Estudio del koala

8/8/2019 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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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



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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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



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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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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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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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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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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Estudio del koala

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