Department of Reproductive Biology,
University of Veterinary Medicine Hannover, Germany
___________________________________________________________________
Induced spermiation and sperm morphology in the
Green Poison Frog, Dendrobates auratus
THESIS
Submitted in partial fulfillment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
at the University of Veterinary Medicine Hannover
by
Christian Lipke
from Hannover
Hannover 2008
Supervisor:
Prof. Dr. S. Meinecke-Tillmann
Advisory Committee:
Prof. Dr. S. Meinecke-Tillmann
Prof. Dr. H. Pröhl
Prof. Dr. K. Eulenberger
1st Evaluation:
Prof. Dr. S. Meinecke-Tillmann,
Department of Reproductive Biology,
University of Veterinary Medicine Hannover
Prof. Dr. H. Pröhl,
Institute of Zoology,
University of Veterinary Medicine Hannover
Prof. Dr. K. Eulenberger,
Zoo Leipzig GmbH
2nd Evaluation:
Prof. Dr. H. Hofer,
Leibniz Institute for Zoo and Wildlife Research (IZW)
To A ntje and m y parents
Es gibt nichts Schöneres, als das Mysteriöse.
Aus ihm entspringt alle wahre Kunst und Wissenschaft.
(Albert Einstein)
Parts of the thesis have already been published or communicated:
LIPKE, C., S. MEINECKE-TILLMANN, and B. MEINECKE (2005):
Induktion
der
Spermiation
und
Spermienmorphometrie
bei
dem
Goldbaumsteiger, Dendrobates auratus (Amphibia, Anura, Dendrobatidae)“.
Abstracts of the 38th annual meeting on physiology and pathology of
Reproduction, the 30th Joint Meeting of Veterinary and Human Medicine and
the Satellite Symposium “Reproductive medicine for the veterinarian”, Zurich,
Switzerland. Schweiz. Arch. Tierheilk. 147:66
LIPKE, C., S. MEINECKE-TILLMANN, and B. MEINECKE (2005):
Ein
Beitrag
zur
Spermiengewinnung
und
-morphologie
sowie
phylogenetischen Einstufung des Goldbaumsteigers, Dendrobates auratus.
Abstracts
of
the
26th
Annual
Conference
of
the
“Deutsche
Veterinärmedizinische Gesellschaft (DVG)”, Berlin, Germany
LIPKE, C., S. MEINECKE-TILLMANN, and B. MEINECKE (2007):
An advanced spermiation protocol in a dendrobatid frog, Dendrobates auratus
(Amphibia, Anura, Dendrobatidae)”. Abstracts of the 40th Annual Meeting on
Physiology and Pathology of Reproduction, the 32nd Joint meeting of
Veterinary and Human Medicine, Berlin, Germany. Reprod. Dom. Anim. 42
(Suppl. 1):19
LIPKE, C., S. MEINECKE-TILLMANN, and B. MEINECKE (2007):
Induzierte Spermiation und Eizellgewinnung bei einem Baumsteigerfrosch,
Dendrobates auratus (Amphibia, Anura, Dendrobatidae). Abstracts of the 33rd
Scientific Meeting of the European Embryo Transfer Association, Hannover,
Germany
LIPKE, C., S. MEINECKE-TILLMANN, W. MEYER, and B. MEINECKE (2007):
The ultrastructure of the spermatozoa of Dendrobates auratus (Amphibia,
Anura, Dendrobatidae). Annual Conference of the European Society for
Domestic Animal Reproduction, Celle, Germany. Reprod. Dom. Anim. 42
(Suppl. 2):80
LIPKE, C., S. MEINECKE-TILLMANN, and B. MEINECKE (2008):
Induced spermiation and sperm morphology in a dendrobatid frog,
Dendrobates
auratus
(Amphibia,
Anura,
Dendrobatidae).
Salamandra
(accepted for publication July 11th, 2008)
LIPKE, C., S. MEINECKE-TILLMANN, W. MEYER, and B. MEINECKE (2008):
Preparation and ultrastructure of spermatozoa from Green Poison Frogs,
Dendrobates auratus, following hormonal induced spermiation (Amphibia,
Anura, Dendrobatidae). Anim. Reprod. Sci.
doi:10.1016/j.anireprosci.2008.06.005
Contents
I
Contents
Chapter 1 Introduction .................................................................................. 1
1.1
Aims of the present study.......................................................................... 2
1.2
Situation in dendrobatid species .............................................................. 3
1.3
Decline of amphibian populations ............................................................ 5
1.3.1
Alien species........................................................................................ 7
1.3.2
Over-exploitation ................................................................................. 8
1.3.3
Land use change ................................................................................. 9
1.3.4
Global change.................................................................................... 10
1.3.5
Contaminants..................................................................................... 11
1.3.5.1
Pesticides.................................................................................... 12
1.3.5.2
Heavy metals .............................................................................. 12
1.3.5.3
Acidification................................................................................ 13
1.3.5.4
Nitrogenous compounds ........................................................... 13
1.3.5.5
Contaminants acting as endocrine disruptors ........................ 14
1.3.6
1.4
Emerging infectious diseases .......................................................... 16
Assisted reproductive technology (ART) in amphibians ...................... 17
1.4.1
Collection of sperm cells .................................................................. 19
1.4.2
Induction of ovulation and spawning .............................................. 19
1.4.3
Artificial fertilization .......................................................................... 21
1.4.4
Preservation of gametes ................................................................... 22
1.5
1.4.4.1
Gamete storage in solutions of high osmolality...................... 22
1.4.4.2
Short term storage of spermatozoa .......................................... 23
1.4.4.3
Cryopreservation........................................................................ 23
The Green Poison Frog, Dendrobates auratus (Girard, 1855) .............. 24
1.5.1
Phylogenetic systematics................................................................. 25
Contents
II
1.5.2
Distribution ........................................................................................ 26
1.5.3
Habitats .............................................................................................. 26
1.5.4
Reproduction ..................................................................................... 28
1.5.4.1
Calling behavior.......................................................................... 28
1.5.4.2
Mating behavior .......................................................................... 28
1.5.4.3
Parental behavior ....................................................................... 29
1.5.4.4
Sex identification........................................................................ 31
Chapter 2 Induced spermiation and sperm morphology in a dendrobatid
frog, Dendrobates auratus (Amphibia, Anura, Dendrobatidae) ................ 33
2.1
Abstract ..................................................................................................... 35
2.2
Introduction............................................................................................... 35
2.3
Materials and methods ............................................................................. 37
2.3.1
Animals............................................................................................... 37
2.3.2
General experimental design for induced spermiation .................. 37
2.3.3
Experiment 1: Sperm morphology ................................................... 38
2.3.4
Experiment 2: Membrane integrity................................................... 39
2.3.5
Experiment 3: hCG dosage............................................................... 39
2.3.6
Experiment 4: Sperm motility ........................................................... 40
2.3.7
Experiment 5: Proportion of recovered spermatozoa .................... 40
2.4
Results....................................................................................................... 40
2.4.1
Experiment 1: Sperm morphology ................................................... 40
2.4.2
Experiment 2: Membrane integrity................................................... 41
2.4.3
Experiment 3: hCG dosage............................................................... 42
2.4.4
Experiment 4: Sperm motility ........................................................... 45
2.4.5
Experiment 5: Proportion of recovered spermatozoa .................... 46
Contents
III
2.5
Discussion................................................................................................. 47
2.6
Acknowledgements .................................................................................. 49
2.7
References ................................................................................................ 49
Chapter 3 Preparation and ultrastructure of spermatozoa from Green
Poison Frogs, Dendrobates auratus, following hormonal induced
spermiation (Amphibia, Anura, Dendrobatidae) ......................................... 55
3.1
Abstract ..................................................................................................... 57
3.2
Introduction............................................................................................... 58
3.3
Materials and Methods ............................................................................. 59
3.3.1
Recovery and fixation of spermatozoa ............................................ 59
3.3.2
Transmission electron microscopy ................................................. 60
3.3.3
Scanning electron microscopy ........................................................ 61
3.4
Results....................................................................................................... 61
3.4.1
General morphology.......................................................................... 61
3.4.2
Acrosomal complex........................................................................... 61
3.4.3
Nucleus............................................................................................... 61
3.4.4
Midpiece ............................................................................................. 64
3.4.5
Flagellum............................................................................................ 64
3.5
Discussion................................................................................................. 68
3.6
Acknowledgements .................................................................................. 69
3.7
Literature cited.......................................................................................... 70
Chapter 4 Discussion.................................................................................. 75
Contents
IV
4.1
Induced spermiation................................................................................. 76
4.1.1
Hormonal stimulation........................................................................ 76
4.1.2
Quantity of recovered spermatozoa ................................................ 77
4.1.3
Sperm motility.................................................................................... 77
4.1.4
Membrane integrity of spermatozoa ................................................ 78
4.2
Sperm structure ........................................................................................ 78
4.3
Concluding remarks ................................................................................. 79
Chapter 5 Summary..................................................................................... 81
Chapter 6 Zusammenfassung .................................................................... 85
Chapter 7 References.................................................................................. 89
Appendix
................................................................................................... 125
Abbreviations
V
Abbreviations
AF
artificial fertilization
ART
assisted reproductive technology
ASL
above sea level
Bd
Batrachochytrium dendrobatidis
cd
conservation dependent
CFC
chlorofluorocarbon
CR
critically endangered
DD
data deficient
DNA
deoxyribonucleic acid
EM
electron microscopy
EN
endangered
FM
fluorescent microscopy
FSH
follicle stimulating hormone
GnRH
gonadotropin-releasing hormone
hCG
human chorionic gonadotropin
ICSI
intra-cytoplasmic sperm injection
IE
internationale Einheiten
IPS
isotonic phosphate-free amphibian saline
IU
international units
IUCN
International Union for Conservation of Nature
lc
least concern
LH
luteinizing hormone
LHRH
luteinizing hormone releasing hormone
LM
light microscopy
LR
lower risk
nt
near threatened
PAH
polycyclic aromatic hydrocarbon
PI
propidium iodide
SEM
scanning electron microscopy
Abbreviations
VI
TEM
transmission electron microcopy
UV
ultraviolet
VU
vulnerable
XB
Xenopus fertilization medium
List of figures and tables
VII
List of figures and tables
Fig. 1-1:
Peeled frogs offered on a public market on Koh Samui Island
(Thailand) together with pork and duck. ............................................ 9
Fig. 1-2:
Dorsal (A, C) and frontal (B, D) view of a newly metamorphosed
control (A, B) R. pipiens and a similar-aged animal exposed to
the nine-pesticide mixture (C, D). ................................................... 15
Fig. 1-3:
Differences in the skin coloration of Dendrobates auratus. (A)
Frog from Costa Rica (black blotches) (B) Frog from Panama
(“Bronze”). ...................................................................................... 25
Fig. 1-4:
Phylogenetic grouping of Dendrobates auratus. ............................. 26
Fig. 1-5:
Distribution of Dendrobates auratus. .............................................. 27
Fig. 1-6:
Courtship in Dendrobates auratus. (A) Female resting front feet
on the back of the male (B) Female placing hind feet on the
back of the male. ............................................................................ 29
Fig. 1-7:
Male Dendrobates auratus carrying one tadpole. ........................... 30
Fig. 2-1:
Typical spermatozoon of Dendrobates auratus. ............................. 41
Fig. 2-2:
Dead spermatozoon of Dendrobates auratus. (A) Sperm head
with defective cell membrane, PI dyed. (B) Cytoplasm
membrane at the head with numerous protrusions. ........................ 42
Fig. 2-3:
Differences in the distribution of the spermiation responses after
single and double hCG stimulation. ................................................ 43
List of figures and tables
VIII
Fig. 2-4:
Number of recovered viable spermatozoa of Dendrobates
auratus after hCG single and double stimulation. ........................... 44
Fig. 2-5:
Percentage of recovered motile sperm cells with typical
morphology using either IPS of XB medium after hCG double
stimulation. ..................................................................................... 45
Fig. 2-6:
Composition of arranged groups of spermatozoa including
accordant morphology in phase-contrast microscopy and
additional PI staining behavior in head defective cells. ................... 46
Fig. 2-7:
Distribution of typically shaped (non-motile and motile) and
abnormally shaped spermatozoa in recovered cloaca flushings. .... 47
Fig. 3-1:
Spermatozoa of Dendrobates auratus. Scanning electron
microscopy. (A) Whole spermatozoon with head and single
flagellum. (B) Head without cytoplasmic drop. (C) Head with
large cytoplasmic drop at posterior region. ..................................... 62
Fig. 3-2:
Schematic illustration of the ultrastructure of the spermatozoon
of Dendrobates auratus. ................................................................. 63
Fig. 3-3:
Spermatozoa of Dendrobates auratus. Transmission electron
microscopy. (A) Longitudinal section of the acrosomal complex
showing
the
thin
conical
acrosomal
vesicle
and
the
subacrosomal cone. (B) Transverse section of the nucleus with
electron-dense chromatin and inclusions. (C, D) Transverse and
longitudinal sections of the nucleus with cytoplasmic remains
containing several mitochondria. (E) Longitudinal section of the
complete headpiece. (F) Longitudinal section of the midpiece
with proximal centriole. (G) Midpiece in longitudinal section. (H)
List of figures and tables
IX
Transverse section of the flagellum. (I) Longitudinal section of
the tail. ....................................................................................... 65-67
Tab. 1-1:
Summary of numbers of dendrobatid species in each Red List
category. ........................................................................................... 5
Tab. 1-2:
Trend of threatened amphibian species between 2003 and
2007. ................................................................................................. 7
Tab. 2-1:
Individual numbers of recovered viable sperm cells from single
and double stimulated frogs per experiment. .................................. 44
X
List of figures and tables
Introduction
1
Chapter 1 Introduction
Introduction
2
1.1
Aims of the present study
The term “spermiation” is used for the process of sperm release into the seminiferous
tubules of mature anurans combined with the transport of sperm cells into the cloaca
and the outside environment. This physiological process was studied by influencing
the endogenous hormonal system of frogs via the administration of different
exogenous substances (induced spermiation). One practical application was the
diagnosis of human pregnancy using male toads (GALLI-MAININI 1947).
Substances as pituitary extracts (HANSEN and SWEAT 1962; LICHT 1973; EASLEY
et al. 1979; MINUCCI et al. 1989) were used for induced spermiation. In addition
gonadotropin-releasing hormone (GnRH), luteinizing hormone releasing hormone
(LHRH; LICHT 1973; EASLEY et al. 1979; MINUCCI et al. 1989; WAGGENER and
CARROLL JR. 1998; ROWSON et al. 2001; IIMORI et al. 2005), luteinizing hormone
(LH; BURGOS and LADMAN 1955; BURGOS and VITALE-CALPE 1967; EASLEY et
al. 1979), follicle stimulating hormone (FSH; BURGOS & LADMAN 1955; MEYER et
al 1961; EASLEY et al. 1979; IIMORI et al. 2005) and human chorionic gonadotropin
(hCG; THORBURG 1952; BURGOS & LADMAN 1955; CHATTERJEE et al. 1971;
EASLEY et al. 1979; MINUCCI et al. 1989; IIMORI et al. 2005) were applied over the
decades to examine their potential to induce spermiation.
The hormonal stimulation of dendrobatid frogs with the aim of sperm recovery was
never reported, although the sperm morphology and ultrastructure in some species of
the Dendrobatidae was analyzed mainly to investigate amphibian phylogeny. In these
studies the gentle instrument of hormonal stimulation was ignored and euthanasia
was preferred.
GARDA et al. (2002) discovered structural evidence for the grouping of the
Dendrobatidae within the Bufonoidea. Furthermore, AGUIAR-JR. et al. (2003) found
similar sperm ultrastructure in Allobates femoralis and Colostethus sp, although in
the meantime Allobates femoralis was placed in the Aromobatidae (FROST 2007).
AGUIAR-JR. et al. (2003) also described in these frogs a biflagellate conformation
only reported before in the leptodactylid frog Telmatobufo australis (PUGIN and
GARRIDO 1981) and Chiromantis xerampelina, a rhacophorid frog (WILSON et al.
1991; JAMIESON 1999). Sperm morphology in Ameerega trivittata and Ameerega
Introduction
3
hahneli (Dendrobatidae) was analyzed by AGUIAR-JR. et al. (2004) finding identical
morphological characteristics except for the expansion of the acrosomal vesicle.
Authors using sperm ultrastructure for phylogenetic analysis suggested a grouping of
the Dendrobatidae within the Bufonoidea neobatrachian lineage mainly because of
the tail structure. These results are in accordance with gene data retrieved from
nuclear and mitochondrial sequences (HILLIS et al. 1993; HAY et al. 1995).
With nearly one third of amphibian species threatened with extinction, the
populations are in an alarming worldwide decline (IUCN 2007). Therefore, the
transfer of reproductive technologies useful for the preservation of endangered
amphibians has to be advanced.
The aim of this study is to establish and transfer methods of assisted reproductive
medicine in a dendrobatid species, the Green Poison Frog, Dendrobates auratus
including the possibility of induced spermiation and recovery of spermatozoa as well
as light, fluorescent and electron microscopic investigations of sperm cells.
Ultrastructural data may be useful for the phylogenetic analysis of the genera within
the Dendrobatidae and the position of the whole family within the Anura. As these
techniques are the fundamentals in future breeding programs for endangered
amphibians, especially members of the Dendrobatidae, all investigations were
planned under the requirement that no animals have to be killed.
1.2
Situation in dendrobatid species
Currently the IUCN Red List of Threatened Species contains data on 234 species of
the Dendrobatidae. Unfortunately 97 species are rated as “data deficient” (DD) due to
inadequate information about distribution and/or population status. A risk assessment
about declines of these species is not reliable, although the biology of these species
might be well studied. With 72 species a large group is rated as “lower risk” (LR)
representing evaluated species that do not satisfy criteria for the higher categories.
The remaining members of the Dendrobatidae (n = 65) are allocated to the
categories “vulnerable” (VU; n = 16), “endangered” (EN; n = 29), and “critically
endangered” (CR; n = 20). It is an alarming fact, that in the majority (n = 56) of these
4
Introduction
species the investigated populations exhibit a negative trend, implying actual
declines (IUCN 2007).
From the genus Dendrobates three species are rated as CR (Dendrobates abditus,
Dendrobates lehmanni and Dendrobates steyermarki) mainly due to their small
extent of occurrence with less than 100 km2 to only 10 km2 and continuing decline in
the extent and quality of their habitats (COLOMA and RON 2004; BOLÍVAR et al.
2004; LA MARCA and SEÑARIS 2004). Living in one single location the population
of D. abditus declined more than 80 % over the last ten years in consequence of
agriculture and livestock farming. The species also could have been affected by the
synergistic effects of chytridiomycosis and climate change (COLOMA and RON
2004). The declining populations of the Red-banded Poison Frog, D. lehmanni, suffer
from habitat fragmentation and loss resulting from agricultural development in
combination with pollution from spraying of pesticides, logging and human
settlement. The species also appears in illegal pet trade (BOLÍVAR et al. 2004).
The species of D. steyermarki is restricted to Cerro Yapacana, at elevations from 600
to 1300 m ASL, in Amazonas state, Venezuela. Intensive open gold mining in the
area, wildfires and illegal collection of specimen are the main threats affecting this
species (LA MARCA and SEÑARIS 2004).
Actually Dendrobates auratus is rated “least concern” (lc) by the IUCN due to its wide
distribution, tolerance of a degree of habitat modification, presumed large population,
and because it is unlikely to be declining fast enough to qualify for listing in a more
threatened category. However, populations are affected by the general loss of
suitably wooded areas and the collection for international pet trade. Over harvesting
especially of the rare morphs might contribute to declines of localized populations.
The USA are a large market for pet trade. Approximately 18500 specimens were
exported to North America during 1991 and 1996. Moreover, the chytrid fungus is
found in museum species of D. auratus, a possible impact of this pathogen on living
specimens is not known (SOLÍS et al. 2004).
With almost 28 % of dendrobatid frogs threatened, meaning rated VU, EN, or CR,
this family mirrors decline ratios of the Amphibia quite exactly. Table 1-1 gives an
overview of the allocation of dendrobatid species in Red List categories.
Introduction
5
Tab. 1-1. Summary of numbers of dendrobatid species in each Red List category
(data from IUCN 2007).
trend
EX
EW
CR
EN
LR
VU
cd
nt
lc
DD
NE
↑
0
0
0
0
0
0
0
0
0
0
=
0
0
0
1
7
0
0
40
2
0
↓
0
0
20
28
8
0
14
8
1
0
?
0
0
0
0
1
0
0
10
94
0
Abbreviations: EX = “extinct”; EW = “extinct in the wild”; CR = “critically endangered”;
EN = “endangered”; VU = “vulnerable”; LR = “lower risk”; cd = “conservation
dependent”; nt = “near threatened”; lc = “least concern”; DD = “data deficient”; NE =
“not evaluated”.
1.3
Decline of amphibian populations
The class Amphibia exists on this planet since about 250 million years and endured
many environmental changes (including ice ages) during this time (RAGE 1997).
However, over the past decades changes without precedent have been noticed.
Species from all six inhabited continents are declining at an alarming rate or even
become extinct (VIAL and SAYLOR 1993). Although biodiversity in general is
declining worldwide, amphibians are not only conventional representatives of the loss
of populations and species (HOULAHAN et al. 2000; ALFORD et al. 2001; WILSON
2002). Amphibians possess an exceptional position compared to other classes of
animals because of disproportional increasing reports of declines and extinctions.
The causes appear simultaneously and over great distances and even in protected
natural habitats. Typical annual changes in population size have to be distinguished
from a systematic decline preceding extinction (PECHMANN et al. 1991;
BLAUSTEIN et al. 1994). ALFORD and RICHARDS (1999) provide an overview of
techniques used in 46 studies for the quantification of frog and salamander
6
Introduction
populations. In breeding habitats predominantly counts of individuals, counts of
pit/fence trapped animals, and counts of egg masses are carried out to obtain
population data.
Annually the IUCN publishes changes in numbers of threatened species in the
current Red List that is available via the worldwide web (IUCN 2007). In 2007 29 %
(1808 species) of all described amphibian species are classified as threatened.
These data is meaningful because with 5915 out of 6199 the vast majority of
described species was evaluated (Tab. 1-2).
Six leading hypotheses are thought to underlie amphibian declines. According to
COLLINS and STORFER (2003) these hypotheses are divided into two classes.
Invading alien species, over-exploitation and land use change are summarized in
class I hypotheses. The basic ecological mechanisms and effects of these processes
on populations are well investigated in part because they affected amphibians for
more than 100 years. For the impacts of global change (including increased
ultraviolet radiation and global warming), increased use of pesticides and other toxic
chemicals and emerging infections diseases, classified in class II hypotheses, only
limited understanding is available.
Amphibians acting as prey, predators and herbivores are important components of
many ecosystems. Due to their clear contribution to tropical dynamics, loss of
amphibian populations will surely affect other organisms (BLAUSTEIN et al. 1994).
In some concrete cases of declines correlations to one or more hypotheses are
demonstrated. A closing explanation for the global losses is still missing, but a
complex interaction of all hypotheses including local particularities can be assumed.
Introduction
7
Tab. 1-2. Trend of threatened amphibian species between 2003 and 2007 (IUCN
2007; modified).
Total
Numbers of other classes of vertebrates are given for comparison.
1.3.1 Alien species
Alien species can cause declines and extinctions of native amphibian populations via
the multiple and partly interacting mechanisms of predation on natives, competition
between one and more life stages, introduction of pathogens and hybridization
(COLLINS and STORFER 2003). Native species lack evolutionary history with aliens,
leading to lack of adaptations and population declines (GILLESPIE 2001).
Amphibians and particularly their eggs and larvae are vulnerable to alien aquatic
predators. Fishes are the most widespread alien predator on amphibians (STEBBINS
and COHEN 1995) and are often placed into habitats to provide game for sport
fishermen (CORY 1963; KNAPP 1996; STEIN et al. 2000). In protected areas
throughout the Sierra Nevada Mountains (USA) the Mountain Yellow-legged Frog,
Rana muscosa, declined or disappeared from lakes by 1910 where non-native trouts
had been introduced since the 1800s (CAREY et al. 2003). During a study of KNAPP
et al. (2007) the predatory fishes in three lakes in this area were removed. The R.
muscosa population density in all lakes increased significantly compared to
populations in control lakes over the same period of time.
Salmonids have been introduced not only in North America (BRADFORD et al. 1993)
but worldwide, including Australia, New Zealand, Europe and Central America
8
Introduction
(BRÖNMARK and EDENHAMN 1994; TOWNSEND 1996; POUGH et al. 1998). Due
to its effectiveness in controlling mosquito populations Mosquitofishes (Gambusia
sp.) are one of the most widespread genera of the Vertebrata. The diet of this fish
includes tadpoles (WEBB and JOSS 1997; GOODSELL and KATS 1999). Every fish
introduction for biological control that has been studied has negative effects on nontarget species (SIMBERLOFF and STILING 1996). It is generally accepted that alien
predators can drive local populations of amphibians to extinction (BRADFORD 1991;
BRADFORD et al. 1994; GAMRADT and KATS 1996; MATTHEWS et al. 2001) or
reduce them to such low numbers that populations will become isolated from others,
and may ultimately disappear (BRADFORD et al. 1993).
The general mechanisms by which alien species can cause amphibian declines are
obvious but dealing with introduced species is difficult. Additionally, aliens can
interact with other factors such as acting as vectors for infectious diseases, resulting
in indirect effects (COLLINS and STORFER 2003).
1.3.2 Over-exploitation
The effect of harvesting on amphibian populations is unclear and reliable data is
missing, but it can be assumed that it is significant. Frogs have been collected for
nutrition purposes for many centuries in some areas. In a single Iowa county (USA)
amphibian populations declined between 1920 and 1992 from 20 million frogs to
50.000. Besides effects from wetland drainage at least one-third of these losses
result from harvesting (LANNOO et al. 1994). Professional frog farming particularly in
Asia is profitable, a variety of products (meat and egg jelly) can be ordered via the
worldwide web (THIAW 2008), but also animals collected in the wild are offered (Fig.
1-1). In North America the farming of frogs is not proven to be economically
successful. Many reputed frog farmers are only distributors of adults, tadpoles and
eggs, harvested in the wild. Sorely a permit for capturing, holding, propagating, and
selling of amphibians, is required in most states of the USA (HELFRICH et al. 2001).
Introduction
9
Fig. 1-1. Peeled frogs offered on a public market on Koh Samui Island (Thailand)
together with pork and duck.
1.3.3 Land use change
MEYER and TURNER (1992) define the terms “land use change” (altering land the
way humans use land) and “land cover change” (alteration of the physical or biotic
nature of a site) separately. According to VITOUSEK (1994) both terms can be
merged as “land use change”. Effects for amphibians are habitat loss, alteration, and
fragmentation.
Since the exponential human population growth at the beginning of the 20th century
predominately in the subtropical and tropical regions, amphibians face loss of natural
habitats by broad-scale changes in land cover and land use, typically in support of
agriculture (GALLANT et al. 2007). In a study of PIHA et al. (2007) landscape
characteristics on the island of Gotland (Sweden) regarding amphibian species
10
Introduction
richness were investigated. Proportions of current and historic arable land are
correlated negatively with amphibian occurrence and richness of species. Also
agricultural ponds can support amphibian populations as far as the parameters water
quality (low total nitrogen concentration), vegetation, and predators (fishes) are
maintained (KNUTSON et al. 2004).
Land use change is accepted to be an important cause for a reduced worldwide
biodiversity. It has to be clarified if the massive loss of amphibian species during the
last decades is related to changes in land use within this period of time.
1.3.4 Global change
Generally the term global change is used for a transformation that occurs on a
worldwide scale e.g. increasing concentrations of carbon dioxide and global warming,
increased nitrogen fixation, increased concentrations of atmospheric gaseous nitrous
oxide, widespread distribution of synthetic organic compounds, increased levels of
UV-radiation (VITOUSEK 1994). The same author added the factors harvesting of
natural populations, land use / land cover and biological invasions by non-native
species. These impacts are mentioned in accordant sub-chapters.
In primary forests at La Selva, Costa Rica, a decline of 75 % in total densities of
amphibians and reptiles inhabiting the leaf litter region is described by WHITFIELD et
al. (2007). These authors found correlations between these declines and an
increased daily minimum temperature between 1982 and 2004 combined with a
constant daily total rainfall between 1970 and 2004 resulting in decreased tree
growth and decreased leaf litter depth. Influences of habitat modifications and
chytridiomycosis are excluded in this study.
Over evolutionary time ultraviolet (UV) radiation has been an important stressor on
organisms (COCKELL and BLAUSTEIN 2001). Large-scale ozone depletion caused
by natural events (impacts of large extraterrestrial material, solar flares and volcano
activity) joined by increased UV radiation occurred regularly in earth history.
However, humanly induced long-term effects via the production of inter alia
chlorofluorocarbons (CFCs) are distinguishable from the natural events that only
Introduction
11
cause significant stratospheric ozone damage for a few years (COCKELL and
BLAUSTEIN 2000).
Particularly amphibian embryos can be killed by UV-B radiation (280-315 nm)
directly. Sublethal effects also in combination with contaminants, pathogens or other
climate changes can also affect these animals. These non-lethal effects appear in
embryos, tadpoles and adults as behavior alteration (NAGL and HOFER 1997; KATS
et al. 2000), decelerated development and growth (BELDEN et al. 2000; PAHKALA
et al. 2000; SMITH at al. 2000) and physiological and developmental malfunctions
(HAYS at al. 1996; FITE et al. 1998; ANKLEY et al. 2002). Hatching rates of
examined embryos from North American (e.g. Rana cascae, Bufo boreas), European
(Bufo bufo) and Australian (Litoria verreauxii, Crinia signifera) frogs, toads and
salamanders shielded from radiation are higher compared to groups of embryos
exposed to ambient UV-B radiation (BLAUSTEIN et al. 1998, 2001). The hatching
success in other amphibian species (e.g. Rana aurora, Bufo calamita, Litoria dentata)
was not affected by radiation (BLAUSTEIN et al. 1998).
Distinct impacts of UV-B radiation may differ between species and even between
populations of the same species. In addition, weather conditions, water chemistry (in
cases of aquatic species) and geography influence the effects of the radiation
(BLAUSTEIN et al. 1998; BLAUSTEIN and KIESECKER 2002).
There are evidences that also UV-A radiation (315-400 nm) leads to mutations and
cell death and damages living organism, especially in combination with other
stressors (BLAUSTEIN et al. 2003). In the newt, Pleurodeles walfl, FERNADEZ and
L´HARIDON (1992, 1994) found that UV-A radiation enhances the toxicity of
polycyclic aromatic hydrocarbons (PAHs).
1.3.5 Contaminants
A variety of different substances which can be assigned to pesticides (e.g.
herbicides, fungicides, and insecticides), fertilizers and other groups of compounds
affect amphibians. These contaminants can act on local scale or globally when
transported atmospherically. They have the potential to be harmful in amphibian
species even in low concentrations (BLAUSTEIN et al. 2003). Impacts of
12
Introduction
contaminants on amphibians in the laboratory are studied extensively, but little is
known about the effects on population level (ALFORD and RICHARDS 1999).
1.3.5.1 Pesticides
More than 98 % of sprayed insecticides and 95 % of herbicides reach a destination
other than their target species, including nontarget species such as amphibians, air,
water, bottom sediments and food (MILLER 2004).
In laboratory experiments many pesticides show lethal and sublethal effects on
amphibians including reduced growth and development as well as developmental
and behavioral abnormalities (BOONE and BRIDGES 2003). Studies carried out
under natural conditions allow the conclusion that pesticides also influence the
declines of whole populations. DAVIDSON et al. (2001) investigated the decline of
California Red-legged Frogs, Rana aurora, ranked “near threatened” (SANTOSBARRERA 2004). The authors concluded, that pesticides carried upwind from the
highly agricultural Central Valley cause the decline of Rana aurora populations.
1.3.5.2 Heavy metals
With industrial and agricultural production the prevalence of heavy metals increases
in surface waters affecting amphibian populations. Metals as aluminum (Al), lead
(Pb), zinc (Zn), cadmium (Cd), mercury (Hg), silver (Ag), copper (Cu), arsenic (As),
manganese (Mn), molybdenum (Mo), and antimony (Sb) induce lethal and a plurality
of non-lethal effects (LEFCORT et al. 1998). In larval amphibians elements may
accumulate at higher levels than in adults. Differences in surface area to volume
ratios and skin permeability are discussed (HALL and MULHERN 1984). However,
examples of older stages being more sensitive exist (FREDA 1991).
Tadpoles of the bullfrog (Rana catesbeiana) exposed to different heavy metals in a
coal ash deposition basin (contaminated with As, Cd, Cr, Cu, Se) and a downstream
drainage swamp show higher incidences of oral deformities compared to control
animals. Due to a reduced ability to graze periphyton as sole food source these
animals possess retarded growth rates (ROWE et al. 1996). CHEN el al. (2006)
Introduction
13
raised Northern Leopard Frogs (Rana pipiens) in water contaminated with
environmentally relevant concentrations of lead from the embryonic stage to
metamorphosis. The authors detected significantly slower growth at 100 µg/l lead
treatment and high rates of lateral spinal curvatures associated with abnormal
swimming behavior but significantly lower maximum swimming speed.
1.3.5.3 Acidification
The effects of heavy metals on amphibians are often connected to acidification,
because elements may be leached from soils in presence of acid water due to a
higher solubility in this medium (BLAUSTEIN et al. 2003). A low water pH in
combination with inorganic monomeric aluminum often acts synergistically and
causes increased embryo mortality (FREDA et al. 1990).
Water acidification not only affects juvenile stages but has also an impact on the
behavior of adult amphibians. In a study of ORTIZ-SANTALIESTRA et al. (2007) a
correlation between acid water (pH 4-5) and the protective egg wrapping behavior of
the dwarf newt, Triturus pygmaeus, was detected. The mean percentage of eggs
wrapped around aquatic plants by female specimen was lower at low water pH. This
alteration in the breeding behavior may lead to reduced reproductive success and
also may have an influence on population level.
1.3.5.4 Nitrogenous compounds
Nitrogenous compounds of human origin in the form of agricultural waste (nitrogen
fertilizers, livestock husbandry) as well as industrial and human effluents enter
aquatic habitats (eutrophication). Both adult amphibians and larval stages are
affected by theses contaminants.
In experimental exposure studies with nitrogenous fertilizers several pathological
effects on tadpoles of five species of amphibians (Rana pretiosa, Rana aurora, Bufo
boreas, Hyla regilla, Ambystoma gracile) occur. The individuals show a reduced
feeding activity together with a less vigorously swimming activity, balance disorders,
paralysis, deformations and edemas. Additionally, in all species exposed to the U.S.
14
Introduction
Environmental Protection Agency-recommended limits of nitrite for warm-water fishes
(5 mg N-NO2- per liter) a high mortality and in species exposed to the recommended
limits of nitrite concentration for drinking water (1 mg N-NO2- per liter) a significant
larval mortality was demonstrated (MARCO et al. 1999). In larvae of the Southern
Leopard Frog (Rana sphenocephala) exposed to nitrate at concentrations of 100 mg/l
during the complete development until the metamorphosis, increased mortality and
reduced growth are detectable (ORTIZ-SANTALIESTRA and SPARLING 2007).
Even after metamorphosis alterations e.g. in the feeding behavior in presence of
nitrogenous fertilizers occur (HATCH et al. 2001).
Nitrogenous compounds also have indirect impact as they promote infections in
amphibians. The trematode parasite Ribeiroia ondatrae sequentially infects birds,
snails, and amphibian larvae, frequently causing severe limb deformities and
mortality. The enrichment with nitrogen and phosphorus enhances the algal and snail
host production consequently leading to emergence of this parasite and higher
intensity of infections in amphibians (JOHNSON et al. 2007).
1.3.5.5 Contaminants acting as endocrine disruptors
Many pesticides and other chemical contaminants can act as endocrine disruptors in
humans and animals (LUTZ and KLOAS 1999; MASUTOMI et al. 2004; HAYES
2005). Amphibians are particularly vulnerable to contaminants due to their highly
permeable skin. Additionally, also most terrestrial amphibians reproduce and pass
through critical hormone-regulated developmental stages in aquatic habitats.
Endocrine disrupting compounds may thus have considerable influences on both
individuals and populations (HAYES et al. 2006).
The herbicide atrazine which is widely used in the USA induces hermaphroditism and
demasculanization of the larynx in the African Clawed Frog, Xenopus laevis. HAYES
at al. (2002) hypothesized a disruption of atrazine on the production of steroids.
In a study of HAYES et al. (2006) the impact of nine pesticides commonly used in the
USA together with atrazine and S-metachlor combined as well as the formulation
Biceps II Magnum containing the latter two herbicides on the Leopard Frog, Rana
pipiens was analyzed. The authors found retarded larval growth and metamorphosis
Introduction
15
resulting in undersized adults and in specimens exposed to low-doses mixtures of all
nine compounds thymus damages leading to immunosuppression, meningitis, otitis
interna and septicemia due to Chryseobacterium meningosepticum infections (Fig.
1-2). Effects of atrazine on the gonads were not detectable because frogs did not
complete sexual differentiation before metamorphosis. However, HAYES at al.
(2002a,b) demonstrated endocrine disrupting effects of this compound as induction
of testicular oogenesis in ranid species.
Fig. 1-2. Dorsal (A, C) and frontal (B, D) view of a newly metamorphosed control (A,
B) R. pipiens and a similar-aged animal exposed to the nine-pesticide mixture (C, D).
The control animal is in good body condition, while the pesticide-treated animal is in
poor body condition because of a generalized gram-negative bacterial infection and
signs of disease: head tilt, unilateral extensor muscle rigidity, anisocoria, and
intermittent recumbency due to a severe otitis interna and meningitis (HAYES et al.
2006; modified).
16
Introduction
A stress response (increased production of corticosterone) induced by contaminants
can also interfere with reproductive hormonal systems (HAYES 2000).
Both reproductive biology and behavior of the Red-spotted Newt (Notophthalamus
viridescens) are affected by the cyclodiene organochlorine insecticide, endosulfan. In
laboratory tests males preferred females unexposed to the compound, probably due
to the impact on female pheromonal glands (PARK et al. 2001).
1.3.6 Emerging infectious diseases
An emerging infectious disease is defined as an affection newly recognized, newly
appeared in a population or rapidly increased in incidence, virulence or geographic
range (DASZAK et al. 2000). The major suspected pathogens involved in global
amphibian declines are the chytrid fungus, Batrachochytrium dendrobatidis (Bd), and
different ranaviruses from the Iridoviridae Family (CAREY et al. 2003; COLLINS et al.
2003).
BERGER at al. (1998) published the first report on Bd detected as a pathogen in
amphibian species in montane rain forests in Queensland (Australia) and Panama.
The authors found the disease associated with mass mortality events and population
declines also in wild and captive frogs in other regions of Australia and Central
America and hypothesized that the Bd infection is the proximal cause for worldwide
amphibian declines. Shortly after that chytrid infections in Europe were demonstrated
in died captive anurans (Oophaga pumilio and Phyllobates vittatus) imported from
French Guiana and captive bred frogs in Germany and Belgium (MUTSCHMANN et
al. 2000). Fungal population genetics and field studies on amphibians suggest that
Bd bargains for a newly introduced invasive pathogen (BRIGGS et al. 2005;
MORGAN et al. 2007). Numerous case studies are investigating the correlation
between the appearance of Bd and mass mortality. LIPS et al. (2006) e.g. describes
an outbreak of chytridiomycosis in Panama combined with losses of biodiversity
across eight families of frogs and salamanders. At the locality of El Copé the fungus
caused an epizootic, as the prevalence increased from zero to high prevalence
rapidly. Moreover, Batrachochytrium dendrobatidis is implicated in the declines of the
endangered Wyoming Toad, Bufo baxteri (CAREY et al. 2003), the Boreal Toad,
Introduction
17
Bufo boreas (MUTHS et al. 2003), and the critically endangered Harlequin Frog,
Atelopus mucubajiensis (LA MARCA et al. 2005). Thus the chytrid fungus is the best
supported pathogen related to amphibian declines (DASZAK et al. 2003).
COLLINS et al. (1988) investigated influences of iridoviruses on population dynamics
in the Arizona Tiger Salamander, Ambystoma tigrinum. These viruses cause
epizootics, but in contrast to Bd, they provoke strong fluctuations in amphibian
populations. As in Bd, genetic analysis of ranaviruses isolated from tiger
salamanders revealed low divergences in DNA sequences in all studied markers,
suggesting a recent spread of the pathogen (JANCOVICH et al. 2005). Although
there is no evidence that ranaviruses alone cause amphibian declines or extinctions
in combination with other threats these pathogens can be harmful to amphibian
populations.
1.4
Assisted reproductive technology (ART) in amphibians
For decades amphibians are common laboratory animals for research and education.
Reproductive studies on amphibians were conducted for basic research involving a
few common species. Numerous embryo studies (KAMIMURA et al. 1976) and
investigations concerning sperm-egg interactions (JAFFE et al. 1985; CAMPANELLA
et al. 1997) were performed because amphibian gametes and embryos were
inexpensive and readily available in large numbers. Large-sized embryos and brief
aquatic larval stages made amphibians ideal for developmental and cellular
differentiation studies (ROTH and OBRINGER 2003).
Despite the large amount of information known regarding a few laboratory species
(most notably Rana pipiens and Xenopus laevis), the reproductive biology and
possibilities for the sucessful application of ARTs in the majority of amphibian
species remain poorly understood. This makes assisted breeding programs
complicated as anurans show the greatest diversity in reproductive strategies among
all of the terrestrial vertebrates, including internal and external fertilization, terrestrial
and aquatic breeding, development with a larval stage and direct external
development, ovoviviparity, mass seasonal breeding and continuous breeding, and
presence or absence of parental care (MICHAEL et al. 2004).
18
Introduction
The Wyoming toad, Bufo baxteri, was rescued from near-extinction by the actions of
the Wyoming toad recovery group, founded in 1987 to coordinate habitat protection,
environmental monitoring and research. One of the first objectives was to estabish a
captive breeding program (BROWNE et al. 2006). As a start ten thousand ex situ
bred toadlets were reintroduced into their natural habitat after the pesticide
treatments in this region had been stopped (DICKERSON et al. 1999; RABB 1999).
In the course of the conservation project a total of fifty thousand tadpoles were
released into the wild. Wyoming toads are now breeding in their natural habitat as
well as in newly established reintroduction sites (BROWNE et al. 2006).
Besides natural breeding strategies, endangered amphibian species could benefit
from integrating assisted reproductive technologies (gentle sperm and oocyte
recovery, artificial fertilization and cryopreservation of gametes and embryos) into
breeding programs. Reports on successful breeding programs for threatened
amphibians with implementations of ART are missing.
Also reproductive cloning as a way of increasing a small number of threatened
individuals is conceivable to conserve the genetic diversity if cloning is guaranteed to
show high rates of success in most of the individuals (HOLT et al. 2004). In mice
success rates of <2 % with nuclear transfer were achieved (reviewed by KUES and
NIEMANN 2004). Treatments with Trichostatin A, an inhibitor of histone deacetylase,
following oocyte activation improved the effectiveness of somatic cell nuclear transfer
in mice from 2 to 5-fold depending on the donor cell (KISHIGAMI et al. 2006).
With their studies on the Northern Leopard frog, Rana pipiens, BRIGGS and KING
(1952) showed that regular post-neurula embryos can be obtained from blastula cell
nuclei transplanted into enucleated eggs. The transfer of adult Rana pipiens
erythrocyte nuclei resulted in feeding tadpoles that survived up to one month
(DIBERARDINO et al. 1986), but adult and fertile frogs were generated only from
embryonic (blastula cell) nuclei (MCKINNELL 1962) or nuclei of intestinal epithelial
cells of feeding larvae (GURDON and UEHLINGER 1966). It is still essential that no
adult cloned frog has yet been produced from a somatic adult donor cell nucleus
(GURDON and BYRNE 2003), although the first described vertebrate clones were
frogs (BRIGGS and KING 1952).
Introduction
19
1.4.1 Collection of sperm cells
Controlled and reliable methods of gamete recovery are meaningful in situations
when natural mating is impossible (in cases of insufficient environmental stimuli) or
unwanted (e.g. when subsequent artificial fertilization is planned).
In many studies concerning cryopreservation and sperm morphology, the recovery of
amphibian sperm cells is achieved after euthanasia and maceration of the testes
(BROWNE et al. 1998; MUGNANO et al. 1998; AGUIAR-JR. et al. 2004).
Disadvantages of this approach are the heterogeneous mixture of recovered cell
types and the fact that one male can only be used once in an experiment
(WAGGENER and CARROLL 1998).
The hormonally induced release of spermatozoa as a non-lethal sperm collection
method is known since the 1940s when spermiation in frogs was induced by
intraperitoneal injections of human chorionic gonadotropin (hCG) for human
pregnancy tests (GALLI-MAININI 1947). A variety of different exogenous substances
as pituitary extracts (EASLEY et al. 1979; MINUCCI et al. 1989), GnRH and LHRH
(EASLEY et al. 1979; MINUCCI et al. 1989; ROWSON et al. 2001; IIMORI et al.
2005), LH (BURGOS and LADMAN 1955; EASLEY et al. 1979), FSH (BURGOS and
LADMAN 1955, EASLEY et al. 1979; IIMORI et al. 2005) and hCG (BURGOS and
LADMAN 1955; CHATTERJEE et al. 1971; EASLEY et al. 1979; MINUCCI et al.
1989; IIMORI et al. 2005) are effective stimulators of spermiation in anurans.
Reports on induced spermiation in dendrobatid species are missing. Also few studies
are available concerning induced spermiation in frogs with the aim of amphibian
conservation. In the endangered Wyoming toad, Bufo baxteri, 75 % of hormonally
treated animals showed spermic urine within 3 h with an average of 1.9 ± 0.9 × 106
sperm cells per ml after intraperitoneal LHRH treatment (OBRINGER et al. 2000).
1.4.2 Induction of ovulation and spawning
In in vitro studies SCHUETZ (1971) demonstrated, that follicular oocytes of Rana
pipiens arrested in the meiotic prophase can be induced to mature and ovulate after
incubation with frog pituitary homogenate. In contrast to the inhibiting effects on
20
Introduction
ovulation in mammals, the gestagene progesterone stimulates the final oocyte
maturation and ovulation in Xenopus laevis (FORTUNE et al. 1975; RASTOGI and
IELA 1999). Without apparent progesterone involvement, both the oocyte maturation
and ovulation of another amphibian species (Rana dybowskii) in vitro is induced by
12-O-tetradecanoylphorbol-13-acetate (TPA) via the activation of protein kinase C in
the follicle wall (KWON et al. 1992).
In captive breeding programs, amphibians may fail to spawn naturally even if
neccessary exogenous stimuli (e.g. temperature management, humidity or
hibernation) are provided (BROWNE et al. 2006). Furthermore, simulated hibernation
increases the risk of bacterial and fungal infections in amphibians (TAYLOR et al.
1999).
Several substances (amphibian hypophyseal extract, GnRH, LHRH and hCG)
possess the potential to induce ovulation and spawning in anuran species in vivo. In
the bull frog, Rana catesbeiana, concentrations of 5.5 and 7.7 mg hypophyseal
extract per kg revealed to be effective to induce spawning (FILHO et al. 1998). In the
same species MCCREERY and LICHT (1983) studied the influences of continuous
GnRH applications on plasma profiles of FSH, LH and the sex steroids testosterone,
estradiol-17β and progesterone at different follicular maturation stages in vitro. The
effectiveness of gonadotropin injection to induce spawning is dependent on the
ovarian stage of the treated animal. Females with preovulatory follicles reacted with
significant higher elevations of plasma LH compared to females with follicles in early
stages.
With the objective of enhancing ART for the conservation of the endangered
Wyoming toad, Bufo baxteri, BROWNE et al. (2006) worked on hormonal stimulation
protocols for induced spawning. Female frogs reacted with release of highest egg
numbers after two primings (500 IU hCG in combination with 4 µg LHRH analogue at
0 h + 100 IU hCG in combination with 0.8 µg LHRH analogue at 72 h) and the final
stimulation (500 IU hCG in combination with 4 µg LHRH analogue) at 168 h.
Introduction
21
1.4.3 Artificial fertilization
Induced recovery of sperm cells and oocytes in conservation projects for amphibians
is only reasonable, when cryopreservation of the gametes or artificial fertilization (AF)
is planned. Since most amphibians practice external fertilization, artificial
insemination is not required (ROTH and OBRINGER 2003).
Generally two methods exist for the preparation of amphibian eggs for fertilization.
Either recovered eggs and sperm cells are incubated together directly in a medium
for fertilization, or the gametes undergo several steps of micromanipulation before
oocytes are fertilized directly by added spermatozoa or by intra-cytoplasmic sperm
injection (ICSI), which also allows the production of transgenic animals (SPARROW
et al. 2000; SMITH et al. 2006). Published data exist only on gamete
micromanipulation of common laboratory frog species (e.g. Xenopus laevis), reports
on exotic or threatened species are missing. In hormonally stimulated and
anesthetized Xenopus laevis, oocytes are recovered via partial ovarectomy followed
by manual defolliculation (HEASMAN et al. 1991). This species oocytes are resistant
to manipulations and microinjections of up to 40 nl water and can be maintained in
culture for several hours. Since surgically recovered oocytes lack the influence of
proteolytic enzymes from the proximal oviduct, their surrounding vitelline membrane
remains impenetrable for sperm cells (HEASMAN et al. 1991), which can be
counteracted by either transferring manipulated oocytes into the body cavity of a host
female (BRUN 1975), or breaking down or removing completely the vitelline
membrane (SUBTELNY and BRADT 1961; ELINSON 1973; KATAGIRI 1974). By
adding progesterone (1 µM final concentration) to the culture medium, the essential
maturation of the oocytes to the second meiotic metaphase is initiated (FORTUNE et
al. 1975; HEASMAN et al. 1991). Subsequent fertilization in X. laevis is
accomplished directly by incubation of mature oocytes with sperm cells from
macerated testes (REINHART et al. 1998) or by ICSI (SMITH et al. 2006).
In assisted breeding programs particularly the described surgical methods are
difficult to realize due to high stress and suffering for the animals, limited number of
specimens and potential small animal size. Moreover, a need for breeding transgenic
animals is not existing. Also sacrificing male amphibians for sperm collection from
22
Introduction
macerated testes is not acceptable within breeding programs. In vivo recovered fresh
gametes of Bufo baxteri were used successfully in AF experiments including direct
incubation for fertilization. Healthy frogs were raised and released into the wild as
part of the Wyoming toad recovery plan (BROWNE et al. 2006).
1.4.4 Preservation of gametes
Since spermiation and spawning in amphibians occur not simultaneously after
hormone treatment, induced spermiation and ovulation have to be synchronized for
AF (BROWNE et al. 2006). In B. baxteri, male specimens react with sperm release
5-12 h after stimulation, female frogs show egg deposition 11.5-17.5 h after hormonal
treatment (BROWNE et al. 2006). Problems with asynchrony and variation of gamete
release for AF can be solved with the application of preservation technologies
(MICHAEL and JONES 2004). In particular the cryopreservation and short-term
storage of sperm from non-commercial amphibian species was object of studies
because of its potential to assist conservation and management of threatened
species both in their natural habit and in captivity (WILDT et al. 1995; BROWNE et al.
2002a; BROWNE et al. 2002b). The banking of cryopreserved gametes provides a
temporary protection against extinction and displays a source of genetically diverse
material (MUGNANO et al. 1998). A wide diversity in biological parameters and
response to cryopreservation in frogs is assumable due to the fact that frogs exhibit
the widest range of reproductive models of all terrestrial vertebrates (MICHAEL and
JONES 2004).
1.4.4.1 Gamete storage in solutions of high osmolality
The osmolality of the storage medium for both eggs and sperm cells, when AF is not
to be carried out immediately after gamete recovery, is of great importance. Eggs of
Xenopus laevis rapidly become unfertilizable when stored in a medium with low
osmolality (WOLF and HEDRICK 1971). The authors present a solution named DB
(0.11 M NaCl, 0.0013 M KCl, 0.00044 M CaCl, pH 7.2), in which oocytes remain
fertilizable for more than 2 h (12-14 h) and also sperm cells keep their fertilization
Introduction
23
capacity for more than 24 h (MORIYA 1976). Moreover, the osmolality of the
circumfluent medium surrounding the egg also affects the swelling of the egg jelly. At
low ionic strength the jelly coat swells fast and fertilizability drops rapidly (DEL PINO
1973; HOLLINGER and CORTON 1980).
1.4.4.2 Short term storage of spermatozoa
As well as the gamete storage in solutions of high osmolality at room temperature, a
short-term storage of amphibian gametes at low temperatures is reasonable when
the availability of male and female gametes for AF is asynchronous (LAHNSTEINER
et al. 1997) or sources of gametes are separated spatially (BROWNE et al. 2001).
This technique is also necessary for transporting material from moribund or recently
dead animals in the field to a cryopreservation facility (BROWNE et al. 2001).
For the 1-3 d storage of sperm cells of Bufo americanus, a medium containing 15 %
ethylene glycol is suitable, when cells are frozen slowly to -20°C (BARTON and
GUTTMAN 1972). Detected average motile sperm percentages ranged from 88 %
(after 1 d storage) to 55 % (after 3 d storage). BROWNE et al. (2002b) tested two
cryoprotectants (Me2SO and glycerol) in two concentrations each (15 % and 20 %) in
either simplified amphibian Ringer (SAR) or 10 % sucrose diluents for storage of
Bufo marinus sperm for 6 d at 0°C and found high sperm motility (8 8 ± 4 %) when
SAR and 15 % glycerol were used.
1.4.4.3 Cryopreservation
Sperm cells are vulnerable to freeze-thaw damage caused by intracellular ice and
osmotic dehydration (MORRIS et al. 1999). A premature activation of spermatozoa
caused by freezing might be responsible for low fertilization capability of thawed frog
sperm (WATSON 2000). Regardless of that, the cryopreservation of sperm is a more
promising method of storing gametes of amphibia than cryopreservation of their
fragile and large eggs (SARGENT and MOHUN 2004). In the first description of
successful sperm cryopreservation and thawing, the authors used frog sperm cells
Introduction
24
incubated in a sucrose solution for vitrification in liquid air (LUYET and HODAPP
1938).
In sperm cells of the cane toad, Bufo marinus, high rates of motility and fertilizing
capacity were described using a simplified amphibian Ringer solution containing
10 % sucrose, 15 % Me2SO and 20 % glycerol serving as cryoprotective additives
(BROWNE et al. 1998). Also in hylid (Litoria nasuta, Litoria lateopalmata, Litoria
peroni, Litoria dentata, Litoria leseuri, Litoria verreauxi and Litoria fallax) and
myobatrachid (Uperiola fusca, Limnodynastes tasmaniensis, Limnodynastes peronii,
Mixophys balbus and Crinia signifera) frogs the same concentrations of
cryoprotectants proved to ensure high motility of frozen and thawed sperm
(BROWNE et al. 2002a). MICHAEL and JONES (2004) verified the cryopreservation
success in Eleutherodactylus coqui sperm via the staining behavior with propidium
iodide and SYBR 14 to detect damages of the cell membrane. In this study fetal
bovine serum (FBS) and glycerol were most effective to maintain the integrity of the
cytoplasm membrane. In all published studies concerning cryopreservation of
amphibian sperm, male specimens were killed to obtain sperm samples from
macerated testes.
1.5
The Green Poison Frog, Dendrobates auratus (Girard, 1855)
The Green Poison Frog with a snout-vent length of 25-45 mm (SILVERSTONE 1975)
is a member of the genus Dendrobates whose Greek meaning (“dendro” = tree,
“bates” = runner) refers to the partial arboreal living of some members of this genus.
The epithet (“auratus” = golden) describes glimmering dermal pigments that outlast
even several month of preservation before the skin of fixed specimens turns to black.
The ground coloration of the smooth skin ranges from blue-green to bright leaf green
with black to brown-bronze blotches (SAVAGE 1968) (Fig. 1-3). In addition a plurality
of polymorph colored populations exist. BIRKHAHN et al. (1994) e.g. characterized a
population with white markings, and even spacious yellow colored variants appeared
at breeders. The origins of the latter variants remain unclear.
The species is diurnal and shows highest rates of activity at the morning after rain
falls (DUNN 1941).
Introduction
25
A
B
Fig. 1-3. Differences in the skin coloration of Dendrobates auratus. (A) Frog from
Costa Rica (black blotches) (B) Frog from Panama (“Bronze”)
1.5.1 Phylogenetic systematics
The Dendrobatidae are a family within the Anura with largely unresolved
phylogenetic relationships (FORD and CANNATELLA 1993). A wide range of
datasets have been used to investigate dendrobatid phylogenetic relationships.
Numerous studies report on external morphological characteristics (NOBLE 1931;
INGER 1967; LYNCH 1971, 1973; DUELLMAN and TRUEB 1986; FORD 1993;
FORD and CANATELLA 1993) and behavioral observations (ZIMMERMANN and
ZIMMERMANN 1988; TOFT 1995) possibly applicable to clarify dendrobatid
systematic relationships. Increasingly cytogenetic and molecular markers are used to
fill the gaps in previous data sets (MORESCALCHI 1973; RASOTTO et al., 1987; DE
SÁ and HILLIS 1990; BOGART 1991; HILLIS et al. 1993; HAY et al. 1995;
SUMMERS et al. 1999; CLOUGH and SUMMERS 2000; VENCES et al. 2000,
GRANT et al. 2006). The current phylogenetic grouping of D. auratus is reflected by
Fig. 1-4.
Introduction
26
Class: Amphibia
Order: Anura
Family: Dendrobatidae
Subfamily: Dendrobatinae
Genus: Dendrobates
Species: Dendrobates auratus (Girard, 1855)
Synonym: Dendrobates auratus - Dunn, 1931
Fig. 1-4. Phylogenetic grouping of Dendrobates auratus (FROST 2007; modified).
1.5.2 Distribution
The first description of Dendrobates auratus was carried out on Isla Taboga, Bahia
de Panama, Panama (GIRARD 1855). Populations extend from South Nicaragua to
Columbia including Costa Rica and Panama from sea-level to 800 m elevation, see
figure 1-5 (SAVAGE 1968; SAVAGE 2002). At the locality of El Copé, Panama,
Green Poison Frogs were found at 1450 m elevation (BIRKHAHN et al. 1994),
furthermore frogs originally from Taboga or Tobagilla Island were introduced on the
Hawaiian Island Oahu in 1932 (DALY et al. 1992).
1.5.3 Habitats
Dendrobates auratus is very adaptable and populates forests as well as humanly
altered areas. Originally it inhabits the leaf layer and lower sections of tree trunks of
wet and seasonal wet-dry tropical forests (WELLS 1978). In the dry and agricultural
land of western Panama and Costa Rica the occurrence of this frog is limited to small
spots of forest partial located along rivers. As long as wet hiding places and small
water ponds for tadpoles are reachable the species also lives in gardens, parks and
plantations. In this environments e.g. coconut shells, tin cans or plastic canisters
Introduction
27
instead of water filled tree holes or bromeliads are accepted for tadpole deposition.
The number of individuals of a population appears to be limited by the availableness
of these places for tadpoles but not by the food supply (OSTROWSKI and MAHN
2008). In another dendrobatid frog, Oophaga pumilio, PRÖHL (2002) demonstrated a
relationship between tadpole-rearing sites and population density.
Nicaragua
200 km
Costa Rica
Panama
Colombia
Hawaiian Islands
Ecuador
Native extant
Introduced
Fig. 1-5. Distribution of Dendrobates auratus. (SOLÍS et al. 2004; modified)
28
Introduction
1.5.4 Reproduction
The reproductive behavior in the Green Poison Frog is complex and includes
particular mating behavioral patterns combined with a terrestrial oviposition and
fertilization mode as well as parental care performed by the male. The courtship is
initiated by the vocal advertisement of male frogs.
1.5.4.1 Calling behavior
The advertisement call of males is a 2-4 s long buzzing sound with low frequency.
Calling frogs are distinguishable by their slightly distended vocal sac. Vocalization in
individuals is observed not to be continuously during the breeding season and
seldom at fixed locations suggesting that no long-term territories are maintained
(WELLS 1978).
1.5.4.2 Mating behavior
The courtship behavior in the field (predominantly on Barro Colorado Island, Canal
Zone, Panama) and in captivity is described in detail by WELLS (1978).
Courtship begins with the female approaching a calling male and a snout-to-snout
contact of the partners in some cases. While the male moves through the leaves over
the ground the female follows in a short distance (< 30 cm). DUNN (1941) observed
Dendrobates auratus on Taboga Island and reported that several females follow one
calling male frog. In cases when the female lags or disappears (e.g. under a leaf) the
male frog stops, turns toward the female and calls until she begins moving again.
Touching the male with her snout the female contacts the male. Should the female
not respond to his calls the male takes over the approaching and contacting
behavior.
The pursuit is interrupted consistently by specific tactile interactions in which the
female plays the more active role. During these interactions she jumps on the male
placing the front feet on the back and gently prods his vent and back region (Fig. 16A). Also climbing and sitting on the male and drumming the toes of the hind feet on
the males back occurs (Fig. 1-6B). Other described interactions include female
Introduction
29
encircling of the male, female head-rubbing on the males chin and moving the front
feet up and down while facing each other. WELLS (1978) observed five courtships in
the field that lasted 65-135 min. The frogs covered distances of 5-6 m and visited 10
sites before reaching the definite sheltered oviposition site. Oviposition in the natural
habitat itself was not observed, but the animals remain hidden for approx. 40 min
until the pair separates.
In captivity three to nine black eggs (2 mm diameter) surrounded by a transparent
jelly coat (6 mm outer diameter) are deposited on a plain underlay, e.g. a bromeliad
leaf, and fertilized by the male. By sitting down directly onto the egg clutch (at least
for 30 min) the male moisturizes the clutch and as a result jelly coats swell as well as
the moisture around the eggs increases (LANGE 1981; ZIMMERMANN 1974).
♀
♂
♂
A
♀
B
Fig. 1-6. Courtship in Dendrobates auratus. (A) Female resting front feet on the back
of the male (B) Female placing hind feet on the back of the male (WELLS 1978;
modified).
1.5.4.3 Parental behavior
Parental care is only performed by male frogs and consists of clutch attendance,
exploration and tadpole deposition. The clutch is frequented and irrigated up to four
30
Introduction
times per day for 50 min (ZIMMERMANN 1974) as the male sits on the eggs while
working his legs in and out of the jelly mass.
Male frogs explore for potential egg deposition sites either immediately after a
tadpole transport or independently from carrying a tadpole. They visit several waterfilled tree-holes including diving and moving around the perimeter of each pool
(SUMMERS 1989).
Fig. 1-7. Male Dendrobates auratus carrying one tadpole (→ indicates head of the
larva).
Ten to 13 day after fertilization tadpoles of Dendrobates auratus hatch. The male
uses visual and/or tactile impulses to detect the moment when the offspring can be
carried. Then it climbs on the eggs with a distinctive posture indicating an arched
Introduction
31
back and pressed together ankles. Tadpoles reach the back of the male via wriggling
over the hind legs which are flattened in the jelly. Only one tadpole is carried at once
(WELLS 1978; own observations; Fig. 1-7), but SENFFT (1936) reported that two or
three tadpoles can be transported.
When the adult enters a water pond and floats vertically with spread legs the tadpole
leaves the back and swims free. This action ends the parental care behavior (EATON
1941). Males can coordinate the care for more than one clutch at a time. WELLS
(1978) observed a male frog visiting two clutches (5 m distance) containing larvae in
different stages of development.
1.5.4.4 Sex identification
The identification of the gender in the Green Poison Frog is not obvious, because the
species lacks a distinct and reliable external sex dimorphism. Generally female frogs
possess a greater length of the body and are more corpulent (OSTROWSKI and
MAHN 2008). The second, third and fourth finger disks are wider in male frogs from a
Costa Rican population. Additionally the width of the third finger disk, expressed as a
percentage of the snout-vent length, differs between male and female frogs. In
specimens from Taboga Island (Panama) these differences are marginal; animals
from this population are only slightly sexually dimorphic (SILVERSTONE 1975).
Behavioral observations including calling, clutch attendance and larvae transport are
male specific. These methods are highly reliable for sexing but display of the
behavior has to be awaited.
Sexing via cytogenetic analysis is conceivable but appears to be problematic since
there is no uniform type of genetic sex determination in anurans. Moreover,
amphibian sex chromosomes, if present, are generally very weakly differentiated
making cytogenetic sex identification difficult (HAYES 1998; MATSUBA and MERILÄ
2006).
The chromosomes in Rana pipiens males and females are morphologically identical
(DIBERARDINO 1962); sex is determined by a small number of genes on otherwise
identical X and Y chromosomes (Wright and Richards 1983). In few species sex
chromosomes have been identified by gross morphology and/or banding patterns.
32
Introduction
Male heterogamy (XY/XX type) is present e.g. in Rana esculenta (SCHREMPP and
SCHMID 1981), Eupsophus migueli (ITURRA and VELOSO 1981) and Gastrotheca
riobambae (SCHMID et al. 1983). Female heterogamy (ZZ/ZW) was shown e.g. in
Xenopus laevis (WALLACE et al. 1999) and Pyxicephalus adspersus (SCHMID
1980).
Knowledge of the cytogenetic conditions in dendrobatid frogs is missing. The gentle
and reliable possibility of sex identification, also as secondary effect, via gamete
appointment of hormonal stimulated animals was never performed before in
members of the Dendrobatidae.
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
33
Chapter 2 Induced spermiation and sperm morphology in a dendrobatid
frog, Dendrobates auratus (Amphibia, Anura, Dendrobatidae)
Christian Lipke, Sabine Meinecke-Tillmann, Burkhard Meinecke
Department of Reproductive Biology,
University of Veterinary Medicine Hannover
Accepted for publication by
Salamandra
on July 11th, 2008
34
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
The extent of Christian Lipke’s contribution to the article is evaluated
according to the following scale:
A. has contributed to collaboration (0-33%)
B. has contributed significantly (34-66%)
C. has essentially performed this study independently (67-100%)
1. Design of the project including design of individual experiments:
C
2. Performing of the experimental part of the study:
C
3. Analysis of the experiments:
C
4. Presentation and discussion of the study in article form:
C
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
2.1
35
Abstract
In recent decades an alarming worldwide decline of amphibian populations has been
observed. Because of the world wide amphibian decline, studies concerning the
fundamentals for effective captive breeding programs are important. Frog sperm
recovery, in general, is performed post-mortem after testis dissection. In this study
typical dendrobatid sperm as well as irregularly shaped spermatozoa are described
after gentle hormonal stimulation with human chorionic gonadotropin (hCG), cloaca
lavage and light microscopic as well as fluorescent microscopic evaluation. Starting
from this technique two different hCG stimulation protocols are tested to increase the
obtained overall sperm number. Furthermore, two sperm media, isotonic phosphatefree amphibian saline (IPS) and Xenopus fertilization medium (XB), are compared
regarding sperm motility. The typical spermatozoon of Dendrobates auratus consists
of a filiform and curved head and a single flagellum without any accessory structures.
Gained overall sperm number after double injection of 100 IU hCG at intervals of one
hour is nearly ninefold higher in comparison to single injection of 100 IU hCG. In XB
medium 10.5 % of the viable spermatozoa are motile, whereas 1.2% of viable sperm
cells in IPS are motile. These gentle techniques will be useful for breeding and
conservation
programs
including
sexing,
spermatological
examination,
cryopreservation of spermatozoa, artificial fertilization and the build of gamete banks
for endangered and/or fragile anurans.
2.2
Introduction
With 29 % of the species threatened with extinction, amphibian populations are
declining dramatically worldwide (IUCN 2007). Therefore, the transfer of reproductive
technologies useful for the preservation of endangered amphibians, primarily those
originating from mammalian species, have to be promoted. The release of
spermatozoa into the seminiferous tubules of mature anurans (spermiation)
combined with the transport of sperm cells into the cloaca and the outside
environment has been investigated primarily for studying the physiology of involved
36
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
hormones. One application for this procedure was the diagnosis of human pregnancy
using male toads (GALLI-MAININI 1947). In frogs, a variety of different exogeneous
substances as pituitary extracts (EASLEY et al. 1979; MINUCCI et al. 1989),
gonadotropin-releasing hormone (GnRH) and luteinizing hormone releasing hormone
(LHRH) (EASLEY et al. 1979; MINUCCI et al. 1989; ROWSON et al. 2001; IIMORI et
al. 2005), luteinizing hormone (LH) (BURGOS and LADMAN 1955; EASLEY et al.
1979), follicle stimulating hormone (FSH) (BURGOS and LADMAN 1955; EASLEY et
al. 1979; IIMORI et al. 2005) and human chorionic gonadotropin (hCG) (BURGOS
and LADMAN 1955; CHATTERJEE et al. 1971; EASLEY et al. 1979; MINUCCI et al.
1989; IIMORI et al. 2005) have been tested in vivo over the decades to examine their
potential to induce spermiation.
Studies on induced spermiation in dendrobatid frogs have yet to be conducted. In
examinations investigating the sperm structure in the Dendrobatidae, this gentle
instrument was ignored and euthanasia was preferred (GARDA et al 2002; AGUIARJR. et al. 2004). Also few reports are available concerning induced spermiation in
frogs with the aim of amphibian conservation. In the endangered Wyoming Toad
(Bufo baxteri) an average of 1.9 ± 0.9 × 106 sperm cells per ml were recovered after
intraperitoneal LHRH treatment (OBRINGER et al. 2000).
Due to annual variability within amphibian populations and missing long-term field
studies, at the beginning the worldwide amphibian crisis was not easily detected
(BLAUSTEIN et al. 1994). In 1997, the Declining Amphibian Population Task Force
(DAPTF; Species Survival Commission of the IUCN) attested a significant decline in
amphibian populations worldwide (HEYER 1997). The reasons for such losses are
manifold and can be divided into two classes of hypotheses. Class I hypotheses
(alien species, overexploitation, land use change) are well understood and have
interfered with amphibians for more than a century. New and understudied threats
such as global change, contaminants and emerging infectious diseases have been
merged into the group of class II hypotheses (COLLINS and STORFER 2003).
ETEROVICK (2005) reported exemplarily a dramatic change in the anuran
assemblages at four sites in Brazil between 1971-76 and 1996-2000. Also the
dendrobatid frog, Ameerega flavopicta, common between 1971-76, was completely
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
37
missing during hers 1996-2000 study. Populations of other anurans (including the
Dendrobatidae) will inevitably decline, therefore, working on assisted reproductive
methods in small frogs may prove necessary to aid in the conservation of biodiversity
in the tropics. The general aim of this study was to transfer the technique of induced
sperm recovery in common frog species to the small and fragile dendrobatid species
such as Dendrobates auratus. The online databases AMPHIBIA WEB (2008) and
“Amphibian Species of the World” (FROST 2007) were inquired for the taxonomy
used within the article.
2.3
Materials and methods
2.3.1 Animals
Male specimens of the Green Poison Frog (Dendrobates auratus; n = 12) purchased
from a local breeder were used for the study described herein. The frogs were fertile,
had an average weight of 2.7 g, and were housed in terraria together with female
individuals prior to the experiments. Terraria were ventilated in the bottom and upper
sections consisting of fine grids and were equipped with sufficient lighting. An easy to
clean Plexiglas® plate formed the floor of the terraria. Frogs had access to refugia in
plants and under half coconuts, as well as permanent access to water. Flightless
Drosophila melanogaster fruit flies dredged with vitamin powder were provided every
second day.
2.3.2 General experimental design for induced spermiation
Frogs were carefully immobilized in a wet gauze bandage and received one or two
injections of human chorionic gonadotropin (100 IU hCG, Ovogest® 1500, Intervet,
Unterschleissheim, Germany) each into the dorsal lymph sac at intervals of one hour.
For injection purposes single-use fine dosage syringes with integrated needle
(Omnican® F, 0.30 x 12 mm, Braun, Melsungen, Germany) were used. Sperm
recovery was performed via cloacal lavages at different time intervals, depending on
the selected stimulation protocol. Using single hCG injection cloacal lavages were
38
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
carried out 15, 30, 45, 60, 90 and 120 min after the stimulation. In double stimulation
experiments sperm cells were recovered 5, 15, 30, 45, 75, 90, 120 and 240 min after
the first hCG injection. With a fine bulbous tip cannula (0.7 x 30 mm, Heiland,
Hamburg, Germany) spermatozoa were washed out of the cloaca. Two media,
isotonic phosphate-free amphibian saline (IPS, 111.3 mM NaCl, 3.3 mM KCl, 1.4 mM
CaCl2, 1.2 mM NaHCO3, 3.0 mM D-Glucose, pH 7.4 at 23°C, 220 mosmol/kg;
COSTANZO et al. 1998) and Xenopus fertilization medium (XB, 41.3 mM NaCl, 1.2
mM KCl, 0.3 mM CaCl2, 0.1 mM MgCl2, 1.9 mM NaOH, 0.5 mM Na2HPO4, 2.5 mM
HEPES, pH 7.8 at 20°C, 84 mosmol/kg; HOLLINGER and C ORTON 1980) were
compared regarding sperm motility. Unless otherwise stated, chemicals were
obtained from Sigma-Aldrich, Taufkirchen, Germany. Between these treatments the
frog was placed in a separate box for resting. To detect intracloacal pre-treatment
spermatozoa, a control sample was attained immediately before the first hormone
administration. All flushings were centrifuged for 5 min at 173 × g and 20 µl of the
sediments were placed on non-coated microscopic glass slides and covered with a
cover slip for light microscopic evaluation (BX60, Olympus, Hamburg, Germany;
800×). All animals rested between the experiments for seven to ten days. Statistics
were performed using the program SAS® for Microsoft Windows (Version 9.1).
2.3.3 Experiment 1: Sperm morphology
In this first study the morphology of the spermatozoa describing the majority of the
recovered sperm cells (typically shaped spermatozoa) was investigated using phasecontrast microscopy (800×). Twelve sperm samples from three different stimulated
(100 IU hCG) animals were processed with a microscopic video system linked to the
measurement unit (Kappa Messfadenkreuz MFK II, Gleichen, Germany). The
measurements were repeated after the production and digitalization of video prints
from a color video printer (Sony Mavigraph UP-1850 EPM). All digital images were
processed using the program ImageJ (NIH). Segmented line measurements of the
whole sperm cell and the head and tail regions were carried out. Additionally, surface
area and perimeter of the sperm head were calculated.
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
39
2.3.4 Experiment 2: Membrane integrity
After morphological differentiation into the groups “typically shaped” (cells with filiform
and curved head, slight or no cytoplasm drop and intact flagellum) and “abnormally
shaped spermatozoa” (cells with major rest of cytoplasm drop, immature round-head,
defective head or tail), the integrity of the cytoplasm membrane of recovered cells
(n = 949) from four frogs was investigated using the dye propidium iodide (PI) that
penetrates only strongly damaged membranes (BERTHELSEN 1981). Binding to the
DNA of membrane defective spermatozoa and emitting red fluorescent light, sperm
cells with an intact outer membrane were distinguishable from membrane-defective
cells (HARRISON and VICKERS 1990). Cloaca lavages were centrifuged for 2 min at
173 × g before 1 µl PI solution (1 mg/ml; Sigma-Aldrich, Taufkirchen, Germany) was
added to 199 µl sperm containing sediment. Following a second centrifugation (2 min
at 173 × g) 20 µl of the sediments were deposited on non-coated microscopic glass
slides and were covered with a cover slip. Sperm cells were evaluated by phasecontrast microscopy and fluorescent microscopy (excitation wavelength of 520-550
nm, emission wavelength of 610 nm; BX60 microscope, 800×, Olympus, Hamburg,
Germany). Individual spermatozoa were classified firstly by their head surface
structure in phase-contrast microscopy (smooth vs. protrusions) and subsequently by
their PI staining behavior. The correlation between the dying behavior seen in
fluorescent microscopy and the phase-contrast microscopic cell membrane
morphology were calculated to estimate the viability of the spermatozoa in the
following studies from phase-contrast microscopy.
2.3.5 Experiment 3: hCG dosage
Two dosages of hCG (100 IU vs. twice 100 IU; diluted with 50 µl sterile water each)
were compared to induce spermiation in Dendrobates auratus. Male frogs were
divided into two groups consisting of six animals each. The first group was tested
with 100 IU hCG at least twice. A double stimulation with 100 IU hCG at intervals of
one hour was performed at least twice with every male frog within the second group.
Two different media (IPS, XB) were equally used in both groups. One hour after
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
40
sperm collection, sediment samples from all points of time were processed
seperately under the light microscope. All typically shaped spermatozoa (non-motile
and motile cells were pooled to exclude medium effects on sperm motility) under the
coverslip were counted. Differences in the spermiation response were determined
using the Wilcoxon Rank-Sum Test (SAS 9.1).
2.3.6 Experiment 4: Sperm motility
To investigate possible effects on sperm motility the two different sperm media IPS
and XB were compared. A group of eight male frogs was hormonally stimulated
(twice 100 IU hCG). Cloaca flushings from all points of time were performed with
either IPS or XB per experiment. Both media were used in every individual up to four
times. Only typically shaped viable sperm cells demonstrating a pulsating flagellum
were considered as motile, although in some cases altered cells showed a
movement of the tail. Statistical analysis was performed using the Signed Rank Test
(SAS 9.1).
2.3.7 Experiment 5: Proportion of recovered spermatozoa
Viable motile and non-motile spermatozoa with typical morphology, as well as cells
with altered conformation were analysed. These aberrant sperm cells were divided
into four groups: (1) major rest of cytoplasm drop, (2) immature round-head, (3)
defective head with faulty membrane integrity, and (4) defective tail. With equal
usage of both sperm media (excluding medium effects on sperm motility) for every
individual frog, data of 30 successful double stimulation experiments (total number
15011 spermatozoa) were pooled for analysis (Signed Rank Test; SAS 9.1).
2.4
Results
2.4.1 Experiment 1: Sperm morphology
Spermatozoa of Dendrobates auratus consisted of a filiform, curved head and a
single flagellum. The heads possessed in some cases a slight cytoplasm drop, which
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
41
was clearly distinguishable from large cytoplasm drops belonging to cells grouped
within irregular shaped spermatozoa. The surface of the cell membrane at the head
region appeared consistently smooth and glossy in phase-contrast microscopy. A
midpiece was not visible. No undulating membrane was present at the tail (Fig. 2-1).
Typically shaped sperm cells with intact cytoplasm membrane (n = 39) from three
different
frogs
were
selected
for
measurement
purposes.
The
complete
spermatozoon of Dendrobates auratus had an average length of 56.1 ± 5.5 µm,
whereas 21.1 ± 2.7 µm was allotted to the head and 35.0 ± 4.2 µm to the tail. The
head had a width of 2.0 ± 0.2 µm and the surface area of the headpiece was
calculated with 36.0 ± 5.7 µm2. The perimeter of the sperm head averaged 44.0 ± 5.9
µm.
10 µm
Fig. 2-1. Typical spermatozoon of Dendrobates auratus. Note the smooth and glossy
surface of the cell membrane at the head. Phase-contrast microscopy, 800×.
2.4.2 Experiment 2: Membrane integrity
All dead sperm cells (n = 78) with DNA-bound propidium iodide (PI) emitting red light
in fluorescent microscopy had been previously detected in phase-contrast
microscopy. The membrane showed in these cells numerous bullous protrusions in
42
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
the head area which were distributed equably (Fig. 2-2). For the vast majority
(99.2 %) of spermatozoa ranked membrane intact (n = 864) in phase-contrast
microscopy this observation was verified by the PI staining behavior. As a result of
this examination, viable spermatozoa and membrane-defect sperm cells mentioned
below were differentiated using phase-contrast light microscopy.
10 µm
A
10 µm
B
Fig. 2-2. Dead spermatozoon of Dendrobates auratus. (A) Sperm head with
defective cell membrane, PI dyed (fluorescent microscopy, 800×). (B) Cytoplasm
membrane at the head with numerous protrusions (→) (light microscopy, 800×).
2.4.3 Experiment 3: hCG dosage
In 11 out of 15 experiments (73.3 %) with frogs stimulated with a single injection of
100 IU hCG, sperm cells were obtained. Thirty-one out of 32 sperm collection
attempts (96.9 %) were successful when double stimulation with 100 IU hCG each
was performed. All pre-treatment samples (0 min) were aspermic. An average
number of 42.6 ± 73.0 viable sperm cells were collected after single hCG stimulation
(2.1 ± 3.7 × 103 cells per ml) between 15 min and 120 min, whereas an average
number of 382.6 ± 409.8 viable sperm cells (19.1 ± 20.5 × 103 cells per ml) were
found during experiments with double hCG administration between 5 min and 240
min (Fig. 2-3). Both individual and intraindividual variations in the spermiation
response were noticeable. Four single stimulated frogs showed no or low sperm
release, whereas two frogs reacted with moderate spermiation. After double
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
43
stimulation in two individuals low and moderate and in four individuals high
spermiation responses were detectable. Table 2-1 displays the individual numbers of
Recovered intact spermatozoa (n)
released sperm cells of single and double stimulated frogs.
400
300
single
200
double
100
0
contr. 5 min
15
min
30
min
45
min
60
min
75
min
90
min
120
min
240
min
Fig. 2-3. Differences in the distribution of the spermiation responses (only
membrane-intact spermatozoa with typical morphology, non-motile and motile sperm
cells were pooled) after single and double hCG stimulation. IPS and XB medium was
used equally in both groups.
The average distribution of spermatozoa during the recovery process after single
hCG administration showed a low single peak 30 min following the stimulation. The
first samples were positive at 15 min and in some cases few cells were observed at
90 min and later. In frogs receiving a double stimulation, the sperm response began
30 min following the first injection, increased until 75 min (117.3 ± 107.6 sperm cells)
for a first peak before decreasing again. After 2 hours, the maximum sperm release
was noticeable (147.9 ± 201.1 viable spermatozoa). Analog to the averaged
calculations, the individual spermiation responses showed a biphasic characteristic in
18 out of 32 successful experiments with double stimulation. This stimulation method
was more effective (P<0.05) than the single injection of 100 IU hCG inducing
spermiation in D. auratus (Fig. 2-4).
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
44
Tab. 2-1. Individual numbers of recovered viable sperm cells (motile and non-motile
were pooled) from single and double stimulated frogs per experiment.
100 IU hCG single
frog
1
2
3
viable sperm 60 10 12
cells per
37
5 356
experiment
83
4
0
0
0
5 6
2 3
0 3
1
7
11
413
522
209
874
416
340
60 7.5 184 0 1 3 397.9
x
100 IU hCG double
8
9
10
11
12
1073
0
1164
251
6
159
32
1802
30
35
205
3
874
35
34
1274
149
801
1464
1133
562
434
99
4
50
362.9 205.1 1168.7 140.5 20.5
Note the individual and intraindividual variation of the spermiation response.
Recovered spermatozoa (n)
800
600
*
400
200
0
100 IU hCG single
100 IU hCG double
Fig. 2-4. Number of recovered viable (motile and non-motile) spermatozoa of
Dendrobates auratus after hCG single and double stimulation (* P<0.05).
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
45
2.4.4 Experiment 4: Sperm motility
Using IPS medium in 9 out of 19 experiments no motile spermatozoa with typical
morphology were counted during the whole recovery procedure. On average, 5
motile (maximum 39) sperm cells were recovered. Gained average motile sperm
percentages ranged in individual frogs from 0 to 4.8% ( x =1.2 %). In contrast, in 16
out of 17 experiments with XB medium, motile spermatozoa were found with a mean
of 39 (maximum 198). Percentages of motile spermatozoa ranged in individuals from
8.1 to 16.7 % ( x =10.5 %) (Fig. 2-5). The XB medium provided a significantly higher
number (P<0.05) and percentage (P<0.001) of motile spermatozoa coming from
cloaca flushings of hormonal stimulated dendrobatid frogs than the IPS medium. No
significant correlation could be observed in the distribution of non-motile cells in the
two different media.
Recovered spermatozoa (%)
15
***
10
5
0
IPS
XB
Fig. 2-5. Percentage of recovered motile sperm cells with typical morphology using
either IPS of XB medium after hCG double stimulation (*** P<0.001).
46
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
2.4.5 Experiment 5: Proportion of recovered spermatozoa
Due to the complexity of the used classification for spermatozoa, morphology and PI
staining behavior are summarized in Figure 2-6.
typical sperm
abnormal sperm
(motile & non-motile)
head defect
motile
non-motile
large
cytoplasm drop
round
head
tail
defect
Fig. 2-6. Composition of arranged groups of spermatozoa including accordant
morphology in phase-contrast microscopy and additional PI staining behavior in head
defective cells.
The sperm cell population consisting of viable but non-motile cells amounted to
73.5 % and 5.8 % were motile. With 79.3 %, most sperm cells had a typical
morphology and were viable. All other cells were divided into four groups of
abnormally shaped sperm cells. A large remainder of cytoplasm at the head region
was found in 5.1 % (18.6 ± 14.5) of the sperm, 1.6 % (5.9 ± 3.8) of the cells showed a
rounded and non-elongated headpiece, 4.9 % (22.8 ± 20.8) had a defective cell
membrane (membrane protrusions, rarely entire breaks of the head) and in 9.2 %
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
47
(34.2 ± 24.7) of all cells, tail alterations were visible (breaks or elongations rarely).
Figure 2-7 gives an overview of the constitution of sperm sub-populations together
800
80
700
70
600
60
500
50
400
40
300
30
200
20
100
10
0
Recovered spermatozoa (%)
Recovered spermatozoa (n)
with photographs of representative cells.
0
non-motile motile
plasm
drop
round
head
head
defect
tail defect
Fig. 2-7. Distribution of typically shaped (non-motile and motile) and abnormally
shaped spermatozoa in recovered cloaca flushings (data from all moments of
recovery were pooled). Absolute numbers and percentage of sperm cells were
gained with equal usage of both sperm media for every individual frog after hCG
stimulation (100 IU twice).
2.5
Discussion
This study was designed to offer the ability to manipulate spermiation in future
assisted breeding programs for endangered dendrobatid species. Using the nonendangered Green Poison Frog (Dendrobates auratus) as a research model, a
gentle sperm collection method was established for the potential adoption in other
small tropical frogs, in particular endangered dendrobatid frogs such as the closely-
48
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
related Dendrobates tinctorius, listed as vulnerable, the endangered Oophaga
speciosa or the critically endangered Oophaga lehmanni (IUCN 2007). The
subcutaneous application of hCG into the dorsal lymph system was performed before
only in large and common laboratory frogs belonging to other families other than the
Dendrobatidae, as Rana pipiens (BURGOS and LADMAN 1955), Rana catesbeiana
(EASLEY et al. 1979) and Rana esculenta (MINUCCI et al. 1989) representing the
family Ranidae. In toads, the family Bufonidae, more invasive intraperitoneal
injections of hCG were done in Bufo melanostictus (CHATTERJEE et al. 1971) and
Bufo marinus (IIMORI et al. 2005) to characterize the sperm release. A ventral
dermal administration of LHRH in Bufo americanus and Bufo valliceps resulted in the
release of sperm containing urine (ROWSON et al. 2001), but similar effects of hCG
have not been demonstrated yet.
Low numbers of recovered spermatozoa and low percentages of motile sperm cells
in D. auratus compared to other amphibian species are noticeable, although in
myobatrachid frogs similar percentages of motile sperm cells were observed
(EDWARDS et al. 2004), however sperm suspensions were obtained from
macerated testes. Despite little or no forward progressive motility and a low general
motility, the authors of this study detected high rates of fertilization in Limnodynastes
tasmaniensis. Although no data exist on released sperm numbers under natural
mating conditions, low concentration is likely due to the special terrestrial fertilization
mode of dendrobatid species. A high sperm number placed directly on only few laid
eggs not only means waste of resources but increases the risk of polyspermy. From
this it follows that released numbers of spermatozoa from frog species fertilizing high
numbers of eggs in waters cannot be compared to the condition found in the
Dendrobatidae.
Both sperm media sufficiently supported the integrity of the outer cell membrane. No
significant differences in the appearance of morphologically altered spermatozoa
were observed. Higher sperm motility in the XB medium without energy source
means a rapid loss of cellular energy and should not take place before contacting the
egg jelly coat. This medium is adequate to study movement characteristics of sperm
cells and with the addition of an energy source it may be useful in fertilization
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
49
experiments. The correlation of PI staining behavior and the occurrence of specific
cytoplasm membrane alterations seen in phase-contrast microscopy is obvious but
should be verified. Particularly early and small defects on the outer membrane might
not be noticeable when using phase-contrast microscopy alone.
The external morphology of the spermatozoa of Dendrobates auratus is similar to
that of Ameerega trivittata and Ameerega hahneli (AGUIAR-JR. et al. 2004) and
Ameerega flavopicta (GARDA et al. 2002), except for the absence of accessory tail
structures such as an undulating membrane. It is also similar to the spermatozoa of
Allobates femoralis and Colostethus sp. (AGUIAR-JR. et al. 2003) except for the
presence of two flagella with accessory tail structures in the latter two species. In
Rana and Xenopus spermatozoa (BERNARDINI et al. 1986; LEE and JAMIESON
1993) as well as in D. auratus (LIPKE et al. 2007) one flagellum composed of an
axoneme is present.
Although a non-invasive hormone stimulation method is preferable in assisted
breeding programs for endangered anurans, the hCG double injection is effective in
inducing spermiation and never resulted in negative consequences for the animals
during the study. The rapid decline in frog populations worldwide shows the necessity
of research in the field of amphibian reproduction. Results of this project could be
useful in breeding programs including assisted mating, evaluating male fertility,
artificial fertilization and cryopreservation of gametes and embryos as well as in
veterinary practise for sexing in species lacking external sex dimorphism.
2.6
Acknowledgements
The authors wish to thank Edita Podhajsky and Monika Labsch (both from the
Department of Reproductive Biology, University of Veterinary Medicine Hannover) for
their technical assistance during the experiments.
2.7
References
AGUIAR-JR., O., A.A.GARDA, A.P. LIMA, G.R. COLL, S.N. BÁO and S.M. RECCOPIMENTEL (2003):
50
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
Biflagellate spermatozoon of the poison-dart frogs Epipedobates femoralis and
Colostethus sp. (Anura, Dendrobatidae).
J. Morphol. 255, 114-121
AGUIAR-JR., O., A.P. LIMA, S.N. BÁO and S.M. RECCO-PIMENTEL (2004):
Sperm ultrastructure of the Brazilian Amazon poison frogs Epipedobates trivittatus
and Epipedobates hahneli (Anura, Dendrobatidae).
Acta Zool. 85, 21-28
AMPHIBIA WEB (2008):
Information on amphibian biology and conservation. [web application]. Berkeley,
California: AmphibiaWeb. Available: http://amphibiaweb.org/. (Accessed: Feb 19,
2008)
BERNARDINI, G., R. STIPANI and G. MELONE (1986):
The ultrastructure of Xenopus spermatozoon.
J. Ultrastruct. Mol. Struct. Res. 94, 188-194
BERTHELSEN, J.G. (1981):
Vital staining of spermatozoa performed by the patient.
Fertil. Steril. 35, 86-88
BLAUSTEIN, A.R., D.B. WAKE and W.P. SOUSA (1994):
Amphibian declines: judging stability, persitence and susceptibility of populations to
local and global extinctions.
Conserv. Biol. 8, 60-71
BURGOS, M.H. and A.J. LADMAN (1955):
Effect of purified gonadotropins upon release of spermatozoa in the frog, Rana
pipiens.
Proc. Soc. Exp. Biol. Med. 88(3), 484-487
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
51
CHATTERJEE, A., S. BHADURI and A.K. BASUMALLIK (1971):
The ability of hCG in the induction of spermination in toads devoid of an endogenous
source of gonadotropin.
Endokrinologie 58(1), 119-120
COLLINS, J.P. and A. STORFER (2003):
Global amphibian declines: sorting the hypotheses.
Divers. Distrib. 9, 89-98
COSTANZO, J.P., J.A. MUGNANO, H.M. WEHRHEIM and R.E. LEE-JR. (1998):
Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant
frogs.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 275, 713-719
EASLEY, K.A., D.D. CULLEY JR., N.D. HORSEMAN and J.E. PENKALA (1979):
Environmental influences on hormonally induced spermiation of the bullfrog, Rana
catesbeiana.
J. Exp. Zool. 207, 407-416
EDWARDS, D.L., M.J. MAHONY and J. CLULOW (2004):
Effects of sperm concentration, medium osmolality and oocyte storage on artificial
fertilisation success in a myobatrachid frog, Limnodynastes tasmaniensis.
Reprod. Fertil. Dev. 16, 347-354
ETEROVICK, P.C. (2005):
Amphibian declines in Brazil: An overview.
Biotropica 37(2), 166-179
FROST, D.R. (2007):
52
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
Amphibian species of the world: an online reference. Version 5.1 (10 October, 2007).
Electronic database accessible at
http://research.amnh.org/herpetology/amphibia/index.php.
Am. Mus. Nat. Hist., New York, USA
GALLI-MAININI, C. (1947):
Pregnancy test using the male toad.
J. Clin. Endocrinol. 7, 653-658
GARDA, A.A., G.R. COLLI, O. AGUIAR-JR., S.M. RECCO-PIMENTEL and S.N. BÁO
(2002):
The ultrastructure of the spermatozoa of Epipedobates flavopictus (Amphibia, Anura,
Dendrobatidae), with comments on its evolutionary significance.
Tissue Cell 34, 356-364
HARRISON, R.A.P. and S.E. VICKERS (1990):
Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa.
J. Reprod. Fertil. 88, 343-352
HEYER, R. (1997):
Declining amphibian populations task force.
Species 29:66
HOLLINGER, T.G. and G.L. CORTON (1980):
Artificial fertilization of gametes from the South African clawed Frog, Xenopus laevis.
Gamete Res. 3:45-57
IIMORI, E., M.J. D’OCCHIO, A.T. LISLE and S.D. JOHNSTON (2005):
Testosterone secretion and pharmacological spermatozoal recovery in the cane toad
(Bufo marinus).
Anim. Reprod. Sci. 90, 163-173
IUCN (2007):
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
53
2007 IUCN Red List of Threatened Species. <www.iucnredlist.org>. Downloaded on
21 December 2007
LEE, M.S.Y. and B.G.M. JAMIESON (1993):
The ultrastructure of the spermatozoa of bufonid and hylid frogs (Anura, Amphibia) Implications for phylogeny and fertilization biology.
Zool. Scr. 22, 309-323
LIPKE, C., S. MEINECKE-TILLMANN, W. MEYER and B. MEINECKE (2007):
The ultrastructure of the spermatozoa of Dendrobates auratus (Amphibia, Anura,
Dendrobatidae).
Reprod. Domest. Anim. 42 (Suppl. 2):80
MINUCCI, S., L. DI MATTEO, G. CHIEFFI BACCARI and R. PIERANTONI (1989):
A gonadotropin releasing hormone analog induces spermiation in intact and
hypophysectomized frogs, Rana esculenta.
Experientia 45, 1118-1121
OBRINGER, A.R., J.K. O'BRIEN, R.L. SAUNDERS, K. YAMAMOTO, S. KIKUYAMA
and T.L. ROTH (2000):
Characterization of the spermiation response, luteinizing hormone release and sperm
quality in the American toad (Bufo americanus) and the endangered Wyoming toad
(Bufo baxteri).
Reprod. Fertil. Dev. 12, 51-58
ROWSON, A.D., A.R. OBRINGER and T.L. ROTH (2001):
Non-invasive treatments of luteinizing hormone-releasing hormone for inducing
spermiation in American (Bufo americanus) and Gulf Coast (Bufo valliceps) toads.
Zoo Biol. 20, 63-74
54
Induced spermiation and sperm morphology in a dendrobatid frog, D. auratus
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
55
Chapter 3 Preparation and ultrastructure of spermatozoa from Green
Poison Frogs, Dendrobates auratus, following hormonal induced
spermiation (Amphibia, Anura, Dendrobatidae)
Christian Lipke, Sabine Meinecke-Tillmann, Wilfried Meyer, Burkhard Meinecke
Department of Reproductive Biology,
University of Veterinary Medicine Hannover
Accepted for publication by
Animal Reproduction Science
on June 13th, 2008
56
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
The extent of Christian Lipke’s contribution to the article is evaluated
according to the following scale:
A. has contributed to collaboration (0-33%)
B. has contributed significantly (34-66%)
C. has essentially performed this study independently (67-100%)
1. Design of the project including design of individual experiments:
C
2. Performing of the experimental part of the study:
C
3. Analysis of the experiments:
C
4. Presentation and discussion of the study in article form:
C
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
3.1
57
Abstract
Few ultrastructural studies have been performed on members of the Dendrobatidae,
although such investigations can be useful for the understanding of reproductive
patterns, as a diagnostic method for males in breeding programs for endangered
amphibians and for phylogenetic analysis. The sperm ultrastructure of the Green
Poison Frog, Dendrobates auratus, from Panama is described following induced
spermiation in living animals. To date only testicular spermatozoa in other
dendrobatid frogs have been analysed. Moreover, an electron microscopic
preparation method (transmission und scanning electron microscopy) for dendrobatid
sperm cells in low concentration is presented.
Sperm cells from stimulated frogs (100 IU human chorionic gonadotropin, hCG, twice
at an interval of 1 h) were recovered via cloaca lavage using 600 µl isotonic
phosphate-free amphibian saline (IPS). Centrifuged flushings (5 min, 173 × g) were
deposited on microscopic slides. Adherent spermatozoa were treated with Karnovsky
fixative (overnight, 4°C). After postfixation (2 h, 1 % osmium tetroxide), samples were
dehydrated in series of ascending acetones (30-100 %). For transmission electron
microscopy sperm cells were encapsulated using Epon and 1.5 % 2,4,6tris(dimethylaminomethyl)phenol (DMP 30). Ultrathin sections (70 nm) were cut and
stained with uranyl acetate (30 min) and lead citrate (5 min).
Sperm cells are filiform with a 21.1 ± 2.7 µm long and arcuated head and a single tail
(35.0 ± 4.2 µm length). Their acrosomal complex is located at the anterior portion of
the head and consists of the acrosomal vesicle which has low electron density, and
the subjacent electron-dense subacrosomal cone. In transverse section, the nucleus
is circular (1.9 ± 0.2 µm diameter) and conical in longitudinal section. It is surrounded
by several groups of mitochondria. The chromatin is highly condensed and electrondense but shows numerous electron-lucent inclusions. A short midpiece has a
mitochondrial collar with a proximal and a distal centriole. The latter gives rise to the
axoneme which alone forms the flagellum. The sperm ultrastructure of D. auratus
differs from that of other Dendrobatidae because of the absence of a nuclear space
and the absence of the undulating membrane associated with an axial fibre. This tail
conformation is found in the Ranoidea but not in the Bufonoidea. These results show
58
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
that the spermatozoa of D. auratus are the first within the Dendrobatidae without
accessory tail structures. Methods of using sperm samples from hormonal treated
frogs for ultrastructural studies is not only reasonable to examine e.g. amphibian
phylogeny without killing frogs threatened with extinction but allows investigations in
the field of assisted reproduction and male fertility for example in conservation
programs for endangered amphibians.
3.2
Introduction
Sperm ultrastructure has been used as an alternative for the phylogenetic analysis of
many taxa including amphibians (JAMIESON et al. 1993; MEYER et al. 1997;
SCHELTINGA and JAMIESON 2001).
The Dendrobatidae are among the families within the Anura with largely unresolved
phylogenetic relationships (FORD and CANNATELLA 1993). Classification of genera
within the Dendrobatidae has undergone many changes including establishing new
genera and shifting species among them (MYERS 1987; SCHELTINGA and
JAMIESON 2003; FROST et al. 2006; GRANT et al. 2006). Online databases like
“AMPHIBIA WEB” (2008) and “Amphibian Species of the World” (FROST 2007)
provide access to the latest taxonomy. These online sources were inquired for the
taxonomy used within the article.
Numerous studies reported external morphological characteristics possibly adaptive
to clarify dendrobatid systematic relationships (DUELLMAN and TRUEB 1986; FORD
1993; FORD and CANNATELLA 1993), as well as behavioural observations
(ZIMMERMANN and ZIMMERMANN 1988). Increasingly, cytogenetic and molecular
markers have been used to fill the gaps in previous datasets (CLOUGH and
SUMMERS 2000; VENCES et al. 2000), but no well-established clades have been
characterized.
The ultrastructure of Ameerega flavopicta spermatozoa was described by GARDA et
al. (2002) who found structural evidence for the grouping of the Dendrobatidae within
the Bufonoidea. These results are in accordance with gene data retrieved from
nuclear and mitochondrial sequences (HILLIS et al. 1993; HAY et al. 1995).
Furthermore, AGUIAR-JR. et al. (2003) examined the sperm ultrastructure of
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
59
Allobates femoralis and Colostethus sp. and noted their similarity to each other,
although in the meantime Allobates femoralis was placed in the Aromobatidae
(FROST 2007). Sperm ultrastructure in two other species of the Dendrobatidae,
Ameerega trivittata and Ameerega hahneli was analysed by AGUIAR-JR. et al.
(2004). These two species had identical morphological characteristics except for the
expansion of the acrosomal vesicle. In all sperm cells described in dendrobatid
species the flagellum consisted of an axoneme, an undulating membrane and an
axial fibre. All authors using sperm ultrastructure structure for phylogenetic analysis
suggested a grouping of the Dendrobatidae within the Bufonoidea neobatrachian
lineage mainly because of the tail structure.
The aim of the present work is to describe the ultrastructure of mature sperm of
Dendrobates auratus. For recovery of spermatozoa induced spermiation was used.
So far in dendrobatid frogs sperm ultrastucture was investigated only after autopsy in
testicular tissue. A new method for the preparation of these low concentration sperm
samples resulting from induced spermiation was established. Data may be useful for
the phylogenetic analysis of the genera within the Dendrobatidae and the position of
the whole family within the Anura. The preparation procedure for sperm samples
from hormonal stimulated frogs for electron microscopic purposes may also be
reasonable in breeding programs for endangered amphibians.
3.3
Materials and Methods
3.3.1 Recovery and fixation of spermatozoa
Four 18-month-old males of D. auratus from Panama were purchased from a local
breeder. The mature frogs were fertile, had an average weight of 2.7 g and were
housed in terraria together with females before the experiments. Spermatozoa were
obtained from the living animals using induced spermiation. The frogs got two
injections of 100 IU human chorionic gonadotropin (Ovogest® 1500, Intervet,
Unterschleissheim, Germany) at an interval of 1 h into the dorsal lymph sac. 75-120
min after the first hCG injection spermatozoa were washed out of the cloaca with 600
µl isotonic phosphate-free amphibian saline (IPS, 111.3 mM NaCl, 3.3 mM KCl, 1.4
60
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
mM CaCl2, 1.2 mM NaHCO3, 3.0 mM D-Glucose, pH 7.4 at 23°C, 220 mosmol/kg;
COSTANZO et al. 1998) using a fine bulbous tip cannula (LIPKE et al. 2007). Unless
otherwise stated, chemicals were obtained from Sigma-Aldrich, Taufkirchen,
Germany. The flushings were centrifuged for 5 min at 173 × g and 20 µl of the
sediments were deposited on non-coated microscopic glass slides and were covered
with a cover slip for light microscopic evaluation (BX60, Olympus, Hamburg,
Germany; 800×). Samples containing more than 600 spermatozoa were selected for
electron microscopic purposes. After overlaying the slide with Karnovsky fixative
(KARNOVSKY 1965), the coverslip was removed manually. Slides with adherent
sperm were kept overnight at 4°C.
3.3.2 Transmission electron microscopy
Slides with fixed samples were rinsed four times in 0.1 M cacodylate buffer (pH 7.2,
Serva, Heidelberg, Germany) and kept overnight at 4°C. Postfixation was performed
for 2 h in 1 % osmium tetroxide (Roth, Karlsruhe, Germany) before the spermatozoa
on the slide were rinsed again four times for 10 minutes in cacodylate buffer.
Subsequently the material was dehydrated in a series of ascending acetones (30-100
%; Merck, Darmstadt, Germany) and embedded in Epon resin (Serva, Heidelberg,
Germany). The cells were infiltrated with propylene oxide (Serva, Heidelberg,
Germany) for 45 minutes, with propylene oxide and Epon for 1 h and with Epon alone
for 105 min. The spermatozoa were encapsulated by placing a gelatine capsule filled
with Epon and 1.5 % 2,4,6-tris(dimethylaminomethyl)phenol (DMP 30, Serva,
Heidelberg, Germany) onto the slide. The samples were kept at 35°C and then at
45°C for 24 h each and for 5 days at 65°C for polymer isation. Using liquid nitrogen
the blocks were detached from the surface of the slides. Ultrathin sections (70 nm)
were cut with an ultramicrotome Ultracut E (Leica, Wetzlar, Germany) and stained
with uranyl acetate (Merck, Darmstadt, Germany) for 30 min and lead citrate for 5
min. Sections were observed in an EM 10A transmission electron microscope (Zeiss,
Oberkochen, Germany) at 60 kV.
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
61
3.3.3 Scanning electron microscopy
After rinsing six times in 0.1 M cacodylate buffer (pH 7.2) the fixed samples were
kept overnight at 4°C. Postfixation was performed for 2 h in 1 % osmium tetroxide
and the samples were washed again four times for 10 min in cacodylate buffer. The
cells were dehydrated in a series of ascending acetones (30-100 %), before the
sections were cut and gold-coated. Examination was performed with a DMS 950
scanning electron microscope (Zeiss, Oberkochen, Germany) at 10 kV.
3.4
Results
3.4.1 General morphology
With scanning electron microscopy, the spermatozoa of D. auratus consisted of a
21.1 ± 2.7 µm long head and a single 35.0 ± 4.2 µm long flagellum (Fig. 3-1A). The
head was filiform and curved and possessed a distinctive cytoplasmic drop in some
cases (Fig. 3-1B and C). A midpiece was not clearly distinguishable. No undulating
membrane was present on the tail. In Figure 3-2 a schematic reconstruction of the
spermatozoon is shown.
3.4.2 Acrosomal complex
The acrosomal complex, located at the anterior portion of the head, was 1.8 ± 0.3 µm
long. It was composed of a narrow acrosomal vesicle of low electron density and an
electron-dense subacrosomal cone below. The subacrosomal cone formed a conical
cap covering the front region of the nucleus (Fig. 3-3A).
3.4.3 Nucleus
The nucleus was cylindrical and elongated with conical ends (1.9 ± 0.2 µm diameter
in transverse section). Its anterior part was covered by the subacrosomal cone.
Electron-dense chromatin filled the nucleus completely except for numerous electronlucent inclusions that were distributed uniformly (Fig 3-3A and B).
62
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
At the arcuated part of the head in most cases the nucleus was partly surrounded by
electron-lucent cytoplasmic formings containing several mitochondria (Figs. 3-3C and
D). These structures were completely absent in some spermatozoa (Fig. 3-3E).
2 µm
B
2 µm
A
5 µm
C
Fig. 3-1. Spermatozoa of Dendrobates auratus. Scanning electron microscopy.
(A) Whole spermatozoon with head and single flagellum. 2602×. (B) Head without
cytoplasmic drop. 6376×. (C) Head with large cytoplasmic drop at posterior region.
5796×.
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
63
Fig. 3-2. Schematic illustration of the ultrastructure of the spermatozoon of
Dendrobates auratus.
64
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
3.4.4 Midpiece
A midpiece was only distinguishable in transmission electron microscopy because
there was no visible intersection on the sperm cell surface of the nucleus and the
midpiece region.
This area connected head and tail of the spermatozoon, and contained the proximal
and distal centrioles, axoneme and mitochondria. A nuclear fossa (AGUIAR-JR. et al.
2004) as well as a paraxonemal rod (JAMIESON et al. 1993) was absent. The
proximal centriole (Fig. 3-3F) and the distal centriole were inserted in the anterior part
of the midpiece and lay 90° to each other. The latte r gave rise to the axoneme.
Several round mitochondria, whose inner membrane was compartmentalised into
numerous cristae, were scattered in the midpiece.
A mitochondrial collar of cytoplasmic extensions (AGUIAR-JR. et al. 2004) was also
visible in D. auratus sperm. It formed a smooth cavity termed the flagellar fossa with
the free flagellum in its centre (Fig. 3-3G).
3.4.5 Flagellum
The flagellum in D. auratus consisted of an axoneme that showed the typical 9+2
microtubule arrangement in transverse section (Fig. 3-3H and I). No accessory
structures as axial fibre or undulating membrane were present.
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
65
nu
nu
in
in
0.5 µm
B
sc
av
0.5 µm
A
D
cr
nu
nu
mt
in
in
cr
C
mt
0.5 µm
1 µm
66
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
F
nu
pc
5 µm
E
0.2 µm
0.5 µm
nu
mt
ax
ff
ff
G
ax
H
0.3 µm
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
67
ax
I
0.2 µm
Fig. 3-3. Spermatozoa of Dendrobates auratus. Transmission electron microscopy.
(A) Longitudinal section of the acrosomal complex showing the thin conical
acrosomal vesicle and the subacrosomal cone. 50000×. (B) Transverse section of
the nucleus with electron-dense chromatin and inclusions. 63000×. (C, D) Transverse
and longitudinal sections of the nucleus with cytoplasmic remains containing several
mitochondria. 50000×, 40000×. (E) Longitudinal section of the complete headpiece.
Note the absence of circumjacent cytoplasmic formings. 6300×. (F) Longitudinal
section of the midpiece with proximal centriole. 63000×. (G) Midpiece in longitudinal
section. Note the mitochondria within the cytoplasmic extensions forming the flagellar
fossa. 50000×. (H) Transverse section of the flagellum. Note the microtubule
structure of the axoneme. 16000×. (I) Longitudinal section of the tail. 63000×.
Abbreviations: av = acrosomal vesicle; ax = axoneme; cr = cytoplasmic remains; ff =
flagellar fossa; in = inclusion; mt = mitochondrion; nu = nucleus; pc = proximal
centriole and sc = subacrosomal cone.
68
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
3.5
Discussion
Ultrastructure of testicular spermatozoa previously was described in dendrobatid
frogs only after killing the animals and dissecting the testes. In the present study
mature sperm cells from hormone-stimulated frogs were analysed. The spermatozoa
of D. auratus are similar to those of Ameerega trivittata, Ameerega hahneli (AGUIARJR. et al. 2004) and Ameerega flavopicta (GARDA et al. 2002), except for the
absence of a nuclear space and any accessory tail structures such as an undulating
membrane and the axial fiber. They are also similar to the sperm cells of Allobates
femoralis and Colostethus sp. (AGUIAR-JR. et al. 2003) except for the presence of
two flagella in those species.
The acrosomal complex in D. auratus is shorter than in all other described
dendrobatid species and consists of a thin and narrow acrosomal vesicle and a
subacrosomal cone. A nuclear space is completely missing in D. auratus. In contrast,
this structure between the subacrosomal cone and nucleus is present in all members
of the Dendrobatidae previously described, A. trivittata, A. hahneli (AGUIAR-JR. et al.
2004), A. flavopicta (GARDA et al. 2002), A. femoralis and Colostethus sp. (AGUIARJR. et al. 2003).
Our results illustrate a compact nucleus, which is completely filled with chromatin
despite numerous nuclear inclusions containing granular material. Nuclear lacunae
that lack material are not present. Both structures are common in frog spermatozoa
and were described in dendrobatid (GARDA et al. 2002; AGUIAR-JR. et al. 2004),
hylid (LIN et al. 2000), microhylid (SCHELTINGA et al. 2002), ranid (POIRIER and
SPINK 1972) and ascaphid (JAMIESON et al. 1993) frogs. Numerous glycogen
inclusions are visible in sperm cell nuclei of Rana pipiens and Rana clamitans
(POIRIER and SPINK 1972), which differ from the inclusions in D. auratus regarding
the grade of granulation. However, in Ameerega flavopicta (GARDA et al. 2002) the
nuclear inclusions appear similar to those of D. auratus spermatozoa but are less
frequent. In both species they might be homologous to nuclear vacuoles that contain
nuclear material not involved in chromatin condensation during sperm development
as described in Hyla chinensis (LIN et al. 2000).
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
69
Cytoplasmic structures surrounding the nucleus and containing mitochondria were
present in some spermatozoa indicating potentially different stages in sperm
maturation. In mature sperm cells these cytoplasmic structure may have been
retracted to the midpiece containing residual mitochondria.
The structure of the midpiece in D. auratus spermatozoa with two centrioles and a
mitochondrial collar is similar to that of all other members of the Dendrobatidae. The
arrangement of cytoplasmic extensions containing mitochondria and forming a collar
around the anterior free end of the flagellum is a synapomorphy of bufonid,
leptodactylid and hylid families (LEE and JAMIESON 1993).
A sperm tail consisting of a single axoneme as described here for Dendrobates
auratus is present in Rana and Xenopus spermatozoa (BERNARDINI et al. 1986;
LEE and JAMIESON 1993), but this condition has not been reported previously in
any member of the Dendrobatidae or Bufonoidea.
A diversity of this attribute within the family can be assumed and should be analysed
in future projects. Ultrastructural sperm studies in a broad range of dendrobatid
species and other amphibian families together with behavioural observations, studies
on morphological characteristics and molecular methods may also be useful to
investigate amphibian phylogeny.
Our methods using sperm samples from hormonal stimulated living dendrobatid frogs
for ultrastructural studies are not only applicable to investigate aspects of amphibian
phylogeny, but demonstrate the basis for future studies concerning functional
characteristics in spermatozoa and assisted reproduction in amphibians. The
possibility of ultrastructural sperm evaluation is also valuable for diagnostic purposes
in male frogs in breeding programs for endangered amphibians. With 29 % of the
taxons species threatened with extinction, amphibian populations are in a dramatic
worldwide decline (IUCN 2007). Therefore, the appliance of gentle reproductive
methods avoiding killing endangered animals has to be promoted.
3.6
Acknowledgements
We thank Kerstin Rohn (Department of Pathology, University of Veterinary Medicine
Hannover Foundation) for technical assistance preparing the sperm samples for
70
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
electron microscopy and Edita Podhajsky (Department of Reproductive Biology,
University of Veterinary Medicine Hannover Foundation) for assistance in drawing
the schematic illustration.
3.7
Literature cited
AGUIAR-JR., O., A.A. GARDA, A.P. LIMA, G.R. COLLI, S.N. BÁO and S.M. RECCOPIMENTEL (2003):
Biflagellate spermatozoon of the poison-dart frogs Epipedobates femoralis and
Colostethus sp. (Anura, Dendrobatidae).
J. Morphol. 255, 114-121
AGUIAR-JR., O., A.P. LIMA, S.N. BÁO and S.M. RECCO-PIMENTEL (2004):
Sperm ultrastructure of the Brazilian Amazon poison frogs Epipedobates trivittatus
and Epipedobates hahneli (Anura, Dendrobatidae).
Acta Zool. 85, 21-28
AMPHIBIA WEB (2008):
Information on amphibian biology and conservation. [web application]. Berkeley,
California: AmphibiaWeb. Available: http://amphibiaweb.org/. (Accessed: Feb 19,
2008)
BERNARDINI, G., R. STIPANI and G. MELONE (1986):
The ultrastructure of Xenopus spermatozoon.
J. Ultrastruct. Mol. Struct. Res. 94, 188-194
CLOUGH, M. and K. SUMMERS (2000):
Phylogenetic systematics and biogeography of the poison frogs: evidence from
mitochondrial DNA sequences.
Biol. J. Linnean Soc. 70, 515-540
COSTANZO, J.P., J.A. MUGNANO, H.M. WEHRHEIM and R.E. LEE-JR. (1998):
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
71
Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant
frogs.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 275, 713-719
DUELLMAN, W.E. and L. TRUEB (1986):
Biology of amphibians.
McGraw-Hill, New York, USA.
FORD, L.S. (1993):
The phylogenetic position of the dart-poison frogs (Dendrobatidae) among anurans An examination of the competing hypotheses and their characters.
Ethol. Ecol. Evol. 5, 219-231
FORD, L.S. and D.C. CANNATELLA (1993):
The major clades of frogs.
Herpetol. Monogr. 7, 94-117
FROST, D.R. (2007):
Amphibian species of the world: an online reference. Version 5.1 (10 October, 2007).
Electronic database accessible at
http://research.amnh.org/herpetology/amphibia/index.php.
Am. Mus. Nat. Hist., New York, USA
FROST, D.R., T. GRANT, J. FAIVOVICH, R.H. BAIN, A. HAAS, C.F.B. HADDAD,
R.O. DE SÁ, A. CHANNING, M. WILKINSON, S.C. DONNELLAN, C.J.
RAXWORTHY, J.A. CAMPBELL, B.L. BLOTTO, P. MOLER, R.C. DREWES, R.A.
NUSSBAUM, J.D. LYNCH, D.M. GREEN and W.C. WHEELER (2006):
The Amphibian tree of life.
Bull. Am. Mus. Nat. Hist. 297, 1-291
72
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
GARDA, A.A., G.R. COLLI, O. AGUIAR-JR., S.M. RECCO-PIMENTEL and S.N. BÁO
(2002):
The ultrastructure of the spermatozoa of Epipedobates flavopictus (Amphibia, Anura,
Dendrobatidae), with comments on its evolutionary significance.
Tissue Cell 34, 356-364
GRANT, T., D.R. FROST, J.P. CALDWELL, R. GAGLIARDO, C.F.B. HADDAD,
P.J.R. KOK, D.B. MEANS, B.P. NOONAN, W.E. SCHARGEL and W.C. WHEELER
(2006):
Phylogenetic systematics of dart-poison frogs and their relatives (Amphibia:
Athesphatanura: Dendrobatidae).
Bull. Am. Mus. Nat. Hist. 299, 1-262
HAY, J.M., I. RUVINSKY, S.B. HEDGES and L.R. MAXSON (1995):
Phylogenetic relationships of amphibian families inferred from DNA sequences of
mitochondrial 12S and 16S ribosomal RNA genes.
Mol. Biol. Evol. 12, 928-937
HILLIS, D.M., L.K. AMMERMAN, M.T. DIXON and O. DE SÁO (1993):
Ribosomal DNA and the phylogeny of frogs.
Herpetol. Monogr. 7, 118-131
IUCN (2007):
2007 IUCN Red List of Threatened Species. <www.iucnredlist.org>. Downloaded on
21 December 2007
JAMIESON, B.G.M., M.S.Y. LEE and K. LONG (1993):
Ultrastructure of the spermatozoon of the internally fertilizing frog Ascaphus truei
(Ascaphidae: Anura: Amphibia) with phylogenetic considerations.
Herpetologica 49, 52-65
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
73
KARNOVSKY, M.J. (1965):
A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron
microscopy.
J. Cell Biol. 27, 137
LEE, M.S.Y. and B.G.M. JAMIESON (1993):
The ultrastructure of the spermatozoa of bufonid and hylid frogs (Anura, Amphibia) Implications for phylogeny and fertilization biology.
Zool. Scr. 22, 309-323
LIN, D.J., Y.L. YOU and G. ZUMMO (2000):
Spermiogenesis in Hyla chinensis.
Acta Zool. Sinica 46, 376-384
LIPKE, C., S. MEINECKE-TILLMANN and B. MEINECKE (2007):
An advanced spermiation protocol in a dendrobatid frog, Dendrobates auratus
(Amphibia, Anura, Dendrobatidae).
Reprod. Dom. Anim. 42 (Suppl. 1), 19
MEYER, E., B.G.M. JAMIESON and D.M. SCHELTINGA (1997):
Sperm ultrastructure of six Australian hylid frogs from two genera (Litoria and
Cyclorana): phylogenetic implications.
J. Submicrosc. Cytol. Pathol. 4, 443-451
MYERS. C.W. (1987):
New generic names for some neotropical Poison Frogs (Dendrobatidae).
Pap. Avul. Zool. 36, 301-306
POIRIER, G.R. and G.C. SPINK (1972):
Spermatozoal glycogen in two species of Rana.
Z. Zellforsch. 129, 272-277
74
Preparation and ultrastructure of spermatozoa from Green Poison Frogs, D. auratus
SCHELTINGA, D.M. and B.G.M. JAMIESON (2001):
Ultrastructure of the spermatozoon of Leiopelma hochstetteri (Amphibia, Anura,
Leiopelmatidae).
Zoosystema 23, 157-171
SCHELTINGA, D.M. and B.G.M. JAMIESON (2003):
Spermatogenesis and the mature spermatozoon: Form, function and phylogenetic
implications. In: Jamieson, B.G.M., (Ed.), Reproductive biology and phylogeny of
anura.
Science Publishers, Inc., Enfield, USA, pp. 119-251
SCHELTINGA, D.M., B.G.M. JAMIESON, D.P. BICKFORD, A.A. GARDA, S.N. BÁO
and K.R. MCDONALD (2002):
Morphology of the spermatozoa of the Microhylidae (Anura, Amphibia).
Acta Zool. 83, 263-275
VENCES, M., J. KOSUCH, S. LÖTTERS, A. WIDMER, K.H. JUNGFER, J. KOHLER
and M. VEITH (2000):
Phylogeny and classification of poison frogs (Amphibia: Dendrobatidae), based on
mitochondrial 16S and 12S ribosomal RNA gene sequences.
Mol. Phylogenet. Evol. 15, 34-40
ZIMMERMANN, H. and E. ZIMMERMANN (1988):
Ethotaxonomie und zoogeographische Artengruppenbildung bei Pfeilgiftfröschen
(Anura: Dendrobatidae).
Salamandra 24, 125-160
Discussion
75
Chapter 4 Discussion
Discussion
76
Dendrobatid frogs represent small tropical amphibian species which have never been
studied concerning amphibian conservation in combination with reproductive biology
before, although almost 28 % of these species are threatened. Therefore,
transferring
reproductive
technologies
for
the
preservation
of
endangered
amphibians, primarily those originating from mammalian species is reasonable.
The technique of hormonal induced spermiation for sperm recovery was tested and
improved in the Green Poison Frog, Dendrobates auratus. Resulting sperm samples
were analyzed to estimate the quality of ejaculates. Two different sperm media (IPS,
XB) were compared regarding sperm motility. Unpublished results also show the
possibility to recover oocytes from hCG stimulated female individuals. The results will
be useful for artificial fertilization purposes and gamete preservation in amphibian
conservation programs. In addition to important research in the field of ecology
investigating causes for amphibian declines, projects utilizing reproductive methods
apply a practical approach to conserve biodiversity.
4.1
Induced spermiation
4.1.1 Hormonal stimulation
The subcutaneous double application of 100 IU hCG into the dorsal lymph system
resulting in sperm release in Dendrobates auratus, revealed a more effective method
for gentle induction of spermiation than single injections of hCG. In dendrobatid
species no comparable studies exist.
Subcutaneous applications of hCG into the dorsal lymph system are common only in
large laboratory frogs as Rana pipiens (BURGOS and LADMAN 1955), Rana
catesbeiana (EASLEY et al. 1979) and Rana esculenta (MINUCCI et al. 1989). More
invasive intraperitoneal administrations of hCG were done in the toads Bufo
melanostictus (CHATTERJEE et al. 1971) and Bufo marinus (IIMORI et al. 2005) to
characterize the sperm release. Ventral dermal application of LHRH resulted in Bufo
Americanus and Bufo valliceps releasing of sperm-containing urine (ROWSON et al.
2001), but similar effects of hCG have not been verified.
Discussion
77
4.1.2 Quantity of recovered spermatozoa
Between 5 min and 240 min after hCG double stimulation an average viable sperm
cell concentration of 19.1 ± 20.5 × 103 per ml was detected. The variability of these
results refers to high individual differences in the hCG responsiveness. From all hCG
treated male specimens of D. auratus, spermatozoa were recovered. All ejaculates
demonstrate direct hormonal influenced reactions, since all pre-treatment samples
were aspermic.
IIMORI et al. (2005) succeeded to recover viable sperm numbers of up to 24.9 1 ±
14.1 × 106 in Bufo marinus after injection of 2000 IU hCG. Since no data exist on
released sperm numbers in members of the Dendrobatidae neither in spermiation
experiments nor under natural mating conditions all estimates on the effectiveness of
the spermiation procedure remain speculative. It seems reasonable to assume that
low concentrations display a physiological condition within the terrestrial fertilization
mode of D. auratus, meaning not only saving resources but also decreasing the risk
of polyspermy. Therefore, it is likely that recovered numbers of spermatozoa in this
species suffice to fertilize eggs in in vitro experiments.
4.1.3 Sperm motility
In the XB medium sperm cells showed a significant higher percentage of motility
compared to spermatozoa in the IPS medium. Since XB contained no energy source
this medium is adequate to study movement characteristics of sperm cells but is
inapplicable for storage and fertilization purposes due to rapid loss of cellular energy.
With addition of an energy source it appears to be useful in future fertilization
experiments as well as IPS medium. Though low percentages of recovered motile
sperm cells in the Green Poison Frog compared to other mostly aquatic amphibian
species are noticeable.
With a maximum of 66.2 %, IIMORI et al. (2005) found high rates of motility sperm of
Cane Toads, Bufo marinus, after hCG stimulation. By contrast, in myobatrachid frogs
percentages of motile sperm cells similar to these found in D. auratus were observed
(EDWARDS et al. 2004). Results of this study indicate that amphibian ejaculates with
Discussion
78
little or no forwards progressive motility and a low motility possess the potential to
fertilize eggs.
4.1.4 Membrane integrity of spermatozoa
Both IPS and XB sperm medium sufficiently supported the integrity of the outer cell
membrane of dendrobatid spermatozoa. The obvious correlation between PI staining
behavior and occurrence of specific cytoplasm alterations has to be verified. The
expressiveness of this method is limited since small defects in the cell membrane
might not be noticeable in LM, although the current approach is reasonable to
estimate the quality of recovered sperm samples and to diagnose subfertility in rare
amphibian species.
4.2
Sperm structure
Mature sperm cells from hormone-stimulated frogs were analyzed in this study. For
all previous projects investigating sperm ultrastructure in dendrobatid species
animals were killed to remove the testes. Ultrastructural sperm studies are useful to
both
characterize
spermatological
standards
and
investigate
phylogenetic
relationships.
Size and external morphology of the spermatozoa of Dendrobates auratus are similar
to that of other members of the Dendrobatidae. However, light microscopic and
electron microscopic examinations revealed distinct differences in the acrosomal
complex, the subacrosomal area and the sperm tail.
In D. auratus the acrosomal complex in Dendrobates auratus is shorter than in other
described dendrobatid species consisting of a thin and narrow acrosomal vesicle and
a subacrosomal cone. Such acrosomal arrangements are present in non-dendrobatid
frogs e.g. Bufo arenarum (BURGOS and FAWCETT 1956), Batrachyla spp.
(GARRIDO et al. 1989), Odontophrynus cultripes (BÁO et al. 1991), Litoria spp. and
Cyclorana spp. (MEYER et al., 1997), Physalaemus spp. (AMARAL et al. 1999) and
Pseudopaludicola falcipes (AMARAL et al. 2000). The nuclear space, a common
Discussion
79
structure between subacrosomal cone and nucleus in all dendrobatid frogs, is
completely missing in D. auratus.
The sperm tail conformation in Dendrobates auratus consisting of a single axoneme
without accessory structures differs from all conditions investigated in the
Dendrobatidae to date, but is seen in Rana and Xenopus spermatozoa
(BERNARDINI et al. 1986; LEE and JAMIESON 1993). Sperm cell tails of the
dendrobatid frogs Ameerega trivittata, Ameerega hahneli (AGUIAR-JR. et al. 2004)
and Ameerega flavopicta (GARDA et al. 2002) exhibit an undulating membrane
connecting an axial fibre with the axoneme. In Allobates femoralis and Colostethus
sp. (AGUIAR-JR. et al. 2003) even two of such complex sperm tails are present.
A diversity of these attributes within the Dendrobatidae can be assumed and should
be analyzed in future projects. In a broad range of amphibian species ultrastructural
sperm studies and other amphibian families together with behavioral observations,
studies on morphological characteristics and molecular methods will be useful to
investigate amphibian phylogeny.
4.3
Concluding remarks
In the Green Poison Frog, Dendrobates auratus, it was demonstrated that hCG
possesses the potential to induce spermiation. A stimulation regime with 100 IU hCG
each at an interval of one hour was most effective to recover high numbers of intact
spermatozoa. The IPS and also the XB medium plus glucose appear to be suited for
the short term storage of sperm cells and for fertilization purposes. A first insight on
spermatological standards (sperm number, motility, irregular formed spermatozoa)
has been established. These data will be useful to estimate the quality of future
sperm samples.
With knowledge that hCG stimulation is also capable to induce spawning in female
individuals of D. auratus (own studies, unpublished), the requirements for assisted
reproduction programs for endangered dendrobatid frogs are given. In addition,
ultrastructural studies expanded the knowledge on the diversity of sperm morphology
within the Dendrobatidae.
80
Discussion
However, further studies in dendrobatid frogs are needed to elucidate the
responsiveness on hormonal stimulation, spermatological parameters and sperm
ultrastructure in order to create adequate protocols for every species in assisted
breeding programs for endangered amphibians.
Summary
81
Chapter 5 Summary
82
Summary
Christian Lipke - Induced spermiation and sperm morphology in the Green Poison
Frog, Dendrobates auratus
In general induced spermiation in amphibians is performed to investigate endocrine
conditions and rarely with the aim of conservation. The operability of hormonal
induced spermiation in dendrobatid frogs using human chorionic gonadotropin (hCG)
as well as spermatological studies including light (LM), fluorescent (FM), and electron
microscopic (EM) investigations remains to be determined.
In the light of worldwide amphibian declines alternative investigations to the studies
exploring causes for the massive losses examinations in the field of reproduction
were performed. In the Green Poison Frog, Dendrobates auratus, two different
spermiation protocols were carried out to characterize differences in the spermiation
response between 5 min and 240 min after injections of hCG into the dorsal lymph
sac. In addition, two sperm media, isotonic phosphate-free amphibian saline (IPS)
and Xenopus fertilization medium (XB) were compared regarding motility of released
sperm cells. The morphology of spermatozoa was described using LM and both
transmission (TEM) and scanning electron microscopy (SEM). A preparation method
for single sperm cells in low concentration for electron microscopic purposes was
established. Sperm membrane integrity was examined by propidium iodide (PI)
staining behavior in fluorescent microscopy and membrane morphology evaluated in
light microscopy. Sperm cells with altered conformation were also quantified and
divided into four groups (1) major rest of cytoplasm drop; (2) immature round-head;
(3) defective head with faulty membrane integrity; (4) defective tail.
A double stimulation of 100 IU hCG each at intervals of one hour was most effective
inducing spermiation in D. auratus (96.9 %) joined with distinct higher total numbers
of sperm compared to single injections of 100 IU hCG. With XB a medium was tested
providing a significantly higher number (P<0.05) and percentage (P<0.001) of motile
spermatozoa coming from cloaca flushings of hormonal stimulated dendrobatid frogs
than the IPS medium. With 79.3 %, the majority of all sperm cells showed a typical
morphology and viability. Recovered typically formed spermatozoa of D. auratus
(total length: 56.1 ± 5.5 µm) consisted of curved, filiform head and a single flagellum
Summary
83
lacking accessory structures. For EM sperm samples were deposited on microscopic
slides and fixated with Karnovsky fixative. Thusly handling and further preparation of
sperm cell samples in low concentration was achieved. Typical structures for
dendrobatid sperm cell as the acrosomal complex consisting of acrosomal vesicle
and subacrosomal cone, nuclear inclusions containing granular material and a short
midpiece showing a mitochondrial collar with a proximal and a distal centriole were
found. Admittedly, with D. auratus the first member of the Dendrobatidae lacking an
undulating membrane attached to the axoneme was described. Results also indicate
a correlation between morphological alterations of the cytoplasm membrane and the
PI staining behavior. Observation concerning the membrane state of spermatozoa
seen in light and fluorescent microscopy conformed. Cells showing numerous bullous
protrusions in the head area in LM were evaluated membrane-defect in PM.
A controlled and gentle method for dendrobatid sperm cell recovery in vivo combined
with spermatological investigations demonstrate the pre-conditions for assisted
amphibian breeding programs as a strategy to confront the worldwide amphibian
declines. Possible applications within these projects are assisted mating, artificial
fertilization and preservation of gametes and embryos. Implementation of induced
spermiation in veterinary practice may be useful for sexing in species lacking external
sex dimorphism or evaluating male fertility. The finding of a unique sperm tail
conformation for dendrobatid frogs suggest not only a diversity of this feature within
the Dendrobatidae but may also contribute together with behavioral observations,
studies on morphological characteristics and molecular methods to understanding of
amphibian phylogeny.
84
Summary
Zusammenfassung
85
Chapter 6 Zusammenfassung
Zusammenfassung
86
Christian Lipke - Induzierte Spermiation und Spermienmorphologie bei dem
Goldbaumsteigerfrosch, Dendrobates auratus
Die induzierte Spermiation wird bei Amphibien fast ausschließlich durchgeführt, um
endokrine Zusammenhänge zu untersuchen, nur selten steht der Artenschutz im
Vordergrund.
Ein
Nachweis
der
Anwendbarkeit
Spermiation
bei
Baumsteigerfröschen
nach
der
hormonell
Applikation
von
induzierten
humanem
Choriongonadotropin (hCG) sowie spermatologische Untersuchungen einschließlich
lichtmikroskopischer, fluoreszenzmikroskopischer und elektronenmikroskopischer
Studien stehen noch aus.
Vor dem Hintergrund des weltweiten Amphibiensterbens wurden zu den Arbeiten,
welche
Gründe
der
massiven
Verluste
zu
klären
versuchten,
alternative
reproduktionsbiologische Studien durchgeführt. Es wurden Unterschiede bei der
induzierten Spermienabgabe des Goldbaumsteigerfrosches, Dendrobates auratus,
zwischen 5 min und 240 min nach der Injektion von hCG in den dorsalen Lymphsack
charakterisiert. Darüber hinaus wurden zwei Medien, eine isotonische phosphatfreie
Kochsalzlösung sowie ein „Xenopus fertilization“ Medium (XB) hinsichtlich ihrer
Fähigkeit, die Motilität abgegebener Spermien zu gewährleisten, untersucht. Die
Beschreibung
der
Spermienmorphologie
erfolgte
mit
licht-
sowie
transmissionselektronen- und rasterelektronenmikroskopischen Verfahren. Dazu
wurde eine Methode zur elektronenmikroskopischen Probenaufarbeitung für niedrig
konzentrierte Einzel-Zellen etabliert. Die Membranintegrität der Spermatozoen wurde
anhand des Färbeverhaltens mit Propidiumjodid (PI) sowie der Morphologie der
äußeren Zellmembran im Lichtmikroskop untersucht. Neben typisch geformten Zellen
wurden auch pathologische Spermienformen quantifiziert und in vier Gruppen
eingeteilt: (1) großer Zytoplasmatropfen; (2) unreife Rundzelle; (3) Kopfdefekt mit
Schädigung der Zytoplasmamembran; (4) Schwanzdefekt.
Die effektivste Gewinnung von Spermien bei D. auratus (96,9 %) konnte nach
doppelter Stimulation mit jeweils 100 IE hCG im Abstand von einer Stunde erreicht
werden. Unter Verwendung des XB Mediums wurden signifikant höhere absolute
(P<0,05) und relative (P<0,001) Anzahlen motiler Spermien aus den Kloaken
Zusammenfassung
87
hormonell behandelter Frösche gewonnen als unter Verwendung des IPS Mediums.
Die Mehrzahl der Spermatozoen wies eine typische Morphologie und Viabilität auf
(79,3 %). Diese Zellen (Gesamtlänge: 56,1 ± 5,5 µm) bestehen aus einem
gebogenen, filiformen Kopfstück sowie einem einzelnen Spermienschwanz ohne
akzessorische Strukturen. Für die ultrastrukturellen Untersuchungen wurden die
Spermienproben auf Objektträger gegeben und mit Karnovksy-Lösung fixiert. Auf
diese
Weise
konnte
eine
praktische
Handhabung
für
die
weiteren
Vorbereitungsschritte der Elektronenmikroskopie von Einzelzell-Proben in nur
geringer Konzentration erreicht werden. Bei den ultrastrukturellen Studien wurden die
für Spermien der Dendrobaten typischen Strukturen (akrosomaler Komplex
bestehend aus akrosomalem Vesikel und subakrosomalem Konus, nukleäre
Inklusionen aus granulärem Material, kurzes Mittelstück mit proximaler und distaler
Zentriole) gefunden. Allerdings konnte mit D. auratus der erste Vertreter der
Baumsteigerfrösche beschrieben werden, dessen Spermien-Flagellae lediglich aus
einem Axonem ohne undulierende Membran bestehen. Die Ergebnisse deuten
außerdem auf eine Korrelation zwischen morphologischen Veränderungen der
äußeren Zellmembran gewonnener Spermatozoen und dem PI Färbeverhalten hin.
Zellen, die lichtmikroskopisch zahlreiche blasige Auftreibungen im Kopfbereich
besaßen, konnten mit Hilfe der PI Färbung als membrandefekt identifiziert werden.
Die kontrollierte und schonende Spermiengewinnung beim Baumsteigerfrosch in vivo
bietet, verbunden mit der Möglichkeit spermatologischer Untersuchungen, die
Voraussetzungen für assistierte Zuchtprogramme, mit denen auf das weltweite
Amphibiensterben reagiert werden kann. Assistierte Paarung, künstliche Befruchtung
und Konservierung von Gameten und Embryonen stellen mögliche Anwendungen
innerhalb solcher Projekte dar. Die Durchführung der induzierten Spermiation im
Rahmen der tierärztlichen Praxis kann sich bei der Geschlechtsbestimmung solcher
Amphibienarten als sinnvoll herausstellen, die keinen äußerlich erkennbaren
Geschlechtsdimorphismus aufweisen. Eine genauere Diagnostik der männlichen
Infertilität wird ebenfalls ermöglicht. Die Entdeckung einer bislang einzigartigen
Struktur des Spermienschwanzes bei einem Baumsteigerfrosch weist nicht nur auf
eine Heterogenität dieses Merkmals auf Familienniveau hin, sondern kann
88
Zusammenfassung
gemeinsam mit Verhaltensbeobachtungen, Studien zur äußeren Morphologie und
molekularbiologischen Methoden dazu beitragen, die Phylogenie der Amphibien zu
klären.
References
89
Chapter 7 References
References
90
AGUIAR-JR., O., A.A. GARDA, A.P. LIMA, G.R. COLLI, S.N. BÁO and S.M. RECCOPIMENTEL (2003):
Biflagellate spermatozoon of the poison-dart frogs Epipedobates femoralis and
Colostethus sp. (Anura, Dendrobatidae).
J. Morphol. 255, 114-121
AGUIAR-JR., O., A.P. LIMA, S.N. BÁO and S.M. RECCO-PIMENTEL (2004):
Sperm ultrastructure of the Brazilian Amazon poison frogs Epipedobates trivittatus
and Epipedobates hahneli (Anura, Dendrobatidae).
Acta Zool. 85, 21-28
ALFORD, R.A., P.M. DIXON and J.H.K. PECHMANN (2001):
Global amphibian population declines.
Nature 414, 449-500
ALFORD, R.A. and S.J. RICHARDS (1999):
Global amphibian declines: a problem in applied ecology.
Annu. Rev. Ecol. Syst. 30, 133-165
AMARAL, M.J.L.V., A.P. FERNANDES, S.N. BÁO and S.M. RECCO-PIMENTEL
(1999):
An ultrastructural study of spermiogenesis in three species of Physalaemus (Anura,
Leptodactylidae).
Biocell 23, 211-221
AMARAL, M.J.L.V., A.P. FERNANDES, S.N. BÁO and S.M. RECCO-PIMENTEL
(2000):
The
ultrastructure
of
spermatozoa
Leptodactylidae).
Amphibia-Reptilia 21, 498-502.
in
Pseudopaludicola
falcipes
(Anura,
References
91
ANKLEY, G.T., S.A. DIAMOND, J.E. TIETGE, G.W. HOLCOMB, K.M. JENSEN, D.L.
DEFOE and R. PETERSON (2002):
Assessment of the risk of solar ultraviolet radiation to amphibians. 1. Dosedependent induction of hindlimb malformations in the northern leopard frog.
Environ. Toxicol. Chem. 17, 2530-2542
BÁO, S.N., G.C. DALTON and S.F. OLIVEIRA (1991):
Spermiogenesis in Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae):
Ultrastructural and cytochemical studies of proteins using E-PTA.
J. Morphol. 297, 303-314
BARTON, H.L. and S.L. GUTTMAN (1972):
Low temperature preservation of toad spermatozoa (Genus Bufo).
Texan. J. Sci. 23, 363-370
BELDEN, L.K., E.L. WILDY and A.R. BLAUSTEIN (2000):
Growth, survival, and behaviour of larval longtoed salamanders (Ambystoma
macrodactylum) exposed to ambient levels of UV-B radiation.
J. Zool. London 251, 473-479
BERGER L., R. SPEARE, P. DASZAK, D.E. GREEN, A.A. CUNNINGHAM, C.L.
GOGGIN, R. SLOCOMBE, M.A. RAGAN, A.D. HYATT, K.R. MCDONALD, H.B.
HINES, K.R. LIPS, G. MARANTELLI and H. PARKES (1998):
Chytridiomycosis causes amphibian mortality associated with population declines in
the rain forests of Australia and Central America.
Proc. Natl. Acad. Sci. USA 95(15), 9031-9036
BERNARDINI, G., R. STIPANI and G. MELONE (1986):
The ultrastructure of Xenopus spermatozoon.
J. Ultrastruct. Mol. Struct. Res. 94, 188-194
92
References
BIRKHAHN, H., V. KÜLPMANN and K. WASSMANN (1994):
Zur Variabilität des Goldbaumsteigers.
Aquar. Terrar. Zeitschr. 47 (9), 570-576
BLAUSTEIN, A.R., L.K. BELDEN, A.C. HATCH, L.B. KATS, P.D. HOFFMAN, J.B.
HAYS, A. MARCO, D.P. CHIVERS and J.M. KIESECKER (2001):
Ultraviolet radiation and amphibians. In: C.S. Cockell, A.R Blaustein, (Eds.),
Ecosystems, evolution and ultraviolet radiation.
Springer, New York, USA, pp. 63-79
BLAUSTEIN, A.R. and J.M. KIESECKER (2002):
Complexity in conservation: lessons from global decline of amphibian populations.
Ecol. Letters 5, 597-608
BLAUSTEIN, A.R., J.M. KIESECKER, D.P. CHIVERS, D.G. HOKIT, A. MARCO, L.K.
BELDEN and A. HATCH (1998):
Effects of ultraviolet radiation on amphibians: field experiments.
Am. Zool. 38, 799-812
BLAUSTEIN, A.R., J.M. ROMANSIC, J.M. KIESECKER and A.C. HATCH (2003):
Ultraviolet radiation, toxic chemicals and amphibian declines.
Div. Distrib. 9, 123-140
BLAUSTEIN, A.R., D.B. WAKE and W.P. SOUSA (1994):
Amphibian declines: Judging stability, persistence, and susceptibility of populations
to local and global extinctions.
Conserv. Biol. 8, 60-71
BOGART, J.P. (1991):
The influence of life history on karyotypic evolution in frogs. In: Green, D.M.,
Sessions, S.K., (Eds.), Amphibian cytogenetics and evolution.
References
93
Academic Press, San Diego, USA, pp. 233-255
BOLÍVAR, W., F. CASTRO, and S. LÖTTERS (2004):
Dendrobates lehmanni. In: IUCN 2007. 2007 IUCN Red List of Threatened Species.
<www.iucnredlist.org>. Downloaded on 14 May 2008.
BOONE, M.D. and C.M. BRIDGES (2003):
The problem of pesticides: implications for amphibian populations. In: Semlitsch,
R.D. (ed.), Amphibian conservation.
Smithsonian Institution Press, Washington, D.C., USA
BRADFORD, D.F. (1991):
Mass mortality and extinction in a high-elevation population of Rana muscosa.
J. Herpetol. 25, 174-177
BRADFORD, D.F., D.M. GRABER and F. TABATABAI (1994):
Population declines of the native frog, Rana muscosa, in Sequoia and Kings Canyon
National Parks, California.
Southwestern Naturalist 39, 323-327
BRADFORD, D.F., F. TABATABAI and D.M. GRABER (1993):
Isolation of remaining populations of the native frog, Rana muscosa, by introduced
fishes in Sequoia and Kings Canyon National Parks, California.
Conserv. Biol. 7, 882–888
BRIGGS, R. and T.J. KING (1952):
Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs.
Zoology 38, 455-463
BRIGGS, C.J, V.T. VREDENBURG, R.A. KNAPP and L.J. RACHOWICZ (2005):
94
References
Investigating the population-level effects of chytridiomycosis, an emerging infectious
disease of amphibians.
Ecology 86, 3149-3159
BRÖNMARK, C. and P. EDENHAMN (1994):
Does the presence of fish affect the distribution of tree frogs (Hyla arborea)?
Conserv. Biol. 8, 841-845
BROWNE, R.K., J. CLULOW and M. MAHONY (2001):
Short-term storage of cane toad (Bufo marinus) gametes.
Reproduction 121, 167-173
BROWNE, R.K., J. CLULOW and M. MAHONY (2002a):
The short-term storage and cryopreservation of spermatozoa from hylid and
myobatrachid frogs.
Cryo Letters 23, 129-136
BROWNE, R.K., J. CLULOW, M. MAHONY and A. CLARK (1998):
Successful recovery of motility and fertility of cryopreserved cane toad (Bufo
marinus) sperm.
Cryobiology 37(4), 339-345.
BROWNE, R.K., J. DAVIS, M. POMERING and J. CLULOW (2002b):
Storage of cane toad (Bufo marinus) sperm for 6 days at 0°C with subsequent
cryopreservation.
Reprod. Fertil. Dev. 14, 267-273
BROWNE, R.K., J. SERATT, C. VANCE and A. KOUBA (2006):
Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered
Wyoming toad (Bufo baxteri).
Reprod. Biol. Endocrinol. 4(34): 1-11
References
95
BRUN, R. (1975):
Oocyte maturation in vitro: Contribution of the oviduct to total maturation in Xenopus
laevis.
Experimentia 31, 1275-1276
BURGOS, M.H. and D.W. FAWCETT (1956):
An electron microscope study of spermatid differentiation in the toad Bufo arenum
Hensell.
J. Biophys. Biochem. Cytol. 2, 223-240
BURGOS, M.H. and A.J. LADMAN (1955):
Effect of purified gonadotropins upon release of spermatozoa in the frog, Rana
pipiens.
Proc. Soc. Exp. Biol. Med. 88(3), 484-487
BURGOS, M.H. and R. VITALE-CALPE (1967):
The mechanism of spermiation in the toad.
Am. J. Anat. 120, 227-252
CAMPANELLA C., R. CAROTENUTO, V. INFANTE, G. MATURI and U. ATRIPALDI
(1997):
Sperm-egg interaction in the painted frog (Discoglossus pictus): an ultrastructural
study.
Mol. Reprod. Dev. 47(3), 323-333
CAREY, C. and M.A. ALEXANDER (2003):
Climate change and amphibian declines: is there a link?
Div. Distr. 9(2), 111-121
CAREY, C., D.F. BRADFORD, J.L. BRUNER, J.P.COLLINS, E.W. DAVIDSON, J.E.
LONGCORE, M. OUELLET, A.P. PESSIER and D.M. SCHOCK (2003):
96
References
Biotic factors in amphibian population declines. In: C. Linder, S.K. Krest, and D.W.
Sparling (eds.). Amphibian decline: An integrated analysis of multiple stressor
effects.
SETAC Press, Boca Raton, Florida, USA
CHATTERJEE, A., S. BHADURI and A.K. BASUMALLIK (1971):
The ability of hCG in the induction of spermiation in toads devoid of an endogenous
source of gonadotropin.
Endokrinologie, 58(1), 119-120
CHEN, T.H., J.A. GROSS and W.H. KARASOV (2006):
Sublethal effects of lead on northern leopard frog (Rana pipiens) tadpoles.
Environ. Toxicol. Chem. 25(5), 1383-1389
CLOUGH, M. and K. SUMMERS (2000):
Phylogenetic systematics and biogeography of the poison frogs: evidence from
mitochondrial DNA sequences.
Biol. J. Linnean Soc. 70, 515-540
COCKELL, C.S and A.R. BLAUSTEIN (2000):
Ultraviolet spring and the ecological consequences of catastrophic impacts.
Ecology Letters 3, 77-81
COCKELL, C.S and A.R. BLAUSTEIN (2001):
Ecosystems, evolution and ultraviolet radiation.
Springer, New York, USA
COLLINS, J.P., N. COHEN, E.W. DAVIDSON, J.E. LONGCORE and A. STORFER
(2003):
References
97
Global amphibian declines: an interdisciplinary research challenge for the 21st
century. In: M.J. Lannoo (ed.). Status and conservation of U.S. amphibians, Vol. 1.
Conservation essays.
University of California Press, Berkeley, USA, pp. 43-52
COLLINS, J.P., T.R. JONES and H.A. BERNA (1988):
Conserving genetically distinctive populations: the case of the Huachua tiger
salamander (Ambystoma tigrinum stebbinsi). In: R.C. Szaro, K.C. Severson, and
D.R. Patton (eds.). Management of amphibians, reptiles, and small mammals in
North America.
USDA Forest Service GTR-RM-166, Rocky Mountain Forest and Range Experiment
Station, Fort Collins, USA, pp. 45-53
COLLINS, J.P. and A. STORFER (2003):
Global amphibian declines: sorting the hypotheses.
Divers. Distrib. 9, 89-98
COLOMA, L.A. and S. Ron (2004):
Dendrobates abditus. In: IUCN 2007. 2007 IUCN Red List of Threatened Species.
<www.iucnredlist.org> Downloaded on 14 May 2008.
CORY, L. (1963):
Effects of introduced trout on the evolution of native frogs in the high Sierra Nevada
Mountains.
Proc. Int. Congress Zool. 16, 172
DALY, J. W., S. I. SECUNDA, H. M. GARRAFTO, T. F. SPANDE, A. WISNIEWSKI,
C. NISHIHIRA and J. F. COVER JR. (1992):
Variability in alkaloid profiles in neotropical Poison Frogs (Dendrobatidae): Genetic
versus environmental determinants.
Toxicon 30 (8), 887-898
98
References
DASZAK, P., A.A. CUNNINGHAM and A.D HYATT (2000):
Emerging infectious diseases of wildlife-threats to biodiversity and human health.
Science 287, 443-449
DASZAK, P., A.A. CUNNINGHAM and A.D HYATT (2003):
Infectious disease and amphibian population declines.
Div. Distrib. 9(2), 141-150
DAVIDSON, C., H.B. SHAFFER and M.R. JENNINGS (2001):
Declines of the California red-legged frog: climate, UV-B, habitat, and pesticides
hypotheses.
Ecol. Appl. 11, 464-479
DEL PINO, E.M. (1973):
Interactions between gametes and environment in the toad Xenopus laevis (Daudin)
and their relationship to fertilization.
J. Exp. Zool. 185, 121-132
DE SÁ, R.O. and D.M. HILLIS (1990):
Phylogenetic relationships of the pipid frogs Xenopus and Silurana: An integration of
ribosomal DNA and morphology.
Mol. Biol. Evol. 7, 365-376
DIBERARDINO , A. (1962):
The karyotype of Rana pipiens and investigation of its stability during embryonic
differentiation.
Dev. Biol. 5, 101-126
DIBERARDINO, M.A., N. HOFFNER ORR and R.G. MCKINNELL (1986):
Feeding tadpoles cloned from Rana erythrocyte nuclei.
Proc. Natl. Acad. Sci. USA 83(21), 8231-8234
References
99
DICKERSON, K. (1999):
Pesticides and the Wyoming toad. In: Bender, M., Balis-Larsen, M. (Eds.), U.S. Fish
& Wildlife Service Endangered Species Bulletin, vol. 24
USFWS, Washington, USA, pp. 20-21
DUELLMAN, W.E. and L. TRUEB (1986):
Biology of amphibians.
McGraw-Hill, New York, USA
DUNN, E. R. (1941):
Notes on Dendrobates auratus.
Copeia 2, 88-93
EASLEY, K.A., D.D. CULLEY JR., N.D. HORSEMAN and J.E. PENKALA (1979):
Environmental influences on hormonally induced spermiation of the bullfrog, Rana
catesbeiana.
J. Exp. Zool. 207, 407-416
EATON, T. H. Jr. (1941):
Notes on the life history of Dendrobates auratus.
Copeia 2, 93-95
EDWARDS, D.L., M.J. MAHONY and J. CLULOW (2004):
Effects of sperm concentration, medium osmolality and oocyte storage on artificial
fertilisation success in a myobatrachid frog, Limnodynastes tasmaniensis.
Reprod. Fertil. Dev. 16, 347-354
ELINSON, R.P. (1973):
Fertilization of frog body cavity eggs enhanced by treatments affecting the vitelline
coat.
J. Exp. Zool. 183, 291-302
100
References
FERNANDEZ, M. and J. L´HARIDON (1992):
Influence of lighting conditions on toxicity and genotoxicity of various PAH in the newt
in vitro.
Mutation Research 298, 31-41
FERNANDEZ, M. and J. L´HARIDON (1994):
Effects of light on the cytotoxicity and genotoxicity of benzo(a)pyrene and an oil
refinery effluent in the newt.
Environ. Mol. Mutagen 24, 124-136
FILHO, O.P.R., S.L. LIMA, D.R. DE ANDRADE and J.T. DE SEIXAS FILHO (1998):
Study of Bull-Frog spawning, Rana catesbeiana, using induced reproduction.
R. Bras. Zootec 27(2), 216-223
FITE, K.V., A.R. BLAUSTEIN, L. BENGSTON and H.E. HEWITT (1998):
Evidence of retinal light damage in Rana cascadae: a declining amphibian species.
Copeia 1998, 906-914
FORD, L.S. (1993):
The phylogenetic position of the dart-poison frogs (Dendrobatidae) among anurans An examination of the competing hypotheses and their characters.
Ethol. Ecol. Evol. 5, 219-231
FORD, L.S. and D.C. CANNATELLA (1993):
The major clades of frogs.
Herpetol. Monogr. 7, 94-117
FORTUNE, J.E., P.W. CONCANNON, W. HANSEL (1975):
Ovarian progesterone levels during in vitro oocyte maturation and ovulation in
Xenopus laevis.
Biol. Reprod. 13, 561-567
References
101
FREDA, J. (1991):
The effects of aluminum and other metals on amphibians.
Environ. Pollut. 71, 305-328.
FREDA, J., V. CAVDEK and D. MCDONALDS (1990):
Role of organic complexation in the toxicity of aluminium to Rana pipiens embryos
and Bufo americanus tadpoles.
Can. J. Fisheries Aquatic Sci. 47, 217-224
FROST, D.R. (2007):
Amphibian species of the world: an online reference. Version 5.1 (10 October, 2007).
Electronic database accessible at
http://research.amnh.org/herpetology/amphibia/index.php.
Am. Mus. Nat. Hist., New York, USA.
GAMRADT, S.C. and L.B. KATS (1996):
Effect of introduced crayfish and mosquitofish on California newts.
Conserv. Biol. 10, 1155-1162
GALLANT, A.L., R.W. KLAVER, G.S. CASPER and M.J. LANNOO (2007):
Global rates of habitat loss and implications for amphibian conservation.
Copeia 4, 967-979
GALLI-MAININI, C. (1947):
Pregnancy test using the male toad.
J. Clin. Endocrinol. 7, 653-658
GARDA, A.A., G.R. COLLI, O., AGUIAR-JR., S.M. RECCO-PIMENTEL and S.N.
BÁO (2002):
The ultrastructure of the spermatozoa of Epipedobates flavopictus (Amphibia, Anura,
Dendrobatidae), with comments on its evolutionary significance.
102
References
Tissue Cell 34, 356-364
GARRIDO, O., E. PUGIN and B. JORQUERA (1989):
Sperm morphology of Batrachyla (Anura: Leptodactylidae).
Amphibia-Reptilia 10, 141-149
GILLESPIE, G.R. (2001):
The role of introduced trout in the decline of the spotted tree frog (Litoria spenceri) in
southeastern Australia.
Biol. Conserv. 100, 187-198
GIRARD, C. (1855):
Abstract of a report to Lieut. J.M. Gillis, U.S.N., upon the reptiles collected during
U.S.N. astronomical expedition to Chili.
Proc. Acad. Nat. Sci. Phil 7, 226-227.
GOODSELL, J.A. and L.B. KATS (1999):
Effect of introduced mosquitofish on Pacific treefrogs and the role of alternative prey.
Conserv. Biol. 13, 921-924
GRANT, T., D.R. FROST, J.P. CALDWELL, R. GAGLIARDO, C.F.B. HADDAD,
P.J.R. KOK, D.B. MEANS, B.P. NOONAN, W.E. SCHARGEL and W.C. WHEELER
(2006):
Phylogenetic systematics of dart-poison frogs and their relatives (Amphibia:
Athesphatanura: Dendrobatidae).
Bull. Am. Mus. Nat. Hist. 299, 1-262
GURDON, J.B. and J.A. BYRNE (2003):
The first half-century of nuclear transplantation.
Proc. Natl. Acad. Sci. USA 100(14), 8048-8052
References
103
GURDON, J.B. and V. UEHLINGER (1966):
"Fertile" intestine nuclei.
Nature 210, 1240-1241
HALL, R.J. and B.M. MULHERN (1984):
Are anuran amphibians heavy metal accumulators? In: Seigel, R.A., Hunt, L.E.,
Knight, J.L., Malaret, L.,Zuschlag, N.L. (Eds.), Vertebrate Ecology and Systematics A Tribute to Henry S. Fitch
Mus. Nat. Hist. University of Kansas, Lawrence, pp. 123-133
HANSEN, K.L. and D.E. SWEAT (1962):
Spermiation in Rana P. pipiens in response to heteroplasmatic pituitary materials.
Quart. J. Fla. Acad. Sci., 25(2), 109-120
HATCH, A.C., L.K. BELDEN, E. SCHEESSELE and A.R. BLAUSTEIN (2001):
Juvenile amphibians do not avoid potential lethal levels of urea on soil substrate.
Environ. Toxicol. Chem. 20, 2328-2335
HAY, J.M., I. RUVINSKY, S.B. HEDGES and L.R. MAXSON (1995):
Phylogenetic relationships of amphibian families inferred from DNA sequences of
mitochondrial 12S and 16S ribosomal RNA genes.
Mol. Biol. Evol. 12, 928-937
HAYES, T.B. (1998):
Sex determination and primary sex differentiation in amphibians: Genetic and
developmental mechanism.
J. Exp. Zool. 281, 373-399.
HAYES, T.B. (2000):
Endocrine disruption in amphibians. In: Sparling, D.W., G. Linder, and C. Bishop,
(Eds.), Ecotoxicolology of amphibians and reptiles.
104
References
SETAC Press, Pensacola, USA. Pp. 573-593
HAYES, T.B. (2005):
Welcome to the revolution: integrative biology and assessing the impact of endocrine
disruptors on environmental and public health.
J. Integr. Comp. Biol. 45, 321-329
HAYES, T.B., P. CASE, S. CHUI, D. CHUNG, C. HAEFFELE, K. HASTON, M. LEE,
V.P. MAI, Y. MARJUOA, J. PARKER and M. TSUI (2006):
Pesticide mixtures, endocrine disruption, and amphibian declines: are we
underestimating the impact?
Environ. Health Perspect. 114(Suppl. 1), 40-50
HAYES, T.B., A. COLLINS, M. LEE, M. MENDOZA, N. NORIEGA, A.A. STUART and
A. VONK (2002):
Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low
ecologically relevant doses.
Proc. National Acad. Sci. USA 99, 5476-5480
HAYES, T.B., K. HASTON, M. TSUI, A. HOANG, C. HAEFFELE and A. VONK
(2002a):
Atrazine-induced hermaphroditism at 0.1 ppb in American leopard frogs (Rana
pipiens): laboratory and field evidence.
Environ. Health Perspect. 111, 568-575
HAYES, T.B., K. HASTON, M. TSUI, A. HOANG, C. HAEFFELE and A. VONK
(2002b):
Feminization of male frogs in the wild.
Nature 419, 895-896
References
105
HAYS, J.B., A.R. BLAUSTEIN, J.M. KIESECKER, P.D. HOFFMAN, I. PANDELOVA,
A. COYLE and T. RICHARDSSON (1996):
Developmental responses of amphibians to solar and artificial UV-B sources: a
comparative study.
Photochem. Photobiol. 64, 449-456
HEASMAN, J., S. HOLWILL and C.C. WYLIE (1991):
Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited
molecules. In: Kay, B., Peng, K., Benjamin, H., (Eds.), Methods in cell biology, vol.
36, Xenopus laevis: Practical uses in cell and molecular biology.
Academic Press, New York, USA. pp. 213-229
HELFRICH, L.A, R.J. NEVES and J. PARKHURST (2001):
Commercial frog farming.
Virginia Cooperative Extension, Publication 420-255
HILLIS, D.M., L.K. AMMERMAN, M.T. DIXON and O. DE SÁO (1993):
Ribosomal DNA and the phylogeny of frogs.
Herpetol. Monogr. 7, 118-131
HOLLINGER, T.G. and G.L. CORTON (1980):
Artificial fertilization of gametes from the South African clawed frog, Xenopus laevis.
Gamete Res. 3, 45-57
HOULAHAN, J.E., C.S. FINDLEY, B.R. SCHMIDY, A.J. MEYER and S.L. KUZMIN
(2000):
Quantitative evidence for global amphibian declines.
Nature 404, 752-755
IIMORI, E., M.J. D’OCCHIO, A.T. LISLE and S.D. JOHNSTON (2005):
106
References
Testosterone secretion and pharmacological spermatozoal recovery in the cane toad
(Bufo marinus).
Anim. Reprod. Sci. 90, 163-173
INGER, R.F. (1967):
The development of a phylogeny of frogs.
Evolution 21, 369-384.
ITURRA, P. and A. VELOSO (1981):
Evidence for heteromorphic sex chromosomes in male amphibians (Anura:
Leptodactylidae).
Cytogenet. Cell. Genet. 31, 108-110
IUCN (2007):
2007 IUCN Red List of Threatened Species. <www.iucnredlist.org>. Downloaded on
21 December 2007.
JAFFE, L.A., R.T. KADO and L. MUNCY (1985):
Propagating potassium and chloride conductances during activation and fertilization
of the egg of the frog, Rana pipiens.
J. Physiol. 368, 227-242
JAMIESON, B.G.M. (1999):
Spermatozoal phylogeny of the vertebrata. In: Gagnon, C., (Ed.), The male gamete:
From basic science to clinical applications.
Cache River Press, Vienna, USA. pp. 303-331
JANCOVICH J.K.,E.W. DAVIDSON, N. PARAMESWARAN, J. MAO, V.G.
CHINCHAR, J.P. COLLINS, B.L. JACOBS and A. STORFER (2005):
Evidence for emergence of an amphibian iridoviral disease because of humanenhanced spread.
References
107
Mol. Ecol. 14(1), 213-224
JOHNSON, P.T.J., J.M. CHASE, K.L. DOSCH, R.B. HARTSON, J.A. GROSS, D.J.
LARSON, D.R. SUTHERLAND and S.R. CARPENTER (2007):
Aquatic eutrophication promotes pathogenic infection in amphibians.
Proc. Nat. Acad. Sci. U.S.A. 104(40), 15781-15786
KAMIMURA M., K. IKENISHI, M. KOTANI and T. MATSUNO (1976):
Observations on the migration and proliferation of gonocytes in Xenopus laevis.
J. Embryol. Exp. Morphol. 36, 197-207
KATAGIRI, C. (1974):
A high frequency of fertilization in premature and mature coelomic toad eggs after
enzymic removal of vitelline membrane.
J. Embryol. Exp. Morphol. 31, 573-587
KATS, L.B., J.M. KIESECKER, D.P. CHIVERS and A.R. BLAUSTEIN (2000):
Effects of UV-B radiation on antipredator behavior in three species of amphibians.
Ethology 106, 921-932
KISHIGAMI, S., E. MIZUTANI, H. OHTA, T. HIKICHI, N. VAN THUAN, S.
WAKAYAMA, H. BUI and T. TERUHIKO (2006):
Significant improvement of mouse cloning technique by treatment with trichostatin A
after somatic nuclear transfer.
Biochem. Biophys. Res. Commun. 340(1), 183-189
KNAPP, R.A. (1996): Nonnative trout in natural lakes of the Sierra Nevada: An
analysis of their distribution and impacts on native aquatic biota.
Sierra Nevada ecosystem project: final report to Congress. University of California,
USA. Vol. III, pp. 363-407
108
References
KNAPP, R.A., D.M. BOIANO and V.T. VREDENBURG (2007):
Removal of nonnative fish results in population expansion of a declining amphibian
(mountain yellow-legged frog, Rana muscosa)
Biol. Conserv. 135(1), 11–20
KNUTSON, M.G, W.B. RICHARDSON, D.M. REINEKE, B.R. GRAY, J.R.
PARMELEE and S.E. WEICK (2004):
Agricultural ponds support amphibian populations.
Ecol. Appl. 14(3), 669-684
KUES, W.A. and H. NIEMANN (2004):
The contribution of farm animals to human health.
Trends Biotechnol. 22, 286-294
KWON, H.B., K.J. CHANG, Y.R. YOO, C.C. LEE and A.W. SCHUETZ (1992):
Induction of ovulation and oocyte maturation of Amphibian (Rana dybowskii) ovarian
follicles by protein kinase C activation in vitro.
Biol. Reprod. 47, 169-176
LAHNSTEINER, F., T. WEISMANN and R.A. PATZNER (1997):
Aging processes of rainbow trout semen during storage.
The Progressive Fish-culturist 59, 272-279
LA MARCA, E., K.R. LIPS, S. LÖTTERS, R. PUSCHENDORF, R. IBAÑEZ and S.
RON (2005):
Catastrophic population declines and extinctions in neotropical harlequin frogs
(Bufonidae: Atelopus).
Biotropica 37, 190-201
LA MARCA, E. and C. SEÑARIS (2004):
References
109
Dendrobates steyermarki. In: IUCN 2007. 2007 IUCN Red List of Threatened
Species. <www.iucnredlist.org>. Downloaded on 14 May 2008.
LANGE, J. (1981):
Beitrag zur Zucht von Smaragd-Baumsteigerfröschen (Dendrobates auratus).
Z. Kölner Zoo 24(1), 6-8
LANNOO, M.J., K. LANG, T. WALTZ and G.S. PHILLIPS (1994):
An altered amphibian assemblage: Dickinson County, Iowa, 70 years after Frank
Blanchard’s survey.
Am. Midland Naturalist 131, 311-319
LEE, M.S.Y. and B.G.M. JAMIESON (1993):
The ultrastructure of the spermatozoa of bufonid and hylid frogs (Anura, Amphibia) Implications for phylogeny and fertilization biology.
Zool. Scripta 22, 309-323
LEFCORT, H., R.A. MEGUIRE, L.H. WILSON and W.F. ETTINGER (1998):
Heavy metals alter the survival, growth, metamorphosis, and antipredatory behavior
of Columbia spotted frog (Rana luteiventris) tadpoles.
Arch. Environ. Contam. Toxicol. 35, 447-456
LICHT, P. (1973):
Induction of spermiation in anurans by mammalian pituitary gonadotropins and their
subunits.
Gen. Comp. Endocrinol. 20, 2-529
LIPS, K.R, F. BREM, R. BRENES, J.D. REEVE, R.A. ALFORD, J. VOYLES, C.
CAREY, L. LIVO, A.P. PESSIER and J.P. COLLINS (2006):
Emerging infectious disease and the loss of biodiversity in a neotropical amphibian
community.
110
References
PNAS 103(9), 3165-3170
LUTZ, I. and W. KLOAS (1999):
Amphibians as a model to study endocrine disruptors: I. Environmental pollution and
estrogen receptor binding.
Sci. Total Environ. 255, 49-57
LUYET, B.J. and E.L. HODAPP (1938):
Revival of frog’s spermatozoa vitrified in liquid air.
Proc. Soc. Exp. Biol. Med. 39, 433-434
LYNCH, J. (1971):
Evolutionary relationships, osteology and zoogeography of leptodactyloid frogs.
Univ. Kansas Mus. Nat. Hist. Misc. Pub., Lawrence, USA, 53, pp. 1-238.
LYNCH, J. (1973):
The transition from archaic to advanced frogs. In: Vial, J.L., (Ed.), Evolutionary
biology of the anurans: contemporary research on major problems.
Univ. Missouri Press, Columbia, USA, pp. 133-182.
MARCO, A., C. QUILCHANO and A.R. BLAUSTEIN (1999):
Sensitivity to nitrate and nitrite in pond-breeding amphibians from the Pacific
Northwest.
Environ. Toxicol. Chem. 18, 2836-2839
MASUTOMI N., M. SHIBUTANI, H. TAKAGI, C. UNEYAMA, K.Y. LEE and M.
HIROSE (2004):
Alteration of pituitary hormone-immunoreactive cell populations in rat offspring after
maternal dietary exposure to endocrine-active chemicals.
Arch. Toxicol. 78, 232-240
References
111
MATSUBA, C. and J. MERILÄ (2006):
Genome size variation in the common frog Rana temporaria.
Hereditas 143, 157-160
MATTHEWS, K.R., K.L. POPE, H.K. PREISLER and R.A. KNAPP (2001):
Effects of nonnative trout on Pacific treefrogs (Hyla regilla) in the Sierra Nevada.
Copeia 1130-1137
MCCREERY, B.R. and P. LICHT (1983):
Induced ovulation and changes in pituitary responsiveness to continuous infusion of
gonadotropin-releasing hormone during the ovarian cycle in the Bullfrog, Rana
catesbeiana.
Biol. Reprod. 29, 863-871
MCKINNELL, R.G. (1962):
Intraspecific nuclear transplantation in frogs.
J. Hered. 53, 199-207
MEYER, E., B.G.M. JAMIESON and D.M. SCHELTINGA (1997):
Sperm ultrastructure of six Australian hylid frogs from two genera (Litoria and
Cyclorana): phylogenetic implications.
J. Submicrosc. Cytol. Pathol. 4, 443-451
MEYER, R.K., J. LEONORA and W.H. MCSHAN (1961):
Effectiveness of sheep FSH and LH preparations on spermiation in the frog. In:
Albert A, editor. Human pituitary gonadotropins.
Thomas, Springfield, Illinois. pp. 326-328
MEYER, W.B. and B.L. TURNER (1992):
Human population growth and global land-use/cover change.
Annual Rew. Ecol. System. 23, 39-62
112
References
MICHAEL, S.F., C. BUCKLEY, E. TORO, A.R. ESTRADA and S. VINCENT (2004):
Induced ovulation and egg deposition in the direct developing anuran
Eleutherodactylus coqui
Reprod. Biol. Endocrinol. 2(6), 1-5
MICHAEL, S.F. and C. JONES (2004):
Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog,
Eleutherodactylus coqui.
Cryobiology 48, 90-94
MILLER, G.T. (2004):
Sustaining the Earth, 6th edition.
Thompson Learning, Inc. Pacific Grove, California, USA, pp 211-216.
MINUCCI, S., L. DI MATTEO, G. CHIEFFI BACCARI and R. PIERANTONI (1989):
A gonadotropin releasing hormone analog induces spermiation in intact and
hypophysectomized frogs, Rana esculenta.
Experientia 45, 1118-1121
MORESCALCHI, A. (1973):
Amphibia. In: Chiarelli, A.B., Capanna, E., (Eds.), Cytotaxonomy and vertebrate
evolution.
Academic Press, New York, USA, pp. 233-348
MORGAN, J.A.T, V.T. VREDENBURG, L.J. RACHOWICZ, R.A. KNAPP, M.J.
STICE, T. TUNSTALL, R.E. BINGHAM, J.M. PARKER, J.E. LONGCORE, C.
MORITZ, C.J. BRIGGS and J.W. TAYLOR (2007):
Population genetics of the frog-killing fungus Batrachochytrium dendrobatidis.
PNAS 104(34), 13845-13850
MORIYA, M. (1976):
Required salt concentrations for successful fertilization of Xenopus laevis.
References
113
J. Fac. Sci. Hokkaido Univ. Ser. Zool. 20, 272-276
MORRIS, G.J., E. ACTON and S. AVERY (1999):
A novel approach to sperm cryopreservation.
Hum. Reprod. 14, 1013-1021
MUGNANO, J.A., J.P. COSTANZO, S.G. BEESLEY and R.E. LEE-JR. (1998):
Evaluation of glycerol and dimethylsulfoxide for the cryopreservation of spermatozoa
from the wood frog (Rana sylvatica).
Cryo Letters 19, 249-254
MUTHS, E., P.S. CORN, A.P. PESSIER and D.E. GREEN (2003):
Evidence for disease-related amphibian decline in Colorado.
Biol. Conserv. 110, 357-365.
MUTSCHMANN, F., L. BERGER, P. ZWART and C. GAEDICKE (2000):
Chytridiomycosis on amphibians - first report from Europe.
Berliner und Münchener Tierärztliche Wochenschrift 113(10), 380-383
NAGL, A.M. and R. HOFER (1997):
Effects of ultraviolet radiation on early larval stages of the Alpine newt, Triturus
alpestris, under natural and laboratory conditions.
Oecologica 110, 514-519
NOBLE, G.K. (1931):
The biology of amphibia.
McGraw-Hill, New York, USA
OBRINGER, A.R., J.K. O'BRIEN, R.L. SAUNDERS, K. YAMAMOTO, S. KIKUYAMA
and T.L. ROTH (2000):
114
References
Characterization of the spermiation response, luteinizing hormone release and sperm
quality in the American toad (Bufo americanus) and the endangered Wyoming toad
(Bufo baxteri).
Reprod. Fertil. Dev., 12, 51-58
ORTIZ-SANTALIESTRA, M.E., A. ADOLFO, M.J. FERNANDEZ-BENEITEZ and M.
LIZANA (2007):
Effects of ammonium nitrate exposure and water acidification on the dwarf newt: The
protective effect of oviposition behaviour on embryonic survival.
Aquat. Toxicol. 85(4), 251-257
ORTIZ-SANTALIESTRA, M.E. and D.W. SPARLING (2007):
Alteration of larval development and metamorphosis by nitrate and perchlorate in
Southern Leopard Frogs (Rana sphenocephala).
Arch. Environ. Contam. Toxicol. 53, 639-646
OSTROWSKI, T. and T. MAHN (2008):
Artbeschreibung Dendrobates auratus. - Dendrobase.de - Eine Online-Datenbank
der Familie Dendrobatidae (Anura).
www.dendrobase.de/show.php?gattung=Dendrobates&art=auratus; Stand: März
2008
PAHKALA, M., A. LAURILA and J. MERILÄ (2000):
Ambient ultraviolet-B radiation reduces hatchling size in the common frog, Rana
temporaria. Ecography 23, 531-538
PARK, D., S.C. HEMPLEMAN and C.R. PROPPER (2001):
Endosulfan exposure disrupts pheromonal systems in the red-spotted newt: a
mechanism for subtle effects of environmental chemicals.
Environ. Health Persp. 109, 669-673
References
115
PECHMANN, J.H., D.E. SCOTT, R.D. SEMLITSCH, J.P. CARDWELL, L.J. VITT and
J.W. GIBBONS (1991):
Declining amphibian populations: The problem of separating human impacts from
natural fluctuations.
Science 253, 892-895
PIHA, H., M. LUOTO and J. MERILÄ (2007):
Amphibian occurrence is influenced by current and historic landscape characteristics.
Ecol. Appl. 17(8), 2298-2309
POUGH, F.H., R.H. ANDREWS, J.E. CADLE, M.L. CRUMP, A.H. SAVITZKY and
K.D. Wells (1998):
Herpetology.
Prentice Hall, New Jersey, USA.
PRÖHL, H. (2002):
Population differences in female resource abundance, adult sex ratio and male
mating success in Dendrobates pumilio.
Behav. Ecol. 13, 175-181
PUGIN, E. and O. GARRIDO (1981):
Morfologica espermatica en algunas especies de anuros pertenecientes al bosque
temperado del sur de Chile. Ultraestructura comparada.
Medio Ambiente 5, 45-57
RABB, G.B. (1999):
The amphibian crisis. In: Roth, T.L., Swanson, W.F., Blattman, L.K., (Eds.), The 7th
World Conference on Breeding endangered species proceedings.
Cincinnati Zoo & Botanical Garden, Cincinnati, USA, pp. 23-30
RAGE, J.C. (1997):
116
References
Palaeobiological and palaeogeographical background of the European herpetofauna.
In: Gasc, J.P., Cabela, A., Crnobrnja-Isailovic J., Dolmen, D., Grossenbacher, K.,
Haffner, P., Lescure, J., Martens, H., Martinez Rica, J.P., Maurin, H., (Eds.), Atlas of
amphibians and reptiles in Europe.
Mus. Nat. Hist. Nat., Paris, France, pp. 23-28
RASOTTO, M.B., P. CARDELLINI and M. SALA (1987):
Karyotypes of five Dendrobatidae (Anura, Amphibia).
Herpetologica 43, 177-182
RASTOGI, R.K. and L. IELA (1999):
Female reproduction system, amphibians. In: Knobil, E., NEILL, J.D., (Eds.),
Encyclopedia of Reproduction Vol. 2.
Academic Press, London, UK, pp. 183-190
REINHART, D., J. RIDGWAY and D.E. CHANDLER (1998):
Xenopus laevis fertilisation: analysis of sperm motility in egg jelly using video light
microscopy.
Zygote 6, 173-182
ROTH, T.L. and A.R. OBRINGER (2003):
Reproductive research and the worldwide amphibian extinction crisis. In: Holt W.V.,
Pickard A.R., Rodger J.C., Wildt D.E., (Eds.), Reproductive science and integrated
conservation.
Cambridge University Press, Cambridge, UK, pp. 359-375
ROWE, C.L., O.M. KINNEY, A.P. FIORI and J.D. CONGDON (1996):
Oral deformities in tadpoles (Rana catesbeiana) associated with coal ash deposition:
effects on grazing ability and growth.
Freshwater Biol. 36, 723-730
References
117
ROWSON, A.D., A.R. OBRINGER and T.L. ROTH (2001):
Non-invasive treatments of luteinizing hormone-releasing hormone for inducing
spermiation in American (Bufo americanus) and Gulf Coast (Bufo valliceps) toads.
Zoo Biol. 20, 63-74
SANTOS-BARRERA, G., C. PEARL and G. HAMMERSON (2004):
Rana aurora. In: IUCN 2007. 2007 IUCN Red List of Threatened Species.
<www.iucnredlist.org>. Downloaded on 05 May 2008.
SARGENT, M.G. and T.J. MOHUN (2005):
Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis.
Genesis 41, 41-46
SAVAGE, J. M. (1968):
The dendrobatid frogs of Central America.
Copeia 4, 745-776
SAVAGE, J. M. (2002):
The amphibians and reptiles of Costa Rica. A herpetofauna between two continents,
between two seas.
The University of Chicago Press, Chicago
SCHEMPP, W. and M. SCHMID (1981):
Chromosome banding in Amphibia. VI. BrdU-replication patterns in Anura and
demonstration of XX/XY sex chromosomes in Rana esculenta.
Chromosoma 83, 697-710
SCHMID, M. (1980):
Chromosome banding in Amphibia. V. Highly differentiated ZW/ZZ sex chromosomes
and exceptional genome size in Pyxicephalus adspersus (Anura, Ranidae).
Chromosoma 80, 69-96
118
References
SCHMID, M., T. HAAF, B. GEILE and S. SIMS (1983):
Chromosome banding in Amphibia. VIII. An unusual XY/XX-sex chromosome system
in Gastrotheca riobambae (Amphibia, Hylidae).
Chromosoma 88, 69-82
SCHUETZ, A.W. (1971):
In vitro induction of ovulation and oocyte maturation in Rana pipiens ovarian follicles:
effects of steroidal and non-steroidal hormones.
J. Exp. Zool. 178, 377-385
SENFFT, W. (1936):
Das Brutgeschäft des Baumsteigerfrosches (Dendrobates auratus Girard) in
Gefangenschaft.
Zool. Garten 8, 122-131
SIMBERLOFF, D. and P. STILING (1996):
How risky is biological control?
Ecology 77, 1965-1974
SILVERSTONE, P. A. (1975):
A revision of the poison-arrow frogs of the genus Dendrobates Wagler.
Los Angeles Cty. Nat Hist. Mus. Sci. Bull. 21, 1-55
SMITH, S.J., L. FAIRCLOUGH, B.V. LATINKIC, D.B. SPARROW and T.J. MOHUN
(2006):
Xenopus laevis transgenesis by sperm nuclear injection.
Nat. Protoc. 1, 2195-2203
SMITH, G.R., M.A. WATERS and J.E. RETTIG (2000):
Consequences of embryonic UV-B exposure for embryos and tadpoles of the plains
leopard frog.
References
119
Conserv. Biol. 14, 1903-1907
SOLÍS, F., R. IBÁÑEZ, C. JARAMILLO, G. CHAVES, J. SAVAGE, G. KÖHLER, K.H.
JUNGFER and W. BOLÍVAR (2004):
Dendrobates auratus. In: IUCN 2007. 2007 IUCN Red List of Threatened Species.
<www.iucnredlist.org>. Downloaded on 07 May 2008.
SPARROW, D.B., B. LATINKIC and T.J. MOHUN (2000):
A simplified method of generating transgenic Xenopus.
Nucleic Acids Res. 28(4), 1-4
STEBBINS, R.C. and N.W. Cohen (1995):
A natural history of amphibians.
Princeton University Press, New Jersey, USA
STEIN, B.A., L.S. KUTNER and J.S. ADAMS (2000):
Precious heritage: the status of biodiversity in the United States.
Oxford University Press, Oxford, UK
SUBTELNY, S. and C. BRADT (1961):
Transplantations of blastula nuclei into activated eggs from the body cavity and from
the uterus of Rana pipiens. II. Development of the recipient body cavity eggs.
Dev. Biol. 3, 96-114
SUMMERS, K. (1989):
Sexual selection and intra-female competition in the green poison-dart frog,
Dendrobates auratus.
Anim. Behav. 37, 797-805
SUMMERS, K., L.A. WEIGT, P. BOAG and E. BERMINGHAM (1999):
The evolution of female parental care in poison frogs of the genus Dendrobates:
Evidence from mitochondrial DNA sequences.
120
References
Herpetologica 55, 254-270
TAYLOR S.K., E.S. WILLIAMS, E.T. THORNE, K.W. MILLS, D.I. WITHERS and A.C.
PIER (1999):
Causes of mortality of the Wyoming toad.
Wildlife Disease 35(1):49-57
THIAW, W.B (2008):
Jurong Frog Farm Webpage. <www.jurongfrogfarm.com.sg/main.html>. Accessed on
22 April 2008.
THORBURG, J.V. (1952):
The use of Xenopus laevis, Bufo bufo, and Rana esculenta as test animals for
gonadotrophic hormones.V. Experience with the Galli Mainini spermiation reaction as
a routine test for pregnancy diagnosis and quantitative assay of chorionic
gonadotrophin.
Acta Endocrinol. 11, 147-155
TOFT, C.A. (1995):
Evolution of diet specialization in poison dart frogs (Dendrobatidae).
Herpetologica 51, 202-216
TOWNSEND, C.R. (1996):
Invasion biology and ecological impacts of brown trout Salmo trutta in New Zealand.
Biol. Conserv. 78, 13-22
VENCES, M., J. KOSUCH, S. LÖTTERS., A. WIDMER, K.H. JUNGFER, J. KOHLER
and M. VEITH (2000):
Phylogeny and classification of poison frogs (Amphibia: Dendrobatidae), based on
mitochondrial 16S and 12S ribosomal RNA gene sequences.
Mol. Phylogenet. Evol. 15, 34-40
References
121
VIAL, J.L. and L. SAYLOR (1993):
The status of amphibian populations.
IUCN/SSC Declining Amphibians Populations Task Force. Gland, Switzerland. The
World Conservation Union
VITOUSEK, P.M. (1994):
Beyond global warming: Ecology and global change.
Ecology 75, 1861-1876
WAGGENER, W.L. and E.J. CARROLL JR. (1998):
A method for hormonal induction of sperm release in anurans (eight species) and in
vitro fertilization in lepidobatrachus species.
Develop. Growth. Differ. 40(1), 19-25
WAKAYAMA, T. and R. YANAGIMACHI (2001):
Mouse cloning with nucleus donor cells of different age and type.
Mol. Reprod. Dev. 58, 376-383
WALLACE, H., G.M. BADAWY and B.M. WALLACE (1999):
Amphibian sex determination and sex reversal.
Cell. Mol. Life. Sci. 55(6-7), 901-909.
WATSON, P.F. (2000):
The causes of reduced fertility with cryopreserved semen.
Anim. Reprod. Sci. 60, 481-492
WEBB, C. and J. JOSS (1997):
Does predation by the fish Gambusia holbrooki (Atheriniformes: Poeciliidae)
contribute to declining frog populations?
Australian Zoologist 30, 316-324
WELLS, K. D. (1978):
122
References
Courtship and parental behaviour in a Panamanian Poison-Arrow Frog (Dendrobates
auratus).
Herpetologica 34 (2), 148-155
WHITFIELD, S.M., K.E. BELL, T. PHILIPPI, M. SASA, F. BOLANOS, G. CHAVES,
J.M. SAVAGE and M.A. DONNELLY (2007):
Amphibian and reptile declines over 35 years at La Selva, Costa Rica.
PNAS 104(20), 8352-8356
WILDT, D., B. PUKAZHENTHI, J. BROWN, S. MONFORD, J. HOWARD and T.
ROTH (1995):
Spermatology for understanding, managing and conserving rare species.
Reprod. Fertil. Dev. 7, 811-824
WILSON, E.O. (2002):
The future of life.
Knopf, New York, USA
WILSON, B.A., G. VAN DER HORST and A. CHANNING (1991):
Scanning electron microscopy of the unique sperm of Chiromantis xerampelina
(Amphibia-Anura).
Electr. Microsc. Soc. South Africa 21, 255-256
WOLF, D.P. and J.L. HEDRICK (1971):
A molecular approach to fertilization. II. Viability and artificial fertilization of Xenopus
laevis gametes.
Dev. Biol. 25, 348-359
WRIGHT, D.A. and C.M. RICHARDS (1983):
Two Sex-Linked Loci in the Leopard Frog, Rana pipiens.
Genetics 103(2), 249-261
References
ZIMMERMANN, H. (1974):
Die Aufzucht des Goldbaumsteigers Dendrobates auratus.
Aquarien Magazin 12, 526-531
ZIMMERMANN, H. and E. ZIMMERMANN (1988):
Ethotaxonomie und zoogeographische Artengruppenbildung bei Pfeilgiftfröschen
(Anura: Dendrobatidae).
Salamandra 24, 125-160.
123
Appendix
125
Appendix
126
Appendix
Erklärung
Hiermit erkläre ich, dass ich die Dissertation „Induced spermiation and sperm
morphology in the Green Poison Frog, Dendrobates auratus“ selbständig verfasst
habe.
Ich habe keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten
(Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat
von mir unmittelbar oder mittelbar entgeltliche Leistungen für Arbeiten erhalten, die
im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.
Ich habe die Dissertation an folgender Institution angefertigt:
Institut für Reproduktionsbiologie, Stiftung Tierärztliche Hochschule Hannover
Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen
ähnlichen Zweck zur Beurteilung eingereicht.
Ich versichere, dass ich die vorstehenden Angaben nach bestem Wissen vollständig
und der Wahrheit entsprechend gemacht habe.
Hannover, den 28.05.2008
Christian Lipke
Appendix
127
Acknowledgements
• I express my gratitude to my supervisor Prof. Dr. Sabine Meinecke-Tillmann for
entrusting me with this interesting work and for her continuous academic guidance,
confidence, motivation, and patience. I am especially grateful, that she always finds
time to share ideas or give an advice and an always open door in case of problems.
• Many thanks to Prof. Burkhard Meinecke for providing a room for the housing of my
dendrobatid frogs and for his unrestricted support and patience.
• I thank Prof. Dr. Heike Pröhl and Prof. Dr. Klaus Eulenberger, from my tutorial
group, for thoughtful discussions, comments and suggestions.
• Special thanks to Prof. Dr. Wilfried Meyer for his kind support and encouragement
during the ultrastructural studies of my PhD project.
• Thanks to Kerstin Rohn for introducing me in the preparation of samples for
electron microscopy and the operation of electron microscopes as well as solving
arising problems.
• I also thank Dr. Silja Ebeling, Edita Podhajsky and Monika Labsch for the
interesting discussions and qualified help in many issues concerning laboratory work.
• I thank all past and present colleagues from the Department of Reproductive
Biology, University of Veterinary Medicine Hannover, who make this workplace as
friendly and pleasant as it is. Nice memories with you will join me livelong.
• I am grateful to Sigrid Faber, the secretary of the PhD program at the University of
Veterinary Medicine Hannover, for her enthusiastic dedication to the PhD program
and for her concern to avoid and solve the day-to-day problems of a PhD student.
128
Appendix
• I thank Dr. Martin Beyerbach for helpful discussions and statistical advice.
• I am especially grateful to my mother for funding my veterinary studies and being
caring and understanding throughout my life.
• Special thanks to my wife Antje for always listening, trusting in me, and giving me
strength. The support I got from her and also her family was of great value.