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5-2005

Primary Sex Reversal in Female Betta splendens

following Treatments with Temperature and
Trenbolone Acetate.
Tracy S. Smith
East Tennessee State University

Follow this and additional works at: http://dc.etsu.edu/etd

Recommended Citation
Smith, Tracy S., "Primary Sex Reversal in Female Betta splendens following Treatments with Temperature and Trenbolone Acetate."
(2005). Electronic Theses and Dissertations. Paper 982. http://dc.etsu.edu/etd/982

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 Primary Sex Reversal in Female Betta splendens
Following Treatments with Temperature and Trenbolone Acetate

A thesis
presented to
the faculty of the Department of Biology
East Tennessee State University

In partial fulfillment
of the requirements for the degree
Master of Science in Biology

by
Tracy S. Smith
May 2005

J. Leonard Robertson, Ph.D., Chair

Thomas F. Laughlin, Ph.D.
David Currie, Ph.D.

Keywords {Betta splenden, trenbolone acetate, sex reversal,

intersex, aquaculture, fishery}
 ABSTRACT

Primary Sex Reversal in Female Betta splendens

Following Treatments with Temperature and Trenbolone Acetate

by

Tracy S. Smith

Sex reversal in fish can occur as a result of disturbances in natural conditions or laboratory
manipulations. Trenbolone Acetate (TBA), a synthetic anabolic-androgenic steroid used mainly in
the cattle industry, was applied to female Betta splendens, a sexually dimorphic fish. Previously,
females were analyzed for secondary sex characteristics after treatment with TBA, increased
temperature, or a combination of both. This study focused on primary sex characteristics by
histological examination. Gonads were surgically removed and prepared following basic
histological techniques. Following H&E staining, sections were observed for signs of ovarian
regression, as well as signs of testicular formation. The presence of TBA significantly reduced the
size of the gonad and the number and size of vitellogenic oocytes, indicating a loss of ovarian
function. Temperature had a synergistic effect on TBA, as more oocytes were atretic and
interstitial tissue was more abundant, some with clear spermatocysts. Temperature alone had no
significant effect.

2
 ACKNOWLEDGEMENTS

First and foremost, I thank God for leading me toward my dreams and giving me the
strength to face each day.
They say it takes a village to raise a child. Then, by the same token, it must take a faculty
to graduate a student. The faculty of the Department of Biological Sciences has been the key to
my success here at E.T.S.U. My appreciation starts with my graduate committee, who were a
constant source of support and encouragement.
Dr. Currie, thank you for sticking with me even after my course changed. I know you
were not always “in the loop,” but your input was greatly appreciated. Dr. Laughlin, even though
you had barely settled into your faculty position, thank you for jumping into my project without
hesitation. Your knowledge of fish was certainly an asset to our team. And to my chair, Dr.
Robertson, let me first thank you for your willingness to take on this lead position in my time of
need. Your guidance and constant encouragement pushed me toward what sometimes seemed
like and intangible goal. You put in so much time and energy, and I credit much of my success to
your belief in me.
Thank you to Dr. Johnson and Dr. Goyal, who fought for an extension of my G.A. status.
Because of you, I was able to continue teaching labs, which was the highlight of my graduate
career. Dr. Moore, Dr. McIntosh, Dr. Stewart, and many others in the department have shaped
my career with your knowledge, your advice, and your continued support. A special thank you
goes to Mary Howell, who started this project and provided helpful advice and guidance. Thanks
to Lois Hyder and Kristi Tipton, who have been so willing to help me along the way. And finally,
my appreciation is extended to the contributors of the Fraley Memorial Award. This funding,
awarded to me in 2002, made my research possible.
Outside of school, I owe a huge debt of gratitude to the best parents a daughter could ask
for. Without your love and support, I would not be where I am or who I am today. Thank you
for giving me the opportunity and the courage to pursue my dreams.

3
 CONTENTS

Page

ABSTRACT ..................................................................................................................... 2

ACKNOWLEDGMENTS ................................................................................................. 3

LIST OF TABLES ........................................................................................................... 6

LIST OF FIGURES ......................................................................................................... 7

Chapter

1. INTRODUCTION .................................................................................................... 8

Ovarian Structure and Function.............................................................................. 8

Testicular Structure and Function........................................................................... 10

Hormonal Manipulations........................................................................................ 10

Betta splendens...................................................................................................... 12

Present Study......................................................................................................... 14

2. METHODS ............................................................................................................... 15

Previous Research.................................................................................................. 15

Controls................................................................................................................. 15

Gonad Retrieval..................................................................................................... 16

Histological Techniques ......................................................................................... 16

Analysis ................................................................................................................. 16

3. RESULTS ................................................................................................................. 17

Data Analysis ........................................................................................................ 17

Results ................................................................................................................. 17

Histological Analysis ............................................................................................. 23

4
Chapter Page

4. DISCUSSION ........................................................................................................ 34

Environmental Studies .......................................................................................... 35

Surgical Techniques .............................................................................................. 36

Hormonal Manipulations ....................................................................................... 37

Natural Steroids ........................................................................................ 38

Synthetic Steroids ...................................................................................... 39

Other Considerations ................................................................................. 40

Conclusions .......................................................................................................... 42

5. REFERENCES ......................................................................................................... 43

APPENDICES .................................................................................................................. 49

Appendix A: Tail length data from Howell (2000) ................................................. 49

Appendix B: Data from histological analysis .......................................................... 50

VITA ................................................................................................................................ 64

5
 LIST OF TABLES

Table Page

1. Descriptive statistics of length and width of gonad .................................................. 18

2. Descriptive statistics of vitellogenic oocyte counts ................................................... 19

3. Descriptive statistics of vitellogenic oocyte size ........................................................ 21

6
 LIST OF FIGURES

Figure Page

1. Chemical structure of Testosterone (A) and Trenbolone Acetate (B).......................... 11

2. Interaction plot of mean gonad length and width from 72C, 72T, and 82T groups...... 18

3. Dot plot with mean number of vitellogenic oocytes from 72C, 72T, and 82T. ............ 20

4. Dot plot with mean size of vitellogenic oocytes from 72C, 82C, 72T, and 82T.......... 21

5. Linear regression graph of gonad length vs. change in tail length............................... 22

6. Ovary from a 72C Betta showing typical morphology ............................................... 23

7. An atretic follicle (corpora lutea) from a 72C Betta ................................................. 24

8. A testis from a control male Betta............................................................................. 25

9. Comparison of gonads .............................................................................................. 27

10. Ovary from a 72T Betta showing several atretic follicles ........................................... 28

11. Ovary from a 72T Betta with large lumen area and branching ducts........................... 28

12. Ovaries from 72T Bettas exhibiting testicular tissue .................................................. 29

13. Ovary from 82T Betta with many atretic follicles ...................................................... 30

14. Ovary from 82T Betta with extensive lumen area and branching ducts....................... 30

15. Comparison of control testis to 82T gonad ............................................................... 31

16. Gonad from 82T group with nearly complete transformation to testicular tissue........ 32

17. Comparison of control testis to fully transformed 82T ovary ..................................... 33

7
 CHAPTER 1

INTRODUCTION

Within the animal kingdom, fish have evolved into a class with an unprecedented array of
physical and behavioral traits. These characteristics have generated many research opportunities;
among these is the study of reproductive mechanisms and behavior.
The male testis and the female ovary are most often found in separate individuals
(dioecious); however, there are numerous cases of hermaphroditism. Further classification
distinguishes protogynous hermaphrodites (female to male progression) from protoandrous
hermaphrodites, as well as cases of intersex when both male and female gametes are present
(Hoar 1957b, Yamamoto 1969, Redding and Patiño 1993).
Fish reproductive cycles are dependent on a variety of factors including, but not limited to,

environmental cues (i.e. light, temperature) and hormonal cues. Endocrine function in fish is

primarily based on a hypothalamic/pituitary axis, similar to that of other vertebrates. The

gonadotropins control ovarian and testicular function in part by stimulation of gonadal steroids,

which also regulate secondary sex characteristics (i.e. size, color, fin display, etc.). Some

generalizations can be made about the reproductive biology of teleost fish, but it should be noted

that specifics of structure and hormonal influence vary from species to species (Redding and

Patiño 1993).

Ovarian Structure and Function

Oogenesis occurs within the paired lobes of the ovary, which are joined by a common
oviduct. In a manner similar to higher vertebrates, oogonia develop within a protective follicular
layer, which, in later stages of maturation, may be clearly composed of inner granulosa cells and
outer thecal layers. An important stage in oocyte development is that of vitellogenesis, or the
deposition of yolk into the oocyte. This growth process is highly dependent on hormonal

8
communication between the ovary and the liver which produces the protein vitellogenin (Hoar
1969, Moyle and Cech 1982, Liley and Stacey 1983, Nagahama 1983, Redding and Patiño 1993,
Patiño and Takashima 1995).
Female fish produce many hormones including testosterone which is a precursor for 17β-
Estradiol, the dominant female hormone. These hormones function in all stages of oogenesis,
ovulation, breeding, in some cases gestation, and the induction of secondary sex characteristics
(Baggett et al. 1956, Hoar 1957b, Liley 1969, Liley and Stacey 1983, Nagahama 1983, Redding
and Patiño 1993).
Following ovulation, the follicular cells transform into a corpus luteum, in some cases
similar to that of higher vertebrates (Hisaw 1947). However, this is a misnomer in many species
as the atretic follicle has little if any endocrine function in oviparous species. These “corpora
lutea” can be pre-ovulatory (reabsorption of yolk) or post-ovulatory, in which granulosa cells may
have multiple duties (Hoar 1957b, Hoar 1969, Nagahama 1983).
Khoo (1975) reported the formation of oogonia from the atretic remains of follicles.
While this report highlights a difference from corpora lutea of higher vertebrates, Hoar (1965 as
cited in Hoar 1969) offers a more relative description.
Hoar suggests,
that estrogen synthesis was one of the responsibilities of the granulosa from the
earliest stages of vertebrate phylogeny…. [thus], it seems entirely reasonable that
the same granulosa cells (in some fish) may have specialized in hormone
production to the point where corpora atretica become functional corpora lutea –
even though they may synthesize estrogen rather than progesterone as their
hormone (Hoar 1965 as cited in Hoar 1969).
Despite obvious differences, one similarity found in all vertebrates is the methodical distribution of
hormones. Exogenous manipulation of the system can potentially disturb ovarian function at
many levels.

9
 Testicular Structure and Function

Spermatogenesis occurs within the scattered lobules of the paired testes. Each lobule
contains a number of cysts filled with germ cells developing at the same rate. Collection of
mature spermatozoa begins as cysts rupture into the lumen of the testis and culminates with
collection in the vas deferens (Hoar 1969, Moyle and Cech 1982, Nagahama 1983, Redding and
Patiño 1993, Koulish et al. 2002).
Cysts are surrounded by interstitial tissue (i.e. Sertoli cells, Leydig cells), much of which is
endocrine in function. The prominence and arrangement of such tissue varies considerably among
species (Hoar 1957b, Hoar 1969, Nagahama 1983, Redding and Patiño 1993, Koulish et al.
2002). Though a variety of hormones have been identified from the piscine testis, testosterone
and 11-ketotestosterone are recognized as the key androgens (Liley and Stacey in Hunter and
Donaldson 1983). Other hormones may play minor roles in spermatogenesis, secondary sex
characteristics or they may exist as precursors in a biochemical pathway (Liley 1969, Yamamoto
1969). Research has shown that even minute disturbances in hormonal balance can impede
reproductive success in both males and females.

Hormonal Manipulations

Much has been learned about the production and function of hormones in fish by
manipulations of both endogenous and exogenous androgens and estrogens. More recently,
synthetic hormones have emerged as important research tools. For example, testosterone in its
natural state functions both to masculinize (androgenic) and to build tissue (anabolic). These
effects can be observed from early development through sexual maturation. Through research,
many synthetic variants of testosterone have been synthesized, ranging from those with
predominantly anabolic effects to those focused on androgenic activities. Among those is
trenbolone acetate (TBA) which is classified as an anabolic-androgenic steroid. Being derived
from testosterone, TBA contains a basic 4-ring steroid structure with some modifications (Farber
1991, Galvez et al. 1996). Figure 1 illustrates the structural differences between testosterone and

10
synthetic TBA. These minor side-group substitutions in steroid structure become apparent in the
phenotypic characteristics of the treated organisms.
The cattle industry uses several synthetic hormones, including trenbolone acetate to
increase meat production (Henricks et al. 1982, Bartle et al. 1992). During metabolism, TBA is
broken down to water-soluble metabolites which may be lost in both urine and feces. While those
lost have less activity, many forms of these metabolites retain a portion of their androgenic and
anabolic potential (Heitzman 1983, Rico 1983, Lange et al. 2002, Buiarelli et al. 2003).

A B

Figure 1 Chemical structure of Testosterone (A) and Trenbolone Acetate (B)

Even trace amounts of residual hormone have the potential to affect many aspects of the
environment. Run-off from farmland may enter local waterways, extending the impact to aquatic
life (Lange et al. 2002). Because fish reproductive function is highly sensitive to hormonal
fluctuations, primary and secondary sex characteristics can be observed in affected populations.
In mosquitofish, Gambusia affinis, intersexuality, based on secondary characteristics, was found
to be an indicator of environmental disruption (Bortone and Davis 1994). In this particular case
the factor studied was effluent from a paper mill. Many other endocrine disruptors have been
shown to have similar potential (Jobling et al. 1998). In undifferentiated fry, 50mg TBA/ kg feed
or immersion in TBA induced 100% male populations in both channel catfish, Ictalurus
Punctatus, and blue tilapia, Oreochromis aurea (Galvez et al. 1995, Galvez et al. 1996,

11
Fitzpatrick et al. 2000). Research reported that the synthetic androgen may bind to gonad
receptors promoting male-oriented gonadogenesis (Galvez et al. 1995, Galvez et al. 1996).
In addition to its masculinizing effects, TBA was also shown to inhibit ovarian function.
In female hamsters, TBA has been shown to prevent follicle development and ovulation (Zarkawi
et al. 1991). The researchers proposed that FSH production may have been inhibited. A similar
effect was noted in adult channel catfish, Ictalurus Punctatus (Davis et al. 2000). Ovarian
function was disrupted and testicular tissue was formed, but functional males were not produced
(Davis et al. 2000).
Though the mechanism is unclear, the phenotypic effects obtained from TBA treated fish
are potentially beneficial if used in a controlled manner, similar to other hormone treatments used
in aquaculture. The techniques applied in fisheries allow for the production of single sex
populations, which may be economically beneficial due to size, coloration, or rate of maturation.
The timing of sexual maturity and spawning events can also be controlled for maximum efficiency
(Hunter and Donaldson 1983, Galvez et al. 1995, Fitzpatrick et al. 2000).
Many studies of sex reversal by steroid treatment involve the use of young fry with
undifferentiated gonads. Many species are not sexually dimorphic at this stage, making sex
identification nearly impossible. Using a species with clear differences in secondary sexual
characteristics, such as Betta splendens, allows for treatment of a known sex and the evaluation of
potential sex reversal both on primary and secondary characteristics.

Betta splendens

Originating in Siam, Thailand, Betta splendens, more commonly called the Siamese
Fighting Fish, have been used for research since the mid-1800s. Betta splendens (Bettas) are a
fresh-water species inhabiting small ponds and rice paddies in the wild and aquariums worldwide
(Gordon and Axelrod 1968). Bettas prefer shallow waters because they frequently need to
surface. Due to an inefficiency in the gills to provide enough oxygen from the water, Bettas use a
highly vascular organ known as the labyrinth to extract oxygen from surface air. This accessory
organ is similar to a primitive lung in function (Gordon and Axelrod 1968).

12
 In the wild, Bettas tend to live in groups, mostly female with a dominant male
(Jaroensutasinee and Jaroensutasinee 2001a). This dominance by aggressive behavior in defense
of territory secures access to females for reproduction (Gordon and Axelrod 1968, Rhoad et al.
1975, Jaroensutasinee and Jaroensutasinee 2001a). It was this aggressive behavior that led to
Bettas’ original popularity in fighting contests, but more recent popularity stems from the wide
array of colors and fin displays that breeders have introduced to aquarists (Gordon and Axelrod
1968).
Both wild and domesticated Bettas are sensitive to their environment. Even small changes
in pH, salinity, and temperature, among other factors, can be detrimental, both metabolically and
reproductively. Temperature is especially important as Bettas prefer with water temperatures
around 80ºF(27ºC). This is a few degrees warmer even than that suggested for tropical fish.
Studies by Goodrich and Taylor (cited in Gordon and Axelrod 1968) showed that 80ºF was ideal
for nest building and breeding behavior of males. Females also bred more often at 80ºF as
opposed to 60ºF or 70ºF. Temperatures too far above 80ºF may cause developmental problems
(Gordon and Axelrod 1968). This sensitivity has produced many studies dealing with the effects
of temperature on development and reproductive success in this species.
Bettas are known for their extravagant courtship and mating behavior. The results of
many behavioral studies can be attributed to basic secondary sex characteristics that differ
between males and females. Bettas are sexually dimorphic, with differences in size and color.
Compared to males, females are small and lack the vibrant colors. Males are not only larger and
rich in color, but they sport longer, showy fins. Breeding has produced a wide variety of colors
and displays in male Bettas, the sex most preferred commercially (Robertson and Sale 1975,
Jaroensutasinee and Jaroensutasinee 2001b).
Besides these physical differences, there exist some behavioral sexual dimorphisms in
Bettas. Males, as previously mentioned, are very aggressive especially in matters of reproductive
success. Many studies have focused on the precise timing and details of the fight display and the
impending pugilistic struggle. Often these fights occur to protect a bubble nest built by the male
Betta in preparation for mating. During spawning, fertilized eggs (up to 400-500 per female) are
gathered by the male and placed in the bubble nest. The male furnishes the primary care of the
fry, producing new bubbles frequently to provide needed oxygen. In the wild, males must defend
13
the nest from predators, including other male and female Bettas (Gordon and Axelrod 1968,
Robertson and Sale 1975, Jaroensutasinee and Jaroensutasinee 2001a). This sexual dimorphism
allows for studies of the effects of environmental, hormonal, chemical, and social manipulations
on male and female characteristics.

Present Study

The purpose of this study was to examine the effects of TBA, increased temperature and a
combined treatment on primary sex characteristics of female Betta splendens. This investigation
involved the histological analysis of Bettas; this included all treatment groups, as well as control
female and male Bettas. The study involved the identification of ovarian regression (atretic
follicles, decrease in number and/or size of oocytes, and decrease in overall gonad size) and/or
testicular formation.
Several major questions and hypotheses were addressed in this research, as follows.
Question 1: Is there a difference in ovarian morphology between fish kept at 72ûF and fish
kept at 82ûF? Specifically, are there signs of ovarian regression and/or testicular formation?
Question 2: Is there a difference in ovarian morphology in fish treated with TBA?
Specifically, are there signs of ovarian regression and/or testicular formation?
Question 3: Is there an increased difference in ovarian morphology in fish treated with
TBA at 82ûF? Specifically, are there signs of ovarian regression and/or testicular formation?

14
 CHAPTER 2
METHODS

In a previous research endeavor by Howell (2000) female Betta splendens were used and
preserved in 95% ethanol. These fish, along with the data collected by Howell, were used for
histological examination and analysis (Data provided in Appendix 1).

Previous Research

Female Bettas were separated into four aquaria; two at 72ºF and two at 82ºF. Light, pH,
and salinity were all standardized and initial caudal fin length recorded for each Betta. The
hormone trenbolone acetate (TBA) was introduced through the diet. An untreated food supply
was fed to the control groups at each temperature and TBA treated pellets were fed to
experimental Bettas at each temperature. All fish were fed a standard amount (5 pellets/day) for a
30-day period. Final caudal fin length was recorded and other secondary sex characteristics were
noted. Surviving Bettas were preserved in 95% ethanol and refrigerated (Howell 2000).

Controls

The number of preserved control Bettas at 72ºF and 82ºF (N=3, N=1, respectively) was
low. Several female Bettas were purchased for this study from a local pet supply to supplement
the histological observations. These fish had been maintained in an aquarium at room temperature
(near 72ºF). For the purposes of this research, control females purchased locally and preserved
control females (72ºF) will be considered synonymous.
Several male Bettas were also purchased for comparison of normal testis morphology.
Male Bettas and female Bettas were all processed by the same protocol. All purchased Bettas
were anesthetized and euthanized in 95% ethanol.

15
 Gonad Retrieval

All gonads were surgically removed using standard techniques and immediately placed in
Gilson’s fluid. The fixative was changed several times over approximately one week (Jones
1966).

Histological Techniques

Following fixation, gonads were dehydrated through a series of ethanol and xylene.
Paired or single gonads were set in paraffin and sectioned (10µm) using a A-0 microtome.
Sections were mounted on Fisherbrand Superfrost Plus slides and stained with a standard
Hematoxylin and Eosin series (Jones 1966). Slides were sealed with Cytoseal© XYL mounting
medium.
Some problems were encountered in sectioning control gonads from both temperature
groups. Due to size and stability of the gonads, adjustments were made to the original protocol
to ensure quality slides. Tissues were fixed in formalin, a formaldehyde solution commonly used
as a preservative (Cole et al. 1999). The dehydration and staining protocol was the same.

Analysis

Sections were observed under a compound light microscope, as well as a phase

microscope. Measurements and general morphology were recorded for two random sections per
slide. Photographs were taken using a Nikon (M-355) camera attached to a phase microscope.

16
 CHAPTER 3
RESULTS

Data Analysis

Lengths and widths of gonads were recorded along with the number and size of
vitellogenic oocytes for random sections from control and treated groups (See Appendix B for full
data set). These sets of data were analyzed using a computerized statistics program (Minitab 14.0
version). A one-way ANOVA followed by multiple comparisons was used to compare the means
of each group. These tests took into account the presence of replicates and unequal observations
(n’s).

Results

All tables and figures use 72C, 82C, 72T, and 82T to refer to 72ºF Control group, 82ºF
Control group, 72 ºF Treated group, and 82 ºF Treated group, respectively. A one way ANOVA
was used to compare mean lengths and widths of gonads from the 72C, 72T, and 82T groups.
The 82C group was not used in this statistical comparison because accurate measurements were
not possible with available sections. There was a significant difference in lengths (p=0.044) as
well as widths (p<0.001) among the 3 groups. Individual t-tests revealed a significant difference
in width between the 72C and 72T groups (p=0.018), indicating a significant decrease in size of
the gonad caused by the addition of TBA in the diet. Table 1 contains the descriptive statistics
from lengths and widths of gonads. Figure 2 illustrates the decrease in gonad size from the
control to treated groups of both temperature groups. The combination treatment caused no
significant change in gonad length and width (p=0.972, p=0.378, respectively). No comparison
could be made between the two temperature groups.

17
Table 1 Descriptive statistics of length and width of gonad

Treatment N Mean SE Standard Minimum Median Maximum

Groups Deviation

72C Length 4 6912 1753 3507 2625 6903 11215

Width 4 3301 283 566 2738 3210 4044

72T Length 12 4478 216 749 3544 4604 5326

Width 12 1863 106 367 1448 1759 2529

82T Length 8 4498 495 1400 1779 4589 6031

Width 8 1620 240 678 511 1702 2710

Interaction of Mean Values of Gonad Length and Width

8000

▲ Length
7000

▄ Width
6000

5000

4000

3000

2000

1000

0
72C 72T 82T

Figure 2 Interaction plot of mean gonad length and width from 72C, 72T, and 82T groups

18
 A one-way ANOVA was also used to compare mean number of vitellogenic oocytes in
gonads from the 72C, 72 T, and 82T groups. The 82C group was not used in this statistical
comparison because a minimum number of accurate counts was not possible with the available
sections. There was a significant difference in the number of vitellogenic oocytes (p=0.000)
between the 3 groups. The main significance was between 72C and 72T (p=0.001), suggesting a
loss of ovarian function (based on a decrease in vitellogenic oocytes) in the presence of TBA.
The 82T group contained very few vitellogenic oocytes. However, in undamaged longitudinal
sections where counts could be made for statistical analysis, no vitellogenic oocytes were present.
This suggests that the combination of TBA and increased temperature has a synergistic effect on
ovarian dysfunction (based on vitellogenic oocyte counts). No statistical comparison could be
made between the two temperature groups. Table 2 contains the descriptive statistics from the
number of vitellogenic oocytes. Figure 3 illustrates the decrease in number of vitellogenic oocytes
from the control to treated groups of both temperature groups.

Table 2 Descriptive statistics of vitellogenic oocyte counts

Treatment N Mean SE Standard Minimum Median Maximum

Groups Deviation
72C 4 52.88 3.85 7.70 44.50 52.26 62.50

72T 12 2.549 0.941 3.259 0 1.557 10.636

82T 6 0 0 0 0 0 0

19
 Individual Value Plot of # of Vitellogenic Oocytes
70

60

50
Number of Oocytes

40

30

20

10

72CN 72TN 82TN

Figure 3 Dot plot with mean number of vitellogenic oocytes from 72C, 72T, and 82T

The size of vitellogenic oocytes was also compared by a one-way ANOVA in gonads from
the 72C, 82C, 72 T, and 82T groups. There was a significant difference in the size of vitellogenic
oocytes (p=0.002) between the 4 groups. Specifically, there was a significant difference in size
between the 72C and 72T (p=0.001), suggesting a loss of ovarian function (based on a decrease in
size of vitellogenic oocytes) in the presence of TBA. There was only one Betta in each of the
82C and 82T groups that contained measurable vitellogenic oocytes which were used for
statistical analysis. Statistical significance could not be determined with n values of 1; however,
there was a dramatic decrease in mean vitellogenic oocyte size between the 72C and 82C (444.2,
375.01), 82C and 82T (375.01, 156.61), and 72T and 82T (321.2, 156.61). This suggests that an
increase in temperature, the presence of TBA, and the combination of TBA and increased
temperature, all have an effect on ovarian dysfunction (based on decreases in vitellogenic oocyte
size). Table 3 contains the descriptive statistics from the size of vitellogenic oocytes. The
decrease in size of vitellogenic oocytes can be seen in Figure 4.
20
Table 3 Descriptive statistics of vitellogenic oocyte size

Treatment N Mean SE Standard Minimum Median Maximum

Groups Deviation
72C 6 444.2 10.6 26 410.9 441.9 479.4

82C 1 375.01 - - 375.01 375.01 375.01

72T 10 321.2 24.3 76.8 177.0 319.9 410.7

82T 1 156.61 - - 156.61 156.61 156.61

Individual Value Plot of Size of Vitellogenic Oocytes

500

450
Size of Oocytes (microns)

400

350

300

250

200

150
72CS 82CS 72TS 82TS

Figure 4 Dot plot with mean size of vitellogenic oocytes from 72C, 82C, 72T, and 82T

21
 When the 72C group was compared to the testis control group a significant difference was
noted in the width (p=0.005). This supports the observation that ovaries generally occupy 50-
60% of total body weight, whereas testes occupy only about 12%. The significant difference in
control sizes suggests that the decreases in size seen with the treatments may reflect not only a
loss of ovarian function but also a structural shift toward testicular composition.
A comparison was also made between the mean change in tail length as recorded in
previous research and the size of the gonad. Although a correlation between secondary and
primary sex characteristics was expected, there appeared to be little correlation between the two
measures. External (secondary) sex characteristic changes did not indicate a change in primary
sex morphology. Figure 5 shows a linear regression graph of this data.

Scatterplot of gonad length vs. change in tail length

5
gonad length (mm)

0 2 4 6 8 10 12 14
tail length (mm)

Figure 5 Linear regression graph of gonad length vs. change in tail length

22
 Histological Analysis

Slides from the 72C (and purchased control females) were analyzed for normal ovarian
morphology. Oocytes from different stages of development were observed in all slides. See
Figure 6 for control ovarian morphology. The 72C ovaries contained many yolk-filled, nearly ripe
oocytes. This was a clear indication of vitellogenesis and of typical estrogenic function.

L
T

T
P
A
20µm

L 10µm

P
T
S

Figure 6 Ovary from a 72C Betta showing typical morphology. A. Several stages of development
(40X); B. (100X); Lumen (L); Oil drop stage (O); Primary yolk globule stage (P); Secondary yolk
globule stage (S); Tertiary yolk globule stage (T); Perinucleolus stage (arrow).

23
 There were also a few atretic follicles (corpora lutea), both pre-ovulatory and post-
ovulatory. A typical atretic follicle is shown in Figure 7. Cells within the granulosa and theca
layers become hypertrophied and eventually fold inward into the follicular atrium. Vacuolization
of oocyte remnants was also common.

2µm

Figure 7 An atretic follicle (corpora lutea) from a 72C Betta. The hypertrophied granulosa and
theca layers have begun to fold inward toward the atrium (A). Some yellow lutein cells can be
seen though they are not equivalent to the mammalian corpora lutea. (200X).

Ovaries from the 82C group were also analyzed for changes in normal ovarian
morphology. The control ovaries were very large and delicate in structure, which led to
difficulties during fixation and sectioning; however, basic morphology could still be observed.
Very little observable change was noted from the 72C group. All stages of oocyte development
through tertiary yolk globule stage were observed. There were more atretic follicles in the group
compared to the 72C group. There were no fully intact sections of the 82C group due to the
histological problems encountered; therefore, statistical comparison was not possible.

24
 Several male Bettas were used to observe normal testicular morphology. The lobular
divisions separated by small ducts could clearly be seen in Figure 8. At a higher magnification
individual spermatocysts could be seen. Spermatogonia, spermatocytes, and spermatids were
observed within these spermatocysts, though discrimination between 1º and 2º spermatocytes was
not possible at this magnification.

ST

SC

IT

ST
IT

SC
ST
ST
SG
SG

SG

B
A 25µm
125µm

Figure 8 A testis from a control male Betta. A. 40X; B. 400X, Individual spermatocysts are
visible within the divided lobules of the testis. The stages of sperm development are not clearly
defined at this magnification, but general labels have been applied. Interstitial tissue (IT);
Spermatocytes (SC); Spermatogonia (SG); Spermatids (ST).

25
 The groups treated with TBA (72T and 82T) were examined for changes in normal
ovarian morphology and possible formation of testicular tissue. As previously stated, there was a
significant (p= 0.044, p=0.000) difference in the length and width, respectively, of transforming
gonads between the 72C group and 72T group. Figure 9 compares gonads from these two
groups. A major difference noted was the decrease in number of yolky or vitellogenic oocytes.
Because vitellogenesis is used as an indicator of estrogenic and, thus, ovarian function, it was
likewise used as a measure of ovarian disruption (Zerulla et al. 2002). There was a significant (p=
0.001) difference in the number of vitellogenic oocytes in the 72C and 72T. This indicated a
change in normal ovarian morphology and function in the presence of TBA. In addition, a greater
number of atretic follicles was noted. Figure 10 shows atretic follicles at various stages of
development.
Division of the lobular ovary by lumen was also noted. This segregation into small lobular
units was more common in the testis. Figure 11 shows this division of the gonad. Tissue found
between the follicles or interstitial tissue was a common feature in many of the 72T gonads.
Interstitial tissue is normally associated with the testis. Within this tissue, in some gonads, were
small clusters of cells similar to spermatocysts. Though identification of specific stages of
developing germ cells was not possible, there were clearly signs of early testicular formation
characteristic of the control males analyzed. The testicular formation and possible spermatocysts
in 72T Bettas are shown in Figure 12.
There was a difference in the amount of ovarian regression and/or testicular formation
within the 72T group. Some gonads were primarily ovarian though they showed signs of
regression. Others exhibited both ovary regression and traces of interstitial tissue with possible
testicular characteristics. Finally there were a few gonads that contained obvious ovarian
regression as well as clearly testicular cysts forming in the interstitial tissue. This pattern of early
to late transition has been noted in several hermaphroditic species, including the honeycomb
grouper (Epinephelus merra: Bhandari et al. 2003).

26
 L
T

T
P
A
20µm

* *
T
*

O * O

P
B
20µm

*
* *
IT
* *

C IT
20µm

Figure 9 Comparison of gonads. A. Ovary from 72C, oocytes from all stages of development
present; B. Ovary from 72T contains few vitellogenic oocytes and an increasing number of atretic
follicles; C. The 82T gonad contains few healthy oocytes (non-atretic) and increasing amounts of
interstitial tissue with possible testicular components. All 40X. Interstitial Tissue (IT); Lumen
(L); Oil drop stage (O); Primary yolk globule stage (P); Tertiary yolk globule stage (T);
Perinucleolus stage (arrow); Atretic oocytes (*).

27
 20µm
*
*
*
* *
*
*
*
O
*
*
P
*
*

Figure 10 Ovary from a 72T Betta showing several atretic follicles (40X). Oil drop stage (O);
Primary yolk globule stage (P); Perinucleolus stage (arrow); Atretic oocytes (*).

20µm
*
* *
*
*
*

* L
*
*
*
* *

Figure 11 Ovary from a 72T Betta with large lumen area and branching ducts (40X). Lumen (L);
Perinucleolus stage (arrow); Atretic oocytes (*).

28
 T *

*
*
* C

A
2µm

*
* *

* C

B *
L 10µm

Figure 12 Ovaries from 72T Bettas exhibiting testicular tissue. A. 200X; B. 100X; Spermatocysts
(C); Lumen (L); Tertiary Yolk Globule Stage (T); Atretic oocytes (*).

The TBA treated ovaries at 82 ºF were analyzed for changes to normal ovarian function
and possible testicular formation. There was not a significant change between 72T and 82T
(length: p= 0.972, width p=0.378). This indicated that temperature did not significantly increase
the effects of TBA on the size of the gonad. Morphologically there were many differences
between the two treated groups (72T and 82T). A comparison between the morphology of the
treated groups is shown in Figure 9. Most noticeable is the lack of vitellogenic oocytes in most
sections observed. This clearly indicated a major disruption in ovarian function. Many more
29
follicles at all stages of development were atretic (See Figure 13). Again, the presence of a high
number of pre-ovulatory atretic follicles is indicative of ovarian disturbances. An increase in
lumen area was also noted, as seen in Figure 14.

* *
IT *
*
*
* IT
IT *
L *
*
*
* *
20µm

Figure 13 Ovary from 82T Betta with many atretic follicles (40X). Interstitial tissue (IT); Lumen
(L); Atretic follicles (*).

*
*
* * *
* *

L *
* *
*

*
20µm

Figure 14 Ovary from 82T Betta with extensive lumen area and branching ducts (40X). Lumen
(L); Atretic follicles (*).

30
 Interstitial tissue was even more common in the 82T group. Many sections appeared as
compact as the testicular control slides. Within the interstitial tissue were nests of spermatocysts,
some more clear than others. A testis control is compared to an 82T gonad in Figure 15. The
similarity between nests of spermatocysts was clear, though stages of sperm development are not
easily identified with the equipment used.

IT
IT
SC
ST
ST SC SG

ST
ST
A
SG 25µm

*
* *

C
B 20µm

Figure 15 Comparison of control testis to 82T gonad. A. Control testis showing spermatocysts at
various stages of development (400X); B. Partially transformed gonad from 82T with atretic
oocytes and spermatocysts (200X). Spermatocysts (C); Interstitial tissue (IT); Lumen (L);
Spermatocytes (SC); Spermatogonia (SG); Spermatids (ST); Atretic oocytes (*).

31
 Like the 72T group, the 82T group exhibited a gradient of transformation. Some gonads
had little, if any, testicular tissue. Others had a nearly 50:50 ratio of ovarian to testicular tissue,
while a few had transformed completely into testicular tissue. Figure 16 shows a fully
transformed gonad that consists of primarily testicular tissue, with the exception of a few atretic
oocytes. A magnified section of the transformed gonad compared to a testis control can be seen
in Figure 17. The cluster-like division varied from that seen in the control, but spermatocysts
were clearly defined. Again, identification of specific stages of sperm development was difficult,
but cysts similar to those containing spermatids in the control could be seen near the lumen in the
82T gonads. The cellular matter in the lumen was unidentified but could be mature spermatozoa.

L
L

*
C

A 250µm
C

C B
250µm

Figure 16 Gonad from 82T group with nearly complete transformation to testicular tissue; A.
40X; B. expanded view. Lumen (L); Spermatocysts (C); Atretic Oocyte (white arrow, *).

32
 IT
IT
SC
ST
ST SC SG

ST
ST
A SG 25µm

SG IT
SC

ST ST

L
B 20µm

Figure 17 Comparison of control testis to fully transformed 82T ovary. A. Control testis showing
spermatocysts at various stages of development (400X); B. Ovary from 82T showing
spermatocysts and open lumen area (200X). Interstitial tissue (IT); Lumen (L); Spermatocytes
(SC); Spermatogonia (SG); Spermatids (ST).

33
 CHAPTER 4

DISCUSSION

Studies of hermaphroditic species, such as the marbled swamp eel, Synbranchus

marmoratus, have revealed the morphological changes involved with natural sex reversal, mainly

the formation of testicular lobules within the ovarian structure (Harrington 1971, Lo Nostro et al.

2003, Lo Nostro et al. 2004). Studies of sex reversal were not limited to hermaphroditic species.

Other studies have revealed many factors that may help determine the phenotypic sex of

gonochoristic species. The focus of these studies varied from environmental factors to surgical

techniques or hormonal manipulations. More recent studies have used synthetic drugs and

hormones, with the common goal of improving fishery management and commercial fish

production. Sex reversal studies have also been used to indicate the effects of pollutants, or other

stress factors, on various species of fish (Bortone and Davis 1994). The present study examined

temperature, as well as the introduction of the synthetic hormone, TBA. The following literature

review highlighted some research that has shaped the study of sex reversal in gonochoristic

species.

34
 Environmental Studies

There are several environmental factors that have been studied in regard to sex reversal in

fish, including length of day, light intensity, temperature, water quality, and social environment

(Chen and Yeung 1983). Among these, temperature has been the focus of many studies that

examined a number of teleost species. Research, similar to studies in reptiles and amphibians, has

shown that during early development in fish, there existed a labile period when temperature can

affect sexual differentiation (Strussman and Nakamura 2002).

In the Atlantic Silverside, Menidia menidia, high temperatures induced sex differentiation

at an earlier stage of development (Conover and Fleisher 1986). Other studies focused on

resulting sex ratios following various temperature treatments. Exposure to high temperatures

during early development resulted in an increased male population in sockeye salmon

(Oncorhynchus nerka, Azuma et al. 2004), southern flounder (Paralichthys lethostigma,

Luckenbach et al. 2003), and Nile tilapia (Oreochromis niloticus, Baras et al. 2001). Southern

flounder also exhibited a shift toward the male phenotype in lower temperature treatments,

suggesting a mid-range temperature protocol for maximizing the commercially desired female

phenotype (Luckenbach et al. 2003). While high temperature often initiates a male-oriented shift,

the channel catfish underwent sex reversal to phenotypic females, following treatment with

increased temperature (Patiño et al. 1996). Changes in normal temperature at any stage of

development are potentially detrimental to fish. Many studies report high mortality rates of fry,

which could skew results (Chan and Yeung 1983, Patiño et al. 1996, Baras et al. 2001,

Luckenbach et al. 2003, Azuma et al. 2004).

In recent studies, the mechanisms of sex reversal, due to temperature, have been explored.

Some studies suggested that hormone production, specifically that of estrogens, was reduced by

high temperatures. These studies further suggested that the increased temperature directly

affected cytochrome P450 aromatase, which is necessary in the conversion of androgens into
35
estrogens. The decrease of estrogen production has been shown to cause oocyte apoptosis,

which led to testicular formation (Strussman and Nakamura 2002, Uchida et al. 2004).

Temperature, however, is not a factor for sex reversal in all fish species. Lucas (1968 as

cited in Chan and Yeung 1983) investigated many environmental factors on Betta sex ratios and

found temperature to be the only factor not causing a significant change. In the first part of this

study, Howell (2000) found a significant difference in tail lengths between 72T and 82T. of

Bettas. However, histological examination of Betta gonads from 72T and 82T revealed little, if

any, morphological changes. The latter results supported those found by Lucas.

Surgical Techniques

Early studies of sex reversal in fish involved surgical removal of reproductive tissues. This

included removal of partial gonads and complete castration (Yamamoto 1969). Researchers

suggested that fragments of gonad, male or female, contained germ cells with the potential to

mature into either eggs or sperm (Chan and Yeung 1983). In cases of ovariectomy, follicles,

which are the major site of estradiol production, are no longer present. Without needed

estrogens, the germ cells were under the influence of naturally occurring androgens that have the

potential to shift gonadogenesis from ovary to testis (Hoar 1969). Lowe and Larkin (1975)

suggested that the bipotentiality remained in the oviduct and ovarian wall, which may be present

following an ovariectomy. It has also been suggested that the presence of a few follicular or

granulosa cells may have produced enough estrogen to maintain the female gonad (Lowe and

Larkin 1975).

This surgical technique was used by U.S. researchers, Noble and Kumph (1937 In

Yamamoto 1969) who removed ovaries from female Bettas. Though their success rate was

extremely low, this team was able to produce a regenerated testis in a genotypically female fish.

These results were later duplicated in Germany (Yamamoto 1969). Later, Lowe and Larkin
36
(1975) repeated this procedure with much higher success rate. Of 245 female Bettas

ovariectomized, 42.4% formed a testis (22.9% regenerated an ovary, 34.7% died; Lowe and

Larkin 1975). Matings from these reversals resulted in both male and female progeny (Lowe and

Larkin 1975), which discredited earlier views that male sex in Bettas is heterogametic (Yamamoto

1969). These results suggested a polygenic sex determination controlled by several genes (Lowe

and Larkin 1975). Though Bennington (1936) described possible sex chromosomes within the

spermatogonia of the Betta, these sex-determining genes may be found throughout autosomal

chromosomes (Lowe and Larkin 1975).

Hormonal Manipulations

As early as the 1930s studies involving sex determination and differentiation in fish began

observing the use of sex steroids. As basic mechanisms were understood, studies of sex steroids

focused on the phenotypic benefits observed in specific species (Hunter and Donaldson 1983).

Processes controlled naturally by hormones could be manipulated to affect timing of development

and spawning, as well as gamete supply, and even sex ratios of offspring. Secondary and

functional sex reversal has been achieved in many species. This has benefited the commercial fish

market with both desired size of fish for consumption and desired appearance for ornamental fish

(Patiño 1997). Many studies have analyzed the use of natural steroids, synthetic steroids, and

even steroidal or chemical contamination of the environment and their effects on fish

reproduction. Some of these studies have been described in the following sections, which led to

the design of the present study.

37
Natural Steroids

According to a review by Pandian and Sheela (1995), the most commonly used natural

steroids for studies of fish reproduction are 17β-estradiol and 17α-methyltestosterone. Together,

these two hormones accounted for treatment protocols in 40 species of fish (Pandian and Sheela

1995). Studies of hermaphroditic species, such as the protoandrous black porgy, revealed high

levels of 17β-estradiol during sex reversal (Chang et al. 1994).

The use of 17β-estradiol during treatments of gonochoristic species has been successful at

inducing both direct and indirect feminization. Direct feminization involved treatment of

undifferentiated males with estrogens, while indirect feminization treated with testosterone first to

produce neomales, or genetic females displaying a male phenotype. These neomales (XX) are

later mated with normal females (XX) producing a female brood (Piferrer 2001). In the Atlantic

halibut, direct feminization was produced by 17β-estradiol and indirect feminization was produced

by 17α-methyldihydrotestosterone (Hendry et al. 2003). Direct reversal has been observed in a

number of other species following treatments with estradiols. (Pandian and Sheela 1995,

Odontesthes bonariensis: Strüssman et al. 1996, Oryzias latipes: Papoulias et al. 1999, Danio

rerio: Örn et al. 2003, Pseudobagres fulvidraco: Park et al. 2004).

Hormonal masculinization has been observed following treatment with 17α-

methyltestosterone. Studies have explored the use of this hormone through immersion and

dietary supplement. Functional males have been produced with 17α-methyltestosterone in many

species, including the silver barb (Puntius gonionotos: Pongthana et al. 1999), Eurasian perch

(Perca fluviatilis: Rougeot et al. 2002), European sea bass (Dicentrarchus labrax L.: Blázquez et

al. 1995), and rainbow trout (Oncorhynchus mykiss: Feist et al 1995). Treatment with 11β-

hydroxyandrosterone also produced functional males in rainbow trout (Feist et al. 1995) and

Florida red tilapia (Oreochromis niolticus: Desprez et al. 2003).

38
 Natural testosterone has been the subject of several studies of sex reversal in fish. Badura

and Friedman (1988) observed female Bettas following interperitoneal injections of testosterone.

This study found degeneration of the ovarian structure and proliferation of possible testicular

tissue, as well as changes in the secondary sex characteristics that had previously been reported.

Though sex reversal had been successful, the study did not explore the functionality of reversed

gonads (Badura and Friedman 1988).

Like other species, Bettas naturally use several androgens during testis formation (Leitz

and Reinboth 1987, Leitz 1987). Pandian and Sheela (1995) report the following order of

androgens based on their reviews of sex reversal: Miboterone > 19-nor-ethynyl-testosterone >

17α-methyltestosterone > testosterone (Pandian and Sheela 1995).

Synthetic Steroids

The review by Pandian and Sheela (1995) reported more experiments using synthetic

estrogens and androgens than their natural counterparts. This use was attributed to the efficiency

of production, as well as the decreased rate of metabolism of synthetic hormones (Pandian and

Sheela 1995). In some cases, as in that of channel catfish, natural hormones yielded no significant

sex reversal, and synthetic hormones were employed. After several trials, treatment with

trenbolone acetate proved to be the first successful technique for masculinizing channel catfish

(Galvez et al. 1995).

Trenbolone acetate (TBA), a synthetic steroid, has been reported to have a much higher

anabolic and androgenic potential that testosterone (Galvez et al. 1996). Commonly used to

increase weight gain in cattle (Henricks et al. 1982), TBA has become a steroid of interest to the

fishery industry. The anabolic and androgenic characteristics exploited in cattle have been

mimicked in several fish species (Galvez et al. 1995, Galvez et al. 1996, Davis et al. 2000,

Fitzpatrick et al. 2000). The techniques used to masculinize the channel catfish were also
39
successful in the blue tilapia (Galvez et al. 1996). In this study, a comparison was made to 17α-

methyltestosterone, which produced a significantly lower percentage of males compared to TBA

(Galvez et al. 1996). Neither the channel catfish nor the blue tilapia was observed long enough to

record functionality of the reversed gonads by spawning. Another study revealed that treatment

of channel catfish with TBA did not produce functional males, despite significant morphological

changes (Davis et al. 2000).

Many previous experiments with TBA introduced the synthetic hormone through dietary

supplements. Fitzpatrick et al. (2000) observed masculinization in Nile tilapia following

immersion in TBA. Both techniques have been shown to be effective at sex reversal. Studies

involving masculinization of fish by TBA treatment have included commercially important species

for consumption. To date there have been no reports of the use of TBA for masculinization of

ornamental fish such as Betta splendens. Research was needed to explore the androgenic and

anabolic effects of TBA treatment on primary and secondary sex characteristics.

Other considerations

Several problems were revealed with hormonal treatments like those previously discussed.

Each method of administration has disadvantages ranging from unequal treatments to physical

damage. A review of literature revealed that types and dosages of hormones have caused

formation of intersex gonads, sterility, and increased mortality rates (Pandian and Sheela 1995).

While synthetic hormones have increased success rates and decreased cost and time requirements

for treatments, the general public remains cautious. Any manipulation of fish destined for

consumption should be thoroughly examined and possibly regulated (Hunter and Donaldson 1983,

Patiño 1997).

Despite some difficulties, the successful protocols for sex reversal have greatly benefited

the fishery industry. Economically important species for consumption have been treated for
40
production of desired monosex populations. Ornamental fish have also been treated to produce

desired phenotypes (Hunter and Donaldson 1983). Beyond commercial benefits, sex reversal

techniques have the potential to enhance environmental studies. The sensitivity of fish

reproductive functions has made them excellent indicators of environmental stress, such as

pollutants (Bortone and Davis 1994, Jobling et al. 1998, Dutta and Maxwell 2003, Patiño et al

2003).

The present study differs from much of the previous research because of the

developmental stage used for treatment and examination. Many studies use early fry because of

the sexual bipotentiality (Chan and Yeung 1983); however, this study used sexually differentiated

adult Bettas. This developmental stage was chosen to take into account the sexually dimorphic

secondary sex characteristics. Based on these characteristics, it was assumed that the Bettas used

were sexually mature at the time of treatment. The high mortality rate in studies using fry was not

seen in this study. Other studies suggested that temperature and hormonal manipulation had no

effect on sex reversal prior to or following the sensitive time during early development (Conover

and Fleisher 1986). This statement, among others, prompted the need for studies on sex reversal

in adult fish species.

The results of this study have implications in the fishery industry, not only for the

production of phenotypically male Bettas, but also for the use of TBA in other species of fish.

The use of adult Bettas has a bearing on studies of affected populations of fish in contaminated

areas. Successful sex reversal of these adult gonads may also add to current studies of fertility

and hormone functions of many species. These results may apply to treatment or contamination

from TBA, as well as other synthetic hormones and chemicals.

41
 Conclusions

Statistical analysis of temperature effects on gonad size was not possible due to problems

encountered in the histological techniques. Morphological examination revealed little if any

difference in ovarian structure. This indicates that temperature alone causes no significant change

in primary sex characteristics of Bettas.

The addition of TBA to the diet caused a statistically significant decrease in gonad length

and width. There was also a statistically significant decrease in the total number and size of

vitellogenic oocytes. Morphologically, there was a dramatic change in structure size and

composition (type of oocytes present). These results taken together indicate that the synthetic

hormone TBA has the potential to cause significant changes to primary sex characteristics of

Bettas.

Gonads from 72T and 82T revealed a significant change in gonad size. Statistical

comparison could not be performed on total vitellogenic oocyte number or size (n=1). The 82T

group contained few, if any, vitellogenic oocytes and many more atretic follicles. This group also

exhibited much more interstitial tissue including visible spermatocysts. These data suggest that

the combination of TBA treatment and increased temperature may enhance the morphological

changes with the female Betta reproductive system.

These techniques may be effective on similar species; however, studies on dosage, timing,

and side effects would need to be performed. Future studies should also consider change in body

mass and gonad mass (gonadosomatic index: DeMartini and Lau 1999, Lone et al. 2001) as a

biological indicator. A comparison of morphological changes occurring at various points during a

broadened treatment timetable may enable a better understanding of the effects of TBA on the

reproductive tract. These studies could be followed by histochemical analysis to explore the

mode of action for TBA. Finally, studies are needed to explore the lasting effect of TBA and the

continuation or regression of sex-reversed characteristics.

42
 CHAPTER 5

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48
 APPENDICES

APPENDIX A

Tail length data from Howell (2000)

Initial tail Change in Initial tail Change in

length Final tail tail length length Final tail tail length
* (mm) length (mm) * (mm) length (mm)
Fish (mm) Fish (mm)
172C1 5.9 8.5 2.6 282C1 9.2 11 1.8
172C2 8.2 8.4 0.2 282C2 10 11.5 1.5
172C3 7.8 8.4 0.6 282C3 9.5 9.9 0.4
172C4 8.5 8.7 0.2 282C5 8.1 8.2 0.1
172C5 7.9 8.1 0.2 282C6 9 11.6 2.6
172C6 6.8 7.2 0.4 282T4 8 20.6 12.6
172T1 6.7 14.8 8.1 372C1 11 12.3 1.3
172T2 8.1 14.7 6.6 372T1 8.2 10 1.8
172T3 7.5 17.1 9.6 372T2 10.7 21.8 11.1
172T6 8.8 19.3 10.5 372T3 11.4 21.8 10.4
182C1 5.8 10.1 4.3 372T4 7.8 9.9 2.1
182C2 6.4 7.1 0.7 372T5 10.1 18.5 8.4
182C3 6.2 7.1 0.9 372T6 11 19.1 8.1
182C4 6.4 7.2 0.8 382T1 9 22 13
182C5 8.1 11.1 3 382T2 11.7 24.4 12.7
182C6 8.1 11.3 3.2 382T3 10.8 24.2 13.4
182T1 7.8 10.3 2.5 382T4 11 23.9 12.9
182T2 6.9 18.5 11.6 382T5 10.7 24.8 14.1
182T3 8.2 18 9.8 382T6 10.3 22.4 12.1
182T4 7.2 14.5 7.3 472C1 8 8 0
182T6 7.9 10.2 2.3 472C2 7.9 7.9 0
272C3 7.7 7.9 0.2 472C3 8.7 8.9 0.2
272C4 10.1 10.7 0.6 472C4 7.8 7.9 0.1
272T2 8.3 17.2 8.9 472C5 7.9 8.1 0.2
272T3 8.4 20.9 12.5 472C6 7.7 7.8 0.1
272T5 8.9 17.3 8.4

*Fish ID number: 1st digit = set number (fish were treated in 4 sets due to aquarium space); 2nd &
3rd digit = refer to temperature (72 or 82ºF); 4th digit/letter = C (control), T (treated with TBA);
5th digit = fish number

49
 APPENDIX B

Data from histological analysis

Data from Control Female Bettas (72C: preserved and OC: purchased)

Total # Diameter of Random Vitellogenic Oocytes

* Length Width of V
#1 #2 #3 #4 #5 AVG
Slide # (µm) (µm) Oocytes
OC1 n/a n/a n/a n/a n/a n/a n/a n/a n/a
OC2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
OC3#1a n/a 2725 n/a 460.6 539 480.2 539 529.2 509.60
OC3#1b n/a 2750 n/a 499.8 470.4 490 333.2 539 466.48
OC3#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
OC3#3a n/a n/a n/a 470.4 499.8 441 431.2 529.2 474.32
OC3#3c n/a n/a n/a 509.6 480.2 392 490 548.8 484.12
OC3#4a n/a n/a n/a n/a n/a n/a n/a n/a n/a
OC3#4b 2625 n/a n/a 480.2 499.8 470.4 431.2 431.2 462.56
OC3#5 n/a n/a n/a n/a n/a n/a n/a n/a n/a
OC4#1 n/a n/a n/a n/a n/a n/a n/a n/a n/a
OC4#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
OC4#3 n/a n/a n/a 352.8 343 333.2 333.2 294 331.24
OC4#4a n/a n/a n/a 529.2 578.2 313.6 343 372.4 427.28
OC4#4b n/a n/a n/a 617.4 372.4 460.6 568.4 352.8 474.32
OC7#1b 6350 2725 54 333.2 490 352.8 441 401.8 403.76
OC7#1b 6325 2900 58 441 529.2 303.8 362.6 480.2 423.36
OC7#2c 6900 2950 64 343 460.6 352.8 490 480.2 425.32
OC7#2g 6725 3125 60 529.2 362.6 480.2 460.6 382.2 442.96
OC7#3a 8250 2800 62 441 372.4 450.8 460.6 470.4 439.04
OC7#3b 7925 2625 61 431.2 372.4 441 382.2 460.6 417.48
OC7#4a 8075 2550 66 509.6 490 362.6 548.8 333.2 448.84
OC7#4b 8100 2575 67 392 382.8 372.4 480.2 323.4 390.16
OC7#5c 8875 1950 44 303.8 480.2 382.2 480.2 431.2 415.52
OC7#5d 8875 1975 43 499.8 352.8 441 421.4 411.6 425.32
OC7#6c 3875 3250 27 372.4 450.8 509.6 499.8 333.2 433.16
OC7#6e 3900 3475 30 352.8 480.2 382.2 421.4 421.4 411.6
OC7#7c 6400 3825 59 333.2 509.6 509.6 392 441 437.08
OC7#7d 6450 3800 62 460.6 401.8 372.4 519.4 450.8 441.00
OC7#8b 6375 3825 61 323.4 460.6 401.8 490 411.6 417.48
OC7#8d 6550 3925 61 558.6 401.8 441 421.4 539 472.36
OC8#1b 6950 2625 40 470.4 352.8 607.6 509.6 519.4 491.96
OC8#1d 6825 2600 41 441 490 509.6 392 519.4 470.40
50
 Total # Diameter of Random Vitellogenic Oocytes
* Length Width of V
#1 #2 #3 #4 #5 AVG
Slide # (µm) (µm) Oocytes
OC8#2c 6800 2550 42 431.2 460.6 519.4 401.8 490 460.60
OC8#2e 7000 2550 43 401.8 509.6 588 509.6 597.8 521.36
OC8#3b 4650 n/a n/a 548.8 372.4 529.2 450.8 499.8 480.20
OC8#3c 4625 n/a n/a 362.6 460.6 558.6 499.8 490 474.32
OC8#4a 7587 2587.1 37 478.3 456.5 369.6 413.1 413.1 426.12
OC8#4e 7348.1 2391.4 44 456.5 391.3 413.1 565.2 500 465.22
OC8#5b 7956.8 2217.5 41 521.8 391.3 434.8 500 500 469.58
OC8#5c 7065.5 2304.4 10 521.8 500 347.8 456.5 586.9 482.60
OC8#6a 7456.8 3826.2 73 500 456.5 369.6 434.8 478.3 447.84
OC8#6b 7739.4 4695.8 77 478.3 434.8 413.1 543.5 500 473.94
OC8#7b n/a 4674.1 n/a 456.5 326.1 413.1 565.2 434.8 439.14
OC8#7c n/a 4956.7 n/a 500 347.8 521.8 543.5 369.6 456.54
OC8#8c 7478.6 4804.5 76 478.3 434.8 500 369.6 500 456.54
OC8#8e 7609 4869.8 71 478.3 500 500 500 478.3 491.32
OC9#1b 8543.8 3913.2 63 391.3 500 456.5 565.2 391.3 460.86
OC9#1d 8413.4 3913.2 62 500 391.3 478.3 456.5 413.1 447.84
OC9#2c 9391.7 n/a n/a 413.1 369.6 565.2 500 434.8 456.54
OC9#2e 9413.4 n/a n/a 456.5 500 369.6 456.5 434.8 443.48
OC9#3a 9174.3 n/a n/a 500 347.8 587 464.8 500 479.92
OC9#3c 9326.5 n/a n/a 369.6 391.3 413.1 565.2 456.5 439.14
OC9#4b 13000.5 n/a n/a 391.3 369.6 500 500 456.5 443.48
OC9#4d 12609.2 n/a n/a 391.3 500 391.3 565.2 391.3 447.82
OC9#5a 12870.1 n/a n/a 413.1 434.8 456.5 608.7 391.3 460.88
OC9#5e 12913.6 n/a n/a 434.8 434.8 391.3 478.3 456.5 439.14
OC9#6a 12913.6 n/a n/a 521.8 434.8 500 369.6 500 465.24
OC9#6d 12804.9 n/a n/a 500 456.5 369.6 413.1 456.5 439.14
OC9#7b 12804.9 3565.4 n/a 500 478.3 608.7 391.3 500 495.66
OC9#7c 12826.6 4782.8 n/a 456.5 478.3 565.2 434.8 391.3 465.22
72C6b#1 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72C6b#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72C6b#3b n/a n/a 44 445.7 510.9 543.5 500 369.6 473.94
72C6b#3c n/a n/a 45 423.9 467.4 391.3 500 478.3 452.18
Average 8148.28 3266.03 52.75 451.65
*Slide # Key: OC = ovary control; 72C = 72ºF Control; a/b/c = ovary lobes (if separated during
dehydration); # = slide # (multiple slides made for each ovary); final letter = section number on each
slide chosen at random

51
 Data from Control Female Bettas (82C)

Total # Diameter of Random Vitellogenic Oocytes

* Length Width of V
Slide # (µm) (µm) Oocytes #1 #2 #3 #4 #5 AVG
82C1a#1 n/a n/a n/a n/a n/a n/a n/a n/a n/a
82C1a#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
82C1a#3 n/a n/a n/a n/a n/a n/a n/a n/a n/a
82C1a#4a n/a n/a n/a 510.9 347.8 423.9 326.1 326.1 386.96
82C1a#4b n/a n/a n/a 315.2 337 369.6 369.6 423.9 363.06
Avg 375.01
*Slide # Key: 82C = 82ºF Control; a/b/c = ovary lobes (if separated during dehydration); # = slide #
(multiple slides made for each ovary); final letter = section number on each slide chosen at random

52
 Data from Control Male Bettas (TC)

*
Slide # Length (µm) Width (µm)
TC1-1b1 2456.6 847.9
TC1-1b2 2500.1 804.4
TC1-2b1 2282.7 869.6
TC1-2b2 2500.1 760.9
TC1-3c1 2500.1 934.8
TC1-3c2 2521.8 934.8
TC2-1 n/a n/a
TC2-2 n/a n/a
TC3-1 n/a n/a
TC3-2 n/a n/a
TC3-3 n/a n/a
TC3#1 n/a n/a
TC3#2 n/a n/a
TC3#4 n/a n/a
TC6#1c1 1989.2 500.0
TC6#1c2 1706.6 445.7
TC6#2a1 2543.6 510.9
TC6#2a2 2271.8 380.5
TC6#3 n/a n/a
TC6#4b1 2032.7 510.9
TC6#4b2 1934.9 423.9
TC6#5b1 1739.2 445.7
TC6#5b2 1880.5 587.0
TC6#6e 2130.5 380.5
TC6#6g 2108.8 510.9
Avg 2193.7 615.5
*Slide # Key: TC = testis control; # = slide # (multiple slides made
for each testis); final letter/# = section number on each slide

53
 Data from Treated Bettas (72T)

Total Diameter of Random Vitellogenic Oocytes

* Length Width # of
Slide # (µm) (µm) VO’s #1 #2 #3 #4 #5 AVG
72T1#1a 2826.20 1271.80 n/a n/a n/a n/a n/a n/a n/a
72T1#1c 3021.90 1315.30 n/a n/a n/a n/a n/a n/a n/a
72T1#2a 4021.90 1652.20 n/a n/a n/a n/a n/a n/a n/a
72T1#2b 3521.90 1760.90 n/a n/a n/a n/a n/a n/a n/a
72T1#3 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T1#4a 4413.20 1587.00 1 282.60 n/a n/a n/a n/a 282.60
72T1#4b 4239.30 1587.00 1 282.60 n/a n/a n/a n/a 282.60
72T1#5b 4369.70 2021.80 1 456.50 n/a n/a n/a n/a 456.50
72T1#5d 4456.70 2087.00 1 413.10 n/a n/a n/a n/a 413.10
72T1#6c 4739.30 1347.90 0 n/a n/a n/a n/a n/a n/a
72T1#6d 4869.80 1652.20 0 n/a n/a n/a n/a n/a n/a
72T1#7b 5108.90 1565.30 1 500.00 n/a n/a n/a n/a 500.00
72T1#7c 5195.90 1478.30 1 521.80 n/a n/a n/a n/a 521.80
72T2a#1a n/a 2000.10 6 206.50 195.60 239.10 217.40 n/a 214.65
72T2a#1b n/a 2043.60 7 n/a n/a n/a n/a n/a n/a
72T2a#2a n/a 2108.80 3 152.20 206.50 217.40 n/a n/a 192.03
72T2a#2c n/a 2043.60 3 n/a n/a n/a n/a n/a n/a
72T2a#3a n/a n/a 1 228.30 n/a n/a n/a n/a 228.30
72T2a#3c n/a n/a 2 206.50 173.90 n/a n/a n/a 190.20
72T2a#4a n/a 1739.20 0 n/a n/a n/a n/a n/a n/a
72T2a#4b n/a 1804.40 0 n/a n/a n/a n/a n/a n/a
72T2a#5e n/a 1913.10 0 n/a n/a n/a n/a n/a n/a
72T2a#5g n/a 2000.10 0 n/a n/a n/a n/a n/a n/a
72T2a#6c n/a 1675.00 4 184.40 152.20 173.90 184.80 n/a 173.82
72T2a#6f n/a 1728.30 3 152.20 163.10 184.80 n/a n/a 166.70
72T2a#7b n/a 1546.40 3 228.70 185.10 185.10 n/a n/a 199.63
72T2a#7d n/a 1677.10 4 206.50 173.90 184.80 163.10 n/a 182.07
72T2b#1a 4326.30 1663.10 3 87.00 97.90 119.60 n/a n/a 101.50
72T2b#1e 2152.30 728.30 0 n/a n/a n/a n/a n/a n/a
72T2b#2b 3695.80 2130.50 0 n/a n/a n/a n/a n/a n/a
72T2b#2d 3782.80 2195.70 0 n/a n/a n/a n/a n/a n/a
72T2b#3a 3674.10 2217.50 2 119.60 97.80 n/a n/a n/a 108.70
72T2b#3d 3978.40 2326.20 3 108.70 97.80 108.70 n/a n/a 105.06
72T2b#4 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2b#5a 3565.40 2347.90 5 174.20 217.80 141.30 250.00 228.30 202.32
72T2b#5b 3761.00 2217.50 3 250.00 206.50 250.00 n/a n/a 235.50
72T3a#1b 5413.30 n/a n/a 336.90 250.00 260.90 n/a n/a 282.60
72T3a#1c 5304.60 n/a n/a 315.20 304.40 271.80 250.00 n/a 285.35
72T3a#2a 5087.20 2021.80 n/a 304.40 271.80 260.90 n/a n/a 279.03
72T3a#2b 5152.40 1956.60 n/a 260.90 250.00 260.90 n/a n/a 257.27
54
 Total Diameter of Random Vitellogenic Oocytes
* Length Width # of
Slide # (µm) (µm) VO’s #1 #2 #3 #4 #5 AVG
72T3a#3b 5304.60 2087.00 3 217.80 205.00 293.50 n/a n/a 238.77
72T3a#3c 5348.00 2000.10 3 293.50 228.70 250.00 n/a n/a 257.40
72T3a#4 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T3a#5c 5478.50 n/a n/a 217.40 293.50 n/a n/a n/a 255.45
72T3a#5d 5522.00 n/a n/a 315.20 260.90 282.60 n/a n/a 286.23
72T3a#6b n/a n/a n/a 337.00 304.40 293.50 n/a n/a 311.63
72T3a#6e n/a n/a n/a 347.80 217.40 n/a n/a n/a 282.60
72T3a#7d n/a n/a n/a 369.60 195.60 326.10 n/a n/a 297.10
72T3a#7f n/a n/a n/a 369.60 206.50 282.60 n/a n/a 286.23
72T3b#1b n/a 2043.60 n/a n/a n/a n/a n/a n/a n/a
72T3b#1d n/a 2021.80 n/a n/a n/a n/a n/a n/a n/a
72T3b#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T3b#3b n/a 2021.80 n/a n/a n/a n/a n/a n/a n/a
72T3b#3c n/a 2021.80 n/a n/a n/a n/a n/a n/a n/a
72T3b#4c n/a 2152.30 n/a n/a n/a n/a n/a n/a n/a
72T3b#4d n/a 2217.50 n/a n/a n/a n/a n/a n/a n/a
72T6#1 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T6#2a 4848.00 2347.90 n/a 206.50 141.30 163.10 n/a n/a 170.30
72T6#2b 4869.80 2369.70 n/a 206.50 184.80 152.20 n/a n/a 181.17
72T6#3a 4956.70 2326.20 n/a 206.50 195.70 195.70 n/a n/a 199.30
72T6#3b 4935.00 2304.40 n/a 141.30 n/a n/a n/a n/a 141.30
72T6#4c1 5869.80 2021.80 2 445.70 168.10 n/a n/a n/a 306.90
72T6#4c2 4913.20 3369.70 6 489.20 500.00 391.30 489.20 413.10 456.56
72T6#5c1 5739.40 1891.40 2 521.80 173.90 n/a n/a n/a 347.85
72T6#5c2 4956.70 3391.40 7 434.80 391.30 586.90 565.20 543.50 504.34
72T6#6c1 5456.70 1739.20 1 391.30 n/a n/a n/a n/a 391.30
72T6#6c2 4956.70 3326.20 7 608.70 521.80 587.00 500.00 326.10 508.72
72T6#7d1 5869.80 1934.90 2 326.10 195.70 n/a n/a n/a 260.90
72T6#7d2 5108.90 3304.50 4 543.50 500.00 434.80 217.40 n/a 423.93
72T6#8a1 5978.50 1913.10 4 608.70 282.60 413.10 500.00 n/a 451.10
72T6#8a2 n/a 3130.60 n/a n/a n/a n/a n/a n/a n/a
72T6#9c1 5826.30 2108.80 5 500.00 521.80 543.50 478.30 608.70 530.46
72T6#9c2 n/a 2978.40 n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#1b 2978.40 1608.80 0 n/a n/a n/a n/a n/a n/a
72T2-IIa#1c 3369.70 1500.10 0 n/a n/a n/a n/a n/a n/a
72T2-IIa#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#3 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#4 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#5 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#6 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#7 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#8 n/a n/a n/a n/a n/a n/a n/a n/a n/a
55
 Total Diameter of Random Vitellogenic Oocytes
* Length Width # of
Slide # (µm) (µm) VO’s #1 #2 #3 #4 #5 AVG
72T2-IIa#9a 3956.70 2087.00 n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#9d 3913.20 2043.60 n/a n/a n/a n/a n/a n/a n/a
72T2-IIa#10 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIb#1 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIb#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIb#3 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIb#4 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIb#5 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIb#6 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIb#7b 4261.00 n/a n/a 282.60 n/a n/a n/a n/a 282.60
72T2-IIc#1 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIc#2 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIc#3 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIc#4c n/a 1304.40 n/a n/a n/a n/a n/a n/a n/a
72T2-IIc#4e n/a 1304.40 n/a n/a n/a n/a n/a n/a n/a
72T2-IIc#5b 3217.50 1043.50 0 n/a n/a n/a n/a n/a n/a
72T2-IIc#5c 3108.80 1065.30 0 n/a n/a n/a n/a n/a n/a
72T2-IIc#6 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T2-IIc#7 n/a n/a n/a n/a n/a n/a n/a n/a n/a
72T1-IIIa#1d n/a 2804.50 n/a 326.10 608.70 478.30 391.30 n/a 451.10
72T1-IIIa#1e n/a 2782.70 n/a 326.10 413.10 434.80 500.00 n/a 418.50
72T1-IIIa#2c 3848.00 1804.40 1 413.10 n/a n/a n/a n/a 413.10
72T1-IIIa#2e 3717.50 1739.20 1 391.30 n/a n/a n/a n/a 391.30
72T1-IIIa#3c n/a 2304.40 n/a 391.30 413.10 260.90 413.10 456.50 386.98
72T1-IIIa#3d n/a 2282.70 n/a 413.10 478.30 326.10 369.60 n/a 396.78
72T1-IIIa#4b n/a 2195.70 n/a 347.80 413.10 173.90 n/a n/a 311.60
72T1-IIIa#4f n/a 2326.20 n/a 565.20 347.80 347.80 304.40 239.10 360.86
72T1-IIIb#1e n/a 2565.30 n/a 347.80 391.30 543.50 304.40 434.80 404.36
72T-IIIb#1f n/a 2521.80 n/a 543.50 304.40 304.40 434.80 304.40 378.30
72T-IIIb#2a 6500.30 2413.10 4 173.90 326.10 434.80 173.90 n/a 277.18
72T-IIIb#2b 5935.00 3195.80 4 173.90 434.80 326.10 195.70 n/a 282.63
72T-IIIb#3a 5739.40 n/a n/a 391.30 217.40 195.70 n/a n/a 268.13
72T-IIIb#3d 5717.60 2934.90 n/a 239.10 282.60 413.10 n/a n/a 311.60
72T2-III#1e1 4021.90 1652.20 0 n/a n/a n/a n/a n/a n/a
72T2-III#1e2 3761.00 1869.60 0 n/a n/a n/a n/a n/a n/a
72T2-III#2c1 3891.50 1478.30 0 n/a n/a n/a n/a n/a n/a
72T2-III#2c2 3630.60 1869.60 0 n/a n/a n/a n/a n/a n/a
72T2-III#3d1 3913.20 1478.30 0 n/a n/a n/a n/a n/a n/a
72T2-III#3d2 3391.40 1826.20 0 n/a n/a n/a n/a n/a n/a
72T2-III#4c1 3956.70 1608.80 0 n/a n/a n/a n/a n/a n/a
72T2-III#4c2 3804.50 1804.40 0 n/a n/a n/a n/a n/a n/a
72T2-III#5b1 3934.90 1587.00 0 n/a n/a n/a n/a n/a n/a
56
 Total Diameter of Random Vitellogenic Oocytes
* Length Width # of
Slide # (µm) (µm) VO’s #1 #2 #3 #4 #5 AVG
72T2-III#5b2 3891.50 1717.50 0 n/a n/a n/a n/a n/a n/a
72T3-IIa#1b 5761.10 n/a n/a 434.80 413.10 500.00 500.00 n/a 461.98
72T3-IIa#1c 5608.90 n/a n/a 521.80 500.00 521.80 413.10 n/a 489.18
72T3-IIa#3b 6913.30 1500.10 4 500.00 434.80 304.40 543.50 304.40 417.42
72T3-IIa#3c 6891.60 1500.10 4 391.30 478.30 500.00 413.10 304.40 417.42
72T3-IIa#4a 6652.40 1630.50 5 478.30 500.00 326.10 391.30 347.80 408.70
72T3-IIa#4b 6609.00 1652.20 4 326.10 326.10 391.30 456.50 347.80 369.56
72T3-IIa#5b 6348.10 n/a n/a 347.80 369.60 326.10 326.10 n/a 342.40
72T3-IIa#5c 6369.80 1652.20 n/a 391.30 413.10 521.80 347.80 391.30 413.06
72T3-IIa#6a 6478.50 1608.80 6 369.60 434.80 413.10 347.80 391.30 391.32
72T3-IIa#6c 6239.40 1739.20 n/a 347.80 326.10 326.10 n/a n/a 333.33
72T3-IIa#7c n/a 1652.20 n/a 543.50 456.50 434.80 326.10 282.60 408.70
72T3-IIa#7g n/a 1456.60 n/a 500.00 456.50 521.80 391.30 413.10 456.54
72T3-IIa#8b n/a 1521.80 n/a 326.10 500.00 413.10 391.30 369.60 400.02
72T3-IIa#8e n/a 1587.00 n/a 347.80 521.80 413.10 391.30 391.30 413.06
72T3-IIa#9b n/a 1674.00 n/a 413.10 521.80 478.30 n/a n/a 471.07
72T3-IIa#9e n/a 1674.00 n/a 391.30 521.80 456.50 n/a n/a 456.53
72T3-IIb#1a 4000.20 1521.80 6 304.40 456.50 521.80 239.10 260.90 356.54
72T3-IIb#1c 3913.20 1630.50 n/a 478.30 369.60 282.60 n/a n/a 376.83
72T3-IIb#2b n/a n/a n/a 608.70 326.10 n/a n/a n/a 467.40
72T3-IIb#2d n/a n/a n/a 500.00 326.10 n/a n/a n/a 413.05
72T3-IIb#3c 4000.20 1934.90 4 587.00 369.60 500.00 260.90 n/a 429.38
72T3-IIb#3e 4000.20 n/a n/a 608.70 n/a n/a n/a n/a 608.70
72T3-IIb#4a 4413.20 1934.90 7 456.50 500.00 413.10 478.30 369.60 443.50
72T3-IIb#4c n/a n/a n/a 391.30 456.50 413.10 260.90 n/a 380.45
72T3-IIb#5b 3717.50 1695.70 7 413.10 413.10 456.50 304.40 587.00 434.82
72T3-IIb#5e 3804.50 1695.70 7 608.70 369.60 369.60 391.30 326.10 413.06
72T3-IIb#6d 3543.60 1587.00 8 280.60 326.10 369.60 478.30 500.00 390.92
72T3-IIb#6f 3521.90 1587.00 7 478.30 543.50 326.10 304.40 543.50 439.16
72T3-IIb#7a 3521.90 1695.70 n/a 434.80 326.10 652.20 304.40 369.60 417.42
72T3-IIb#7b 3565.40 1652.20 n/a 658.20 326.10 478.30 347.80 n/a 452.60
72T3-IIb#8a 5130.60 1956.60 7 347.80 391.30 282.60 304.40 304.40 326.10
72T3-IIb#8c 5087.20 1891.40 6 391.30 326.10 391.30 282.60 304.40 339.14
72T3-IIb#9d 5130.60 1891.40 7 282.60 413.10 347.80 260.90 195.70 300.02
72T3-IIb#9f 5065.40 1956.60 5 347.80 347.80 282.60 347.80 304.40 326.08
72T5-II#1a 4652.40 n/a n/a n/a n/a n/a n/a n/a n/a
72T5-II#1e 2804.50 n/a n/a n/a n/a n/a n/a n/a n/a
72T5-II#2d1 3869.70 1413.10 n/a 347.80 n/a n/a n/a n/a 347.80
72T5-II#2d2 4543.70 1304.40 0 n/a n/a n/a n/a n/a n/a
72T5-II#3b1 3456.70 n/a n/a n/a n/a n/a n/a n/a n/a
72T5-II#3b2 4304.50 1478.30 1 282.60 n/a n/a n/a n/a 282.60
72T5-II#4a1 3239.30 n/a n/a 217.40 260.90 n/a n/a n/a 239.15
57
 Total Diameter of Random Vitellogenic Oocytes
* Length Width # of
Slide # (µm) (µm) VO’s #1 #2 #3 #4 #5 AVG
72T5-II#4a2 4217.60 1556.60 0 n/a n/a n/a n/a n/a n/a
72T5-II#5b1 2869.70 n/a n/a 304.40 239.10 n/a n/a n/a 271.75
72T5-II#5b2 4087.10 1413.10 1 239.10 n/a n/a n/a n/a 239.10
72T5-II#6d1 2630.50 n/a n/a n/a n/a n/a n/a n/a n/a
72T5-II#6d2 2891.40 1521.80 1 369.60 n/a n/a n/a n/a 369.60
72T3-III#1a 4848.00 2108.80 9 500.00 260.90 347.80 413.10 391.30 382.62
72T3-III#1c 4630.60 2043.60 10 434.80 369.60 326.10 326.10 391.30 369.58
72T3-III#2d1 5565.40 1652.20 21 434.80 304.40 304.40 500.00 326.10 373.94
72T3-III#2d2 5130.60 1782.70 8 326.10 347.80 347.80 195.70 217.40 286.96
72T3-III#3a 5326.30 2434.90 12 369.60 478.30 326.10 282.60 434.80 378.28
72T3-III#3b 5217.60 2500.10 12 500.00 413.10 347.80 391.30 195.60 369.56
72T3-III#4b1 5108.90 1587.00 n/a 304.40 347.80 347.80 217.40 217.40 286.96
72T3-III#4b2 5348.00 1630.50 10 304.40 217.40 391.30 369.60 391.30 334.80
72T3-III#5a 5087.20 2500.00 11 391.30 413.10 456.50 391.30 304.40 391.32
72T3-III#5d 5130.60 2413.10 11 282.60 326.10 413.10 413.10 369.60 360.90
72T3-III#6b 4956.70 2652.30 11 413.10 304.40 347.80 304.40 347.80 343.50
72T3-III#6e 5108.90 2587.10 11 326.10 413.10 326.10 326.10 304.40 339.16
72T4-III#1a1 6152.40 1413.10 0 n/a n/a n/a n/a n/a n/a
72T4-III#1a2 2261.10 1195.70 1 260.90 n/a n/a n/a n/a 260.90
72T4-III#2f1 5391.50 1521.80 0 n/a n/a n/a n/a n/a n/a
72T4-III#2f2 4608.90 1456.60 0 n/a n/a n/a n/a n/a n/a
72T4-III#3d1 5652.40 1695.70 n/a n/a n/a n/a n/a n/a n/a
72T4-III#3d2 5782.80 1456.60 0 n/a n/a n/a n/a n/a n/a
72T4-III#4d1 5522.00 1565.30 0 n/a n/a n/a n/a n/a n/a
72T4-III#4d2 5348.00 1500.10 0 n/a n/a n/a n/a n/a n/a
72T4-III#5b1 n/a 1608.80 0 n/a n/a n/a n/a n/a n/a
72T4-III#5b2 4065.40 1587.00 0 n/a n/a n/a n/a n/a n/a
72T5-III#1b 3891.50 1847.90 0 n/a n/a n/a n/a n/a n/a
72T5-III#1c 3891.50 1869.60 0 n/a n/a n/a n/a n/a n/a
72T5-III#2a 4087.10 1913.10 0 n/a n/a n/a n/a n/a n/a
72T5-III#2g 3978.40 1934.90 0 n/a n/a n/a n/a n/a n/a
72T5-III#3a 3782.80 1891.40 0 n/a n/a n/a n/a n/a n/a
72T5-III#3b 3869.70 1847.90 0 n/a n/a n/a n/a n/a n/a
72T5-III#4c 3869.70 1804.40 0 n/a n/a n/a n/a n/a n/a
72T5-III#4d 3761.00 1804.40 0 n/a n/a n/a n/a n/a n/a
72T5-III#5c 3848.00 1804.40 0 n/a n/a n/a n/a n/a n/a
72T5-III#5d 3978.40 1804.40 0 n/a n/a n/a n/a n/a n/a
72T5-III#6a 3695.80 1695.70 0 n/a n/a n/a n/a n/a n/a
72T5-III#6e 3782.80 1717.50 0 n/a n/a n/a n/a n/a n/a
Avg 4545.27 1898.40 Avg. 340.53
*Slide # Key:72T =72ºF Treated; Roman numeral =trial #; a/b/c =ovary lobes (if separated during
dehydration); # =slide # (multiple slides made for each ovary); final letter/# =section number chosen at random
58
 Data from Treated Bettas (82T)

* Length Width Total # of Diameter of Random Vitellogenic Oocytes

Slide # (µm) (µm) VO’s #1 #2 #3 AVG
82T1#2 n/a n/a n/a n/a n/a n/a n/a
82T1#3 n/a n/a n/a n/a n/a n/a n/a
82T1#4 n/a n/a n/a n/a n/a n/a n/a
82T2#1a1 4000.2 1674 0 n/a n/a n/a n/a
82T2#1a2 7282.9 1260.9 0 n/a n/a n/a n/a
82T2#2c1 5391.5 1630.5 0 n/a n/a n/a n/a
82T2#2c2 5413.3 1391.4 0 n/a n/a n/a n/a
82T2#3b 6369.8 1630.5 0 n/a n/a n/a n/a
82T2#3g 6174.2 1695.7 0 n/a n/a n/a n/a
82T2#4c 6609 1760.9 0 n/a n/a n/a n/a
82T2#4d 6674.2 1674 0 n/a n/a n/a n/a
82T2#5c 5674.1 1717.5 0 n/a n/a n/a n/a
82T2#5g 5826.3 1739.2 0 n/a n/a n/a n/a
82T2#6b 6522 1695.7 0 n/a n/a n/a n/a
82T2#6f 6435 1695.7 0 n/a n/a n/a n/a
82T4#1 n/a n/a n/a n/a n/a n/a n/a
82T4#2 n/a n/a n/a n/a n/a n/a n/a
82T4#3 n/a n/a n/a n/a n/a n/a n/a
82T4#4 n/a n/a n/a n/a n/a n/a n/a
82T6#1b 1424 402.2 n/a n/a n/a n/a n/a
82T6#1d 1347.9 500 n/a n/a n/a n/a n/a
82T6#2a 1750.1 521.8 n/a n/a n/a n/a n/a
82T6#2d 1956.6 597.9 n/a n/a n/a n/a n/a
82T6#3a 2032.7 500 n/a n/a n/a n/a n/a
82T6#3d 2163.1 543.5 n/a n/a n/a n/a n/a
82T6#4 n/a n/a n/a n/a n/a n/a n/a
82T4-IIa#1c n/a 2869.7 n/a n/a n/a n/a n/a
82T4-IIa#1d n/a 2891.4 n/a 173.9 152.2 n/a 163.050
82T4-IIa#2a n/a 3108.8 n/a 152.2 119.6 108.7 126.833
82T4-IIa#2e n/a 2934.9 n/a 195.7 173.9 152.2 173.933
82T4-IIa#3a n/a 3000.1 n/a 152.2 195.7 n/a 173.950
82T4-IIa#3d n/a 2956.6 n/a 217.4 n/a n/a 217.400
82T4-IIa#4c n/a 3130.6 n/a 173.9 130.4 108.7 137.667
82T4-IIa#4f n/a 2978.4 n/a 108.7 108.7 n/a 108.700
82T4-IIa#5d n/a 3021.9 n/a 152.2 130.4 n/a 141.300
82T4-IIa#5g n/a 2978.4 n/a 130.4 195.7 173.9 166.667
82T4-IIb#1b 6065.5 2282.7 0 n/a n/a n/a n/a

59
 * Length Width Total # of Diameter of Random Vitellogenic Oocytes
Slide # (µm) (µm) VO’s #1 #2 #3 AVG
82T4-IIb#1c 6304.6 2217.5 0 n/a n/a n/a n/a
82T4-IIb#2a 6022 2304.4 0 n/a n/a n/a n/a
82T4-IIb#2e 6043.7 2369.7 0 n/a n/a n/a n/a
82T4-IIb#3b 5391.5 2500.1 0 n/a n/a n/a n/a
82T4-IIb#3c 5543.7 2456.6 0 n/a n/a n/a n/a
82T4-IIb#4 n/a n/a n/a n/a n/a n/a n/a
82T4-IIb#5d 4978.5 2608.8 n/a n/a n/a n/a n/a
82T4-IIb#5f 5130.6 2652.3 n/a n/a n/a n/a n/a
82T4-IIb#6b n/a 2434.9 n/a n/a n/a n/a n/a
82T4-IIb#6d n/a 2500.1 n/a n/a n/a n/a n/a
82T4-IIc#1d n/a n/a n/a n/a n/a n/a n/a
82T4-IIc#1e n/a n/a n/a n/a n/a n/a n/a
82T4-IIc#2 n/a n/a n/a n/a n/a n/a n/a
82T4-IIc#3 n/a n/a n/a n/a n/a n/a n/a
82T4-IIc#4 n/a n/a n/a n/a n/a n/a n/a
82T4-IIc#5 n/a n/a n/a n/a n/a n/a n/a
82T1-III#1 n/a n/a n/a n/a n/a n/a n/a
82T1-III#2 n/a n/a n/a n/a n/a n/a n/a
82T1-III#3 n/a n/a n/a n/a n/a n/a n/a
82T1-III#4 n/a n/a n/a n/a n/a n/a n/a
82T1-III#5 n/a n/a n/a n/a n/a n/a n/a
82T1-III#6b 3913.2 782.6 n/a n/a n/a n/a n/a
82T1-III#6d 3869.7 847.9 n/a n/a n/a n/a n/a
82T1-III#7e 4478.4 2304.4 n/a n/a n/a n/a n/a
82T1-III#7f 4326.3 2347.9 n/a n/a n/a n/a n/a
82T1-III#8d 4674.1 2239.2 n/a n/a n/a n/a n/a
82T1-III#8e 4587.1 2239.2 n/a n/a n/a n/a n/a
82T1-III#9 n/a n/a n/a n/a n/a n/a n/a
82T1-III#10 n/a n/a n/a n/a n/a n/a n/a
82T1-III#11 n/a n/a n/a n/a n/a n/a n/a
82T1-III#12 n/a n/a n/a n/a n/a n/a n/a
82T2-III#1a 5826.3 n/a n/a n/a n/a n/a n/a
82T2-III#1d 5956.8 n/a n/a n/a n/a n/a n/a
82T2-III#2b 5891.5 n/a n/a n/a n/a n/a n/a
82T2-III#2c 5869.8 n/a n/a n/a n/a n/a n/a
82T2-III#3d1 5978.5 n/a n/a n/a n/a n/a n/a
82T2-III#3d2 n/a 2065.3 n/a n/a n/a n/a n/a
82T2-III#4c1 6043.7 1478.3 n/a n/a n/a n/a n/a
82T2-III#4c2 n/a 1956.6 n/a n/a n/a n/a n/a
82T2-III#5d1 5891.5 1347.9 n/a n/a n/a n/a n/a
82T2-III#5d2 5522 2217.5 n/a n/a n/a n/a n/a
82T2-III#6a 5087.2 1608.8 n/a n/a n/a n/a n/a
82T2-III#6d 5174.1 1608.8 n/a n/a n/a n/a n/a
60
 * Length Width Total # of Diameter of Random Vitellogenic Oocytes
Slide # (µm) (µm) VO’s #1 #2 #3 AVG
82T2-III#7d 5630.7 1760.9 n/a n/a n/a n/a n/a
82T2-III#7e 5608.9 1869.6 n/a n/a n/a n/a n/a
82T2-III#8c 5935 1608.8 n/a n/a n/a n/a n/a
82T2-III#8e 5717.69 1587 n/a n/a n/a n/a n/a
82T2-III#9a 5935 1739.2 n/a n/a n/a n/a n/a
82T2-III#9e 5717.6 1695.7 n/a n/a n/a n/a n/a
82T2-III#10c1 4326.3 1587 0 n/a n/a n/a n/a
82T2-III#10c2 6065.5 1500.1 n/a n/a n/a n/a n/a
82T3-III#1a1 4391.5 1239.2 0 n/a n/a n/a n/a
82T3-III#1a2 2565.3 804.4 0 n/a n/a n/a n/a
82T3-III#2c1 2956.6 847.9 0 n/a n/a n/a n/a
82T3-III#2c2 3978.4 1174 n/a n/a n/a n/a n/a
82T3-III#3d1 n/a 1260.9 n/a n/a n/a n/a n/a
82T3-III#3d2 3108.8 891.3 0 n/a n/a n/a n/a
82T3-III#4a1 n/a 1217.4 n/a n/a n/a n/a n/a
82T3-III#4a2 3065.3 934.8 0 n/a n/a n/a n/a
82T3-III#5a 3652.3 956.6 0 n/a n/a n/a n/a
82T3-III#5f n/a 847.9 n/a n/a n/a n/a n/a
82T3-III#6 n/a n/a n/a n/a n/a n/a n/a
82T3-III#7a 4391.5 1239.2 0 n/a n/a n/a n/a
82T3-III#7b 4413.2 1195.7 0 n/a n/a n/a n/a
82T4-IIIa#1b 4217.6 2130.5 0 n/a n/a n/a n/a
82T4-IIIa#1e n/a 2108.8 n/a n/a n/a n/a n/a
82T4-IIIa#2c n/a 2413.1 n/a n/a n/a n/a n/a
82T4-IIIa#2e n/a 2347.9 n/a n/a n/a n/a n/a
82T4-IIIa#3c n/a 2456.6 n/a n/a n/a n/a n/a
82T4-IIIa#3d n/a 2500.1 n/a n/a n/a n/a n/a
82T4-IIIa#4b n/a 2695.8 n/a n/a n/a n/a n/a
82T4-IIIa#4e n/a 2739.2 n/a n/a n/a n/a n/a
82T4-IIIa#5b n/a 2717.5 n/a n/a n/a n/a n/a
82T4-IIIa#5c n/a 2761 n/a n/a n/a n/a n/a
82T4-IIIb#1a n/a 1695.7 n/a n/a n/a n/a n/a
82T4-IIIb#1d n/a 1695.7 n/a n/a n/a n/a n/a
82T4-IIIb#2a n/a 1826.2 n/a n/a n/a n/a n/a
82T4-IIIb#2e n/a 1674 n/a n/a n/a n/a n/a
82T4-IIIb#3c n/a 1891.4 n/a n/a n/a n/a n/a
82T4-IIIb#3f n/a 1891.4 n/a n/a n/a n/a n/a
82T4-IIIb#4b 5065.4 1956.6 n/a n/a n/a n/a n/a
82T4-IIIb#4e 5326.3 1978.3 n/a n/a n/a n/a n/a
82T5-III#1a 4587.1 1217.4 n/a n/a n/a n/a n/a
82T5-III#1f 4739.3 1152.2 n/a n/a n/a n/a n/a
82T5-III#2c 4000.2 1413.1 0 n/a n/a n/a n/a
82T5-III#2e 3891.5 1456.6 n/a n/a n/a n/a n/a
61
 * Length Width Total # of Diameter of Random Vitellogenic Oocytes
Slide # (µm) (µm) VO’s #1 #2 #3 AVG
82T5-III#3 n/a n/a n/a n/a n/a n/a n/a
82T5-III#4 n/a n/a n/a n/a n/a n/a n/a
82T5-III#5e n/a 1304.4 n/a n/a n/a n/a n/a
82T5-III#5f n/a 1326.1 n/a n/a n/a n/a n/a
82T5-III#6b n/a 1369.6 n/a n/a n/a n/a n/a
82T5-III#6d n/a 1326.1 n/a n/a n/a n/a n/a
82T5-III#7b 3456.7 1087 n/a n/a n/a n/a n/a
82T5-III#7f 3434.9 1282.7 n/a n/a n/a n/a n/a
Avg 4820.53 1800.47 Avg 156.61
*Slide # Key: 82C = 82ºF Treated (TBA); Roman numeral = trial #; a/b/c = ovary lobes (if separated
during dehydration); # = slide # (multiple slides made for each ovary); final letter/# = section number
on each slide chosen at random

62
Summary of Data Averages for Each Betta in Analysis

Vitellogenic
Oocytes

Betta Length Width Total AVG

ID (µm) (µm) # diameter
OC3 2625 2737.5 n/a 479.416
OC4 n/a n/a n/a 410.947
OC7 6871.875 3017.188 54.938 427.778
OC8 6935.086 3403.75 49.583 427.778
OC9 11214.75 4043.65 62.5 456.026
72C6 n/a n/a 44.5 463.06
82C1 n/a n/a n/a 375.01
72T1 4232.058 1610.558 0.75 409.433
72T2 3617.013 1905.32 2.364 176.962
72T3 5326.325 2054.43 3 276.639
72T6 5306.107 2528.638 4 348.194
72T2 II 3543.614 1494.638 0 282.6
72T1
III 5242.967 2451.592 2.5 403.743
72T2
III 3819.72 1689.19 0 n/a
72T3 II 5051.508 1684.448 6.625 410.749
72T5 II 3630.592 1447.883 0.6 291.667
72T3
III 5121.567 2157.692 10.636 351.465
72T4
III 4976.056 1500.07 0.111 260.9
72T5
III 3869.725 1827.967 0 n/a
82T2 6031.042 1630.5 0 n/a
82T6 1779.067 510.9 n/a n/a
82T4 II 5685.013 2709.895 0 156.611
82T1
III 4308.133 1793.533 n/a n/a
82T2
III 5676.561 1708.767 0 n/a
82T3
III 3613.656 1050.775 0 n/a
82T4
III 4869.767 2193.322 0 n/a
82T5
III 4018.283 1293.52 0 n/a
63
 VITA

Tracy S. Smith

Personal Data: Date of Birth: February 1, 1980

Place of Birth: Marion, Virginia
Marital Status: Single

Education: Public Schools, Marion, Virginia

Radford University, Radford, Virginia;
Biology, B.S., 2001
East Tennessee State University, Johnson City, Tennessee;
Biology, M.S., 2005

Professional
Experience: Lab Assistant, Radford University; Radford, Virginia,
2000-2001
Teaching Assistant, Radford University; Radford, Virginia,
2000-2001
Graduate Assistant, East Tennessee State University, Department of Biological
Sciences, 2001-2004
Academic Instructor, Upward Bound, East Tennessee State University,
2002-2005
Temporary Faculty, East Tennessee State University, Department of Biological
Sciences, 2005

Honors and
Awards: Fraley Memorial Award, East Tennessee State University, 2002.

64