Storage of cane toad (Bufo marinus) sperm for 6 days at 0°C with subsequent cryopreservation

2002 ◽  
Vol 14 (5) ◽  
pp. 267 ◽  
Author(s):  
R. K. Browne ◽  
J. Davis ◽  
J. Clulow ◽  
M. Pomering
Keyword(s):  
Sperm Motility ◽  
Membrane Integrity ◽  
Bufo Marinus ◽  
Cane Toad ◽  
Before And After ◽  
Cryopreserved Sperm ◽  
High Concentrations ◽  
Toad Bufo ◽  
Sperm Recovery

We investigated the recovery of motility of cane toad (Bufo marinus) sperm after storage for 6 days at 0°C (on ice) and after subsequent cryopreservation. Sperm suspensions were prepared from testes macerated in either simplified amphibian Ringer (SAR) or 10% (w/v) sucrose diluents, with 15% or 20% (v/v) glycerol or Me2SO as cryoprotectants, and were stored for 6 days. Alternatively, suspensions were prepared in either SAR or 10% (w/v) sucrose diluent and stored for 6 days, after which some of these suspensions were kept in diluents alone or, alternatively, had 15% or 20% (v/v) glycerol or Me2SO added. All treatments (suspensions) were then cryopreserved. Sperm motility was measured at Day 1 and Day 6 (before and after cryopreservation). A substantial and variable proportion (range 0%–40%) of sperm was immotile in suspensions immediately after preparation from testes macerates. Sperm stored in either SAR or 10% (w/v) sucrose diluent maintained approximately 75% motility for 6 days, but few sperm survived cryopreservation. After storage and cryopreservation, recovery of motility was substantially higher with Me2SO than in glycerol. However, both cryoprotectants exhibited toxicity at high concentrations. Glycerol was more toxic than Me2SO in 10% (w/v) sucrose than in SAR diluent, both before and after cryopreservation. The addition of some cryoprotectants to suspensions before storage gave greater recovery both before and after cryopreservation. After cryopreservation, the highest rate of sperm recovery was in suspensions with 10% (w/v) sucrose and 15% (v/v) Me2SO added prior to storage (mean (±SEM) 46 ± 7% relative to initial; 39 ± 6% absolute). Sperm were also stored for 6 days at 0°C in suspensions or testes (with suspensions then prepared from testes) and cryopreserved. Sperm maintained higher recovery and membrane integrity both before and after cryopreservation when stored in suspensions rather than in testes.

Short-term storage of cane toad (Bufo marinus) gametes

Reproduction ◽  
2001 ◽  
pp. 167-173 ◽  
Author(s):  
RK Browne ◽  
J Clulow ◽  
M Mahony
Keyword(s):  
Sperm Motility ◽  
Storage Temperature ◽  
Bufo Marinus ◽  
Cane Toad ◽  
Short Term ◽  
Optimal Protocol ◽  
Short Term Storage ◽  
Rare And Endangered ◽  
Toad Bufo ◽  
Term Storage

The responses of cane toad (Bufo marinus) gametes, used as a model for the development of assisted reproduction techniques for rare and endangered amphibians, to short-term storage at temperatures > 0 degrees C were studied. Whole excised testes were stored at 0 degrees or 4 degrees C for 15 days, and sperm motility was measured at excision and after storage for 2, 5, 7, 10, 12 and 15 days. Spermatozoa showed > 50% motility for 7 days at 0 degrees C and for 5 days at 4 degrees C. At 15 days, only spermatozoa stored at 0 degrees C still showed some motility (3%). Sperm suspensions were prepared at 5 day intervals over 30 days in simplified amphibian ringer (SAR) at dilutions of 1:1, 1:5 and 1:10 (w/v) testes:SAR. Aliquots from each dilution were stored at 0 degrees C in Eppendorf tubes opened at 5 day intervals of storage (aerated) or kept sealed (unaerated) (treatments: aerated or unaerated; 5, 10, 15, 20, 25 and 30 days storage). After 30 days, sperm motility and fertilizing capacity were determined. The optimal protocol for sperm storage up to 10 days, as assessed by the retention of fertilizing capacity, was as a 1:5 testis:SAR (w/v) suspension, whereas the longest absolute retention of both motility and fertilizing capacity was observed in concentrated (1:1 dilution), anaerobic suspensions (up to 25-30 days). Oviductal oocytes placed in SAR at 5, 10, 15, 20 and 25 degrees C immediately after ovulation lost viability when cooled rapidly to 5 degrees C and stored for 2 h. However, oocytes retained viability for up to 8 h at the optimum storage temperature of 15 degrees C. Thus, it is concluded that during short-term storage spermatozoa retain viability for longer than oocytes, and that spermatozoa in suspensions retain viability for longer than spermatozoa stored in situ in excised testes.

Z and W sex chromosomes in the cane toad (Bufo marinus)

2009 ◽  
Vol 17 (8) ◽  
pp. 1015-1024 ◽  
Author(s):  
John Abramyan ◽  
Tariq Ezaz ◽  
Jennifer A. Marshall Graves ◽  
Peter Koopman
Keyword(s):  
Sex Chromosomes ◽  
Bufo Marinus ◽  
Cane Toad ◽  
Toad Bufo

An investigation of the allosteric functioning of the haemoglobin of the cane toad, Bufo marinus

1982 ◽  
Vol 73 (4) ◽  
pp. 991-995 ◽  
Author(s):  
Thomas Brittain ◽  
Donald Barber ◽  
Colin Greenwood ◽  
Rufus M. Wells
Keyword(s):  
Bufo Marinus ◽  
Cane Toad ◽  
Toad Bufo

Indirect impacts of invasive cane toads (Bufo marinus) on nest predation in pig-nosed turtles (Carettochelys insculpta)

2006 ◽  
Vol 33 (5) ◽  
pp. 349 ◽  
Author(s):  
J. S. Doody ◽  
B. Green ◽  
R. Sims ◽  
D. Rhind ◽  
P. West ◽  
...  
Keyword(s):  
Nest Predation ◽  
Bufo Marinus ◽  
Cane Toad ◽  
Relative Population ◽  
Monitor Lizard ◽  
Before And After ◽  
Marked Decline ◽  
Study Sites ◽  
Indirect Impacts ◽  
One Year

The cane toad (Bufo marinus) was introduced into Australia in 1935. Because this toxic frog is novel to the Australian fauna, its introduction has impacted native fauna in a variety of ways. We anticipated a severe decline in the yellow-spotted monitor lizard (Varanus panoptes) associated with the arrival of cane toads along the Daly River, Northern Territory, and predicted a simultaneous impact on nest predation in the pig-nosed turtle (Carettochelys insculpta) because the lizard is the chief predator of C. insculpta eggs at the site. We surveyed for monitors and cane toads for five years at two sites before and after the arrival of cane toads, and surveyed for turtle nest predation for three years before, and one year after, the arrival of the toads. Collectively, our data and observations, combined with unpublished reports, indicate that: (1) cane toads arrived at our study sites during the wet seasons of 2003–04 and 2004–05; (2) the lizard V. panoptes readily succumbs to cane toad toxins; (3) . panoptes has experienced a marked decline in relative population numbers coincident with the arrival of the toads at the site; and (4) V. panoptes has been reduced to such low numbers that it is currently no longer a significant predator of pig-nosed turtle eggs.

87 OSMOTIC SENSITIVITY OF OKAPI SPERMATOZOA AND DEVELOPMENT OF CRYOPRESERVATION PROTOCOLS USING CRYOMICROSCOPY

2008 ◽  
Vol 20 (1) ◽  
pp. 124 ◽  
Author(s):  
L. M. Penfold
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Repeated Measures ◽  
Membrane Integrity ◽  
Latin Square ◽  
Crystal Formation ◽  
Plasma Membrane Integrity ◽  
Power Of The Test ◽  
Before And After ◽  
Cooling Effects

Okapi would benefit from artificial insemination with frozen-thawed sperm in cases where aggression prevents mating or where individuals are geographically disparate. Effective sperm cryopreservation is a prerequisite to this goal. Ejaculates (n = 20) were collected from 7 anesthetized adult male okapi housed individually, or with a female for breeding, throughout the year by electroejaculation, and semen and sperm parameters were assessed. Semen aliquots were centrifuged; resuspended in 500 µL of PBS with the osmolarity adjusted to 35, 75, 150, 600, 1200, and 2400 mOsm; and incubated for 30 min before returning to isosmotic conditions. Semen was extended in TEST containing 1%, 2%, or 4% glycerol with or without 0.5% Equex (Minitube, Verona, WI, USA); 5-µL aliquots were cooled in a Latin square design on a Linkam BCS 196 cryomicrostage (Linkam Scientific, Tadworth, Surrey, UK) at 20�C min–1 to –6� –12�C at which point ice crystal formation was induced (seeded), and cooled further to –70�C before warming at 50�C min–1 to 35�C (okapi body temperature). To investigate cooling effects only, raw ejaculate was cooled to –6�C without seeding and warmed to 35�C. Percent sperm motility and plasma membrane integrity (PMI) were recorded before and after treatments. Differences were examined using one-way repeated measures analysis of variance. No differences in motility, total sperm numbers, or percent normal morphology were observed throughout the year (P > 0.05), although the power of the test was low so that negative findings should be interpreted cautiously. Mean semen volume was 1.3 � 0.19 mL, sperm motility was 29 � 3.2%, with a PMI of 39 � 6.8%; 48 � 2.8% were morphologically normal. High proportions of non-motile, plasma membrane-damaged cells were noted in every ejaculate, and whiplash motility, possibly indicating spontaneous capacitation, was observed in several ejaculates 1 h after collection. Motility was dramatically reduced on either side of isosmotic conditions and was more sensitive to osmotic pressure than was plasma membrane integrity. Cooling of raw ejaculate to sub-zero temperatures without freezing did not result in any loss of motility or PMI, indicating cold tolerence. Superior results were obtained when sperm were frozen-thawed in TEST containing 4% glycerol with 0.5% Equex. Findings indicate that okapi semen collected by electroejaculation routinely contain high numbers of non-motile and plasma membrane-damaged spermatozoa, apparently unrelated to season or the length of time since the male was housed with a breeding female. Okapi spermatozoa are remarkably intolerant of departures from isosmotic conditions, indicating a lack of ability to regulate or withstand volume excursions during osmotic stress events; however, cooling to sub-zero temperatures in the absence of cryoprotectant did not reduce percent sperm motility or PMI, indicating resistance to cold shock. Increasing and maintaining proportions of motile, membrane-intact spermatozoa prior to and during cryopreservation will be critical for development of freezing protocols for this species.

Organization of the Vascular Supply to the Brain of the Toad Bufo-Marinus

1990 ◽  
Vol 38 (4) ◽  
pp. 375
Author(s):  
GK Snyder ◽  
B Gannon ◽  
RV Baudinette
Keyword(s):  
Fourth Ventricle ◽  
Third Ventricle ◽  
Vascular Supply ◽  
Bufo Marinus ◽  
Cane Toad ◽  
The Third ◽  
Toad Bufo ◽  
The Brain ◽  
Caudal Rami ◽  
Vascular Corrosion Casts

The vasculature of the brain of the cane toad, Bufo marinus, was studied by means of scanning electron microscopy of vascular corrosion casts. The sole arterial supply to the brain is from branches of the internal carotids. The forebrain is supplied by several branches from the rostra1 ramus of the carotids; the caudal ramus gives rise to a single branch which supplies the mesencephalon and cerebellum. The caudal rami fuse to form a single basilar artery which supplies the medulla. The vascular supply to the choroid plexus of the third ventricle is arterial; the vascular supply to the choroid of the fourth ventricle is entirely venous. Microvascular geometry in the toad brain is specific to the region of the brain examined, ranging from simple long capillaries with few anastomotic connections to much shorter, highly convoluted capillaries with many anastomotic connections.

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