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).

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.

scholarly journals Citrus limon Extract Loaded in Vesicular Systems for the Protection of Oral Cavity

Medicines ◽  
2018 ◽  
Vol 5 (4) ◽  
pp. 108 ◽  
Author(s):  
Maria Manconi ◽  
Maria Manca ◽  
Carla Caddeo ◽  
Giorgia Sarais ◽  
Alessandra Palmieri ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lactobacillus Acidophilus ◽  
Freeze Drying ◽  
Citrus Limon ◽  
Antibacterial Effects ◽  
Bacterial Proliferation ◽  
Freeze Dried ◽  
Ethanol Evaporation ◽  
Dried Extract

Background: The nanoincorporation of the extract of Citrus limon (L.) Osbeck var. pompia into liposomes was aimed at improving its antioxidant and antibacterial effects. Methods: The extract of the rind of Citrus limon (L.) Osbeck var. pompia was obtained by maceration in ethanol, evaporation, and freeze-drying. The extract phytochemical fingerprint was obtained by HPLC and mass spectrometry, and it was determined that gallic acid, neohesperidin, eriocitrin, and neoeriocitrin were the most abundant components. The freeze-dried extract was loaded in liposomes, glycerosomes, and penetration-enhancer-containing vesicles prepared with propylene glycol (PG-PEVs). Results: Capability of the vesicles of improving efficacy of the extract in counteracting oxidative stress was studied in vitro in keratinocytes, along with antimicrobial activity against planktonic cultures of Streptococcus mutans, Lactobacillus acidophilus, and Streptococcus sanguinis. Conclusion: Results showed that the vesicles, especially glycerosomes and PG-PEVs, prevented oxidative damage and cell death, and inhibited bacterial proliferation.

scholarly journals 314 FULL-TERM DEVELOPMENT OF RAT OOCYTES MICROINSEMINATED WITH FREEZE-DRIED SPERMATOZOA

2005 ◽  
Vol 17 (2) ◽  
pp. 307
Author(s):  
M. Hirabayashi ◽  
M. Kato ◽  
S. Hochi
Keyword(s):  
Liquid Nitrogen ◽  
Freeze Drying ◽  
Pure Water ◽  
Full Term ◽  
Female Rats ◽  
Membrane Disruption ◽  
Developmental Potential ◽  
Fresh Freeze ◽  
Freeze Dried

Since freeze-dried spermatozoa can be stored at ambient or refrigerated temperature, the costs required for maintenance and shipping of spermatozoa can be reduced. To date, viable offspring in mice (Wakayama and Yanagimachi 1998 Nat. Biotech. 16, 639) and rabbits (Liu et al. 2004 Biol. Reprod. 70, 1776) have been produced by intracytoplasmic sperm injection (ICSI) using freeze-dried samples. The objectives of the present study were to examine whether freeze-dried rat spermatozoa can participate in full-term development by ICSI, and whether sonication prior to freeze-drying of the spermatozoa influences the offspring rate. Spermatozoa from cauda epididymides of Sprague-Dawley (SD) rats were collected in 10 mM TRIS/HCl buffer supplemented with 50 mM NaCl and 50 mM EGTA. A 2 × 3 factorial-designed experiment was conducted. The sperm suspensions were either sonicated for 10 s using a 10% power output from an ultrasonic cell disruptor or not sonicated. The sperm suspensions were then processed for freeze-thawing (100-μL sample in 1.0-mL cryotube was cooled in liquid nitrogen vapor, stored at -196°C for 48 h, and thawed in a 25°C water bath) and freeze-drying (100-μL sample in 1.5-mL polypropylene tube was frozen in liquid nitrogen for 20 s, lyophilized for 6 h by a freeze-drying apparatus, stored at 4°C for 48 h, and rehydrated with 100 μL ultra pure water), or were subjected to immediate use for ICSI. The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2–4 μm in diameter, as described previously (Hirabayashi et al. 2002 Transgenic Res. 11, 221). The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. The in vivo developmental potential of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa is shown in the table below. Viable rat offspring were produced in all six experimental groups, with the offspring rates at 2.5–35.0%. Sonication treatment of rat spermatozoa to induce membrane disruption and tail/midpiece dissociation from the heads was effective in increasing the offspring rate after ICSI. The positive effect of sperm sonication may be explained as facilitating decondensation of sperm heads by membrane disruption in the spontaneously activating rat oocytes. Thus, successful participation of freeze-dried rat spermatozoa into full-term development was demonstrated by applying the ICSI. Table 1. In vivo development of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa

scholarly journals ASSESMENT OF ANTI-INFLAMMATORY ACTIVITY AND CYTOTOXICITY OF FREEZE DRIED HYDROALCOHOLIC EXTRACT OF Bidens andicola ON ISOLATED NEUTROPHILS

2017 ◽  
Vol 10 (6) ◽  
pp. 160
Author(s):  
Vinueza D ◽  
LÓpez E ◽  
Acosta K ◽  
Abdo S
Keyword(s):  
Freeze Drying ◽  
Water Soluble ◽  
Inflammatory Activity ◽  
Hydroalcoholic Extract ◽  
Cytotoxicity Assays ◽  
Aerial Parts ◽  
Controlled Conditions ◽  
Freeze Dried ◽  
Anti Inflammatory

Objective: The aim of this study was to evaluate anti-inflammatory activity and cytotoxicity in vitro of hydroalcoholic extract of Bidens andicola.Methods: B. andicola hydroalcoholic extract was obtained from aerial parts of B. andicola, following a standardized methodology. Briefly, aerial parts of B. andicola were extracted with ethanol 70% v/v and defatted with n-hexane, hydroalcoholic fraction was concentrated under controlled conditions in a rotary evaporator, and finally the residue was freeze-drying to obtain the hydroalcoholic extract of B. andicola. Anti-inflammatory activity and cytotoxicity assays were carried out using in vitro isolated neutrophils model using stable water-soluble tetrazolium salts.Results and Conclusions: The in vitro anti-inflammatory assay on isolated neutrophils demonstrated that the hydroalcoholic extract showed antiinflammatoryactivity compared to aspirin, with inflammatory inhibition percent values of 80.138±0.729 to hydroalcoholic extract of B. andicola and 82.117±0.762 to aspirin, each tested in five replicates at the concentration of 200 ppm of hydroalcoholic extract or reference. 

Viability of microencapsulated Akkermansia muciniphila and Lactobacillus plantarum during freeze-drying, storage and in vitro simulated upper gastrointestinal tract passage

2018 ◽  
Vol 9 (11) ◽  
pp. 5868-5879 ◽  
Author(s):  
Martín Sebastián Marcial-Coba ◽  
Tomasz Cieplak ◽  
Thiago Barbosa Cahú ◽  
Andreas Blennow ◽  
Susanne Knøchel ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Lactobacillus Plantarum ◽  
Freeze Drying ◽  
Storage Stability ◽  
Upper Gastrointestinal Tract ◽  
Akkermansia Muciniphila ◽  
Freeze Dried ◽  
Upper Gastrointestinal

Microencapsulated and subsequently freeze-dried cells showed acceptable storage stability and enhanced survival during in vitro upper gastrointestinal tract passage.

376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 303
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
N. Maedomari ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Oocyte Meiosis ◽  
Freeze Dried ◽  
Sperm Penetration ◽  
Sperm Factor ◽  
Pre Treatment ◽  
Pronuclear Formation

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P &lt; 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

scholarly journals Effect of Drying Process, Encapsulation, and Storage on the Survival Rates and Gastrointestinal Resistance of L. salivarius spp. salivarius Included into a Fruit Matrix

2020 ◽  
Vol 8 (5) ◽  
pp. 654
Author(s):  
Ester Betoret ◽  
Noelia Betoret ◽  
Laura Calabuig-Jiménez ◽  
Cristina Barrera ◽  
Marco Dalla Rosa
Keyword(s):  
Survival Rate ◽  
Physicochemical Properties ◽  
Freeze Drying ◽  
Molecular Mechanisms ◽  
Survival Rates ◽  
In Vitro Digestion ◽  
Hot Air ◽  
Freeze Dried ◽  
And Storage

In a new probiotic food, besides adequate physicochemical properties, it is necessary to ensure a minimum probiotic content after processing, storage, and throughout gastrointestinal (GI) digestion. The aim of this work was to study the effect of hot air drying/freeze drying processes, encapsulation, and storage on the probiotic survival and in vitro digestion resistance of Lactobacillus salivarius spp. salivarius included into an apple matrix. The physicochemical properties of the food products developed were also evaluated. Although freeze drying processing provided samples with better texture and color, the probiotic content and its resistance to gastrointestinal digestion and storage were higher in hot air dried samples. Non-encapsulated microorganisms in hot air dried apples showed a 79.7% of survival rate versus 40% of the other samples after 28 days of storage. The resistance of encapsulated microorganisms to in vitro digestion was significantly higher (p ≤ 0.05) in hot air dried samples, showing survival rates of 50–89% at the last stage of digestion depending on storage time. In freeze dried samples, encapsulated microorganisms showed a survival rate of 16–47% at the end of digestion. The different characteristics of the food matrix after both processes had a significant effect on the probiotic survival after the GI digestion. Documented physiological and molecular mechanisms involved in the stress response of probiotic cells would explain these results.

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 Freeze-Drying Ethylcellulose Microparticles Loaded with Etoposide for In Vitro Fast Dissolution and In Vitro Cytotoxicity against Cancer Cell Types, MCF-7 and Caco-2

2021 ◽  
Vol 11 (19) ◽  
pp. 9066
Author(s):  
Ahmed A. H. Abdellatif ◽  
Mashari A. Aldhafeeri ◽  
Waleed H. Alharbi ◽  
Fahad H. Alharbi ◽  
Waleed Almutiri ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Freeze Drying ◽  
Chemical Interaction ◽  
In Vitro Cytotoxicity ◽  
Dissolution Experiment ◽  
Freeze Dried ◽  
Fast Dissolution ◽  
Mcf 7

The aim of this study was to improve the solubility of etoposide–ethylcellulose (ET–ETO) microparticles using the freeze-drying technique. Ethylcellulose (EC) microparticles loaded with etoposide (ETO) were prepared with different drug–polymer molar ratios of 1:1, 1:3, 1:6, and 1:20 by the solvent evaporation method. The size of the prepared microparticles was 0.088 µm. The results showed that the amount of ETO encapsulated into the microparticles was 387.3, 365.0, 350.0, and 250 µg/50 mg microparticles for microparticles with drug–polymer ratios of 1:1, 1:3, 1:6, and 1:20, respectively. The FT-IR spectra showed no chemical interaction between ETO and the polymer in the solid state. The results obtained from the dissolution experiment showed that the freeze-dried microparticles were stable in 0.1 N HCl (gastric pH) for 2 h. At pH 7.4, the ETO release was 60 to 70% within the first 15 min and approximately 100% within 30 min. Results from the application of different dissolution models showed that the equations that best fit the dissolution data for the ET–ETO microparticles at pH 7.4 were the Higuchi and Peppas model equations. The in vitro cytotoxicity assay of free ETO and freeze-dried microspheres prepared in this study with a drug–polymer ratio of 1:1 was performed in two mammalian cancer cell lines, MCF-7 (for bone cancer of the mammary organ) and Caco-2 (for mammalian epithelial colorectal adenocarcinoma). The results showed that the half-maximal inhibitory concentrations (IC50 values) for ETO and freeze-dried ET–ETO microparticles were 18.6 µM and 27.1 µM, respectively. In conclusion, freeze-dried ET–ETO is a promising formulation for developing a fast-dissolving form of ETO with a significant antiproliferative activity against the tested cell lines used in this study. It is a promising formulation for local duodenal area targeting.

Sign in / Sign up

Export Citation Format

Share Document