Cooling and freezing of sperm from captive,free-living and endangered squirrel monkey species

Germoplasm banking is an important tool for the preservation of genetic material from Neotropical primates in captivity, and from free living species, especially the endangered ones like Saimiri vanzolinii (Black-headed squirrel monkey), a primate with a low incidence area (870 km² of floodplains) in the southern part of the Mamirauá Sustainable Development Reserve, Brazil. Therefore, in the present study we aimed to develop a sperm cryopreservation protocol comparing sperm cooling in presence (T1) and absence (T2) of egg yolk, and to test freezing protocols to preserve semen from captive (Saimiri collinsi), and free-living (Saimiri vanzolinii, Saimiri cassiquiarensis and Saimiri macrodon) New World primates. Cooling preserved sperm of S. collinsi in all evaluated microscopic parameters, except for sperm motility. No differences were observed among the treatments, indicating that semen of this species can be cooled without egg yolk. Freezing did not affect sperm quality of S. collinsi, except plasma membrane integrity that was negatively affected. Generally, a good maintenance rate was observed between cooling and thawing of semen for the four species, showing the positive translational application of protocols from S. collinsi to the free-living species. Developed freezing protocol proved to be useful for sperm cryopreservation of S. collinsi and in field conditions.     

Cryopreservation of the edible alkalophilic cyanobacterium Arthrospira platensis

Efficient cryopreservation conditions for the edible alkalophilic cyanobacterium Arthrospira (Spirulina) platensis were investigated using a model strain A. platensis NIES-39. As a result, it was found that more than 60% of cells were viable upon thawing, when they had been frozen at a cooling rate of approximately ?1 °C min^?1 in the presence of 10% (v/v) dimethyl sulfoxide. Further examination with other Arthrospira strains showed that many of them had strain-dependent optimal conditions for cryopreservation. For example, the best freezing conditions for A. platensis SAG 21.99 were snap-freezing in liquid nitrogen in the presence of 5% (v/v) dimethyl sulfoxide, while they were slow cooling at approximately ?1 °C min^?1 in the presence of 10% (v/v) methanol for A. platensis NIES-46, NIES-2308 and UTEX 1926. The variety of successful cryopreservation conditions presented in this study is useful when attempting to cryopreserve various Arthrospira strains.     

Cryopreservation of sugarbeet germplasm

Meristems aseptically isolated from shoots developed on sugarbeet (Beta vulgaris L.) inflorescences were precultured on modified MS agar medium containing 19.4 M 6-benzylaminopurine, 6 M triiodobenzoic acid, and supplemented with 5% DMSO. After two days the meristems were transferred to liquid modified MS medium and the cryoprotectants sorbitol and DMSO added in varying concentrations. The meristems were frozen to –40°C and stored in liquid nitrogen. Growth resumed when the meristems were quick-thawed at 39°C.     

Factors Affecting the Outcome of Artificial Insemination Using Cryopreserved Spermatozoa in the Giant Panda (Ailuropoda melanoleuca)

Artificial insemination (AI) is an important component of captive breeding programs for endangered species, such as the giant panda. The panda has been the subject of increasingly successful captive breeding programs involving a compilation of assisted breeding techniques, including AI using cryopreserved spermatozoa. AI implementation is currently hampered by a lack of understanding of the factors that may cause failure. We investigated factors influencing the probability of success of AI for 14 giant panda females housed at the China Center for Research and Conservation of the Giant Panda (CCRCGP) inseminated in a total of 20 instances using cryopreserved spermatozoa from 11 males currently residing in 6 different captive breeding institutions. One of the pandas was the oldest giant panda female to ever successfully conceive from AI (20.5 years old). The success of AI was significantly affected by the timing of AI in relationship to both timing of peak urinary estrogen of the female and percent decline in urinary estrogen between the peak level and the first AI attempt. Our results suggest that the window for successful AI in giant pandas may be narrower than previously suspected, although individual differences in rates of decline in urinary estrogen may reflect some degree of variation in this crucial window across females. Our results are consistent with recent research on pandas and other species that demonstrates the efficacy of cryopreserved spermatozoa for AI and highlights the need for more in‐depth analysis of factors related to female physiology that may influence its success. Zoo Biol 31:561‐573, 2012. © 2011 Wiley Periodicals, Inc.     

Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen–thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2 ± 5.4%, motility following 30 min incubation at 37 °C: 58.5 ± 6.0% and following 3 h incubation: 21.7 ± 7.6%, intact acrosome: 57.1 ± 5.2%, normal morphology: 52.0 ± 5.8% and viability: 67.3 ± 6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.     

Measurement of the apparent diffusivity of ethylene glycol in mouse ovaries through rapid MRI and theoretical investigation of cryoprotectant perfusion procedures

Successful organ cryopreservation will significantly benefit human health and biomedical research. One of the major challenges to this accomplishment is the need for optimization of cryoprotectant agent (CPA) perfusion procedures that involve highly complicated mass transfer processes in organs. The diffusivity of CPA is of critical importance for designing perfusion procedures to minimize the associated toxicity and osmotic damage. However, to date there have been no attempts to measure the CPA diffusivity in organs. In this study, we established a simple CPA diffusion model for relatively small organs, e.g., mouse ovaries, defined the apparent diffusivity () of CPA for these organs, and established a practical approach to measure the value of through magnetic resonant imaging (MRI). Using rapid MRI techniques and water saturation analyses, the distribution of ethylene glycol (EG) concentration in the centric cross-section of mouse ovaries was measured at a series of time points during perfusion, and these data were fit to the integral form of the mass transfer equation in the established model. These fits resulted in a value of for EG in mouse ovaries of 6.1 ± 1.4 × 10⁻⁷ cm²/s (mean ± SD). Based on these results, we proposed a modified perfusion procedure that may improve the survival of small organs or thin tissues during equilibrium cooling processes and assessed its efficiency through theoretical analyses.     

Wheat proteins improve cryopreservation of rat hepatocytes

Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze–thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance‐associated proteins such as WCS120, TaTIL, WCS19, and TaIRI‐2. These improved procedures enhance long‐term storage (2–12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77–93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non‐toxic, natural products that do not require animal serum, and are economical and easy to prepare. Biotechnol. Bioeng. 2009;103: 582–591. © 2009 Wiley Periodicals, Inc.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

Prediction of ice content in biological model solutions when frozen under high pressure

High pressure is, at least, as effective as cryoprotective agents (CPAs) and are used for decreasing both homogenous nucleation and freezing temperatures. This fact gives rise to a great variety of possible cryopreservation processes under high pressure. They have not been optimized yet, since they are relatively recent and are mainly based on the pressure–temperature phase diagram of pure water. Very few phase diagrams of biological material are available under pressure. This is owing to the lack of suitable equipment and to the difficulties encountered in carrying out the measurements. Different aqueous solutions of salt and CPAs as biological models are studied in the range of 0°C down to ‐35°C, 0.1 up to 250 MPa, and 0–20% w/w total solute concentration. The phase transition curves of glycerol and of sodium chloride with either glycerol or sucrose in aqueous solutions are determined in a high hydrostatic pressure vessel. The experimental phase diagrams of binary solutions were well described by a third‐degree polynomial equation. It was also shown that Robinson and Stokes' equation at high pressure succeeds in predicting the phase diagrams of both binary and ternary solutions. The solute cryoconcentration and the ice content were calculated as a function of temperature and pressure conditions during the freezing of a binary solution. This information should provide a basis upon which high‐pressure cryopreservation processes may be performed and the damages derived from ice formation evaluated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009