Quality of rabbit vitrified/thawed transgenic embryos.

The aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous-hFVIII or exogenous-enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate.

The aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8-12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8-12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification.

The aim of this study was to compare two vitrification procedures (VPs), using either ethylene glycol (EG) in combination with dimethylsulfoxide (DMSO, vitrification protocol (VPI)) or Ficoll 70 (vitrification protocol II (VPII)), for rabbit embryo cryopreservation based on their post-thaw survival, cell death and actin cytoskeleton. The pronuclear stage eggs were flushed from the oviducts of the slaughtered New Zealand White rabbit does 19-20h post coitum (hpc) and randomly divided into two groups: intact (control) and microinjected (Mi). Mi embryos or intact embryos were cultured for up to 72hpc (morula stage), and then vitrified using either VPI (VPI+Mi, VPI) or VPII (VPII+Mi, VPII). After 2-3 days at -196 degrees C, the embryos were thawed and cultured until 96-100hpc to assess their development to blastocyst, apoptotic rate (TUNEL assay) and state of actin cytoskeleton (phalloidine-TRITC). Mi procedure reduced blastocyst yield, but it was higher than in either vitrified (VPI) or Mi vitrified (VPI+Mi) embryos. VPI compromised, whereas VPII did not significantly affect blastocyst development compared to intact embryos. Mi and VP both affected the embryo quality increasing TUNEL-index and decreasing the ratio of embryos with high quality actin cytoskeleton compared to control. A higher apoptotic index was recorded in VPI group. A combination of Mi and VP induced an increase in apoptotic rate (10.35% and 7.54% for VPI+Mi and VPII+Mi, respectively) as compared to Mi alone (5.7%). Ratio of embryos belonging to best actin quality (grade I) was different among groups and most of the embryos with grade I actin were in intact (84%), Mi (71%) or VPII (70%) groups. A significantly lower number of embryos with grade I actin quality was observed in VPI (58%), VPI+Mi (54%) or VPII+Mi (66%). These observations indicate that of the vitrification schemes tested, the VPII using EG and ficoll 70 as cryoprotectants, was less harmful than VPI (EG combined with DMSO in vitrification medium).

[Effect of transcutaneous stimulation of diaphragm on systemic and central hemodynamics in patients with chronic bronchitis]. / Obshchaia i tsentralnaia gemodinamika u bolnykh khronicheskim bronkhitom pri primenenii chreskozhnoi élektrostimuliatsii diafragmy.

25 males and 5 females suffering from chronic obstructive bronchitis (COB) underwent transcutaneous diaphragmatic electrostimulation (TDE). As shown by central intrapulmonary hemodynamics, those patients who had no circulatory decompensation benefited from TDE. There was a decrease in the pulmonary hypertension, intensification of general hemodynamics, reduced tonicity of the pulmonary arteriolar bed, improvement of right ventricular function. In circulatory decompensation TDE may cause deterioration of right ventricular function, enhancement of circulation decompensation against unchanged pulmonary hypertension and pulmonary-arteriolar tonicity. The changes in pulmonary artery pressure, total pulmonary vascular resistance, pulmonary-arteriolar elasticity observed after a single TDE session may prompt the validity of further COB treatment according to the above regimen.

[Diaphragmatic muscle fatigue--its diagnosis and treatment]. / Utomlenie diafragmal'noi myshtsy--diagnostika i lechenie.

Transcutaneous stimulation electromyography of the diaphragm was performed in 64 patients suffering from chronic obstructive bronchitis (COB) to evaluate the function of the diaphragmatic muscle. Increased amplitude, area and shorter M-response gave evidence for the muscle fatigue. Changes in the above parameters correlated with the degree of the respiratory insufficiency and pulmonary hypertension. The authors included subcutaneous electrostimulation of the diaphragm in combined treatment of COB patients free of clinical manifestations indicating circulation decompensation. These patients benefited from the stimulation in contrast to COB patients with decompensated circulation who failed to respond to the procedure.