANO ( Trachinotus carolinus ) IN A RECIRCULATING AQUACULTURE SYSTEM. By ELIZABETH A. REYNOLDS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSI TY OF FLORIDA IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Elizabeth A. Reynolds

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3 ACKNOWLEDGMENTS I would like to thank the chair and the members of my committe e for their mentoring and advice, the Mote Marine Laboratory for the use of their facilities and collaboration, and my loving parents, grandparents, husband, and friends who encouraged me and motivated me through this study.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF TABLES ................................ ................................ ................................ ............ 5 LIST OF FIGURES ................................ ................................ ................................ .......... 6 ABSTRACT ................................ ................................ ................................ ..................... 7 CHAPTER 1 MARINE FINFISH AQUACULTURE SYSTEMS AND RELATED REPRODUCTIVE CHALLENGES ................................ ................................ ............ 9 2 SPAWNING BEHAV IOR AND GAMETE AVAILABILITY OF FLORIDA POMPANO ................................ ................................ ................................ ............. 15 Materials and Methods ................................ ................................ ............................ 18 Study Facility and Housing Conditions ................................ ............................. 18 Experimental Procedures ................................ ................................ ................. 19 Hormone Induction of Spawning ................................ ................................ ...... 20 Behavioral Filmi ng Equipment ................................ ................................ .......... 20 Behavioral and Statistical Analysis ................................ ................................ ... 21 Results ................................ ................................ ................................ .................... 22 Male and Female Gamete Production ................................ .............................. 22 Fertilized Egg Yield Across Spawning Cycles ................................ .................. 23 False Spawning Behavior ................................ ................................ ................. 24 True Spawning Behavior ................................ ................................ .................. 25 Comparisons Among True Spawning Rushes. ................................ ................. 26 Discussion ................................ ................................ ................................ .............. 27 Natural Spawning Behavior ................................ ................................ .............. 27 Limited Gamete Quantity ................................ ................................ .................. 28 Impr oving Fertilization Rates ................................ ................................ ............ 31 Conclusion ................................ ................................ ................................ .............. 33 LIST OF REFERENCES ................................ ................................ ............................... 37 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 42

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5 LIST OF TABLES Table page 2 1 Proportion males and females with mature oocytes spawning season ............... 35 2 2 Fertilized egg yield of 2009 spawning season ................................ .................... 35 2 3 Egg diameter and female measurements ................................ ........................... 36

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6 LIST OF FIGURES Figure page 2 1 Spawning behavior video recording timeline ................................ ...................... 34 2 2 Percent individuals with mature gametes ................................ ........................... 34

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7 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ANALYSIS OF SPAWNING BEHAVIOR AND GAMETE AVAILABILITY OF TH E FLORIDA POMPANO ( Trachinotus carolinus ) IN A RECIRCULATING AQUACULTURE SYSTEM. By Elizabeth A. Reynolds August 2010 Chair: Colette M. St. Mary Major: Zoology Marine finfish recirculating aquaculture systems are a viable way to produce environmenta lly sustainable stock. However, one of the biggest challenges production managers face in recirculating systems is that fertilized egg yields are low or inconsistent. An economically valuable fish that is currently in population decline is the Florida po mpano ( Trachinotus carolinus ). This is a preliminary study that focuses on the factors that contribute to the low fertilized egg yields of the Florida pompano in the recirculating aquaculture facility at Mote Aquaculture Research Park, a field station of the Mote Marine Laboratory. In this study, we used video analysis of spawns in combination with sampling and fertilized egg yield data to determine A) if pompano will exhibit natural spawning behavior in a recirculating system, B) does pompano gamete matu rity remain consistent throughout the entire February October spawning season, and C) what are the fertilization rates of pompano spawns in recirculating systems, and how does it compare to other captive pompano fertilization rates. From our data, we dete rmined that pompano spawning behavior is not inhibited by recirculating aquaculture systems. In fact, both false (spawning behavior without the release of

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8 gametes) and true spawning behavior (with the release of gametes) occurred and were quantified. We also found that the production of mature gametes in males is highly variable and possibly the true cause of low fertilized egg yield. A relatively high proportion of males had mature gametes at the beginning (53%, February) and end (36%, September) of the spawning season, but in June, only 7% of males had mature gametes at the time of sampling. Female gamete maturity also peak ed in February and September (70% and 60% of females had mature gametes in these months, respectively) but female gamete maturity did not plunge significantly like the male gamete maturity did. This pattern in gamete maturity could reflect a need for a 90 day break in the middle of the spawning season so adults can produce more mature gametes. Finally, fertilization rates of 0% 57% were much lower than other captive pompano studies and were also variable across the spawning season. Some causes for these low fertilization rates could be smaller egg sizes, small egg fecundity, and sperm limitation. Strong flow rate in the tanks dur ing spawning could also cause fertilization dysfunction.

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9 CHAPTER 1 MARINE FINFISH AQUAC ULTURE SYSTEMS AND R ELATED REPRODUCTIVE CHALLENGES The struggle between the demand for commercial fishing and conservation has proved to be a tumultuous battle. In re sponse to this problem and the ever increasing demand for food fish, the development of recirculating aquaculture systems has been proposed as an environmentally friendly and sustainable solution to the demands for commercial fish. Over the past century, commercial fishing for finfish has increased exponentially (Watson and Pauly 2001). During the 1980s, overfishing was such a threat to marine species that the government passed the Magnuson Stevens Fishery Conservation and Management Act in 1996 that requ ired an end to over exploitation of fish populations and mandatory rebuilding of fish stocks (Rosenberg et al. 2006). Unfortunately, even though there is evidence that biomass in stocks managed under rebuilding plans has the potential to increase, overfis hing still occurs in 45% of these stocks (Rosenberg et al. 2006). Perhaps the most vulnerable species to overexploitation are those that predictably spawn in the same area each season (de Mitcheson et al. 2008). For example, seasonal spawning aggregation s seen in many shallow water grouper such as Nassau grouper ( Epinephelus striatus ), Gag Grouper ( Mycteroperca microlepis ), and Scamp Grouper ( Epiniphelus morio ) are often the target of commercial fishing due to their predictability (Coleman et al. 1996; de Mitcheson et al. 2008). The risk of rapid stock decline is increased as a result. To combat the decline in fish populations due to overfishing and other causes of stock decline (e.g. development, habitat degradation, and pollution), agencies have attem pted to increase marine fish yields through restocking natural populations (increase spawning biomass), fisheries enhancement (overcome recruitment limitation),

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10 and sea ranching (put, grow, and take method) (Bell et al. 2008). All of these supplemental ad ditions first require the production of juveniles (Bell et al. 2008). The principle method of producing juveniles for release is through aquaculture. Aquaculture is by no means a new system for producing fish. Historically, aquaculture involved produci ng fish in inland ponds or coastal cages (Losordo et al. 1998; Holmer et al. 2003). Unfortunately, aquaculture in inland ponds relies on large amounts of water (as much as 1 million gallons per acre to fill a pond, and an equal amount every year to compen sate for evaporation, and seepage; Losordo et al. 1998), a large land area, and its productivity is limited by environmental factors. In addition, the costs of nutrient effluent, shifts in biodiversity, and changes in water chemistry have caused coastal c ages to come under scrutiny (Holmer et al. 2003; Alongi et al. 2003; Islam 2005). The necessity for a sustainable system for fish production is especially important when aquaculture is expected to produce over half the total fish consumed by the world pop ulation by 2030 (FAO 2000.) In the latter half of the 20 th century, aquaculture systems have changed toward environmentally sustainable facilities that have little or no impact on the coastal ecosystems or waterways (Kupren et al. 2008). For many years, scientists have been developing the vision of recirculating aquaculture systems that require less land, have lower water impact, allow environmental control for year round production, and offer the flexibility to be located near prime markets (Masser et al 1992). Recirculating aquaculture systems, or closed system aquaculture, use a recirculating water system instead of a flow through design. In a study comparing the energy use, water dependence, and environmental impact of a recirculating and flow

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11 thr ough trout systems on the same waterway, it was shown that the water use of a 2009). Also, the eutrophication potential of the recirculating system was 26 38% lower t han that of the flow 24 40% more energy than traditional aquaculture systems due to aeration and water treatment, it was acknowledged that early recirculating systems could be engineered using biofilters and airlift designs that would reduce their energy use. Airlift is a cost effective design that provides water movement and aeration (Pfeiffer and Wills 2007). Much work is underway on the exact design of the most effective and cost efficient biofilters, but in fact, many recirculating systems today already use biofiltration and airlift technology as a means of water treatment (Pfieffer and Wills 2007). The technology of recirculating aquaculture systems is rapidly advancing to limit environmental impact and increase cost effectiveness, but there are still many concerns about whether recirculating systems can produce sufficient marine finfish to meet the present and future demands. One of the biggest challenges aquaculture managers are currently facing in recirculating aquaculture systems is the inconsistency of reproductive success (the production of fertilized eggs) (Holt et al. 2007; Cejko et al. 2008; Kucharczyk et al. 2008). The individual factors needed for reproductive success are predictable spawning, the production of viable gametes, spawning participation, and stable fertilization kinetics. Some causes for the reproductive breakdown specificall y in recirculating systems may be due to A) the lack of environmental stimuli, such as

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12 temperature, photoperiod, and lunar phase, B) spatial constraints of depth and volume, and C) a lack of spawning stimuli due to group size, unnatural spawning sex ratios and the absence of hormonal cues. Substantial research has been done on photoperiod manipulation and spawning success in marine finfish aquaculture. By advancing or delaying the annual photoperiod in relation to the natural changes in the environment, r esearchers found that they could control the spawning season of marine finfish (Roberts et al. 1978; Campos Mendoza et al. 2004; Van der Meeren and Ivannikov 2006). In some cases, Atlantic cod were induced to have two spawning seasons each year, instead o f only one, by truncating the annual photoperiod (Van der Meeren and Ivannikov 2006). In many marine species, specific changes in temperature can be an indicator of when to spawn and may actually affect egg quality. For example, Atlantic halibut ( Hippogl ossus hippoglossus) that experienced a temperature drop in late December were induced to spawn in the spring and produced more eggs with a higher fertilization and hatch rate than fish that experienced a temperature rise in late December (Brown et al. 2006 ). These important equipment, and thus fish can be induced to spawn via these techniques. Nonetheless, there remain other problems that have not been adequately address ed in recirculating aquaculture. The problem of poor gamete quality goes deeper to the physiological level. Often females will not ovulate in captivity as they do in their natural habitat. Previous studies have hypothesized that this failure to ovulate i s due to a lack of a luteinizing hormone (LH) surge from the pituitary (Zohar and Mylonas 2001). Standard practices of

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13 spawning fish in captivity include artificial injection of hormones that simulate LH like activity and induce the oocytes to undergo fin al oocyte maturation and ovulation (Shirashi et al. 2005). In standard aquaculture spawning practices, females undergo cannulation, or oocyte biopsy, to examine the stage of the oocytes. As described by Shiraishi et al. (2008), only females with oocytes predominantly in the late stages of vitellogenesis are selected for hormone injection (Ibarra Castro and Duncan 2007). In addition to female examination and injection, males are also examined for milt flow, percent sperm motility, and duration of sperm m otility. For example, in spotted rose snapper ( Lutianus guttatus) males were sampled with haematocrit tubes, the sperm analyzed at 400X magnification, and the sperm was observed for motility (Ibarra Castro and Duncan 2007). Two of the most widely used hormones to induce fish ovulation are human chorionic gonadotropin (HCG) and gonadotropin releasing hormone analog (GnRHa) (Shiraishi et al. 2008). Both hormones are injected into the fish intramuscularly. The exact timing of the induced spawn can be var iable. In an experiment done with chub mackerel ( Scomber japonicas) ovulation occurred about 33 hours post HCG injection and 36 hours post GnRH injection (Shiraishi et al. 2008). However, in red snapper ( Lutjanus campechanus) most females ovulated betw een 24 32 hours post HCG injection and larval survival rate was highest when females spawned during this time period (Bourque and Phelps 2007). These hormone techniques are useful to induce mature fish to release gametes, but little is known about the par ticipation of individuals within the group during spawning. Similarly, little is known about the actual fertilization kinetics in recirculating systems.

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14 Historically, mixed milt fertilization, or fertilization that includes combining eggs and milt from m any individuals in a single container (Wedekind et al. 2007), has been widely used to produce fertilized eggs, with varying success (Hoff et al. 1972; Kloth 1980). However, this method of mixed milt fertilization has been discouraged due to decreased gene tic variation from sperm competition, increased artificial selection, and domestication of the stock population (Wedekind et al. 2007). The restocking of wild fish populations is an important part of management, and both genetic variability and quality of the stock must be maintained through natural spawning. Due to the high water and land requirements of pond aquaculture and the effects of effluent loading on the environment in coastal cages, the most environmentally sustainable option for producing high quantities of fish remains recirculating aquaculture systems. While research has been done on controlling environmental cues and hormonal levels that facilitate spawning, much research is still needed to increase annual fertilized egg yield. The specific questions for future progress in recirculating systems will be can they provide a suitable environment for natural spawning behavior? And can gamete production be optimized or at least controlled to produce consistent yields of fertilized eggs? Once rese archers have consistent production of fertilized eggs, the next step, which has already been undertaken, is to develop cost effective systems to hatch eggs and rear juveniles (for a review of marine finfish larviculture techniques see Lee and Ostrowski 200 1).

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15 CHAPTER 2 SPAWNING BEHAVIOR AN D GAMETE AVAILABILIT Y OF FLORIDA POMPANO Introduction Florida pompano is one species that is not recovering as expected under its overexploitation recovery plan. Economically, Florida pompano is one of the most desirab le marine fish with a price per pound (Retail market prices in 2005 were around $8.00/lb; Main et al. 2007) that is higher than most other US marine and freshwater fish (Weirich et al. 2005). As a fish with such high market demand, it is not surprising th at the pompano stock condition was listed as decreasing due to overfishing for the years 1998 2007, based on catch rate information (FWRI 2008). To limit the impact of overexploitation and decline of natural populations as well as create an economically l ucrative industry, aquaculture techniques for the Florida pompano have been in development for more than 40 years. Unfortunately, a recirculating system that produces reliable yields of fertilized eggs has yet to be developed. Both HCG and GnRHa can be us ed to induce spawning in Florida pompano, and it has been documented that spawning occurs about 36 hours post injection (Weirich et n induce natural spawning when combined with a photoperiod ranging from 11 hours (winter) to 13 hours (summer) (Hoff et al. 1972, 1978a, b; Kloth 1980; Weirich et al. 2005; Main et al. 2007; Weirich and Riley 2007). Unfortunately, even while using these s pawning methods, fertilization rates are highly variable (0% to 91%, Main et al. 2007) and remain a large problem when producing pompano on a commercial scale. While pompano can be conditioned to spawn under artificially controlled conditions of photoperi od, temperature and hormone

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16 injection/implantation, new aquaculture techniques must be developed to improve fertilized egg yield. One possible explanation for low fertilized egg yield in many marine fish, including the Family Carangidae of which pompano ar e a member of (Graham and Castellanos 2005), is that they naturally spawn in large groups (involving 100s of individuals) and in captivity the group size is small and the quarters too confined. These factors may inhibit natural spawning behavior and hence the production of fertilized eggs. Few observations of pompano group spawning in the field have been made, but group spawning behavior is often stereotyped across taxa. Graham and Castellanos (2005) described the spawning behavior of two members of the f amily Carangidae (the permit, Trachinotus falcatus and the yellow jack, Carangoides bartholomaei ), of which pompano is also a member. In permit and yellow jack, Graham and Castellanos (2005) reported schools of at least 300 individuals aggregating about a n hour before sunset. As spawning began, subgroups of the large school broke off as a female swam rapidly toward the surface followed by multiple males and followers. The female would stop approximately 5 m from the surface and vibrated rapidly, releasin g a cloud of eggs, the males released their sperm essentially simultaneously and in very close proximity to the female (Graham and Castellanos 2005). These spawning subgroups are very typical in group spawning species, however data have not been collecte d to determine if females spawn once and then leave the aggregation, or if they spawn multiple times in the same event. In wrasses, for example, females come to the aggregation, and spawn a single clutch of eggs, and then leave (Colin 2010). Replicating t his dramatic spawning

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17 behavior in a tank setting may be an important limitation to high fertilized egg yield in aquaculture. Past attempts to spawn pompano have involved selecting individuals with mature gametes, injecting them with a hormone, and either strip spawning or allowing the individuals to spawn in a separate tank (Hoff et al. 1978; Kloth 1980; Weirich and Riley 2007). While these techniques may produce a larger proportion of fertilized eggs, pompano are especially delicate fish, and susceptibl e to the detrimental effects of excessive handling. To maintain the effects of sexual selection and to limit handling of the fish, natural group spawning behavior was the focus of our study (Forsberg 2008). In this study, the entire group was allowed to participate in the spawn. In this study, I examined whether pompano exhibit natural spawning behavior in a commercial recirculating aquaculture system. This was done using video analysis of spawning and by comparing the number of participants, spawning rush duration, and spawning behavior to the limited descriptions of natural group spawning in carangids. Video analysis of pompano group spawning behavior has never been conducted, and the characterization of pompano spawning behavior in a recirculating s ystem should give an indication of whether pompano are receiving the necessary cues to promote normal spawning behavior. In addition, the proportions of males and females producing mature gametes vs. those adults that are found without mature gametes was a ssessed, and from that analysis, a refined spawning schedule is proposed with the focus on developing a schedule that provides consistent yields of fertilized eggs each season.

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18 Materials and Methods Study Facility and Housing Conditions The work reporte d herein was conducted at the Mote Aquaculture Research Park (MAP) in Sarasota, FL, a field station of the Mote Marine Laboratory. This private marine research organization is dedicated to increasing both marine knowledge and education in the community. Although Mote Marine Laboratory was built in 1955, the Mote Aquaculture Park was established in 2001 and primarily serves as a facility to further the development of sustainable marine and freshwater aquaculture technology. In 2009, Mote Aquaculture Res earch Park housed 20 30 adult pompano in each of two cylindrical indoor recirculating tanks (Group1 and Group2). Our study focuses only on Group1 because a more complete data set from Group1 was collected. The tanks are each 4.57m (15 ft) diameter 1.52m (5 ft) deep with a volume of about 28,000L. To maintain tank clarity and water quality there was a 0.085 m drop filter (Aquaculture Systems Technologies, L.L.C, New Orleans, La ) for solids separation, a 900 L moving bed for biofiltration, and 2 150 W Hig h Output SMART HO UV units (Emperor Aquatics, Inc, Pottstown, PA). The tanks were maintained at a temperature of 28 1.0C during the spawning season. Salinity was held at 35 0.5ppt, D.O. at a was controlled in each tank using Solar 1000 series dimmer (BlueLine Aquatics, San Antonio, TX), a system designed to simulate solar and lunar events. These events closely followed natural solar and lunar patterns of the pompano spawning season of February October. Sex r atios began at 19M:10F during the first sampling and changed to 14M:10F in April.

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19 Experimental Procedures Beginning February 2009 and continuing until October 2009, the pompano were artificially induced to spawn every other month. Handling was restricte d to one day before the spawn. The purpose of this handling was to sample the gametes of the fish and unpaired t tests) were performed to analyze any changes in gamet e maturity throughout the season. During fish handling, the water level of the tanks was dropped by 60%, lab technicians corralled fish and one by one anesthetized the fish using a separate tank filled with water and tricaine methanesulfonate (MS 222) at a concentration of 200mg/L. Once the fish were anesthetized, weight, length and the presence/absence of mature gametes were assessed. The presence of mature oocytes was assessed using a cannulation biopsy. Oocytes recovered from the biopsy were measured and staged. When oocytes were in vitellogenic or post v itellogenic stages, at least 450 m in diameter, and the macronucleus had migrated to one side, the oocytes were considered mature. Females with oocytes that were less than 450 m in diameter were c onsidered to contain no mature gametes. All mature females were injected with a maturation and ovulation advancing hormone during each handling period. Females were either injected with human chorionic gonadotropin (HCG), Ovaplant (active ingredient Salm on gonadotropin sGnRHa), or Ovaprim (active ingredients Salmon gonadotropin sGnRHa and Domperidone, a dopamine inhibitor), see below for more detail. The assessment of males involved determining the amount of milt that could be manually expressed as well as measuring sperm motility using a compound microscope. Percent motility as well as motility duration was recorded when a milt

PAGE 20

20 sample could be obtained. Males were considered to have mature gametes when milt was free flowing when expressed and when spe rm was active for at least 15 sec. Males in which a sample was difficult or impossible to obtain, or whose sperm was not motile under a microscope were categorized as having no mature gametes. Males were injected with Ovaprim twice during the season to i ncrease milt production, as detailed below. After handling, the fish were revived by gill ventilation in the original tank. Hormone Induction of Spawning Various hormones were used over the course of the breeding season in an attempt to evaluate the dif season, mature females were injected intramuscularly at each handling period with HCG, Ovaplant, or Ovaprim. HCG (1000IU/kg body weight) was injected in mature females February, April, and Septe mber. In June, all females were injected with Ovaprim (0.5ml/kg body weight for females with mature oocytes at the time of sampling, 0.25ml/kg body weight for females found without mature oocytes at the time of sampling). In August and October, females w ere injected with Ovaplant based on size (<1500g body weight, 75ug; >1500g body weight, 150ug). In August and October, all males were injected with 0.5ml/kg body weight Ovaprim. Behavioral Filming Equipment Three underwater Neuros OSD (Neuros Technology International, LLC., Chicago, IL) cameras were used to record spawning behavior of the pompano during February and April 2009. The cameras were installed near the bottom of the tanks with a slight upward angle during the handling period. They were mount ed on pvc pipe and secured to the tank so water flow or fish interference would not dislodge the position

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21 during filming. In February, data were captured on VHS tapes, but in April filming was recorded directly to digital thumb drives. Three cameras were set to record from 1500 1900 on February 12, 2009, and from 1330 1700 on April 8, 2009. Behavioral and Statistical Analysis The tapes were analyzed first by identifying possible spawning rushes. During the tape analysis, the duration of the rush, number of participants, relative vertical location at which the rush took place, whether eggs were found shortly after the rush, and period between individual rushes were recorded. A Mann Whitney U statistical test as a difference between the rush intervals, rush durations, and number of participants between the February and April spawns. Observed spawning rush behavior was categorized into false spawning behavior (that which did not result in the release of eggs) a nd true spawning behavior (that which did result in the release of eggs.) Differences between false spawning behavior and true spawning behavior were determined by the measurable aspects of the spawning rushes. During each spawning cycle, both fertilize d and unfertilized eggs were collected at the surface of the tank using a skimmer bar and deposited into an external egg basket. It is important to note that only fertilized eggs are buoyant (Main et al. 2007; Weirich and Riley 2007), however due to the e fficient aeration of the recirculating tank system, unfertilized eggs were also collected using the skimmer bar. The total number of eggs was counted by volumetric estimates post spawn, and the percent fertilized eggs calculated. While not all fertilized eggs were able to be collected via the skimmer bar due to the filtration system in the tank, the calculated percent fertilized eggs is the maximum fertilization rate possible. Some egg samples were aged under the microscope to estimate the spawning time that produced viable eggs.

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22 Results Male and Female Gamete Production Handling data were recorded from February 2009 through October 2009 to characterize variance in gamete production over the 2009 spawning season (Table 2 1 ). On the first handling date i n February 2009, the spawning group had the largest proportion of males with mature gametes across all sampling dates (Figure 2 2) Ten of 19 males had mature gametes at the time of sampling (52%). By the next handling in April 2009, the proportion of ma les with mature gametes significantly decreased mature gametes at the time of sampling (16%). In June 2009, the proportion of males with mature gametes was the lowest of the entire season, with only 1 male out of 14 total males (7%). The number of males with mature gametes increased slightly at the next handling date, August 2009, to 2 males out of 14 total males (14%). By the next sampling date, the proportion of males with mature gametes increased to the second highest value of the sea son. September 2009, 5 males of 14 were found with mature gametes (36% ) The proportion of males with mature gametes on the last sampling date, October 2009, was a moderate 3 males of 14 total males (21%). Thus the proportion of males with mature gamete s in the spawning group was highly variable, with two peaks of male gamete maturity in February and September of the spawning season. Although males were injected with Ovaprim in August and October, there was no measurable difference in the proportion of males found with mature gametes in February June compared to August October (unpaired t test: t=0.09, p=0.9326, df=4, se=0.153).

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23 Female gamete maturity and fecundity was also recorded for the 2009 spawning season (Table 2 1) Egg maturity was determined in part by egg diameter (Table 2 3). Similar to the males, the highest proportion of females with mature oocytes was found in February (70% females with mature gametes) and September (60% of females ). For all months other than February and September, the proportion of females wit h mature oocytes was below 50% (April, 30%; June, 40%; August, 40%; October, 2 0%) (Figure 2 2) Fecundity was also estimated by taking the total number of eggs collected and dividing by the number of females with mature oocytes ( Table 2 2) The largest number of eggs collected in one spawning cycle was 324,000 eggs in September, 2009. During this cycle, 6 females were found to have mature oocytes, and thus, the average numb er of eggs per female was 54,000 eggs. The average numb er of eggs per female across th e 2009 season ranged from 32,500 eggs (August 2009) t o 74,666 eggs (April 2009) per female with mature oocytes. Fertilized Egg Yield Across Spawning Cycles In addition to gamete maturity and hormone injections, the total number of fertilized eggs collected after each spaw ning cycle was recorded (Table 2 2). The two peak time periods in fertilized egg collection corresponded to the months of peak male gamete maturity. The largest numbers of fertilized eggs were collected i n February (112,128 fertilized eggs, 44% total eggs), April (127,904, 57%), and September (139,639, 43%). From February to April, the number of fertilized eggs collected increased slightly (112,128, 44%; 127,904, 57%), even though the numbers of females w ith mature oocytes dropped by more than half; there were no fertilized eggs collected in June. In August, only 47,970 (37%) fertilized eggs were collected, leading up to the

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24 largest yield in September (139,639 fertilized eggs, 43%). In the final cycle, collected in October, the number of fertilized eggs was very low at 20,893 fertilized eggs (29%). False Spawning Behavior Prior to gamete release, the majority of the school swam clockwise against the current. However, rushes, where two to five fish broke out of the school and chased, swam against the current, and stopped mid swim occurred frequently. False spawning rushes often included a sudden speeding up of a few fish to position themselves with their flanks touching. The fish remained closely positi oned for a few seconds, and then the group broke up and the individual fish swam away. The closely swimming group swam either up or down in the water column and often swam in the opposite direction to the rest of the school. Their erratic swimming could be easily seen by flank flashing. None of these rushes included vibrations, often associated with egg release, in any participants. False spawning rushes were captured April 2009 when recording began several hours prior to the first coll ection of eggs at 13:30 (Figure 2 1 ). These false spawning rushes began when one fish paused in the water column with a group of other individuals closely surrounding it, and ended when it began swimming again. During the false spawning rushes, those individuals not direc tly involved either ignored the rush and kept swimming or followed the false spawning group in the direction of the current. By 14:42, the school still swam against the current, but more often they either stopped swimming or swam in the other direction. The school looked like a disorganized group and individuals milling about in any direction. The false spawning rushes in April 2009 began at 14:42 and continued until about 14:57. There were at least 6 false spawning rushes that lasted on average 3.6 sec

PAGE 25

25 (SD=1.397 sec) with an average of 3.4 participants (SD=0.976 sec). The average time interval between false spawning rushes was 2 min 59 sec (SD=2.018 min). These events were undoubtedly false spawning rushes because there were still no eggs found at 15: 08. The true spawning rushes in April 2009, characterized by the female vibrating during release of gametes, began at 16:07 and lasted until 16:37. No false spawning rushes were observed in February 2009 (Figure 2 1 ). True Spawning Behavior A characte ristic true spawning rush involved five or more individuals, with one female in the center of a close group of 4 10 other fish, pausing in the water column. The female then vibrated her whole body for 5 12 sec as she released eggs into the water column. This extended period of female vibration was the most obvious difference between true spawning and false spawning rushes. The group of fish surrounded her and swam slightly behind her while remaining in close contact with her, and the males presumably rel eased their sperm as she released eggs (a milt cloud was difficult to see due to the aeration of the tank). The whole group was either stationary in the water column or swimming very slowly. All true and false spawning rushes occurred between the midleve The focal breeding group was injected between 10:15 and 12:00 on February 11, 2009, and eggs were found approximately 30 hours later, at 17:06 on February 12, 2009, in the 2 4 egg cell stages (eggs in this stage of cell division are 0 15 min post fertilization). Fourteen true spawning rushes were observed between the time fram e of 16:41 and 17:32 (Figure 2 1 ) despite the fact that only 8 females were found with mature oocytes during handling o n February 11, 2009. The average number of participants in each true spawning rush was 8.0 individuals (SD=2.5 fish), and the

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26 average duration of the true spawning rushes was 7.7 sec (SD=2.79 sec). The average time period between true spawning rushes wa s 3 min 54 sec (SD=2.70 min). From this spawn cycle, a volumetric estimate of 112,128 fertilized eggs was found with an estimated maximum fertilization rate of 44%. When the focal breeding group was next induced to spawn on April 8, 2009, the fish were injected between 10:15 and 12:00 on April 7, 2009. Eggs were found on April 8, 2009, approximately 30 hours later, at 16:30. Three cameras were set to rec ord from 13:30 17:00 (Figure 2 1 ). Setting the camera to record earlier provided the opportunity t o view false spawning behavior. Beginning 16:07 and lasting until 16:37, while only 4 females were found with mature oocytes during handling on April 7, 2009, there were 8 recorded true spawning rushes that lasted on average 6.3 sec (SD=1.83 sec) with an average of 5.5 participants (SD=1.5 fish). The average time interval between true spawning rushes was 4 min 53 sec (SD=2.49 min). From this spawn cycle, an estimate of 127,904 fertilized eggs was collected with an estimated maximum fertilization rate of 57%. Comparisons Among True Spawning Rushes. Mann Whitney U tests were conducted to compare the February and April spawn cycles. No differences were found between the true spawning rush intervals (Mann 1 =8, n 2 =13 ) or the true spawning rush durations (Mann 1 =9, n 2 =14). However, significantly more fish participated in the true spawning rushes in February than in April (February mean participants=8, April mean participants=5 .5; Mann Whitney U test: 1 =9, n 2 =14) which correlates to the larger proportion of adults found with mature gametes, and in particular males with mature gametes, in

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27 February relative to April. On average, spawns included nearly as many males as were sperm producing, suggesting either that males with mature gametes spawned repeatedly or that males without mature gametes were participating in spawning rushes. Discussion Natural Spawning Behavior From this study, we can conclude that natural pompano spawning behavior is not inhibited by closed system aquaculture. Clear distinctions between false spawning rushes and true spawning rushes were quantified in the video analysis, although no false spawning rushes were recorded for the Feb ru ary spawning event (Figure 2 1 ). This may have been due to the later start time for recording spawning behavior at 15 : 00 in February and false spawning rushes were recorded in April at 14 : 42. The number of participants for each spawning rush, was as expe cted for group spawning carangids (Graham and Castellanos 2005). Natural observations of spawning groups of a close relative to pompano, the permit, have been observed in which 5 to 9 individuals broke off from the main group and swam toward the surface o f the water, stopped about 15m from the surface, and vibrated while releasing a puff of gametes (Graham and Castellanos, 2005). While the average number of participants was different for the February and April spawns, this was not surprising given the dif fering numbers of males with mature gametes. Also, the group size and sex ratio used in our study appeared to be acceptable for natural pompano spawning behavior. Interestingly, there is strong evidence that females are participating in multiple spawnin g rushes during each spawning cycle. In the February spawning cycle, 14 individual spawning events were observed, and out of 10 females in the group, only 7 were found with mature oocytes. Likewise, in the April spawning cycle, 8 spawning

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28 ru shes were obs erved and only 3 females with mature oocytes were identified. Thus it would appear that on average 2 spawning rushes can be expected per fertile female. In addition, the number of participants in each spawn also leads to the conclusion that males or fema les that are not producing mature gametes are participating in the spawning rushes. For instan ce, in April, only 3 males and 3 females were producing mature gametes, but the spawning rushes consisted of 4 8 individuals. From this information, it is possi ble that males with mature gametes are participating in multiple spawning rushes, or that adults without mature gametes are also participating in spawning behavior. Limited Gamete Quantity An obvious problem to consider further is the low percentage of ind ividuals with mature gametes. The percentage of males with mature gametes dropped significantly over the 2009 spawning seaso n. Low female fecundity of 0 74,6 00 eggs/female is also a major consideration in our study. This fecundity is extremely low compa red to early natural estimates of 630,000 eggs (Finucane 1969), more conservative recent estimations of 133,400 205,500 eggs (Muller et al. 2002), and even captive stock estimates of 123,000 772,000 eggs (Weirich and Riley 2007). The cause of this drop o f individuals with mature gametes could be the frequency of injection and spawning throughout the season. From natural observations of juveniles, pompano appear to have an extended spawning season that stretches from April to October, with a main spawning peak found in spring (April June) and another spawning peak in late summer or early fall (July October) (Finucane 1969). These peaks have been identified based on the documented size of juveniles caught within that time period (Finucane 1969). A support ing study reports young of year (YOY) juvenile pompano were collected off the

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29 coast of Florida between April and November, with the majority of YOY collected in May (Solomon and Tremain 2009). In addition, as many as four size categories of juveniles have been collected at one time (Finucane 1969), indicating that pompano spawn over a protracted period and may spawn multiple times during each spawning season. Therefore, it is entirely possible that while natural populations are able to spawn the entire se ason (April to October), with peaks of spawning in spring and late summer/early fall, that individual fish are spawning less frequently and with some recovery period between spawning activity. Applying this hypothesis to the aquaculture setting, it may be more effective to induce spawning once or serially in quick succession within the natural spawning peaks, but with an extended resting period between peaks. For instance, instead of inducing pompano to spawn every 60 days from February through October, an experiment where pompano are induced to spawn once a month in March, April, and May, are permitted to recover June and July, and then induced to spawn once a month in August, September, and October may yield higher total eggs as well as a higher fertiliza tion success rate. By scheduling serial spawns within the two natural spawning peaks and a 90 day recovery period between the two peaks, both males and females may produce a larger quantity of sperm and eggs available to be induced with hormone implants There is evidence that even with serial spawning in a shortened period of time, fecundity, fertilization rates, and spawning activity are not severely decreased when fish are induced with GnRHa. By GnRHa implantation, pompano were induced to spawn six times from July October, and each spawning cycle lasted 2 6 days with average yields

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30 of 150,000 to 772,000 eggs/female and average percent fertilization (floating eggs) of 81.8 to 96.9% (Weirich and Riley 2007.) Sperm quantity could also be a limiting fa ctor for egg fertilization when fish are repeatedly induced to spawn over a protracted season. This sperm limitation hypothesis is supported by the dual peaks of males gamete maturity found in our study. The dual peaks of males with mature gametes found in February and September are slightly further apart than the supporting data reflects, but both the data in our study and natural observations indicate that the first spawning cycle of the year is the largest (Finucane 1969). Hence the maximum proportion of males with mature gametes found in February as the first spawn of the season corresponds to the natural first spawn in April. In September, the number of males with mature gametes again spiked, correlating to the natural second spike recorded by Finuc ane (1969). Between these two peaks, the number of males with mature gametes drops to such low proportions that the sperm produced is insufficient to give an appreciable yield of fertilized eggs. Another possible explanation for poor gamete maturation in both males and females could be nitrate concentrations in the broodstock tanks. Recirculating aquaculture systems often have a higher nitrate level than natural water systems, and it has been show that elevated levels of nitrate (57 1.52 mg/L) can disru pt endocrine function in female Siberian sturgeon ( Acipenser baeri ) (Hamlin et al. 2008). Measured NO 3 levels in the pompano tanks from August December 2009 averaged from 55.83 mg/L (Nov. 2, 2009) to 103.64 mg/L (Aug. 13, 2009) with a median of 84.62 mg/L These high nitrate levels could have contributed not only to the low female fecundity but also to the low proportion of males with mature gametes. Nitrate concentrations are

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31 now being measured and adjusted more frequently to prevent high levels of nitr ate in the system. Hormone treatments have been used to increase gamete production over a spawning season. GnRHa and HCG implants have been shown to increase spermiation in fish such as the European Sea Bass ( Dicentrarchus labrax) and Japanese eels ( Angui lla japonica) as well as in a variety of freshwater and marine fish (Rainis et al. 2003; Dou et al. 2007). In Pompano, the use of a GnRHa pellet produces greater egg production, higher fertilization rates, and a spawning period of up to 6 days when compar ed to HCG (Weirich and Riley 2007). While GnRHa compounds (Ovaplant and Ovaprim) were used in our study, on both males and females, use was limited and there were no indications of increased spermiation or fecundity during the time which it was used. The most likely reason that the proportion of fish with mature gametes did not increase after the treatments is because the product used was not a slow releasing hormone, and it is sustained release of GnRHa over a period of days to weeks in male fish that re sults in the most consistent elevation of spermiation (Zohar and Mylonas 2001). To increase gamete maturation, a GnRHa product that is slow release, such as a pellet, microsphere, or monolithic implant (Zohar and Mylonas 2001), and that is applied consis tently over the spawning season should be attempted. Improving Fertilization Rates While fertilization rates of field spawning data for pompano and related jacks are unavailable, some studies have focused on another species of group spawners, e.g., the b lueheaded wrasse ( Thalassoma bifasciatum ). The mean fertilization success (defined as percent of total eggs that were successfully fertilized) for 304 field collected spawns of T. bifasciatum was 76.5% (Petersen et al. 1992). In a captive pompano

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32 spawnin g event in which one female was induced to spawn with four males, was taken out of the tank, and another female was induced to spawn with the same four males, an estimated total egg yield of 125,000 for each female was collected, with a fertilization rate of 52% (Kloth 1980). More recently, a study by Weirich and Riley (2007) revealed high female fecundity (on average 234,000 eggs per female) and high fertilization rates of over 70% when pompano were injected with a GnRHa slow release pellet and allowed to spawn in a recirculating system. Maximum fertilization rates in our study were less than 50% in most spawning cycles. One factor that may contribute to low 450 m in diameter mature diameter (Weirich and Riley 2007). While egg diameter is less reliable than macronucleus placeme nt in the determination of egg maturity, s ome of the eggs released by our females may not be sufficiently mature to be fertilized. Another issue that could be hindering fertilization success is the flow rate in the tank. Analysis of natural spawns of T. bifasciatum revealed that fertilization success (percent eggs fertilized) is higher in calmer water currents than in rough, turbulent currents (Petersen et al. 1992). The flow rate of the recirculating system may be a problem especially with smaller egg sizes. In sea urchins, larger eggs were found to have significantly higher fertilization rates than smaller eggs (Levitan 1993), primarily because they are easier to find by sperm in a turbulent or fast moving environment. Recirculating systems may have low fertilization success due to the strong current in the tank which potentially disperses sperm and eggs too quickly. Especially with the

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33 smaller eggs induced in our study, if the flow of the tank was disruptive enough, it could prevent the sperm from f ertilizing the eggs in the brief window of the spawning event. To test the effects of water flow on fertilization rates, pompano should be induced to spawn in the tanks with the aeration turned off, and the fertilization rates should be compared to those achieved when fish are spawned with normal tank aeration and pump systems. Conclusion The factors that hamper the successful and consistent spawning of pompano in recirculating aquaculture systems are still largely unknown. However, from our study, it is clear that the limiting factors of space and external cues in the recirculating system are not hindering natural group spawning behavior in the pompano. In fact, strong evidence for distinctions between false spawning and true spawning behavior have been found, as well as evidence for multiple spawning in both sexes. While pompano exhibit normal spawning behavior in a recirculating aquaculture system, further study must explore the factors of inconsistent gamete production and fertilization rates found i n this species. One method that may improve both these factors is inducing serial spawns within the two natural peaks in the spawning season. In addition, further testing with GnRHa hormones should be done to improve gamete production. Finally, the effe cts of water movement during spawning should be examined.

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34 Figure 2 1. Spawning behavior video recording timeline Figure 2 2 Percent individuals with mature gametes

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35 Table 2 1. Proportion males and females with mature oocytes spawning season Rip e Males % Males with Mature Gametes Ripe Females % Females with Mature Gametes Sex Ratio Injection 2/11/2009 10 52.6 % 7 7 0 .0 % 19M:10F HCG F 4/7/2009 3 15.8 % 3 3 0 .0 % 19M:10F HCG F 6/12/2009 1 7.1 % 4 4 0 .0 % 14M:10F Ovaprim F 8/11/2009 2 14.3 % 4 40 .0 % 14M: 10F Ovaplant F, Ovaprim M 9/14/2009 5 35.7 % 6 6 0 .0 % 14M:10F HCG F 10/13/2009 3 21.4 % 2 2 0 .0 % 14M:10F Ovaplant F, Ovaprim M Table 2 2. Fertilized egg yield of 2009 spawning season Spawning Date Total Number of Eggs Fertilized Eggs Mature Female Fert il ization Rate 2/11/2009 256,000 36 571 43.80% 4/7/2009 224,000 74 666 57.10% 6/12/2009 no eggs none no eggs 8/11/2009 130,000 32 500 36.90% 9/14/2009 323,988 53 998 43.10% 10/13/2009 72,800 36 400 28.70%

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36 Table 2 3. Egg diameter and female measurem ents Sampling Date 2/11/2009 4/7/2009 6/9/2009 Female Weight (g) Length (cm) Egg Diameter (m) Weight (g) Length (cm) Egg Diameter (m) Weight (g) Length (cm) Egg Diameter (m) 1 1020 34.5 424 530 1115 35.1 424 530 1240 37.0 239 318 2 745 31.5 31 8 398 845 32.5 No Sample 995 33.9 398 477 3 2000 45.6 371 477 2195 45.9 504 557 2280 46.1 450 530 4 850 31.6 504 557 925 32.2 318 398 1070 33.8 292 371 5 975 34.2 504 583 1005 34.4 398 504 1035 34.9 504 557 6 670 31.3 398 477 755 32.5 80 133 860 33.0 4 24 504 7 830 33.2 80 133 920 34.2 No Sample 1070 35.3 80 133 8 900 34.1 424 530 1040 35.4 80 133 1215 37.1 80 133 9 705 31.5 398 504 755 32.1 80 133 770 32.2 185 265 10 685 30.7 53 80 780 31.8 80 133 860 32.8 106 159 8/11/2009 9/14/2009 10/13/200 9 1 1510 39.5 53 80 1700 40.1 477 557 1755 40.8 159 212 2 955 34.2 477 530 1365 37.2 504 583 1430 38.2 80 133 3 2515 47.0 530 583 2535 47.2 530 610 2520 47.4 133 212 4 1300 36.3 80 133 1445 37.0 318 398 1560 37.0 371 424 5 1160 36.3 504 557 1195 36. 5 239 292 1240 36.9 345 398 6 985 34.1 477 530 1000 34.2 504 610 1055 32.0 504 583 7 1270 37.4 80 133 1350 37.8 159 292 1460 38.6 212 292 8 1515 39.7 80 133 1620 40.0 318 371 1745 41.1 239 318 9 860 33.5 53 80 895 33.5 371 451 970 35.0 106 159 10 1075 35.0 106 159 1190 35.7 424 504 1280 36.5 504 583

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40 Penney, R.W., Lush, P.L., Wade, A.J., Brown, J.A., and M.P.M. Burton. 2006. Effect of photoperiod manipulation on broodstock spawning, fertilization success, and egg developm ental abnormalities in Atlantic Cod, Gadus morhua World Aquaculture Society, 37: 273 281. Petersen, C.W, Warner, R.R, Cohen, S, Hess, H.C, and A.T. Sewell. 1992. Variable pelagic fertilization success: implications for mate choice and spatial patterns of mating. Ecology, 73:391 401. Pfeiffer, T., and P. Wills. 2007. Prototype recirculating aquaculture system design for juvenile red drum production as part of the Florida Fish and Wildlife Conservation sommissions Hatchery Network Initiative [abstract] International Sustainable Marine Fish Culture Conference and Workshop Book of Abstracts, p. 26. Rainis, S, Mylonas, C.C, Kyriakou, Y, and P. Divanach. 2003. Enhancement of spermiation in European sea bass ( Dicentrarchus labrax ) at the end of the repro ductive season using GnRHa implants. Aquaculture, 219:873 890. Resley, M.J., Nystrom, M.J., and K.L. Main. 2007. Comparison of essential fatty acid profiles in captive and wild Florida pompano Trachinotus carolinus eggs. Abstract Aquaculture 2007, Sa n Antonio, Texas. Roberts, Jr. D.E., Harpster, B.V., and G.E. Henderson. 1978. Conditioning and induced spawning of the Red Drum ( Sciaenops ocellata ) under varied conditions of photoperiod and temperature. World Mariculture Society 9:311 332. Rosenberg, A.A., Swasey, J.H., and M. Bowman. 2006. Rebuilding US fisheries: progress and problems. Frontiers Ecol Environ, 4: 303 308. Shiraishi, T., Ohta, K., Yamaguchi, A., Yoda, M., Chuda, H., and M. Matsuyama. 2005. Reproductive parameters of the chub mackerel Scomber japonicas estimated from human chorionic gonadotropin induced final oocyte maturation and ovulation in captivity. Fisheries Science 71:531 542. Shiraishi, T., Ketkar, S.D., Kitano, H., Nyuji, M., Yamaguchi, A., and M. Matsuyama. 2 008. Time course of final oocyte maturation and ovulation in chub mackerel Scomber japonicas induced by HCG and GnRHa. Fisheries Science 74:764 769. Solomon, J.J. and D.M. Tremain. 2009. Recruitment timing and spatial patterns of estuarine use by young of the year Florida pompano, Trachinotus carolinus in northeastern Florida. Marine Science, 85:133 148. Van der Meeren, T. and V. Ivannikov. 2006. Seasonal shift in spawning of Atlantic cod ( Gadus morhua L.) by photoperiod manipulation: egg quality in relation to temperature and intensive larval rearing. Aquaculture Research, 37: 898 913. Watson, R. and D. Pauly. 2001. Systematic distortions in world fisheries catch trends. Nature, 414: 534 536.

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41 Wedekind, C., Rudolfsen, G., Jacob, A., Urbach, D., a nd R. Muller. 2007. The genetic consequences of hatchery induced sperm competition in a salmonid. Biological Conservation, 137:180 188. Weirich, C., and K.L. Riley. 2007. Volitional spawning of Florida Pompano, Trachinotus carolinus induced via admin istration of gonadotropin releasing hormone analogue. Applied Aquaculture, 19:47 60. Weirich, C., Riley, K., and M. Davis. 2005. Florida pompano: Induced reproduction via pelleted GnRHa and preliminary observations regarding larval production. Global A quaculture Advocate, 8:75 77. Zohar, Y., and C. C. Mylonas. 2001. Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture, 197:99 136.

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42 BIOGRAPHICAL SKETCH Elizabeth Ann Dippon was born in Gainesville, FL i n 1986. When she was three, she and her parents moved to Oregon for her father to pursue a career with the Bureau of Land Management. Elizabeth was raised in Portland and has loved animals and music from a very young age. She has enjoyed playing the piano since age 6 and learned the marimba in h igh s chool. Elizabeth enjoyed much success with mus ic during high school, earning three 2 nd Place Awards at the State Music Festival for Marimba. She graduated as Valedictorian from Westview High School in 2004. She earned a B. A. in m usic p e rformance as well as a B.S. in z oology from the University of Florida in 2008. Elizabeth pursued a n M.S. in z oology in 2010 from the University of Florida. Elizabeth was married to Jesse J. Reynolds in May 2008. Elizabeth and her husband Jes se are very active in health and fitness. They own a youth performance center and personal training studio, Accel Sports, Inc. in Jacksonville, FL as well as a nutritional business, Advocare. Elizabeth lov es helpin g her husband coach a n ationally ranked Olympic Weightlifting team and helping peopl e achieve their physical and financial g oals Elizabeth is committed to helping others in health and personal development and attributes all her accomplishment to her Lord and Savior Jesus Christ. She is also especially th ankful for all the supp ort from her family, her husband her friends and Henry.