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• W1504892 abstract "Kangaroo spermatozoa have proven extremely difficult to cryopreserve such that despitenumerous empirical studies using high concentrations of glycerol and/or DSMO, the bestpost-thaw motility has only been in the order of 10%. The efficacy of glycerol as acryoprotectant for marsupial spermatozoa is somewhat paradoxical, being necessary athigh concentrations for adequate cryopreservation, but at the same time “cytotoxic.” Theunderlying objectives of this project were to understand the causes of cryoinjury tokangaroo spermatozoa and to apply this information in an attempt to develop a reliablemethod for cryopreservation. These objectives were addressed through strategically linkedstudies incorporating the documentation of cryoinjury, hypothesis driven investigations intothe causes of cryoinjury and the application of novel cryopreservation protocols.The ultrastructure and freeze-fracture of caput and cauda epididymal Eastern greyKangaroo (EGK) spermatozoa at 35°C, 4°C and following cryopreservation with andwithout 20% glycerol was investigated. The addition of 20% glycerol resulted in significantdamage to the sperm plasma membrane and mitochondria compared to no glycerol at thesame temperatures (P < 0.05). Following cryopreservation, 20% glycerol significantlyimproved the preservation of the cauda epididymal sperm plasma membrane andmitochondria and reduced the incidence of axonemal damage and periaxonemal spaces.For caput epididymal spermatozoa, glycerol only improved the preservation of the plasmamembrane following cryopreservation (P < 0.05).Freeze fracture microscopy revealed a pattern of helically wound intramembranousparticles in the plasma membrane over the fibre network of the mid piece of the sperm tail.After thawing, the plasma membrane was damaged such that this structure was missing iniipatches, and the helical rows of particles were mal-aligned. On the principal piece,particles were arranged randomly at physiological temperatures; however, upon cooling to4°C with 20% glycerol, the particles become aggregated. Once re-warmed (35°C),particles over the principal piece resumed their random organisation. This finding is furtherevidence of a reversible phase transition of the macropod sperm plasma membrane duringcooling that is not associated with a loss of motility or membrane integrity.In attempt to investigate cryoinjury caused by alternative cryoprotectants the next studiesexamined the effect of cryoprotectants (20% DMSO, a 10% DMSO / 10% glycerol mixture,20% glycerol and 1 M sucrose solution) on kangaroo sperm structure and function, alongwith the effect of varying concentrations of glycerol on sperm mitochondrial function.Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 mins at35°C in each cryoprotectant and the plasma membrane integrity (PMI) and motilityassessed using light microscopy. The same samples were fixed for TEM and theultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerolon the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubatedwith JC-1 in Tris-citrate media at 35°C for 20 mins in a range of glycerol concentrations (0,5, 10, 15 and 20%) and the mitochondrial membrane potential (MMP) and plasmamembrane integrity determined. As expected, incubation of spermatozoa in 20% glycerolfor 10 mins resulted in a significant reduction in motility, PMI and ultrastructural integrity.Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility orPMI but a significant loss of structural integrity when compared to the control spermatozoa(0% Glycerol). However, 20% DMSO was overall less damaging to sperm ultrastructurethan glycerol, 10% glycerol and 10% DMSO, and sucrose. While all glycerolconcentrations had an adverse effect on mitochondrial function, the statistical modelspresented for the relationship between MMP and glycerol predicted that sperm, wheniiiadded to 20% glycerol, would loose half of their initial MMP immediately at 35°C and after19.4 mins at 4°C. Models for the relationship between PMI and glycerol predicted thatsperm would loose half of their initial PMI after 1.8 mins at 35°C and 21.1 mins at 4°C.These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermthen it is best administered at 4°C and that mitochondrial function is more sensitive toglycerol than PMI. Further studies were directed at investigating strategies that reduceexposure of spermatozoa to glycerol during processing and evaluating the cryoprotectiveproperties of 20% DMSO for kangaroo sperm.The first hypothesis to be tested with respect the causes of cryopathology examined thepossibility that the ultrastructural changes that occur to the kangaroo sperm duringepididymidal maturation reduced the tolerance of the sperm cell organelles to respond toosmotic flux, glycerol cytotoxicity and ice-crystal damage. Caput and cauda epididymidalspermatozoa were recovered from red-necked wallabies (Macropus rufogriseus) and EGK.In Experiment 1, caput and cauda epididymal spermatozoa were frozen and thawed usinga standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol.Although cryopreservation of caput epididymidal spermatozoa resulted in a significantincrease in sperm plasma membrane damage, they were more tolerant of the procedurethan spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2,caput and cauda epididymidal spermatozoa were diluted into phosphate-buffered salinemedia of varying osmolarity and their osmotic tolerance was determined. Plasmamembranes of caput epididymidal spermatozoa were clearly more tolerant of hypo-osmoticmedia than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput andcauda epididymidal spermatozoa were incubated in Tris-citrate buffer with and without20% glycerol at 35 and 4°C to examine the cytotoxic effects of glycerol. At bothtemperatures, caput epididymidal spermatozoa showed less plasma membrane damageivcompared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05).The results from these experiments clearly indicate that epididymal maturation of kangaroospermatozoa resulted in a decreased ability to withstand the physiological stressesassociated with cryopreservation.The second hypothesis to be tested with respect the causes of cryopathology examinedwhether filamentous (F) actin associated with the complex cytoskeleton of the kangaroosperm head and tail may be contributing to lack of plasma membrane plasticity and aconsequent loss of membrane integrity during cryopreservation. In the first study, thedistribution of G and F actin within EGK cauda epididymidal spermatozoa was successfullydetected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam),respectively. G-actin staining was most intense in the acrosome but was also observedwith less intensity over the nucleus and mid-piece. F-actin was located in the spermnucleus but was not discernable in the acrosome or sperm tail. To investigate whethercytochalsin D (a known F-actin depolymerising agent) was capable of improving theosmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypoosmoticmedia (61 and 104 mOsm) containing a range of cytochalasin D concentrations(0-200μM). Cytochalsin D had no beneficial effect on the plasma membrane integrity ofsperm incubated in hypo-osmotic media. The results of this study indicated that the F-actindistribution in cauda epididymidal spermatozoa of the EGK was surprising different to thatof the Tammar Wallaby (M. eugenii) and that cytochalsin-D does not appear to improvethe tolerance of EGK cauda epididymidal sperm to osmotic induced injury.The aims of the final study in this thesis were to investigate alternative techniques for thecryopreservation of kangaroo spermatozoa that reduce or eliminate the need for glycerol.This was investigated by (1) freezing sperm with 20% glycerol in pre-packaged 0.25 mLCassou straws in such a way as to enable rapid dilution of the glycerol post-thaw, (2)vinvestigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide(DMA - 10, 15 and 20% v/v) as cryoprotectants and (3) to evaluate the potential use ofultra-rapid small volume vitrification cooling of kangaroo spermatozoa with or withoutcryoprotectant (20% v/v Glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-strawpost-thaw dilution (1: 20) of 20% glycerol produced no significant improvement in postthawviability of kangaroo spermatozoa. Cryopreservation of kangaroo spermatozoa in20% DMSO resulted in only 2% motile sperm and 5% of sperm with an intact plasmamembrane. While kangaroo spermatozoa frozen by ultra-rapid freezing techniquesshowed no evidence of post-thaw viability irrespective of whether cryoprotectant was usedor not, sperm frozen in 10 - 20% DMA showed post-thaw motility and plasma membraneintegrity of 11 - 13% and 18 - 23% respectively. Although still modest compared to othermarsupial and eutherian spermatozoa, these results represent a significant improvementin the development of a cryopreservation procedure for kangaroos and provide the basisfor further investigation using other amide-based cryoprotectants." @default.
• W1504892 created "2016-06-24" @default.
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• W1504892 date "2008-03-01" @default.
• W1504892 modified "2023-09-27" @default.
• W1504892 title "Cryopreservation of Kangaroo Spermatozoa" @default.
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