Short-term storage of cane toad (Bufo marinus) gametes - Bioscientifica
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Reproduction (2001) 121, 167–173 Research
Short-term storage of cane toad (Bufo marinus) gametes
R. K. Browne, J. Clulow and M. Mahony
Department of Biological Sciences, University of Newcastle, University Drive, Callaghan
NSW 2308, Australia
The responses of cane toad (Bufo marinus) gametes, used After 30 days, sperm motility and fertilizing capacity
as a model for the development of assisted reproduction were determined. The optimal protocol for sperm storage
techniques for rare and endangered amphibians, to short- up to 10 days, as assessed by the retention of fertilizing
term storage at temperatures > 0⬚C were studied. Whole capacity, was as a 1:5 testis:SAR (w/v) suspension,
excised testes were stored at 0⬚ or 4⬚C for 15 days, and whereas the longest absolute retention of both motility
sperm motility was measured at excision and after stor- and fertilizing capacity was observed in concentrated
age for 2, 5, 7, 10, 12 and 15 days. Spermatozoa showed (1:1 dilution), anaerobic suspensions (up to 25–30 days).
> 50% motility for 7 days at 0⬚C and for 5 days at 4⬚C. At Oviductal oocytes placed in SAR at 5, 10, 15, 20 and
15 days, only spermatozoa stored at 0⬚C still showed 25⬚C immediately after ovulation lost viability when
some motility (3%). Sperm suspensions were prepared at cooled rapidly to 5⬚C and stored for 2 h. However,
5 day intervals over 30 days in simplified amphibian oocytes retained viability for up to 8 h at the optimum
ringer (SAR) at dilutions of 1:1, 1:5 and 1:10 (w/v) storage temperature of 15⬚C. Thus, it is concluded that
testes:SAR. Aliquots from each dilution were stored at during short-term storage spermatozoa retain viability for
0⬚C in Eppendorf tubes opened at 5 day intervals of stor- longer than oocytes, and that spermatozoa in suspensions
age (aerated) or kept sealed (unaerated) (treatments: aer- retain viability for longer than spermatozoa stored in situ
ated or unaerated; 5, 10, 15, 20, 25 and 30 days storage). in excised testes.
sidered as either short-term storage at temperatures above
Introduction
0⬚C, or long-term storage at temperatures below 0⬚C.
In the last two decades ten species of Australian amphib- Short-term storage of amphibian gametes may be neces-
ians are presumed to have become extinct, and a large sary when the availability of male and female gametes for
number of extant species have decreased markedly in in vitro fertilization is asynchronous (Lahnsteiner et al.,
numbers and distribution (Mahony, 1996). Decreases in 1997) or the sources of gametes are separated spatially.
numbers of amphibians have also been recorded in Short-term storage may also be necessary during transport
Europe, and North and South America, with epidemic to a facility with the capacity to cryopreserve gametes for
pathogens implicated in many cases (Blaustein et al., long-term storage when spermatozoa and oocytes are re-
1994; Berger et al., 1998). For some species, populations moved from moribund or recently dead animals in the
survive as only a few individuals in wild or captive popu- field. There are few reports describing the short-term stor-
lations (Denton et al., 1997). There is considerable scope age of amphibian spermatozoa and oocytes. Rostand
for the application of assisted reproductive technologies to (1946) described the storage of spermatozoa from edible
manage and conserve highly endangered amphibian frog (Rana esculenta) at –4⬚C in a high concentration of
species using models developed for fish. For example, in glycerol for 10 days. Hollinger and Corton (1980) exam-
fish, stored spermatozoa may be used to maintain genetic ined the effect of storage of spermatozoa from African
diversity in small populations (Maitland, 1995; Holt et al., clawed toads (Xenopus laevis) for 1 and 6 days at 4⬚C at
1996) such as that of the Formosan landlocked salmon different concentrations on fertility using dose–response
(Oncorynchus masou formosanus) (Gwo et al., 1999). The relationships, and found a decrease in relative fertility of
use of short-term storage of spermatozoa is practised spermatozoa stored in suspensions to 60% of that of fresh
widely in the conservation of Atlantic sturgeon (Acipenser spermatozoa after 1 day and to 25% after 6 days.
huso) (DiLauro et al., 1994). The capacity of amphibian oocytes for extended short-
The handling and storage of gametes is central to many term storage appears to be lower than that of spermatozoa.
assisted reproductive procedures and broadly may be con- Storage of X. laevis oocytes in low ionic strength solution
(0.05 ⫻ DB) (DB contains 0.11 mol NaCl l–1, 0.0013 mol
KCl l–1, 0.00044 mol CaCl2 l–1, and NaHCO3 to pH 7.2
Email: birkb@cc.newcastle.edu.au (Wolf and Hedrick, 1971)) at 24⬚C resulted in low fertility
© 2001 Journals of Reproduction and Fertility
1470-1626/2001
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via free access168 R. K. Browne et al.
after 30 min, but with higher osmolality (3 ⫻ DB) eggs Petri dishes before allocation to experimental treatments
maintained fertility for 2 h and, in some cases, for 12–14 h (see below). The isotonic osmolality of SAR prevented acti-
(Wolf and Hedrick, 1971). Hollinger and Corton (1980) vation of sperm motility. After experimental treatments,
observed an increase in embryo abnormality and a samples were activated by dilution of one part sperm sus-
decrease in viability with time of oocyte storage before pension to two parts distilled H2O (70 mosmol kg–1). The
fertilization. percentage of motile spermatozoa and the vigour of motil-
The use of short-term storage of spermatozoa in the ity on a scale of 0–4 (Emmens, 1947) in activated samples
conservation and management of wild fish stocks has re- before and after experimental treatments were determined
ceived increased attention (DiLauro et al., 1994; under a phase-contrast microscope (⫻ 400).
Satterfield, 1995; Tiersch et al., 1998). Fish spermatozoa In preparation for the induction of ovulation, female
may be stored as seminal fluid or stripped from the testis toads were conditioned in a temperature-controlled room
by maceration in various diluents (Wayman et al., 1997). under a 12 h light: 12 h dark cycle at 28–30⬚C, and fed
The ratio of dilution of seminal fluid or testis mass to dilu- high protein dog food pellets (15% (w/w) protein (Luv
ent has been shown to affect viability over time, and dilu- Tender Chunks, Friskies Pet Care, Sydney, NSW) twice a
ents of different ionicity, and lipid and protein content week for at least 8 weeks. The high temperature and high
have either reduced or increased sperm storage time in protein diet of this protocol markedly improved the per-
fish (Erdahl and Grahan, 1987). Studies of freshwater fish centage of female toads ovulating after induction. Female
spermatozoa that have similar motility activation mecha- toads held at 21⬚C were induced to ovulate by the injec-
nisms to those of amphibian speramatozoa (activation by tion of six freshly collected male B. marinus pituitaries per
lowered osmolality) may provide a useful model for the female (homogenized in 1 ml of SAR) into their dorsal
short-term storage of amphibian spermatozoa (Hollinger lymph sacs using a 1 ml syringe with a 23 gauge needle
and Corton, 1980; Stoss et al., 1983). However, studies (Browne et al., 1998). Females were killed 8–12 h after
have reported osmotic damage to fish (Billard et al., 1990) induction of ovulation when oocytes were expressed from
and amphibian spermatozoa (Wolf and Hedrick, 1971; the cloaca, and the oocyte masses were removed surgically
Costanzo et al., 1998) only minutes after activation in from the oviducts.
fresh water, for example, only 50% of activated wood frog
(Rana sylvatica) spermatozoa survive after 5–10 min in Experiment 1: storage of whole testes in vitro at 0⬚C and
5 mosmol kg–1 (Costanzo et al., 1998). Cane toad (Bufo 4⬚C
marinus) spermatozoa typically maintains a high degree of
Testes removed from six male toads were placed in
motility and membrane integrity for 4–6 h when activated
1.5 ml Eppendorf tubes and immediately stored on ice
in 40 mosmol kg–1 (R. Browne, unpublished).
(0⬚C) or at 4⬚C in a refrigerator (n = 6 per treatment) with
This study investigated the short-term preservation of
one testis from each toad in each treatment. An initial
gametes from the cane toad (Bufo marinus). The effects
0.01 g sample was removed as a 1.5 mm slice from the
of temperature, aeration and dilution on the motility and
middle of the testes. Subsequently, a 0.75 mm slice from
fertilizing capacity of stored spermatozoa and the short-
the central end of one half and the opposite end of the
term storage of oocytes at different temperatures were
other half (about 10% of the testis mass) was removed at
investigated.
each of the sampling intervals. These samples were then
macerated in 0.01 ml SAR, activated and the sperm motility
was determined as described above.
Materials and Methods
Animals Experiment 2: storage of aerated and unaerated sperm
suspensions at 0⬚C at three dilutions
Wild caught, adult B. marinus were obtained from
Mareeba (17.00⬚S, 145.43⬚E), North Queensland. Testes At 5 day intervals over 30 days, 24 testes (from 12
were collected from males and oocytes from ovulated fe- toads) were individually macerated in 0.1 ml SAR and the
males between September and May. At Mareeba, B. mari- resulting suspensions were pooled and immediately stored
nus is an exotic species that spawns during the tropical on ice. The sperm concentration in the pooled suspension
wet season from October to May. Toads were killed by in- at each time interval was determined with a Neubauer
jection with 5 ml MS222 (Tricaine methanesulfonate, Ruth haemocytometer. From the pooled suspension, 24 ⫻ 0.1 ml
Consolidated Industries P/L., Annandale, NSW) at a con- aliquots were placed in 1.5 ml Eppendorf tubes. Eight of
centration of 2 g l–1 into the dorsal lymph sac and the these aliquots were not diluted any further; eight were di-
heart was removed to ensure death before the surgical luted with 0.4 ml SAR (1:5 dilutions) and eight were di-
removal of testes or oocytes. luted with 0.9 ml SAR (1:10 dilutions), giving a total of
After removal, testes were placed in 10 ml of simplified eight replicates of each dilution at each time interval. All
amphibian ringer (SAR) (Rugh, 1962) (113.0 mmol NaCl l–1, samples were stored at 0⬚C (on ice) between processing.
1 mmol CaCl2 l–1, 2.0 mmol KCl l–1, 3.6 mmol NaHCO3 l–1; Four suspensions of each dilution were exposed to air in a
220 mosmol kg–1, prepared in milli Q water, pH 7.2) in refrigerated room (4⬚C) at 5 day intervals (aerobic) and the
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via free accessShort-term storage of cane toad gametes 169
other four were kept sealed (anaerobic) from day 0. At 30 RF = TF/CF ⫻ 100 (TF: treatment fertilization rate; CF: con-
days after preparation of the first sample, the percentage of trol fertilization rate).
motile, activated spermatozoa in each sample was deter-
mined under a light microscope at ⫻ 400 magnification Statistical analyses
and the fertilizing capacity of each sample was determined.
Percentage fertilization data from Expt 1 was subjected
The fertilization rate of sperm suspensions was deter-
to chi-squared analysis after arcsine transformation be-
mined by flushing the samples with 10 ml distilled water
cause of non-parametric distribution. Motility and fertiliza-
into Petri dishes containing freshly collected oocytes
tion data for Expts 2 and 3 were arcsine transformed
(63 ⫾ 3.2, n = 88; mean ⫾ SEM per dish). Only samples
before testing for normality and homogeneity of variance,
showing some motility were tested for fertilizing capacity.
and then analysed by one-way ANOVA. Individual means
Sub-samples of oocytes from each female incubated with-
were ranked with the Tukey–Kramer HSD (honestly signifi-
out spermatozoa were negative controls for parthenogenetic
cant difference) test. All analyses were performed with JMP
activation (n = 4) and controls were fertilized with optimal
3.2 software package (SAS Institute Inc.).
sperm concentrations as for Expt 1 (n = 4). The mean
sperm concentration per dish was 1.06 ⫾ 0.04 ⫻ 107 ml–1
(mean ± SEM) for all treatments. Results
Experiment 1
Experiment 3: effect of temperature on storage of
Spermatozoa stored in whole testes retained the capac-
oocytes
ity for motility for up to 12 days at 4⬚C and 15 days at 0⬚C.
Oviductal oocytes from different females were used in Motility of > 40%, vigour 3, was evident for up to 7 and
each of three replicated fertilization trials. The fertilization 10 days at 4⬚C and 0⬚C, respectively. Motility values for
rate of the oviductal oocytes in each trial (time 0 controls) spermatozoa from testes maintained at 0⬚C were signifi-
was determined immediately after removal of the oocytes cantly higher than for spermatozoa from testes maintained
from the oviduct, and before division of the oviductal egg at 4⬚C testes, except in treatments after 7 days. Testicular
mass into sub-samples that were stored at 5, 10, 15, 20 spermatozoa at 0⬚C showed a linear decrease in motility
and 25⬚C. Samples of approximately 1500 oviductal for 7 days with a 10% decline between 2, 5 and 7 days,
oocytes (mass 7 g) from each female were stored in five then a 20% decline between 7, 10 and 12 days. In con-
containers containing 100 ml SAR. The temperature of trast, the motility of testicular spermatozoa at 4⬚C dropped
each container was maintained by immersion in a water 40% by day 2, maintained a value of approximately 50%
bath set at the appropriate temperature. Temperature in until day 7, and then declined precipitously to 1.5% at day
the containers was monitored with a mercury thermometer 12 (Fig. 1).
and maintained within ⫾ 1.0⬚C. Fertilization rate was de-
termined at time 0 for controls and at 1 and 2 h, and at 2 h
intervals subsequently for treatments until 12 h had
elapsed. Strips of oviductal oocytes (oviductal eggs of B. 4 100 a
marinus are held in gelatinized strips) from each control
a
Relative percentage motile
(time 0) or treatment container were cut into appropriate
80
lengths (121 ⫾ 22, n = 96; 126 ⫾ 16, n = 96; 137 ⫾ 17.2, 3 a
Degree of motility
n = 63; mean ± SD oocytes for trials 1, 2 and 3, respec- a
tively, n = number of Petri dishes) and placed in three sep- 60 b
arate Petri dishes to determine the fertilization rate. Both 2
c
testes from two male B. marinus were macerated in 0.4 ml 40
SAR, producing an initial sperm concentration of
0.5–1.0 ⫻ 109 ml–1. Fertilization in each Petri dish was 1
20
achieved by the addition of 0.025 ml sperm suspension in
10 ml distilled water at 21⬚C to give a final sperm con-
0 0
centration of 1.25–2.5 ⫻ 106 ml–1. The concentration of 0 2 4 6 8 10 12 14
spermatozoa needed to achieve optimal fertilization rates Days of testes storage
with B. marinus is 1 ⫻ 106 ml–1 (Browne et al., 1998). The
percentage of fertilized oocytes in each Petri dish was Fig. 1. Mean percentage and degree of motility of activated cane
toad (Bufo marinus) testicular spermatozoa. Testes were stored at
determined 3 h after fertilization from the proportion of
0⬚C or 4⬚C before release of spermatozoa by maceration. (䊉) 0⬚C,
oocytes showing first cleavage. Samples of oocytes from percentage motile; (䊏) 0⬚C, degree of motility; (䊊) 4⬚C, percent-
each female were also left without the addition of sperma- age motile; (䊐) 4⬚C, degree of motility. Letters indicate significant
tozoa at each sampling period as negative controls to test differences between 0⬚C and 4⬚C means for the same parameter
for parthenogenetic activation. The relative fertilization (percentage motile or degree of motility) at the same storage
rate (RF) for each oocyte trial was calculated as period: a: P < 0.001; b: P < 0.01; and c: P < 0.05.
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via free access170 R. K. Browne et al.
Experiment 2 Experiment 3
There was no cleavage in parthenogenic control Control (time zero) fertilization rates of fresh oocytes
oocytes. At day 10, concentrated sperm suspensions with untreated spermatozoa for trials 1, 2 and 3 were
showed a significantly (P < 0.05) higher capacity for motil- 88.8 ± 9.0, 84.0 ± 6.6 and 80.9 ± 5.5, respectively (mean
ity than did diluted suspensions. Sperm clumping was ob- ± SD, n = 3). The greatest retention of oocyte viability over
served in activated 1:1 suspensions, and the degree of time was observed at 15⬚C, with > 90% of the initial rela-
clumping increased with storage time and time after acti- tive fertilization rate being maintained at 6 h after ovula-
vation (Fig. 2a). The fertilization rate for controls (t = 0) tion, a rate significantly higher than in the other treatments
was 96.8 ± 0.4% (mean ± SEM). At day 5, there were signif- (P < 0.05). Storage at 5⬚C resulted in a precipitous decline
icantly higher (P < 0.05) relative fertilization rates in 1:5 to zero relative fertilization rate at 2 h (Fig. 4).
dilutions (about 85%) than in the other four treatments
(about 55%). At day 10, the mean values had formed into
100
four significantly different (P < 0.05) fertilization rate clus-
Relative percentage fertilization
ters. In contrast to day 10 percentage motilities, day 10 fer-
80
tilization rates of undiluted suspensions were lower than
they were for 1:5 diluted suspensions. Spermatozoa from
anaerobic undiluted suspensions stored for up to 30 days 60
fertilized oocytes (Fig. 2b). There was a significant
(r2 > 0.50) correlation between mean motility and fertiliza- 40
tion rate at days 0, 5 and 25 of storage (Fig. 3).
20
(a)
80
a 0
Percentage motile
60
0 20 40 60 80
Relative percentage motility
40
Fig. 3. Correlation between mean relative percentage sperm
motility and mean relative percentage fertilization of cane toad
20 b (Bufo marinus) oocytes fertilized by spermatozoa stored in sperm
suspensions for 5 days (䊉) r2 = 0.52 (_____); 10 days (䉫) r2 = 0.02;
15 days (䊐) r2 = 0.23; and 20 days (䉱) r2 = 0.84 (----).
0
0 5 10 15 20 25 30
Days of sperm suspension storage 100 a
(b)
100 a
a
Relative percentage fertilization
Relative percentage fertilization
a
80
80 b
b b
b
60 60 b
c b
40
40
20
c
d c
20
0
0 5 10 15 20 25 30
Days of sperm suspension storage
0
Fig. 2. Mean percentage motility of cane toad (Bufo marinus) acti- 0 1 2 3 4 5 6 7 8 9 10 11 12
vated spermatozoa (a) and relative percentage fertilization of oocytes Time after collection (h)
(b) from stored suspensions at dilutions of 1:1, 1:5, or 1:10 (w/v,
testes: simplified amphibian ringer), at 0⬚C in aerated and unaerated Fig. 4. Relative percentage fertilization of cane toad (Bufo mari-
conditions: (䊐) aerated, 1:1; (䊊) aerated, 1:5; (䉭) aerated, 1:10; (䊏) nus) oocytes held at 5 (䊏), 10 (䉫), 15 (䉱), 20 (䊊) and 25⬚C (䊉) in
unaerated, 1:1; (䊉) unaerated, 1:5; (䉱) unaerated 1:10. Individual simplified amphibian ringer against time after collection (n = 3 for
means or clusters with different letters are significantly different 10, 15 and 20⬚C; n = 2 for 5 and 25⬚C). Pooled SE = 7.6, n = 74.
(P < 0.05). Mean percentage motility pooled SE = 2.48, n = 23. Mean Means that share the same letter are not significantly different
relative percentage fertilization pooled SE = 5.63, n = 21. (P < 0.05).
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via free accessShort-term storage of cane toad gametes 171
used in the present study, even though B. marinus is a
Discussion
tropical–warm temperate species. There are no reports to
The present study showed that it is possible to maintain our knowledge of amphibian or fish (Stoss et al., 1983)
high sperm motility and fertilizing ability after storage of spermatozoa displaying cold shock, a phenomenon that is
testis and sperm suspensions from B. marinus for up to 10 common in eutherian mammal (White, 1993) but not mar-
days in vitro at 0⬚C (on ice), although storage in diluent sus- supial spermatozoa (Taggart et al., 1996). Costanzo et al.
pensions was more effective than storage in whole testes. (1998) demonstrated that the spermatozoa of R. sylvatica
After storage for 10 days of storage, higher sperm motility can survive sub-zero temperatures of –40C with naturally
and fertilizing capacity were maintained in SAR suspen- conferred cryoprotection. Consequently, there may be op-
sions than in whole testes. Some sperm motility and fertiliz- timism for short-term, low temperature storage with other
ing capacity was still evident in some samples after 30 days temperate climate anurans and tropical–warm temperate
of storage. The effect of aerobic versus anaerobic condi- species.
tions had only a minor effect on sperm viability in stored There was a positive correlation (r2 > 0.5) between the
suspensions. There was evidence that maintenance of sper- motility and fertilizing capacity of stored B. marinus sper-
matozoa in suspensions at higher concentrations provided matozoa at the beginning (day 5) and end (day 20) of the
the most favourable conditions for short-term storage. The storage period. The greatest deviation from this correlation
finding that amphibian spermatozoa, in situ in excised occurred on day 10 when the fertilization rate of sperma-
testes or in vitro in suspensions, may be maintained un- tozoa in undiluted suspensions was lower than it was in
frozen for a number of weeks at low temperature indicates 1:5 dilutions, while the opposite was the case for sperm
that it is feasible to recover viable spermatozoa from re- motility. The high fertilizing capacity of the 1:5 dilutions,
cently dead or moribund amphibians and store them for even though they displayed low motility, is explained by
some time. The spermatozoa may then be cryopreserved or the high sperm concentration (107 ml–1) which is one order
used for in vitro fertilization. These findings should be of of magnitude above the saturation concentration of sper-
use in the management of small, captive or wild popula- matozoa needed to achieve 100% fertilization (106 ml–1)
tions that contain valuable or rare animals. and two orders of magnitude higher than that required for
The longevity of unaerated B. marinus sperm suspen- 80% fertilization (105 ml–1) rates in fresh samples (Browne
sions indicates that either metabolic rate is lowered suffi- et al., 1998). The absence of the sperm clumping that oc-
ciently for there to be an adequate supply of oxygen, or curred predominantly in undiluted suspensions may also
that anaerobic metabolism maintains metabolism and cel- have increased the fertilization rate in this treatment.
lular viability at low temperatures in this species. In fish, The present study showed that the motility of spermato-
the effect of gaseous exchange on sperm storage varies zoa retained in whole testes was much higher over time at
widely. Some species require aerobic respiration while 0⬚C than at 4⬚C, with substantial motility (28–44%) re-
spermatozoa from other species may persist with energy tained for 10–12 days at 0⬚C. This period of storage in situ
derived only from glycolysis or stored ATP (Büyükatipoglu in excised B. marinus testes was longer than that reported
and Holtz, 1978). With turbot (Scophthalmus maximus) for some fish species. Testicular spermatozoa only retained
spermatozoa, an atmosphere of air rather than pure oxygen viability for 2 days at 0⬚C in herring (Clupea pallasi)
increases viability with storage (Chereguini et al., 1997); (Dushkina, 1975) and for 18 h at 4⬚C in brown trout
however, an atmosphere of pure oxygen increases the stor- (Salmo trutta), which was far less than for sperm suspen-
age time of rainbow trout (Oncorhynchus mykiss) sperma- sions of the same species (Billard et al., 1981). In amphi-
tozoa (Billard, 1981). Oxygen dependence increases the bians, the testes are the site of sperm storage in males.
potential for damaging oxidative effects on membrane in- Testicular spermatozoa are embedded in Sertoli cells
tegrity and cell metabolism (Ciereszko and Dabrowski, until they are released by testicular hydration induced
1994). For some species, a deeper layer of sperm suspen- by gonadotrophins (Burgos and Vitale-Calpe, 1967).
sions in aerated containers, producing hypoxic conditions, Spermatozoa may be stored for many months in the testes
increases storage time (Clemens and Hill, 1969). It is not until the occurrence of suitable spawning conditions
clear from the present study whether the improved (Lofts, 1974). The reason for the higher survival of sperma-
longevity of B. marinus spermatozoa in concentrated sus- tozoa in whole testes at 0⬚C than at 4⬚C, and the rapid de-
pensions was due to an increased availability of glycolytic crease in sperm motility after 2 days storage at 4⬚C was not
substrate. Dilution of fish sperm suspensions has also re- determined in the present study. However, with testicular
sulted in a reduction of storage life (Stoss and Holtz, 1983) excision, Sertoli and other somatic cells in the testes that
but further studies will be required to determine whether are dependent on aerobic metabolism may die more
the capacity to maintain anaerobic metabolism is a general rapidly at a relatively higher metabolic rate and oxygen
characteristic of amphibian sperm metabolism, and demand. Higher temperature may also reduce enzyme ac-
whether addition of glycolytic substrates improves sperm tivity and promote greater release of toxic metabolites at
storage. 4⬚C. With maceration, a proportion of spermatozoa re-
There was no evidence of cold shock in B. marinus leased are immature with cytoplasmic droplets still pre-
spermatozoa with the rate of cooling of samples on ice sent. Further studies may determine the susceptibility of
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via free access172 R. K. Browne et al.
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