Biomedical subjects

Z Smorag

Publications and source records attributed to Z Smorag.

At least 19 recordsLinked to original sources

PubMed2006

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning.

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

Animals↗

PubMed2005

Sequence analysis of proviral DNA of porcine endogenous retroviruses.

Among all species analyzed, the domestic pig seems to be the most appropriate organ donor for xenotransplantation. Porcine endogenous retroviruses (PERVs) are present in genomes of all pigs and are capable of infecting human cells in vitro thus posing a serious threat for xenotransplantation procedures. Despite the abundant distribution of PERVs integrated with porcine genome, the majority of PERV proviral DNA is not capable of expressing viral proteins unless seriously mutated. The aim of the study was to analyze PERV genome for mutations. The study was performed on blood samples from 146 pigs. Long-range polymerase chain reaction (Long-PCR) was performed with primer sets designed within long terminal repeats (LTRs). Long-PCR products of different molecular weights were obtained: 530 bp (33.1% of individuals), 580 bp (76.7%), 933 bp (100%), and 2900 bp (59.8%). Amplimers of 7200 bp were absent in 12.8% of individuals, indicating the lack of intact proviral DNA. Sequence analysis showed that most PERV proviral DNA was significantly mutated, thus suggesting the inability to express functional viral RNA; however, it cannot be ruled out that compensatory recombination processes could occur enabling replication of defective proviruses.

Animals↗

PubMed2002

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

Animals↗

PubMed2002

Effect of rabbit age on sperm chromatin structure.

The aim of this study was to determine the relationship between the age of male rabbits and the sperm chromatin structure. The studies involved the semen of New Zealand White rabbits between 5 and 28 months of age. A flow cytometry and sperm chromatin structure assay (SCSA) method was used to determine chromatin structure. The results of cytometric chromatin structure assay suggested a relatively high stability of sperm chromatin in the rabbit. Between 6 and 16 months of age, the mean percentage of sperm with damaged chromatin was the lowest and ranged from 1.7 to 2.4%. Decreased sperm chromatin stability was found in ejaculates taken from male rabbits less than 5 months and more than 20 months of age.

Aging↗

PubMed2001

Sperm chromatin structure assay of bulls qualified for artificial insemination.

The goal of our study was to find the relationship between fertility of bulls qualified for AI and the percentage of spermatozoa with abnormal chromatin structure as an independent parameter. We used the frozen semen of 8 mature bulls from one AI center. Each bull was represented by 3 ejaculates collected with at least 2-week intervals. Bull fertility was calculated on the basis of non-return ratio and was expressed as a scale where 100 points represented the average fertility of all the AI center's bulls. Bulls with lower or higher fertility received a lower or higher score respectively. Fertility scores of bulls used in the study ranged from 83 to 104 . Semen was processed according to the SCSA (sperm chromatin structure assay) method and was analyzed by flow cytometry. "Artificial" alpha(t) (alpha(t)=red/green+red fluorescence) and red fluorescence histograms were used for calculation of COMPalpha(t), SDalpha(t), %Red, %PeakR and MeanR parameters. The percentage of spermatozoa with abnormal chromatin ranged from 1.2% to 23.8%. A large variation among ejaculates was found for bulls with lower fertility. Fertility correlated significantly with COMPalpha(t) (-0.50, P < 0.05), SDalpha(t) (-0.55, P < 0.01), %Red (-0.53, P < 0.01), %PeakR (-0.58, P < 0.01) and MeanR (-0.45, P < 0.05). The SCSA method has a practical application in analyzing spermatogenesis disorders in bulls. If regularly applied, it allows us to identify and eliminate ejaculates with a high level of sperm chromatin abnormalities.

Acridine Orange↗

PubMed2001

[On the problem of human cloning].

Somatic cell cloning technique in mammals is still not very efficient, but intensive efforts have been made to improve it. Considering the great biological affinity of humans and animals, the cloning technique can in the not too distant future be applied in human cloning and improved to the point of becoming safe. Even when we make such an assumption, I consider it irrational and dangerous to clone the human in order to make their copies (with human cloning for therapeutic purposes being another problem). Life, which is generated by the union of egg cell and spermatozoon is an unforeseeable combination of genetic possibilities, but at the same time it offers a unique chance for the human being, both as an individual and a species. The creation of life by genetic duplication of an already formed individual means a great reduction not only in the biological sense. Action like this is evidence of extreme egocentrism and totalitarian thinking, and its proponents should first answer the question whether they would consider cloning themselves. An answer in the affirmative would help to establish the underlying reasons for their approval.

Bioethics↗

PubMed2000

Sperm chromatin integrity of bucks transgenic for the WAP bGH gene.

The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13-18 months old. Semen was collected at 1-week intervals and 3-7 ejaculates from each rabbit were examined in total. Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes. The mean value of COMPalpha(t) was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.alpha(t) for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (-0.68, P<0.05) was found between S.D.alpha(t) of NTG males and percentages of pregnant does. The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene. The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks.

Acridine Orange↗

PubMed1997

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (</= 2 microm) or micropipette (</= 30 microm). The procedure resulted in expulsion of the blastocyst through the perforation and formation of an incomplete demi-embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

Journal Article↗

PubMed1995

The effect of co-culture system on developmental capacity of bovine IVM/IVF oocytes.

Two experiments were conducted to compare the influence of different culture systems and the oviduct donor's cycle phase on the developmental potential of co-cultured bovine embryos derived from IVM/IVF oocytes and to establish an efficient freezing method for oviduct epithelial cells. In the first experiment, the effects of media (Menezo B2, synthetic oviduct fluid SOF); sera (no serum, fetal calf serum FCS, human serum HS); and the presence or absence of monolayer of bovine oviduct epithelial cells (BOEC) on developmental capacity of bovine embryos were investigated. In the second experiment, the influence of oviduct donor's hormonal status (superovulated versus unstimulated) and the cryopreservation of oviductal tissue on the support of developmental competence of bovine IVM/IVF-derived zygotes were examined. Oviduct epithelial cells were cryopreserved according to the modified two-step method previously applied to rabbit embryos. For zygotes co-cultured with a monolayer of BOEC the following blastocyst development rates were obtained: 40.1% (63/157); 34.5% (60/174); 13.0% (7/54); and 19.2% (14/73), respectively, in B2 serum-free medium, B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS medium. In the absence of BOEC the rates were 12.3% (10/81); 41.4% (36/87); and 8.9% (6/67), respectively, in B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS. It was shown that the source of oviduct epithelial cells and previous freezing had no influence on the proportion of cleaved zygotes (approximately 70%) or on the percentage of blastocysts (approximately 20%).

Journal Article↗

PubMed1994

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA.

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.

Journal Article↗

PubMed1994

The effective culture method of zona-free rabbit 1-, 2- and 4-cell embryos.

The effect of modified incubation systems on the development capacity of the zona-free rabbit embryos was examined. Embryos at 1-, 2- and 4-cell stages were used. The removal of the zona pellucida was accomplished by the enzymic-mechanical technique. Denuded rabbit embryos were cultured using 3 incubation systems. In the first and the second system the embryos were cultured in microdrops. The difference between these first 2 systems concerned the volume of the microdrops and the kind of paraffin oil used. In the first system the embryos were cultured in 5mul microdrops covered with light or heavy paraffin oil; in the second system embryos were cultured in 40-mul microdrops under light paraffin oil. The third traditional system involved the incubation of embryos in glass capillaries into separated columns of medium. The percentage of blastocysts obtained from 1-cell embryos cultured in the first incubation system was 6.1% with heavy paraffin oil as the covering layer and 29.0% with light paraffin oil. In the second and third incubation systems blastocyst yield was 30.8 and 59.6%, respectively. The percentage of blastocysts obtained from 2-cell and 4-cell stage embryos with heavy paraffin oil was 18.7 and 25.0%, respectively; with light paraffin oil these figures were 40.0 and 50.0%, respectively. In the second incubation system these figures were 49.3 and 72.3%; and in the third incubation system they were 72.9 and 78.3%, respectively. The results of the experiment showed that culture into glass capillaries is undoubtedly an effecient method of culturing of the zona-free rabbit embryos.

Journal Article↗

PubMed1994

Cryopreservation of mammalian ova and embryos by vitrification.

Vitrification is a new approach to oocyte and embryo cryoconservation. It consists in the solidification of a solution caused not by crystallization, but by a drastic increase in viscosity during cooling. The application of this approach to cryoconservation of oocytes and embryos of different species depends upon the development of proper procedures and non-toxic media. From the technical point of view, the vitrification method is simple and relatively easily applicable under field conditions. The authors review the current procedures applied to oocytes and embryos of laboratory and farm animals.

Journal Article↗

PubMed1993

Factors affecting the survival of one- and two-cell rabbit embryos cryopreserved by vitrification.

The effects of equilibration time, glycerol (GLY), and 1,2-propanediol (PROH) concentration, and of vitrification and sucrose solution on the viability of 1- and 2-cell rabbit embryos were investigated. After collection, the embryos were equilibrated for 5 or 10 minutes in phosphate buffered saline (PBS) containing 10% GLY-20% PROH and were exposed for 30 seconds at 4 degrees C or were exposed and vitrified in one of two vitrification solutions 35% GLY-35% PROH or 20% GLY-50% PROH. The in vitro survival rates of 1-cell embryos equilibrated for both 5 and 10 minutes were lower (34.0 and 48.0%, respectively) than those of 2-cell embryos (78.8 and 68.5%, respectively; P<0.01). No differences were noted in the viability of embryos exposed to the 2 vitrification solutions. Following vitrification in a mixture of 35% GLY-35% PROH, the survival rates of 1- and 2-cell embryos were 18.3 and 13.7% and 19.6 and 10.4% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1- and 2-cell embryos vitrified in a solution of 20% GLY-50% PROH were 25.7 and 35.4% and 26.2 and 21.3% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1-and 2-cell embryos stored in 1M sucrose solution were 63.8 and 84.0%, respectively. In conclusion, the viability of vitrified 1- and 2-cell rabbit embryos was reduced as a consequence of their equilibration before vitrification, the exposure to vitrification solution and the dilution in a sucrose solution rather than of the vitrification process itself.

Journal Article↗

PubMed1991

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment.

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.

Animals↗

PubMed1990

The effect of sucrose and trehalose on viability of one- and two-cell rabbit embryos.

The effect of sucrose and trehalose on the viability of one- and two-cell rabbit embryos was investigated. A significant decrease in the viability of one- and two-cell embryos exposed for 30 min. at 20 degrees C was observed. At 38 degrees C none of the two-cell embryos in a sucrose solution survived after 30 min exposure, while approximately 50% of the embryos survived in a trehalose solution. The cleveage rate in culture of two-cell embryos exposed both to 2.0 M or 1.45 M trehalose was significantly lower in comparison with the control group. However the survival rate after transfer of two-cell embryos exposed to 1.45 M trehalose solution at 20 degrees C remained the same as that of the control group.

Journal Article↗

PubMed1989

Stage-dependent viability of vitrified rabbit embryos.

The aim of the work was to determine the susceptibility of rabbit embryos to vitrification at different developmental stages. The experiment was carried out on 676 embryos at 1-, 2- and 8-to 16-cell stages as well as the morula and blastocyst stages. As a vitrification medium, a mixture of 30% 1,2-propanediol + 30% glycerol (Solution I), or 35% 1,2-propanediol + 35% glycerol (Solution II), was used. The embryos were frozen in glass ampules placed in nitrogen vapour for 5 min before being plunged into liquid nitrogen. Dilution after rapid thawing was done in one step in a 1-M sucrose solution. After vitrification in Solution I, none of the 1- or 2-cell embryos survived, whereas the survival rate of 8-to 16-cell embryos, morula and blastocysts, was 23.0, 82.7 and 78.5%, respectively. After vitrification in Solution II, the survival rate of 1-, 2- and 8-to 16-cell embryos was 20.0, 43.8 and 92.9%, respectively. The proportion of live offspring on the Day 28 after transfer of 68 vitrified morula was 26.5% compared with 24.0% in the control group. Thus, the proposed vitrification procedures can be useful in the cryopreservation of rabbit embryos.

Journal Article↗

PubMed1989

Influence of hardening of the zona pellucida on in vitro fertilization of bovine oocytes.

The influence of hardening of the zona pellucida of in vivo matured bovine oocytes on fertilizability was investigated. For the study, 163 preovulatory and 73 postovulatory oocytes recovered from superovulated heifers were used. The preovulatory oocytes, before they were used for in vitro fertilization, consisted of: 1) those cultured in vitro for 4 to 6 h to permit final maturation and 2) those incubated in the rabbit oviduct for 4 to 5 h to permit final maturation and induce hardening of the zona pellucida. A few oocytes served as a control of nuclear maturity and the zona pellucida solubility. Preovulatory and postovulatory oocytes were both inseminated in vitro using frozen-thawed, heparin treated and swim-up separated spermatozoa. Significant differences (P<0.01) were established between fertilization rates of cultured preovulatory oocytes (68.8%) and those incubated in the rabbit oviducts (42.9%), or those recovered from bovine oviducts (40.7%). It can be concluded that hardening of the zona pellucida distinctly influences the fertilizability of oocytes. This factor should be taken into account when considering the source of oocytes or the kind of treatment to be used for in vitro fertilization.

Journal Article↗

PubMed1989

Cell loss in bisected mouse, sheep and cow embryos.

The purpose of this experiment was to determine the loss of cells during the splitting of embryos. The experiment was carried out on early, middle and late stage mouse blastocysts as well as on late stage sheep and cow blastocysts. The embryos were bisected with a metal mioroscalpel. Manipulation of the embryos was done in phosphate buffered saline (PBS) solution with 20% fetal calf serum (FCS) at +37 degrees C or in PBS solution free of Ca(++) and Mg(++) ions. To determine the number of cells present, intact embryos and demi-embryos were placed in acridine orange solution and examined under a fluorescent microscope. The average number of cells in early, middle and late stage mouse blastocysts was 39.4, 54.1 and 71.9, respectively. The loss of cells caused by splitting was 6.4, 7.6 and 9.9, respectively. The average number of cells in late stage sheep blastocysts was 115.3 and in late stage cow blastocysts it was 138.5. In sheep and cows, the loss of cells due to splitting the embryos in a PBS solution free of Ca(++) and Mg(++) ions did not differ from that observed in a PBS solution with 20% FCS. Similarly, bisection carried out at +37 degrees C did not result in lower cell losses, than that done at room temperature.

Journal Article↗