Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution.

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196 degrees C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitro culturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitro without freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitro culturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.

[Clinical experience with a modified method of human embryo vitrification]. / Klinické skúisenosti s modifikovanou metódou vitrifikáicie l'udských embryí.

OBJECTIVE: To obtain clinical experience with a modified method of vitrification used to freeze supernumerary human embryos following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). SETTING: Centre of Assisted Reproduction, 2nd gynaecological-obstetrical clinic of Faculty hospital of L. Pasteur and Medical Faculty of the University of P. J. Safáirik, Kosice. METHODS: A modified method of vitrification was used to freeze 215 human embryos after IVF-ICSI, obtained in 42 cycles. The embryos were frozen at 48 h after in vitro culturing. The modified method of vitrification consisted in the use of a series of solutions with increasing cryoprotectant concentrations (ethylene glycol and sucrose) up to the final concentration of 40% ethylene glycol and 1 M sucrose. A 100 microl pipetting "tip" was used as a freezing container. The embroys were thawed at room temperature and the cryoprotectant was washed out three consecutive steps with sucrose solutions of decreasing concentrations (1 M, 0.5 M, and the pure medium). The morphology of embryos was evaluated before and after thawing. After thawing, the embryos were cultured in vitro for 24 hours and only the embryos with at least one cleaved blastomere were used for transfer. RESULTS: The percentage of embryos which survived thawing and were capable of further development was 69.8% and 48%, respectively. The mean number of transferred and developing embryos was 2.9 +/- 1.4. The percentage of clinical pregnancies per thawing cycle and per transfer was 19.0% and 27.6%, respectively. Seven patients delivered 8 children. CONCLUSION: The results showed that our modification of vitrification procedure of human embryos is reliable and represents a simpler, shorter and cheaper alternative to slow-rate freezing.

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification.

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.

Fetal ependyma in sheep and goat. A scanning electron microscopy study.

Morphology of the surface of apical membranes of ependymal cells has been studied in the whole ventricular system of the brain in goat and sheep fetuses using a scanning electron microscope. The surface structure of the ependymal lining surface in fetuses of small ruminants is very similar to that in adult animals. Supraependymal cells were found only in the lower part of the third cerebral ventricle in goat fetuses, but not in sheep fetuses. In small ruminants the ependymal surface is already regionally differentiated within the end of the first half of prenatal development. Fetal ependyma of goats and sheep, unlike the adult ependyma, is characterized by a high secretory activity, which is independent of sex or age of fetuses.

[Adrenal cortex hormones in the mother and fetus in premature labor]. / Hormóny nadoblickovej kôry matky a plodu pri predcasných pôrodoch.

In a group of 126 pregnant women with imminent premature delivery between the 20th and 37th week of gestation where after weekly intervals the serum cortisol and corticosterone levels from the cubital vein was assessed, it was revealed that in the group of women who aborted or had a premature delivery, as well as in women who had the delivery in term the corticoid values vary greatly: cortisol between 370 and 500 nmol/1 and corticosterone 70 to 240 nmol/1. In both groups there is a marked difference in final values. The latter have in women with premature deliveries a rising trend and in the group where pregnancy proceeded successfully to term a declining trend. Glucocorticoid blood levels from the umbilical arteries in the group of women who had an abortion or premature delivery, as compared with women who delivered their baby in term, no significant differences were recorded. However, the glucocorticoid blood levels from the umbilical veins were significantly higher in women who had premature deliveries or abortions, as compared with women who gave birth to their babies in term. The glucocorticoid levels in amniotic fluid collected during delivery did not manifest significant differences between the two groups of pregnant women.

[Changes in blood levels of progesterone, triiodothyronine and thyroxine and the results of quantitative histological analysis of the placentoma and corpus luteum in cows after prolonged labor]. / Zmeny koncentrácie progesterónu, trijódtyronínu a tyroxínu v krvi a výsledky kvantitatívnej histologickej analýzy placentómu i zltého telieska kráv po protrahovanom pôrode.

Changes in the cell structure of the placentoma and the corpus luteum were subjected to quantitative histological investigation, parallel with the examination of the concentrations of progesterone (P4), triiodotyronine (T3) and tyroxin (T4) during parturition (Oth hour) and two and six hours after parturition. The hormone concentrations were determined by the RIA method. The results of the quantitative histological analysis of the placentomae and the corpus luteum suggest that when the placenta is retained there is a significantly higher number of maintained epithelial cells in the placentoma crypts and a higher number of unchanged small, dark and large, light lutein serraetion cells in the biopsy of the corpus luteum during the period under investigation. The morphological finding significantly correlates with higher concentrations of P4 in the blood of the cows with retained placenta, as compared with those whose placenta was not retained. It can be derived from the pattern of changes in T3 and T4 concentrations, compared with P4 and with the histological finding, that there is a functional relationship between the placentoma, corpus luteum, and the thyroid gland.

[Superovulation after administration of PMSG in cows of various breeds]. / Superovulácia po aplikácii PMSG u kráv roznych plemien.

Superovulation response was studied to i. m. administration of 2500 and 3000 i. u. of PMSG (special product Folligon, Intervet Co.) in 67 breeding cows of the Black-Pied Lowland. Slovak Pied and Slovak Pinzgau breeds within the 9th to 12th days of their sexual cycles. The time which had elapsed from these cows' last calving ranged from 50 to 150 days and the number of their prior calvings ranged from one to ten. The best superovulation effect was obtained after the administration of 2500 i. u. PMSG in the Pinzgau breed: these cows had, on the average, 13.20 +/- 2.36 corpora lutea without non-ovulated follicles. At the PMSG dose increased to 3000 i. u., the number of yellow bodies rose to 18.11 +/- 1.12 and that of non-ovulated follicles to 3.46 +/- 0.46. In the Slovak Pied breed the average number of yellow bodies obtained after administration of 2500 i. u. PMSG was 11.74 +/- 1.27 and that of non-ovulated follicles was 0.44 +/- 0.02. At the PMSG dose of 3000 i. u., the number of yellow bodies increased to 15.49 +/- 1.62 and that of non-ovulated follicles increased up to 5.12 +/- 0.81. In the Black-Pied Lowland breed the lowest response was obtained: the i. m. administration of 2500 i. u. PMSG resulted in the formation of 9.5 +/- 0.84 yellow bodies and 1.16 +/- 0.26 non-ovulated follicles, and at the dose of 3000 i. u. the respective numbers were 13.41 +/- 0.89 and 3.07 +/- 0.39. Comparing the superovulation effect in dependence on age, the response of the cows of the Black-Pied Lowland and Slovak Pinzgau breeds to PMSG administration increases until the age of nine years (from 9.79 and 13.6 yellow bodies, respectively, on the average for the first five years to 12.71 and 17.99, respectively, in the ninth year); in the Slovak Pinzgau breed it decreases from 15.83, recorded in the first five years, to 8.28 +/- 1.68 in the ninth year. The number of non-ovulated follicles grows with age in the Slovak Pinzgau breed from 3.46 +/- 0.46 in the first five years to 3.64 between five and nine years of age and up to 4.44 +/- 0.52 after the ninth year, in the Slovak Pied breed from 0.79 to 4.21 +/- 0.38, and in the Black-Pied Lowland breed from 2.69 to 3.20 +/- 0.22 between the fifth and ninth year.

[Fixed acids during the traditional cesarean section in cows]. / Fixné kyseliny pri konzervatívnom cisárskom reze u kráv

The values of lactic acid, pyruvic acid and L/P index were studied in cows at the traditional Caesarean section. The values of the mentioned metabolites are presented in dependence on the intensity and length of parturient activity that are affected by the anesthesia applied during surgical procedures. An increase of the level of lactic acid was hindered by the anesthesia at the protracted parturient activity. An unequal drop of the level of lactic acid resulted from the interrupted intensive parturient activity and lowered labor of the organisms due to anesthesia during the surgical procedure. The levels of pyruvic acid increase after the anesthetic applied, but already during the surgical procedure a drop of their levels occurs that continues, with certain fluctuations, for 48 hours after the fetus extraction. The L/P index is relevant to the levels of fixed acids that reflect the labor of the organism at the protracted parturient activity and under an oxygen deficit. The increased L/P index after the application of the anesthetic decreases for three hours after the surgical procedure and later on, it maintains approximately the same level.

[Changes of lactic and pyramic acid levels and of the L/P index in cows and calves during parturition]. / Zmeny kyseliny mlieconej, pyrohroznovej a indexu L/P u kráv a teliat pri pôrode

The levels of lactic acid, pyruvic acid and L/P index were studied during parturition in cows and calves. The results demonstrate various values of the metabolites studied in dependence on the length of parturient activity and on the way of finishing the parturition. The level of lactic acid increases with protracted parturient activity and that of puruvic acid decreases. Anesthesia applied at the traditional caesarean section hinders the course of fixed acids, which is reflected in the values of the L/P index. An excessive accumulation of fixed acids in mother's organism at the protracted parturition unfavorably influences the metabolism of fetus by means of the fetoplacental bed. Anesthesia does not affect the levels of the metabolites studied in calves. The values of the L/P index are in the same proportion as the levels of lactic and pyruvic acids are.

[Fixed acids in physiological parturition]. / Fixné kyseliny pri fyziologickom pórode

The levels of lactic acid, pyruvic acid, and L/P index in blood were studied. During physiological parturition in 25 cows it was found that the levels of lactic acid and pyruvic acid showed an increase during parturition, and this increase reached a maximum at the expulsion stage. When foetus is delivered and parturient activity decreases, the levels of the two metabolites decline gradually. The increased values of the L/P index showing the saturation of tissues with oxygen indicate some degree of oxygen starvation during parturition which does not stop when foetus is out but persists some time after parturition.

[Fixed acids in protracted parturition]. / Fixné kyseliny pri protrahovanom pôrode

The levels of lactic acid, pyruvic acid, and L/P index were studied in 15 cows during protracted parturition. All the indices under study respond to the stress to which the organism is exposed as a result of continuing parturient activity:the indices increase in their values, and the peak values occur when the foetus is delivered. The levels of lactic acid and pyruvic acid drop immediately after parturition and the L/P index values decrease from the first hour post partum.