scholarly journals Freeze drying as a method of long-term conservation of mammalian semen – a review

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Iwona Rajska
Keyword(s):  
Freeze Drying ◽  
Genetic Material ◽  
Simultaneous Application ◽  
Preservation Method ◽  
Freeze Dried ◽  
Unlimited Time ◽  
Specialized Equipment

Abstract With the development of biotechnological methods that allow for the manipulation and free exchange of genetic material, there is a need to improve the methods of collecting and storing such material. Until now, freezing in liquid nitrogen has allowed the storage of cells and entire plant and animal tissues for practically unlimited time. Despite this, alternatives are still being sought that will eliminate the constant need to keep samples at low temperature. Lyophilization or freeze drying can be an alternative to standard freezing procedures. The storage of samples (lyophilisates) does not require specialized equipment but only the refinement of the preservation method itself. In the case of cells capable of movement, e.g. sperm, as a result of the lyophilization process, they lose the ability to reach the oocyte in vivo and IVF. However, it is possible to use freeze-dried sperm for in vitro fertilization by ICSI, which is observed on the basis of the results obtained in cleavage, embryo development and production of live born offspring after embryo transfer. Studies on lyophilization of sperm are carried out on many animal species, both laboratory and livestock. This conservation method is seen as the possibility of creating biobanking for genetically valuable and endangered species with the simultaneous application of ICSI. This review article was intended to present the issues of the freeze-drying process of mammalian semen and help in finding solutions that will improve this technique of long-term preservation of biological material.

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 15-24 ◽  
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
A. Takizawa ◽  
N. Maedomari ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Freeze Drying ◽  
Chelating Agents ◽  
Sperm Head ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Freeze Dried ◽  
Boar Sperm

SUMMARYSuccessful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

Repeatability of in vitro digestibility assays of forages when using fresh, frozen or freeze-dried cow faeces instead of sheep rumen liquor as sources of micro-organisms

1995 ◽  
Vol 1995 ◽  
pp. 110-110 ◽  
Author(s):  
S Akhter ◽  
E Owen ◽  
M K Theodorou ◽  
S L Tembo ◽  
E R Deaville
Keyword(s):  
Freeze Drying ◽  
Rumen Fluid ◽  
In Vitro Digestibility ◽  
Two Stage ◽  
Freeze Dried ◽  
Fresh Frozen ◽  
Sheep Rumen ◽  
Micro Organisms

Previous studies (El Shaer, Omed and Axford, 1987; Akhter, Owen, Fall, O'Donovan and Theodorou, 1994) with the two-stage in vitro procedure of Tilley and Terry (1963) have shown a high correlation between digestibilities of forages as determined using either sheep rumen liquor, sheep faeces or cow faeces as the microbial inoculum. In the first study of the of the present investigation one objective was to examine the repeatability of these digestibility measurements when made on different occasions. A second objective was to assess whether the correlations between faecal and rumen fluid based inocula could be improved if microorganisms were obtained from pairs rather than individual animals. The objective in the second study using forages of known in vivo digestibility, was to investigate the effect of freezing or freeze-drying of faeces on the repeatability of digestibilities of forages determined in vitro using micro-organisms from cow faeces.

scholarly journals Cryopreservation enables long-term conservation of critically endangered species Rubus humulifolius

2019 ◽  
Vol 29 (1) ◽  
pp. 303-314 ◽  
Author(s):  
Jaanika Edesi ◽  
Jonne Tolonen ◽  
Anna Liisa Ruotsalainen ◽  
Jouni Aspi ◽  
Hely Häggman
Keyword(s):  
Plant Species ◽  
Common Garden ◽  
Critically Endangered ◽  
Ex Situ ◽  
Plant Biodiversity ◽  
Critically Endangered Species ◽  
Preservation Method

Abstract Ex situ storage plays an important role in the conservation of plant biodiversity. Cryopreservation at ultra-low temperatures (−  196 °C) is the only long-term ex situ preservation method for plant species that cannot be stored in seed banks. In the present study, we developed a cryopreservation protocol for micropropagated Rubus humulifolius (Rosaceae) plants representing currently critically endangered population of the species in Finland. Abscisic acid (ABA) has been found to increase the freezing tolerance of several plant species. Thus, we studied the effect of a 10-day pretreatment with 0, 2 or 4 mg/l ABA in comparison to freshly dissected buds. We also studied how the duration of in vitro subculture affects cryopreservation result. The ABA pretreatment had divergent effect on control and cryopreserved buds: the regeneration of non-cryopreserved control buds increased from 51% to 70%, 90% or 87% while the regeneration of cryopreserved buds decreased from 52% to 35%, 6% or 9% after 0, 2 or 4 mg/l ABA pre-treatments, respectively. Buds from plants subcultured for 1 month had 63% survival, which, however, decreased to 29% or nil% after 2 or 4 months subculture. The regenerated plants were successfully transferred from in vitro to in vivo conditions in common garden. Growing in garden is needed for future restoration of the species in wild. Cryostorage and other ex situ conservation actions carried out in botanical gardens may be of increasing importance as a tool to maintain plant biodiversity in the future.

scholarly journals Taking Advantage of Nature’s Benefits: Soluble and Stable Antigen Straight Out of the Pathogen

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 137
Author(s):  
Didier Clénet ◽  
Léna Clavier ◽  
Benoit Strobbe ◽  
Christel Le Bon ◽  
Manuela Zoonens ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Process Development ◽  
Storage Conditions ◽  
Full Length ◽  
Soluble Form ◽  
Drying Process ◽  
Liquid Form ◽  
Freeze Dried

Integral membrane proteins (MP) exhibit specific tridimensional conformation and topology that define their various functions. Pathogen surface antigens, encompassing many MP, are at the forefront of the viral strategy which is broadly targeted by the host immune response. These antigens are present in equilibrium under different oligomeric forms with distinctive epitopes, and to obtain them in a soluble form and/or stable constitutes a real risk. The solubilization of a full-length MP directly from a pathogen to rapidly obtain a native antigen mimicking the original conformation of the MP at the pathogen surface is the process development reported in this work. Rabies virus (RABV) was used as a model for this demonstration and its full-length glycoprotein (G) was stabilized in amphiphatic polymers (A8-35 amphipols). The stability of the soluble RABV-G was evaluated under various stress conditions (temperatures, agitation and light exposures) and a long-term stable RABV-G formulation, suitable for the freeze-drying process, was defined using a design of experiment approach. RABV-G/A8-35 in liquid form was shown to be antigenically stable at 5 °C and 25 °C for one month, and a dedicated kinetic model predicted its stability up to 1 year at 5 °C. To mitigate the RABV-G/A8-35 sensitivity to mechanical stress, a solid form of RABV-G/A8-35 and a freeze-drying process were considered, resulting in a 2-year thermally stable product at 5 °C, 25 °C and 37 °C. To the best of our knowledge, this is the first time that a natural full-length MP, extracted from a virus and trapped in amphipols, was kept antigenically stable in the long term, in a defined freeze-dried form out of any refrigerated storage conditions. These results described an easy process to obtain a pure, well conformed native-like antigen of interest, from a circulating pathogen which is of concern for diagnostic (quantification/characterization assays), therapeutic and vaccine strategies. After the physical characterization of the protein, the identification of RABV G/A8-35 neutralizing epitopes has been underway before in vivo testing.

scholarly journals Bioactive Flavonoids, Antioxidant Behaviour, and Cytoprotective Effects of Dried Grapefruit Peels (Citrus paradisiMacf.)

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Lucia Castro-Vazquez ◽  
María Elena Alañón ◽  
Virginia Rodríguez-Robledo ◽  
María Soledad Pérez-Coello ◽  
Isidro Hermosín-Gutierrez ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Neuroblastoma Cell ◽  
Therapeutic Strategies ◽  
Citrus Paradisi ◽  
Cytoprotective Effect ◽  
Freeze Dried ◽  
Grapefruit Peel ◽  
Neuroblastoma Cell Lines

Grapefruit (Citrus paradisiMacf.) is an important cultivar of theCitrusgenus which contains a number of nutrients beneficial to human health. The objective of the present study was to evaluate changes in bioactive flavonoids, antioxidant behaviour, andin vitrocytoprotective effect of processed white and pink peels after oven-drying (45°C–60°C) and freeze-drying treatments. Comparison with fresh grapefruit peels was also assessed. Significant increases in DPPH, FRAPS, and ABTS values were observed in dried grapefruit peel samples in comparison with fresh peels, indicating the suitability of the treatments for use as tools to greatly enhance the antioxidant potential of these natural byproducts. A total of thirteen flavonoids were quantified in grapefruit peel extracts by HPLC-MS/MS. It was found that naringin, followed by isonaringin, was the main flavonoid occurring in fresh, oven-dried, and freeze-dried grapefruit peels.In vivoassay revealed that fresh and oven-dried grapefruit peel extracts (45°C) exerted a strong cytoprotective effect on SH-SY5Y neuroblastoma cell lines at concentrations ranging within 0.1–0.25 mg/mL. Our data suggest that grapefruit (Citrus paradisiMacf.) peel has considerable potential as a source of natural bioactive flavonoids with outstanding antioxidant activity which can be used as agents in several therapeutic strategies.

scholarly journals Assessment of three generations of mice derived by ICSI using freeze-dried sperm

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 239-251 ◽  
Author(s):  
Ming-Wen Li ◽  
Brandon J. Willis ◽  
Stephen M. Griffey ◽  
Jimmy L. Spearow ◽  
K. C. Kent Lloyd
Keyword(s):  
Genomic Instability ◽  
Freeze Drying ◽  
Mouse Strains ◽  
Control Group ◽  
Three Generations ◽  
Phenotypic Abnormality ◽  
Freeze Dried ◽  
Two Generations

SummaryAlthough the derivation of mice by intracytoplasmic sperm injection (ICSI) using freeze-dried sperm has been demonstrated previously, a comprehensive analysis of their viability, health, and fertility has not. The purpose of the present study was to determine the extent to which ICSI using freeze-dried sperm stored at 4 °C for 1–2 months from mice on either an inbred (C57BL/6J) or hybrid (B6D2F1/J) genetic background results in genomic instability and/or phenotypic abnormality in mice and two generations of their progeny. Fertilization rates (number of 2-cells per injected oocytes) using ICSI of fresh and freeze-dried sperm were similar within and between mouse strains, although fewer freeze-dried sperm-derived embryos than fresh sperm-derived embryos developed to blastocystsin vitro(C57BL/6J and B6D2F1/J) and liveborn pupsin vivo(B6D2F1/J only). Nevertheless, once born, mice derived by ICSI using freeze-dried sperm in both mouse strains were healthy and reproductively sound. No major differences in litter size, weaning rate, and sex ratio were noted in the two generations of progeny (F2 and F3) of ICSI-derived offspring using freeze-dried sperm compared with that in the natural mating (control) group. Further, there was no evidence that either ICSI or freeze drying induced genomic instability, as determined by microsatellite analysis of the derived mice and subsequent generations when compared with both parental genotypes, nor were there differences in the number or types of pathological changes in any of the three generations of progeny. We conclude that viable, healthy and genomically stable mice can be derived by ICSI using freeze-dried mouse sperm stored in the refrigerator for at least 2 months. Further, because freeze drying is a simpler and more economical technique compared with embryo and sperm cryopreservation, the results of this study justify additional research to continue to develop and enhance the technique for the preservation, storage, and sharing of genetically altered mice.

A Comparison of Spray-Drying and Freeze-Drying for the Production of Stable Silybin Nanosuspensions

2020 ◽  
Vol 20 (6) ◽  
pp. 3598-3603 ◽  
Author(s):  
Yingying Ma ◽  
Jin Gao ◽  
Wankui Jia ◽  
Yangyang Liu ◽  
Lanying Zhang ◽  
...  
Keyword(s):  
Spray Drying ◽  
Freeze Drying ◽  
Drying Methods ◽  
Dry Powders ◽  
Term Stability ◽  
Long Term Stability ◽  
Freeze Dried ◽  
Spray Dried

Spray-drying and freeze-drying are effective approaches to improve the long-term stability of nanosuspensions. This research explored the effect of spray-drying and freeze-drying techniques on PVP K30-stabilized silybin nanosuspensions. The morphology was observed by scanning electron microscopy (SEM): The spray-dried sample was spherical, and the freeze-dried samples were rodlike with smooth surfaces. The redispersibility was studied via dynamic light scattering (DLS): The size, PDI, and zeta of the spray-dried sample were 133.27 nm, 0.214, and 24.37 mV, respectively; the size, PDI, and zeta of the freeze-dried sample were 298.70 nm, 0.114, and 20.98 mV, respectively. The in vitro dissolution was studied, and the two dry powders showed a significant increase compared to silybin. The two dried powders had better long-term stability than the liquid starting material. Overall, spray-drying and freeze-drying are appropriate drying methods for the preparation of silybin nanosuspensions with better stability and dissolution velocity.

scholarly journals Lyophilized alginate-based microspheres containing Lactobacillus fermentum D12, an exopolysaccharides producer, contribute to the strain’s functionality in vitro

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Katarina Butorac ◽  
Jasna Novak ◽  
Barbara Bellich ◽  
Lucrecia C. Terán ◽  
Martina Banić ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Structural Information ◽  
Bacterial Count ◽  
Bacterial Cells ◽  
Whole Genome Analysis ◽  
Enteropathogenic Bacteria ◽  
Freeze Dried ◽  
Precursor Molecules

AbstractLactobacillus (Limosilactobacillus) fermentum D12 is an exopolysaccharide (EPS) producing strain whose genome contains a putative eps operon. Whole-genome analysis of D12 was performed to disclose the essential genes correlated with activation of precursor molecules, elongation and export of the polysaccharide chain, and regulation of EPS synthesis. These included the genes required for EPS biosynthesis such as epsA, B, C, D and E, also gt, wzx, and wzy and those involved in the activation of the precursor molecules galE, galT and galU. Both the biosynthesis and export mechanism of EPS were proposed based on functional annotation. When grown on MRS broth with an additional 2% w/v glucose, L. fermentum D12 secreted up to 200 mg/L of a mixture of EPSs, whose porous structure was visualized by scanning electron microscopy (SEM). Structural information obtained by 1HNMR spectroscopy together with composition and linkage analyses, suggested the presence of at least two different EPSs, a branched heteropolysaccharide containing t-Glcp and 2,6-linked Galf, and glycogen. Since recent reports showed that polysaccharides facilitate the probiotic-host interactions, we at first sought to evaluate the functional potential of L. fermentum D12. Strain D12 survived simulated gastrointestinal tract (GIT) conditions, exhibited antibacterial activity against enteropathogenic bacteria, adhered to Caco-2 cells in vitro, and as such showed potential for in vivo functionality. The EPS crude extract positively influenced D12 strain capacity to survive during freeze-drying and to adhere to extracellular matrix (ECM) proteins but did not interfere Caco-2 and mucin adherence when added at concentrations of 0.2, 0.5, and 1.0 mg/mL. Since the viable bacterial count of free D12 cells was 3 logarithmic units lower after the exposure to simulated GIT conditions than the initial count, the bacterial cells had been loaded into alginate for viability improvement. Microspheres of D12 cells, which were previously analyzed at SEM, significantly influenced their survival during freeze-drying and in simulated GIT conditions. Furthermore, the addition of the prebiotic substrates mannitol and lactulose improved the viability of L. fermentum D12 in freeze-dried alginate microspheres during 1-year storage at 4 °C compared to the control.

322 SPERM PRESERVATION BY FREEZE-DRYING IN ENDANGERED ANIMALS

2015 ◽  
Vol 27 (1) ◽  
pp. 250
Author(s):  
T. Kaneko
Keyword(s):  
Ambient Temperature ◽  
Liquid Nitrogen ◽  
Freeze Drying ◽  
Mouse Oocytes ◽  
Preservation Method ◽  
Freeze Dried ◽  
Normal Shape ◽  
Artificial Activation ◽  
Endangered Animals

Sperm preservation is a useful tool for conservation of endangered animals. Freeze-drying sperm have been studied as new preservation method in various mammals as samples can be preserved in a refrigerator at 4°C or ambient temperature. Sperm preservation by freeze-drying is the ultimate method by which sperm can be stored that neither required specialised cryoprotectants nor constant supply of liquid nitrogen. We established the freeze-drying method that mouse and rat sperm could be preserved long-term at 4°C after freeze-drying using a simple solution containing 10 mM Tris and 1 mM EDTA (TE buffer; 2012 PLoS ONE 7, e35043; 2012 Cryobiology 64, 211–214). Using this method, the fertility of the chimpanzee, giraffe, and jaguar sperm after freeze-drying were estimated. Ejaculated chimpanzee and giraffe and cauda epididymal jaguar sperm were freeze-dried using TE buffer. Sperm were rehydrated with sterile distilled water after storage at 4°C for 1 month. Sperm with normal shape were injected into mouse oocytes in CZB medium with HEPES, and oocytes were then cultured in vitro for 6 to 8 h in the same media. In all animals, pronuclei and sperm tail were observed into oocytes without artificial activation after injection of freeze-dried sperm. When chimpanzee, giraffe, and jaguar sperm were injected into oocytes, 86% (12/14), 100% (12/12), and 96% (22/23) of oocytes formed 2 distinct pronuclei. This study demonstrated that the sperm of various animals could be decondensed into the mouse oocytes after freeze-drying using the same protocol. A further advantage is that freeze-dried sperm can be transported oversea at ambient temperature. Freeze-drying preservation without using liquid nitrogen can be protected strongly valuable gametes of endangered animals even in the event of unexpected accidents and disaster such as earthquakes and typhoons. Freeze-drying of sperm has been applied as a “freeze-drying zoo” for conservation of endangered animals (http://www.anim.med.kyoto-u.ac.jp/reproduction/home.aspx).

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Sign in / Sign up

Export Citation Format

Share Document