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 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.

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 Aqueous Extract of Perilla frutescens var. acuta Relaxes the Ciliary Smooth Muscle by Increasing NO/cGMP Content In Vitro and In Vivo

Molecules ◽  
2018 ◽  
Vol 23 (7) ◽  
pp. 1777 ◽  
Author(s):  
Jaeyong Kim ◽  
Huwon Kang ◽  
Hakjoon Choi ◽  
Ara Jo ◽  
Dooi-Ri Oh ◽  
...  
Keyword(s):  
Aqueous Extract ◽  
Perilla Frutescens ◽  
Ciliary Muscle ◽  
Control Group ◽  
Dependent Manner ◽  
Sprague Dawley ◽  
Eye Fatigue ◽  
Freeze Dried

The leaves of Perilla frutescens var. acuta (PFA) are commonly used as a traditional medicine in Korea, Japan, and China. We previously showed that PFA attenuates eye fatigue by improving visual accommodation through a clinical study. However, detailed mechanisms and chemical compounds have not been studied. In this study, we analyzed the active compounds in an aqueous extract of PFA involved in ciliary muscle relaxation in vitro and in vivo. NMR and MS analyses showed that the PFA extract contained mainly luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide. The composition after freeze-drying and spray-drying was similar. Freeze-dried PFA (50 µg/mL, 100 µg/mL, and 200 µg/mL) increased nitric oxide and cGMP levels in ciliary muscle cells isolated from the eyes of rats. [Ca2+]i decreased in a dose-dependent manner. Furthermore, Sprague-Dawley rats treated with freeze-dried PFA (200 mg/kg, orally) showed significantly increased cGMP levels compared with the control group and irradiated with white light. Our results suggest that PFA extract has the potential to reduce eye fatigue by relaxing ciliary muscles.

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.

Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics

2013 ◽  
Vol 25 (6) ◽  
pp. 935 ◽  
Author(s):  
C. Tomás ◽  
E. Blanch ◽  
A. Fazeli ◽  
E. Mocé
Keyword(s):  
Dna Fragmentation ◽  
Sperm Quality ◽  
Epithelial Cell Line ◽  
Dna Integrity ◽  
Sperm Longevity ◽  
Boar Sperm ◽  
Fragmentation Dynamics ◽  
Freezing Treatment

The aim of this work was to examine how a pre-freezing treatment with cholesterol-loaded cyclodextrins (CLC) affects boar sperm longevity, capacitation dynamics, ability to bind to a porcine telomerase-immortalised oviductal epithelial cell line (TERT-OPEC) in vitro and DNA integrity dynamics after freeze–thawing. Although the samples treated with CLC exhibited lower sperm quality than the control samples (P < 0.05) immediately after thawing, these differences disappeared (P > 0.05) after long-term incubation (26 h at 37 or 16°C). Additionally, the CLC-treated spermatozoa underwent similar capacitation and DNA fragmentation dynamics as the control spermatozoa (P > 0.05). However, CLC-treated spermatozoa were better able to bind to TERT-OPEC in vitro (P < 0.0001). In conclusion, the pre-freezing treatment of boar spermatozoa with CLC enhanced the ability of the spermatozoa to bind to TERT-OPEC in vitro, which could have an effect on the establishment of the sperm reservoir in the ampullary­–isthmic junction in vivo. Additionally, frozen–thawed spermatozoa can be stored at 16°C for at least 6 h without a significant observable decline in sperm quality, which could be beneficial for the transport of thawed diluted doses of spermatozoa from the laboratory to the farm.

scholarly journals 321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD

2005 ◽  
Vol 17 (2) ◽  
pp. 311
Author(s):  
M. Nakai ◽  
K. Kikuchi ◽  
A. Takizawa ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Sperm Head ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Sperm Suspension ◽  
Freeze Dried ◽  
Vitro Development

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P < 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs. Table 1. Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs

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.

scholarly journals Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3309
Author(s):  
Michal Ďuračka ◽  
Kamila Husarčíková ◽  
Mikuláš Jančov ◽  
Lucia Galovičová ◽  
Miroslava Kačániová ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Bovine Semen ◽  
Sperm Dna

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus-induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 108 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus, S. warneri, S. kloosii, and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

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.

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.

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