Estudio del koala
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Transcript of Estudio del koala
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Cryobiology 53 (2006) 218–228
www.elsevier.com/locate/ycryo
0011-2240/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cryobiol.2006.06.001
An investigation into the similarities and diV erences
governing the cryopreservation success of koala
(Phascolarctos cinereus: goldfuss) and common
wombat (Vombatus ursinus: shaw) spermatozoa
S.D. Johnston a,¤, C. MacCallum a,b, D. Blyde b, R. McClean a, A. Lisle c, W.V. Holt d
a
School of Animal Studies, University Of Queensland, Gatton 4343, Australiab Western Plains Zoo, Dubbo 2830, Australiac School of Agronomy and Horticulture, The University of Queensland, Gatton 4343, Australia
d Institute of Zoology, The Zoological Society of London, Regent’s Park, London NW1 4RY, UK
Received 14 February 2006; accepted 6 June 2006
Available online 2 August 2006
Abstract
The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to
investigate reasons for their respective post-thaw survival by examining the sperm’s response to a range of osmotic media
and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting adegree of structural inXexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D
(5M), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of
cell volume changes that typically occur during cryopreservation. In experiment 1, koala (nD4) and wombat (nD 4) sper-
matozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast—3 cm above liquid N2 interface;
slow—6°C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris–citrate glucose buV er with
15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma mem-
branes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala sper-
matozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than
wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When
koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneWcial eV ect on
sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant
of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw sur-vival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but
lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely
to be associated with F-actin induced plasma membrane inXexibility.
© 2006 Elsevier Inc. All rights reserved.
This work was funded by institutional sources.* Corresponding author. Fax: +617 33655644.
E-mail address: [email protected] (S.D. Johnston).
mailto:%[email protected]:%[email protected]:%[email protected]
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S.D. Johnston et al. / Cryobiology 53 (2006) 218–228 219
Keywords: Koala; Wombat; Spermatozoa; Cryopreservation; Osmotic tolerance; F-actin; Cytochalasin D; Marsupial
Studies by Taggart et al. [22,23] have shown that
common wombat (Vombatus ursinus) and Southern
Hairy-nosed Wombat (Lasiorhinus latifrons) sperma-
tozoa are highly tolerant of cryopreservation proce-
dures with post-thaw motility reaching over 90% in
some individual samples. More recently, MacCallum
and Johnston [15] have demonstrated cryopreserva-
tion success (60% post-thaw motility) with cauda epi-
didymidal common wombat spermatozoa recovered
from post-mortem specimens. By contrast, similar
attempts to cryopreserve koala (Phascolarctos cine-
reus) spermatozoa have not been as successful [10,22].
Koala spermatozoa require a much higher glycerol
concentration in the cryoprotectant medium for suc-
cessful cryopreservation, but consistently show onlyhalf the post-thaw motility compared to that of
wombat spermatozoa. With the success of koala arti-
Wcial insemination using fresh semen [thirteen pouch
young, [12]] the future development and implementa-
tion of genome resource banks in this species appears
only limited by the need for improving sperm
cryopreservation technology.
Given the close phylogenetic relationship between
the koala and wombat and the similarity of their
sperm morphologies [7], the relative diV erence in cryo-
preservation success presents an unusual opportunityto identify possible causes and prevention of cryoin-
jury in the spermatozoa of both species. This study
aims to conWrm the relative cryotolerance of two spe-
cies under controlled experimental conditions of dilu-
ent type, cryoprotectant concentration and freezing
rate and seeks to investigate the tolerance of these
spermatozoa to an osmotic challenge.
It has been suggested that the sperm cytoskeleton
in murine species may be anchored to the plasma
membrane in such a way that it resists swelling under
hypo-osmotic environments and which consequently,
predisposes the sperm membrane to cryopreservation
damage [18]. However, the same study also showed
that mouse spermatozoa incubated with 5M
cytochalasin D showed an increased tolerance to a
hypo-osmotic challenge. Noiles et al. [18] postulated
that as cytochalasin D is capable of depolymerizing
Wlamentous (F) actin, then it also has the potential to
plasticise the sperm cytoskeleton, a process that
should confer increased Xexibility on the plasma
membrane. Here we therefore attempt to determine
the relative extent and location of F-actin in koala
and wombat spermatozoa and investigate whether
cytochalasin D can be used to increase sperm mem-
brane tolerance to anisosmotic media in a manner
similar to that described by Noiles et al. [18].
Materials and methods
Animals
Common wombats (nD 4) used in this experi-
ment were part of a captive experimental colony
housed at Western Plains Zoo, Dubbo, New South
Wales. Koalas were part of a captive colony at Lone
Pine Koala Sanctuary (nD 3), and a colony located
at the Zoology Department (nD6) at the University
of Queensland, Brisbane. All wombats (nD 4) andkoalas (nD 9) used in this study were sexually
mature and clinically healthy at the time of semen
collection; wombat and koala husbandry and enclo-
sure design have been described previously [14,1].
This work was conducted with the approval of the
University of Queensland and Zoological Parks
Board animal ethics committees.
Anaesthesia
In preparation for semen collection, wombats wereinitially sedated with 200mg of tiletamine and 200mg
zolazepam intramuscularly (Zoletil®, Virbac, Austra-
lia) either by hand injection using an 18 gauge needle
stick or via blow-pipe device (B31 Blowpipe, Telinject
Australasia, Maribyrnong, Australia) using 2mL
blowpipe projectile syringes. Once sedated, animals
were then masked with isoXurane (Forthane, Abbott
Australasia, Pty. Ltd., Kurnell, Australia) and intu-
bated blindly using a 7.5mm cuV ed endotracheal
tube. Maintenance of gaseous anaesthesia was pro-
vided by 2% isoXurane in oxygen. Koala anaesthesia
for the purposes of electroejaculation has been previ-
ously described by McGowan et al. [16].
Semen collection
Prior to semen collection in the wombat and koala,
the penis was everted from the prepuce and cleaned of
contamination, the rectum emptied of faecal material
and lubricated with 2.5mL Microlax® enema
(Pharmacia AB, Sweden). Semen was then collected
by means of electroejaculation; the procedure for
which has been documented in detail by Johnston
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220 S.D. Johnston et al. / Cryobiology 53 (2006) 218–228
et al. [11]. The electroejaculation probe used in the
wombat was approximately 150mm long and 15mm
in diameter, it consisted of three ventrally located elec-
trodes approximately 80mm in length [14]. The design
and dimensions of the koala rectal probe have been
detailed in Johnston et al. [12].
Sperm evaluation
Sperm concentration (£106/mL) of the original
semen sample was estimated using a calibrated sperm
counting chamber (Makler, SeW-Medical Instruments,
HaWa, Israel). Diluted koala and wombat semen
(1 semen: 10 tris–citrate glucose [3.0g tris buV er, 1.7g
citric acid, 1.25g glucose]) was placed onto a pre-
warmed microscope slide (35°C) with a coverslip and
the motility evaluated using a phase-contrast micro-
scope at a magniWcation of 400 £[13]. All microscopicevaluations of spermatozoa, including % motility
were conducted on a warm stage set at 35°C. Plasma
membrane integrity was determined using a dual Xuo-
rescent staining technique (Sperm Viability Kit;
Molecular Probes Inc, USA). This method used two
vital nucleic stains; SYBR-14 (Wnal concentration
100 nM) permeates intact plasma membranes causing
membrane intact sperm nuclei to Xuoresce green, and
propidium iodide (Wnal concentration 12M), which
permeates membrane-damaged spermatozoa causing
them to Xuoresce red [8]. In experiment 1, the propor-tion of decondensed sperm nuclei, pre and post-cryo-
preservation, were also determined as deWned by
Cummins [4] (Fig. 1).
Experiment 1—Relative cryopreservation success of
wombat and koala spermatozoa
Following collection, wombat (nD 4) and koala
(nD 4) semen was equilibrated to room temperature
(approximately 22 °C) and diluted 1–1 with tris–cit-
rate glucose diluent. The extended semen was then
evaluated for initial percentage motility, percentage
of sperm with intact plasma membranes and the per-
centage of intact sperm nuclei (non-decondensed)
before being cooled to 5°C in a conventional refrig-erator for approximately 1–2 h. After cooling, ali-
quots of semen were prepared for dilution with
pre-chilled (5 °C) tris–citrate glucose containing 15%
egg yolk and either 16 or 28% glycerol so that on
Wnal 1–1 dilution with semen, egg yolk and glycerol
concentrations were 7.5% and either 8 or 14%,
respectively. Semen samples (0.2mL) were drawn
into 0.25 mL straws (IMV Technologies, France),
sealed and frozen in either liquid nitrogen vapour
3–4 cm above the liquid nitrogen interface (fast
freeze) as described by Taggart et al. [23] or at
¡6.0 °C/min (slow freeze) to ¡100 °C in a program-mable freezer (Model–Freeze Control® CL863, Cry-
ologics Pty Ltd., Mulgrave, Australia). Semen straws
were then plunged into liquid nitrogen and stored
overnight before thawing (35°C for 30 s). Post-thaw
percentage motility, the percentage of intact plasma
membranes and percentage of intact sperm nuclei
were assessed immediately on thawing (0 h) and
after 2 h incubation at 35 °C post-thaw. For statisti-
cal analysis estimates of post-thaw survival (%
motility and % plasma membrane-intact) were stan-
dardised by proportioning these estimates as a per-centage of their initial value prior to
cryopreservation. Following an angular transforma-
tion, post-thaw survival characteristics of wombat
and koala spermatozoa were compared via a three-
way ANOVA using the SAS® statistical program
(Version 8.2 © 2001). A nested model was assumed
with subjects nested within species and treatment
nested within subjects. Results are presented as
means and 95% conWdence intervals.
Fig. 1. Fluorescence microscopy of SYBR14 (green) and PI (red) stained koala spermatozoa. Note the green sperm head with an intact
plasma membrane and the red spermatozoon with a damaged plasma membrane. The red sperm head chromatin has also decondensed so
that the nuclear volume has increased substantially. Scale bar—5 m.
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S.D. Johnston et al. / Cryobiology 53 (2006) 218–228 221
Experiment 2—Osmotic tolerance of wombat and
koala spermatozoa
In order to determine the relative osmotic toler-
ance of koala (nD4) and wombat (nD 4) spermato-
zoa, 20L of ejaculated semen was diluted with380L phosphate-buV ered saline (PBS) solution of
various osmolalities (60, 104, 160, 233, 300, 641, 970
and 1410mOsmol/kg). Hyper-osmotic diluents were
prepared by the addition of sucrose (Ajax Chemicals
Pty. Ltd., Australia); hypo-osmotic diluents were
prepared by dilution of PBS media with sterile
distilled water. The osmolality of each diluent was
determined by a vapour pressure osmometer
(Wescor, UT). The pH of treatment diluents ranged
from 6.9 to 7.2. These sperm suspensions were then
incubated for 10 min at 35 °C before centrifugation
at 160 g for 2 min, after which the supernatants wereremoved and the pellets resuspended in 100 L of
300 mOsmol/kg PBS. The percentages of motile
sperm and the percentage of plasma membrane
intact sperm were assessed following the procedure.
For statistical analysis, estimates of the above char-
acteristics were standardised by proportioning these
values as a percentage of those spermatozoal char-
acteristics determined for the 300 mOsmol/kg PBS
medium. After an angular transformation, sperm
characteristics were compared between species and
over the range of osmolality tested via a two-wayANOVA using the SAS® statistical program
(Version 8.2 © 2001). A nested model was assumed
with subjects nested within species and treatments
nested within subjects. Results are presented as
means and 95% conWdence intervals.
Experiment 3—Localisation of F-actin in wombat
and koala spermatozoa
To account for diV erences in cryopreservation
ability of koala and wombat spermatozoa it washypothesised that koala sperm may contain higher
amounts of F-actin than wombat spermatozoa and
that this may aV ect their ability to respond to the
osmotic Xux associated with cryopreservation [18].
Hence, the presence and location of F-actin was
determined in both wombat and koala cauda epidid-
ymal spermatozoa by means of an F-actin antibody
(ab205, Abcam, Cambridge, UK). Small pieces
(1mm3) of cauda epididymidis were Wxed with 3%
paraformaldehyde and 0.1% glutaraldehyde in PBS
for at least 1h. They were subsequently washed in
PBS, dehydrated in ethanol and embedded in LR
White (SPI Supplies and Structure Probe Inc, Aus-
tralia) resin before being polymerised at 60 °C for
24 h. Sections (70–100nm) of cauda epididymidis
were cut using a Reichert Ultracut E ultramicro-
tome. The tissue sections (at least 3 per slide) were
subsequently transferred to drops of water on glassslides and dried on a hotplate at approximately
60 °C. Once dry, slides were stained with Toluidine
Blue O (Spectrum Chemicals and Laboratory Prod-
ucts, California, USA) for 10min at room tempera-
ture, and rinsed with distilled water. The Toluidine
Blue O stain was applied to prevent auto-Xuores-
cence [2,6]. Sections were then re-hydrated in PBS
with the addition of blocking agents (containing
1 mL of 200 mM glycine, 200L of 10% BSA and
200 L of 10% Wsh skin gelatin and made up to
10 mL with PBS). Sections were incubated in the
blocking solution for 5 min and stained overnight at4 °C with 100L of 1:100 diluted F-actin antibody
in PBS. A control treatment involved an overnight
incubation in PBS without the addition of antibody.
Slides were then washed twice in PBS for 5min fol-
lowed by two 5min washes in PBS with blocking
agents. Excess PBS were blotted from the slides
before the addition of either Anti-Mouse IgM
(-chain speciWc)–FITC Antibody (F9259, Sigma,
Australia) or PBS as a control. Slides were then
washed twice in PBS and immersed in PBS before
viewing. Slides from each treatment were kept sepa-rate at all times. Sperm were examined using a E400
Nikon epiXuorescent microscope using a blue exci-
tation cube (590–610nm) and photographed with a
Cool-snap CS monochrome digital video capture
device (Roper ScientiWc, USA) and Image-Pro soft-
ware (MediaCybernetics, USA).
Experiment 4—E V ect of cytochalasin D on the
osmotic tolerance of wombat and koala spermatozoa
To determine whether osmotic tolerance of wom-bat (nD 4) and/or koala spermatozoa (nD 5) could
be improved by incubation with the actin Wlament
depolymerising agent cytochalasin D (Sigma, Aus-
tralia), 20L of semen was diluted in 180L of PBS
of varying osmolality (60, 104, 160, 233, 300, 641,
970 and 1410 or 1840 mOsmol/kg) containing either
0 or 5M cytochalasin D. This solution was then
incubated for 10min at 35 °C and the percentage of
plasma membrane intact and coiled-tailed sperma-
tozoa determined. Coiled tails are limited to hypo-
tonic environments so that only spermatozoa
exposed to the range of 60–300mOsmol/kg were
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222 S.D. Johnston et al. / Cryobiology 53 (2006) 218–228
examined for coiled tails. The eV ect of cytochalasin
D on the percentage of plasma membrane-intact
and coiled-tailed sperm within species across the
osmolality range was compared via a three-way
ANOVA using the SAS® statistical program
(Version 8.2©
2001). A nested model was assumedwith subjects nested within species and treatments
nested within subjects. Results are presented as
means and 95% conWdence intervals.
Results
Experiment 1—Relative cryopreservation success of
wombat and koala spermatozoa
The survival of common wombat and koala sper-
matozoa following cryopreservation are shown in
Table 1. Irrespective of the cooling rate and/or glyc-erol concentration, wombat spermatozoa showed
consistently better post-thaw sperm survival
(% motile, % of intact plasma membranes and % of
intact non-decondensed sperm nuclei) immediately
following thawing (0 h) and after 2 h incubation at
35 °C.
With respect to evaluating the relative success of
the diV erent protocols used in the cryopreservation
of wombat spermatozoa, the following observations
can be made based on the post-thaw survival of spermatozoa after 2h incubation at 35 °C. When
compared to the fast cooling rate, the slow rate of
cooling resulted in consistently higher (P
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S.D. Johnston et al. / Cryobiology 53 (2006) 218–228 223
both a slow rate of cooling and 14% glycerol for
optimal post-thaw survival. This combination of
cooling rate and glycerol concentration were signiW-
cantly better (P < 0.05) than any other, with respect
to post-thaw motility, the proportion of intact
plasma membranes and sperm heads.
Experiment 2—Osmotic tolerance of wombat and
koala spermatozoa
The eV ect of media osmolality on the percent-
age of motile and plasma membrane intact wom-
bat and koala spermatozoa is shown in Fig. 2 and
Table 2. Not surprisingly, these percentages varied
signiWcantly over the range of osmolality tested.
When the motility of koala and wombat sperma-
tozoa were compared with respect to each osmotic
medium, koala spermatozoa were more tolerant of the 160 mOsmol/kg (P < 0.05) medium than the
wombat spermatozoa, but wombat spermatozoa
were better able to maintain motility following
exposure to media of 970 mOsmol/kg (P < 0.05). In
a similar manner, but in terms of intact plasma
membranes, koala sperm were not as tolerant of
hyper-osmotic excursions when diluted in970 mOsmol/kg (P < 0.05) and 1410 mOsmol/kg
(P < 0.05) media.
Experiment 3—Identi Wcation and localisation of
F-actin in wombat and koala spermatozoa
Despite repeated attempts using a variety of tech-
niques, it was not possible to identify F-actin by
Xuorescent microscopy in koala spermatozoa. How-
ever, the wombat sperm head showed strong stain-
ing attributable to the presence of F-actin but there
was no corresponding staining of the midpiece orprincipal piece (Fig. 3).
Fig. 2. The eV ect of osmolality on the mean percentage of motility and plasma membrane integrity of koala and wombat spermatozoa.
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Experiment 4—E V ect of cytochalasin D on the
osmotic tolerance of wombat and koala spermatozoa
The eV ect of using cytochalasin D on wombat
and koala sperm survival (intact sperm plasma
membranes and coiled Xagellae) in response to a
range media of diV ering osmotic pressure is shown
in Table 3. Cytochalasin D had no eV ect on the tol-
erance of the koala and wombat sperm plasma
membrane to hyper or hypo-osmotic media. Simi-
larly, cytochalasin D also had no signiWcant eV ect on
the percentage of coiled Xagellae when spermatozoa
were exposed to hypo-osmotic media. Despite these
observations, cytochalasin D produced a weak but
non-signiWcant improvement in the ability of koala
spermatozoa to coil in response to the 60mOsmol/
kg (PD 0.09) and 140mOsmol/kg (PD 0.06) media.
Discussion
Despite some minor diV erences in sperm compo-
nent dimensions [21], koala and wombat spermato-
Table 2
EV ect of osmolality on the percentage motility and the percentage plasma membrane integrity of koala and wombat spermatozoa
¤ a signiWcant diV erence (P < 0.05) between species at that osmolality.
Species Osmolality (mOsmol/kg] Koala Wombat Species comparison¤
Mean 95% CI Mean 95% CI
% Motile 60 0.1 (0.1–6) 0 (0–5)
104 21 (6–41) 31 (13–51)160 68 (67–97) 50 (29–72) ¤
233 98 (87–99) 87 (69–98)
300 100 (95–100) 100 (95–100)
641 66 (44–85) 88 (70–98)
970 19 (5–39) 61 (39–80) ¤
1410 22 (7–43) 3 (0–14) ¤
% Intact Membranes 60 2 (0–12) 2 (0–5)
104 48 (29–67) 61 (41–79)
160 92 (78–100) 75 (59–90)
233 92 (76–100) 91 (53–90)
300 100 (96–100) 100 (96–100)
641 83 (66–95) 96 (59–94)
970 65 (25–68) 98 (89–100) ¤
1410 69 (50–85) 96 (85–100) ¤
Fig. 3. Location of F-actin in wombat spermatozoa (A) Phase contrast image of the wombat cauda epididymidis showing spermatozoa in
the lumen of the tubule (B) Fluorescent image of same section following staining with the F-actin Anti-Mouse IgM (-chain speciWc)–
FITC antibody; ep, epithelium of the cauda epididymis; lu, lumen of the cauda epididymidal tubule; sp, wombat spermatozoa (Scale bar—
25 m).
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S.D. Johnston et al. / Cryobiology 53 (2006) 218–228 225
zoa share a very similar morphology. This point was
originally recognised and used by Hughes [9] in a
light microscope study to propose a close phyloge-
netic association between these species, a point that
was subsequently conWrmed by others at the ultra-
structural level [3,5,24]. Given their apparent struc-
tural similarity, it is surprising that koala and
wombat sperm respond so diV erently to cryopreser-
vation [10,22,23].
The results of the present study conWrm, under
controlled conditions of freezing rate, glycerol con-
centration and cryopreservation protocol, that ejac-
ulated wombat spermatozoa are better able to
survive cryopreservation than ejaculated koala sper-
matozoa. In fact, the post-thaw survival of wombat
spermatozoa in this and other studies [22,23] is some
of the most impressive of all mammalian spermato-
zoa. Post-thaw motilities of over 80% are regularly
achieved and even cauda epididymidal sperm recov-
ered from necropsied specimens and stored chilled
for 3 days before freezing have resulted in over 60%
post-thaw motility [14].
Taggart et al. [22,23] have stated that the best cryo-
protectant for common and southern hairy-nosed
wombat spermatozoa is tris–citrate egg yolk contain-
ing fructose with 4–8% glycerol in the Wnal concentra-
tion. This conclusion was reached primarily from
studies utilising 25 southern hairy-nosed wombats
but only 1 common wombat. In addition, Taggart
et al. [22,23] used a rapid method of freezing in which
spermatozoa were frozen directly into the liquid
nitrogen vapour either 3 or 6cm above the liquid
interface. Cryopreservation results reported in this
study were somewhat diV erent from those of Taggart
et al. [22,23] in that sperm frozen with the fast freezing
rate and 8% glycerol resulted in signiWcantly inferior
post-thaw survival than those frozen with a slow
freezing rate of 6°C/min and a tris–citrate glucose dil-
uent containing a Wnal concentration of 14% glycerol
[10]. Koala spermatozoa that were frozen using a
rapid freezing rate or with a lower glycerol concentra-
tion (8%), showed either no post-thaw survival or
consistently lower post-thaw motility, plasma mem-
brane integrity and nuclear stability.
Table 3
The eV ect of cytochalasin D on the osmotic tolerance (% intact plasma membranes and % coiled Xagella) of koala and wombat spermato-
zoa
Species Osmolality (mOsmol/kg) Cyto D + Mean 95% CI Cyto D¡Mean 95% CI P
Intact membranes
Koala 60 48 34–63 40 26–55 P > 0.10
104 59 44–73 55 40–69 P > 0.10
160 71 57–83 65 50–78 P > 0.10
230 82 69–92 85 73–94 P > 0.10
300 92 82–98 93 83–98 P > 0.10
640 91 80–97 95 87–100 P > 0.10
920 91 81–98 92 82–99 P > 0.10
Wombat 60 4 0–13 3 0–11 P > 0.10
104 66 50–80 59 43–75 P > 0.10
160 88 75–96 90 77–97 P > 0.10
230 93 82–99 95 86–100 P > 0.10
300 89 77–97 95 85–100 P > 0.10
640 87 73–96 92 81–99 P > 0.10
920 90 78–98 90 78–98 P > 0.10
Coiled X agellae
Koala 60 49 27–70 26 9–47 *0.09
104 49 28–70 23 8–44 *0.06
160 33 14–55 52 30–73 P > 0.10
230 16 3–35 27 10–48 P > 0.10
300 9 1–25 3 0–15 P > 0.10
Wombat 60 56 32–79 64 39–85 P > 0.10
104 69 45–89 57 33–80 P > 0.10
160 16 3–37 15 2–35 P > 0.10
230 6 0–22 4 0–19 P > 0.10
300 9 0–28 3 0–16 P > 0.10
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226 S.D. Johnston et al. / Cryobiology 53 (2006) 218–228
Nuclear instability is a common feature of mar-
supial spermatozoa as the associated nucleoproteins
lack disulphide linkages between adjacent chroma-
tin strands and, therefore, can be easily decondensed
by a variety of laboratory based treatments, includ-
ing exposure to sodium dodecyl sulphate and otherdetergents, air drying and high concentrations of
divalent cations (Ca2+ and Mg2+) [24]. A particular
disturbing Wnding in the present study, with respect
to the long-term establishment of a genome resource
bank for the koala, was the high incidence (50%) of
post-thaw nuclear instability even after the most
successful cryopreservation protocol. This phenom-
enon will need to be addressed for the further devel-
opment of a koala artiWcial insemination procedure
based on the use of frozen-thawed semen. While
nuclear decondensation was also apparent in wom-
bat spermatozoa that had been frozen using a fastfreezing rate (55–60% intact sperm heads), the
majority of sperm frozen slowly exhibited a higher
degree of nuclear stability (82–87% intact sperm
heads). VeriWcation of the fertility of frozen-thawed
wombat spermatozoa awaits the development of an
AI program in this species.
Breed et al. [3] have identiWed signiWcant heteroge-
neity in the structure of koala sperm DNA when
compared to wombat spermatozoa. Many koala sper-
matozoa possess a large nuclear vacuole within their
chromatin matrix and are more susceptible thanwombat spermatozoa to detergent (Triton X-100)
induced chromatin dispersal. These observations are
consistent with the proportionally high incidence of
chromatin instability following cryopreservation of
koala spermatozoa noted in the current study.
Further studies are required to investigate the relative
nuclear instability of koala sperm pre- and post- cryo-
preservation and in identifying diluent additives that
may help to prevent or reduce chromatin damage. It
may also be possible to screen out koala ejaculates
that contain a high proportion of sperm with dam-aged chromatin prior to cryopreservation.
Repeated unprotected freeze-thaw procedures
of tammar wallaby (Macropus eugenii ), brushtail
possum (Trichosurus vulpecula) and opossum
(Monodelphis domestica) spermatozoa, failed to
destabilise the acrosomal membrane or matrix [20].
While not a focus of the present study, koala and
wombat acrosomes observed in this study also
appeared to be highly resilient to cryopreservation
damage. It seems, therefore, that cryopreservation
injury in marsupials may be characterised by
nuclear instability and acrosomal stability,
whereas for eutherian spermatozoa, it is the
nucleus, which remains stable and the acrosome
that is most susceptible [26].
While both wombat and koala spermatozoa
exhibit substantial pleiomorphy of the sperm head
[3,11,25,28] recent studies by MacCallum [14] havesuggested that the extent of sperm head heterogene-
ity in the wombat is not as diverse as the koala. The
range of sperm head morphotypes reported in the
koala ejaculate may be indicative of a greater pro-
portion of abnormal or immature sperm cells in this
species [7,11,25] and consequently, a lower post-
thaw sperm survival rate; this hypothesis requires
further examination.
Miller et al. [17] have compared the fatty acid
composition of koala and wombat cauda spermato-
zoa and found signiWcant diV erences between the
two species. The ratio of unsaturated/saturatedmembrane fatty acids in the koala was approxi-
mately 7.6, substantially higher than that described
for the wombat (1.9) or indeed any other mammal
so far described [27]. While this disparity in the com-
parative ratio of unsaturated/saturated sperm mem-
brane fatty acids in koala and wombat spermatozoa
appears to have has no direct relationship with cold
shock susceptibility [17] it may still contribute to the
relative diV erences in membrane tolerance during
cryopreservation.
The eV ect of osmolality on koala and wombat sper-matozoa examined in the present study revealed that
wombat sperm are more tolerant of hyper-osmotic
excursions than koala sperm and that this may be con-
tributing to a lower post-thaw survival in the koala.
The motility of koala spermatozoa exposed to
1410mOsmol/kg media was signiWcantly greater than
of wombat spermatozoa at the same osmolality. This
was an expected Wnding and may be indicative of a
sub-population of sperm in the koala ejaculate that
are capable of tolerating hyperosmotic environments.
Results from experiments 2 and 4 have shown theeV ect of osmotic injury is most severe when spermato-
zoa are exposed to two rapid Xuxes in osmotic pres-
sure rather than one. For example, if the spermatozoa
are exposed to a hypo-osmotic environment and
examined while remaining at that osmolality, then the
eV ect on sperm survival is less than (experiment 4)
when spermatozoa are returned to medium of
300mOsmol/kg (experiment 2) and evaluated. Two
excursions appear to be too much for the koala sperm
membrane to cope with and structural damage results.
A similar phenomenon has also been reported for
human and ram spermatozoa [5].
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S.D. Johnston et al. / Cryobiology 53 (2006) 218–228 227
Surprisingly, F-actin was not detected in the
koala spermatozoa but was found in the wombat
sperm nucleus. A lack of F-actin in the koala sperm
head may contribute to their susceptibility to decon-
densation following cryopreservation; conversely
the F-actin in wombat sperm may have a role innuclear stabilisation. F-actin has been described in
the cauda epididymidal sperm tail of the tammar
wallaby [19] but not in the head of the mature
sperm. It is also possible that koala sperm do actu-
ally possess F-actin but it was simply not detectable
by the methods employed in this study.
Cytochalasin D is a depolymerisation agent for
F-actin and the addition of this compound to koala
and wombat spermatozoa at the dosage used in this
study did not improve the osmotic tolerance of the
plasma membrane as has been shown in mice [18].
There was slight evidence, although not statisticallysigniWcant, that cytochalasin D improved the
response of the sperm tail to hypo-osmotic media by
allowing the Xagellum to swell and accommodate an
increase in water volume. The fact that cytochalasin
D appeared to have a weak eV ect on koala sperma-
tozoa and not the wombat sperm is particularly sur-
prising given the lack of F-actin found in the koala
sperm. Perhaps F-actin is only present in the koala
sperm in trace amounts such that the dose rate of
cytochalasin D was suYcient to induce a minor
eV ect in the koala sperm but not in the wombat,which had a greater proportion of F-actin. Similarly,
F-actin was also diYcult to locate in the mouse sper-
matozoa [18].
In order to establish a truly functional genome
resource bank in the koala it is particularly impor-
tant to develop an experimental method that
examines the Wne details of each step of the cryo-
preservation process and one that can appropriately
evaluate the relative success and failure of each pro-
tocol. In this regard, the wombat has provided a use-
ful experimental model in trying to understand thesusceptibility of the koala spermatozoa to cryopres-
ervation and it is now possible to identify sperm
chromatin instability and plasma membrane intoler-
ance as critical areas for future research focus.
Acknowledgments
The authors thank Dr. Nilendran Prathalingam
for his help in the calculation of the freezing rate for
the fast freeze protocol described in this study. We
are also grateful to Lone Pine Koala Sanctuary and
Dr. Frank Carrick of the University Queensland for
the use of their captive koalas and Western Plains
Zoo for the use of their captive common wombats.
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