X-Message-Number: 31067
Date: Thu, 18 Sep 2008 20:59:17 -0700 (PDT)
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Subject: the latest on dimethylacetamide
Reprod Fertil Dev. 2008;20(6):724-33.
Dimethylacetamide can be used as an alternative to glycerol for the successful
cryopreservation of koala (Phascolarctos cinereus) spermatozoa.
Zee YP, Holt WV, Gosalvez J, Allen CD, Nicolson V, Pyne M, Burridge M,
Carrick FN, Johnston SD.
The University of Queensland, Gatton, QLD, Australia.
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 degrees 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.
PMID: 18671920
Cryo Letters. 2008 May/June;29(3):199-208.
The effect of cryopreservation process on morphology and fertilising ability of
japanese quail (Coturnix japonica) spermatozoa.
Kowalczyk A. Department of Poultry Breeding, Wroclaw University of
Environmental and Life Sciences, Chelmonskiego, Wroclaw, Poland.
Changes in the morphology and fertilising ability of Japanese quail
spermatozoa were studied after semen dilution, equilibration and
freezing-thawing process in order to determine the optimal diluent,
cryoprotectant and the freezing-thawing method. Subsequent stages of quail
semen
cryopreservation caused significant decline in spermatozoa morphology and their
ability to fertilise the ovum. Semen dilution with Lake's extender alone reduced
the number of morphologically normal spermatozoa and decreased their
fertilising ability. Dimethylacetamide (DMA) was the least detrimental
but equilibration of quail spermatozoa with this cryoprotectant caused further
decline in the number of morphologically normal cells. However, despite these
changes, after artificial insemination with semen equilibrated with DMA 25.8
percent of fertile eggs were obtained. Further loss in the number
of normal spermatozoa was observed following the freezing-thawing process. Of
the two investigated freezing-thawing methods, the rapid rate (60 C/min)
appeared less detrimental to spermatozoa morphology and their ability to
fertilize the ovum than the "slow" rate. Also the number of sperm holes
appearing in the inner perivitelline layer and the number of spermatozoa trapped
in the outer perivitelline layer of the ovum was higher after the rapid than
the 'slow' freezing-thawing procedure. Nevertheless, both rates did not yield
any fertile eggs.
PMID: 18754060
Theriogenology. 2008 Mar 15;69(5):632-8. Epub 2008 Feb 1.
Evaluation of amides and centrifugation temperature in boar semen
cryopreservation.
Bianchi I, Calderam K, Maschio EF, Madeira EM, da Rosa Ulguim R, Corcini CD,
Bongalhardo DC, Correa EK, Lucia T Jr, Deschamps JC, Correa MN. PIGPEL,
Centro de Biotecnologia, Brazil.
Two experiments were conducted to evaluate the use of amides as
cryoprotectants and two centrifugation temperatures (15 or 24 degrees C) in
boar semen cryopreservation protocols. Semen was diluted in BTS, cooled
centrifuged, added to cooling extenders, followed by the addition of various
cryoprotectants. In experiment 1, mean (+/-S.E.M.) sperm motility for 5%
dimethylformamide (DMF; 50.6+/-1.9%) and 5% dimethylacetamide (DMA; 53.8+/-1.7%)
were superior (P<0.05) to 5% methylformamide (MF; 43.2+/-2.4%) and 3% glycerol
(GLY; 38.1+/-2.3%), with no significant difference between MF and
GLY. Sperm membrane integrity was higher (P<0.05) for DMA than for MF or GLY
(50.9+/-1.9, 43.3+/-2.5, and 34.5+/-2.8%, respectively). Sperm membrane
integrity was higher in DMF (47.9+/-2.1%) than in glycerol (34.5+/-2.8%,
P<0.05), but was similar to other treatments (P>0.05). In experiment 2, we
tested MF, DMF, and DMA at 3, 5, and 7%. Sperm motility and membrane integrity
were higher for 5% DMA (53.8+/-1.7 and 50.9+/-1.9%) and 5% DMF (50.6+/-1.9 and
47.9+/-2.1%), in comparison with 7% DMF and all MF concentrations (P<0.05). For
sperm motility and membrane integrity, 5% DMA exceeded
(P<0.05) 3% DM, with greater membrane integrity than 3% DMF (P<0.05). In both
experiments, sperm motility and membrane integrity were superior at 15 degrees C
versus 24 degrees C (P<0.05), with no interaction between centrifugation
temperature and treatments (P>0.05). In conclusion, boar semen was
successfully cryopreserved by replacement of glycerol with amides (especially 5%
DMA) and centrifugation at 15 degrees C, with benefits for post-thaw sperm
motility and membrane integrity.
PMID: 18242674
Theriogenology. 2008 Feb;69(3):384-96. Epub 2007 Nov 26.
The effect of internal and external cryoprotectants on zebrafish (Danio rerio)
embryos.
Lahnsteiner F. Department for Organismic Biology, University of Salzburg,
Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
The study investigated the effects of internal (DMSO, 1,2-propanediol,
glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external
cryoprotectants (glucose, sucrose) on the viability and on morphometric
parameters of zebrafish embryos. From the tested internal cryoprotectants,
DMSO
had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external
cryoprotectants were less toxic then the internal ones. Early ontogenetic stages
were more sensible to cryoprotectant exposure than advanced stages. Two-step
incubation procedures in increasing concentrations of internal
and external cryoprotectants were superior to multiple-step exposure procedures.
All tested vitrification solutions exceeded the tolerance limit of embryos. The
tolerance of zebrafish embryos to cryoprotectants was highly variable in a
concentration range causing approximately 50% embryo mortality.
The width of the perivitelline space showed significant morphometrical changes
due to cryoprotectant exposure. In the germinative tissue non-significant
changes occurred. The yolk did not change morphometrically after exposure to
internal cryoprotectants and showed no sign of dehydration after
exposure to external cryoprotectants. Based on these results the study comes to
the following conclusions: as yolk dehydration was impossible and as
vitrification solutions were over the tolerance limit it seems unlikely that
successful vitrification of zebrafish embryos can be achieved. Under these
considerations slow freezing methods would be a better option as lower
cryoprotectant concentrations can be used and embryos can be dehydrated during
freezing.
PMID: 18031801
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