Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

2008 ◽  
Vol 20 (6) ◽  
pp. 724 ◽  
Author(s):  
Yeng Peng Zee ◽  
William V. Holt ◽  
Jaime Gosalvez ◽  
Camryn D. Allen ◽  
Vere Nicolson ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Membrane Integrity ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Similar Degree ◽  
Sperm Cryopreservation ◽  
Phascolarctos Cinereus ◽  
Degree Of Protection ◽  
Fish Sperm ◽  
Cryopreservation Protocol

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 ± 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 ± 3.5%; P < 0.05) and after 2 h incubation at 35°C (35.8 ± 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.

scholarly journals Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

2017 ◽  
Vol 29 (8) ◽  
pp. 1556 ◽  
Author(s):  
S. Morrow ◽  
J. Gosálvez ◽  
C. López-Fernández ◽  
F. Arroyo ◽  
W. V. Holt ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Subsequent Development ◽  
Xenopus Tropicalis ◽  
Model Organisms ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Cryopreservation ◽  
Sperm Plasma Membrane

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P < 0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

24. A generalized fish sperm cryopreservation protocol of livebearers in poeciliidae

Cryobiology ◽  
2008 ◽  
Vol 57 (3) ◽  
pp. 320
Author(s):  
Qiaoxiang Dong ◽  
Chunli Sun ◽  
Xinyou Su ◽  
Xingen Zhao ◽  
Changjiang Huang
Keyword(s):  
Sperm Cryopreservation ◽  
Fish Sperm ◽  
Cryopreservation Protocol

92 Sperm Cryopreservation in the Burmese Python (Python bivittatus) as a Model for Endangered Snakes

2018 ◽  
Vol 30 (1) ◽  
pp. 185
Author(s):  
C. Young ◽  
N. Ravida ◽  
M. Rochford ◽  
B. Durrant
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Monitoring Program ◽  
Florida Everglades ◽  
Equal Weight ◽  
Sperm Cryopreservation ◽  
Acrosome Integrity ◽  
Python Bivittatus ◽  
Southern Florida ◽  
Cryopreservation Protocol

The Burmese python (Python bivittatus) is listed as vulnerable by the International Union for Conservation of Nature (IUCN). Released pet Burmese pythons have detrimental effects on fauna native to southern Florida and are responsible for localised declines of several species in some parts of the Everglades National Park (IUCN, 2012; 10.2305/IUCN.UK.2012-1.RLTS.T193451A2237271.en). As part of an invasive species monitoring program, Burmese pythons were captured in the Florida Everglades and used as a model for the development of sperm cryopreservation protocols for endangered snakes. Sperm was collected by flushing the vas deferens postmortem and initial motility score (IMS; % motile × speed of progression2), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded before cryopreservation. Sperm was extended in TEST-yolk buffer with final dimethyl sulfoxide (DMSO) or glycerol (GLY) concentrations of 8, 12, or 16%, or combinations of DMSO and GLY with final concentrations of 4:4, 6:6, or 8:8%. Sperm in 500 µL of extender was frozen in vials at 0.3°C/min to –40°C before storage in liquid nitrogen. For each treatment, triplicate vials from each of 3 males were thawed at 37°C for 90 s. Cryoprotectant was removed by centrifugation and the sperm pellet was resuspended in TCM-199+HEPES. Sperm was evaluated at 22°C immediately following resuspension (T0) and at 60 (T60) minutes. All data were expressed as a percentage of initial (%IMS, %IPL, and %IAC). The effects of freeze method on %IMS, %IPL and %IAC were analysed by ANOVA and Tukey’s HSD test. Freeze method significantly affected %IMS at T0 (P = 0.0004) and T60 (P = 0.0001), with sperm frozen in the 6%DMSO:6%GLY and 4%DMSO:4%GLY treatments resulting in the highest %IMS at both T0 (19.4% and 17.7%, respectively) and T60 (26.7% and 14.4%, respectively). Regardless of cryoprotectant concentrations, sperm frozen in a combination of DMSO and GLY exhibited significantly higher %IMS than all treatments of DMSO or GLY alone (P < 0.0001 at T0 and T60). The %IPL was significantly affected by freeze method at T0 (P < 0.0001) and T60 (P = 0.0266). Sperm frozen in 8%DMSO:8%GLY and 6%DMSO:6%GLY retained greater %IPL at both T0 (69.1% and 65.7%, respectively) and T60 (47.8% and 49.9%, respectively). Acrosome integrity was significantly affected by freeze method at T0 (P < 0.0001) and sperm frozen in 8% DMSO resulted in the greatest %IAC (56.4%). In addition, all DMSO and DMSO:GLY treatments preserved a significantly greater proportion of intact acrosomes than GLY alone (P < 0.0001). To simplify these analyses and to determine the best overall freeze method for this species, a sperm quality index (SQI) was calculated, giving equal weight to each of the 3 measured indicators of cryosurvival. The SQI analysis revealed that Burmese python sperm frozen at 0.3°C/min in either 6%DMSO:6%GLY or 4%DMSO:4%GLY exhibited significantly higher post-thaw viability at T0 and T60 than all other treatments. This study represents the first comparative, comprehensive attempt to develop a sperm cryopreservation protocol for any snake species.

Differential resistance of mammalian sperm chromatin to oxidative stress as assessed by a two-tailed comet assay

2011 ◽  
Vol 23 (5) ◽  
pp. 633 ◽  
Author(s):  
María Enciso ◽  
Stephen D. Johnston ◽  
Jaime Gosálvez
Keyword(s):  
Oxidative Stress ◽  
Comet Assay ◽  
Disulfide Bonds ◽  
Homo Sapiens ◽  
Chromatin Condensation ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Phascolarctos Cinereus ◽  
Sperm Dna ◽  
Marsupial Species

Protamines of eutherian species are cysteine-rich molecules that become cross-linked by disulfide bonds during epididymal transit, whereas the protamines of most marsupial species lack cysteine residuals. The present study made use of the differences in protamine structure between eutherian and metatherian mammal spermatozoa to examine the comparative resistance of sperm DNA to oxidative damage in three eutherian species (Mus musculus, Homo sapiens, Sus domesticus) and three metatherian species (Vombatus ursinus, Phascolarctos cinereus, Macropus giganteus). Sperm DNA fragmentation of samples exposed to increasing concentrations of hydrogen peroxide was assessed by means of the two-tailed comet assay. The sperm DNA of the marsupial species studied were significantly more sensitive to oxidative stress than the spermatozoa of eutherian species. Such susceptibility is consistent with the lack of disulfide cross-linking in marsupial sperm chromatin and suggests that the oxidation of thiols to disulfides for chromatin condensation during epididymal transit in eutherian mammals is likely to be important in order to provide stability and protect these cells from the genotoxic effects of adverse environments.

scholarly journals Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2153
Author(s):  
Irfan Zidni ◽  
Yun Ho Lee ◽  
Jung Yeol Park ◽  
Hyo Bin Lee ◽  
Jun Wook Hur ◽  
...  
Keyword(s):  
Previous Experiment ◽  
Activity Index ◽  
Medium Composition ◽  
Freezing Rate ◽  
Dilution Ratio ◽  
Sperm Cryopreservation ◽  
Ringer’S Solution ◽  
Potential Market ◽  
Spotted Halibut ◽  
Cryopreservation Protocol

The spotted halibut is species that has a high potential market value in Korea, but the supply of seed is unstable because of the limited milt production of males. The objective of this research was to explore different aspects, such as CPAs, diluents, dilution ratio, and freezing rates, to develop an optimal sperm cryopreservation. The parameters assessed were movable sperm ratio, sperm activity index, survival rate, and DNA damage. The CPAs tested in this research were propylene glycol, dimethyl sulfoxide (DMSO), methanol, ethylene glycol, and glycerol. Different diluents, including 300 mM sucrose, 300 mM glucose, Stain’s solution, and Ringer’s solution, were investigated. The previous experiment showed that the optimal CPA for cryopreservation was DMSO with a concentration of 15% with 300 mM as diluent. To determine the effect of the dilution ratio, sperm was diluted to 1:1, 1:2, 1:10, 1:100, and 1:1000 with 300 mM sucrose containing DMSO at a final concentration of 15%. Lastly, the optimal freezing rate of the sperm was evaluated with four different freezing rates (−1, −5, −10, and −20 °C/min). Post-thaw sperm motility was higher with a dilution ratio lower than 1:2, and the freezing rate was less than −5 °C/min. In conclusion, these findings represent the development of a cryopreservation protocol for spotted halibut.

scholarly journals Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Reproduction ◽  
2004 ◽  
Vol 127 (5) ◽  
pp. 547-556 ◽  
Author(s):  
R E Spindler ◽  
Y Huang ◽  
J G Howard ◽  
P Wang ◽  
H Zhang ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Giant Panda ◽  
Calcium Ionophore ◽  
Sperm Cryopreservation ◽  
Management Tools ◽  
Giant Pandas ◽  
Before And After ◽  
Cryopreservation Protocol ◽  
Acrosomal Integrity

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze–thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (− 40 and − 100 °C/min) cryopreservation by incubation in HEPES-buffered Ham’s F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean±s.e.m. sperm motility post-thaw (56.1 ± 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 ± 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 ± 1.7% versus cryopreserved–thawed, 81.7 ± 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 ± 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 ± 1.1%). Frozen–thawed sperm preincubated without accelerators underwent capacitation (49.6 ± 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 ± 1.4%) and without accelerators (9 h: 41.2 ± 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

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