Comparison of two methods (left carotid artery and abdominal aorta) for surgical implantation of radiotelemetry devices in CD-1 mice.

The goal of this study was to compare two surgical methods, the left carotid (LC) and the abdominal aorta (AA), for mouse instrumentation with telemetry devices, to determine the best method for measuring cardiovascular (CV) parameters by radiotelemetry in freely moving mice. Surgery success rate, postsurgical recovery rate, clinical parameters, CV data (baseline and response to nicotine) and circadian rhythm measurements were compared between these techniques. Brains of LC-implanted mice were evaluated for potential ischaemia by direct observation of the Circle of Willis anatomy and histopathology. For this purpose, a total of 31 CD-1 male mice were instrumented with PA C20 devices (10 with LC and 21 with AA). Mortality, morbidity, physical examination, body weight (BW), water and food consumption (W/FC), mean blood pressure (MBP) and heart rate (HR) were monitored daily during the recovery period (10 days). CV baseline data were recorded continuously during two periods of four days, and finally, both LC- and AA-implanted mice received an acute subcutaneous administration of 1 mg/kg nicotine; BP and HR were recorded during 5 h after nicotine administration. Results showed that, in LC-implanted mice, 80% survived surgery and recovered well. In contrast, only 57% of mice implanted with the AA technique survived surgery and some presented lethal complications. Both techniques had similar recovery times for BW and W/FC, comparable return to normal circadian rhythm (day 6 post-surgery) and similar CV baseline values. No significant differences were observed in CV response to nicotine between both groups of implanted CD-1 mice. No histopathological changes suggestive of ischaemia were noted in the brain of mice implanted in the LC. Six out of the eight LC-implanted mice remained in good health and had good pressure signal for at least 100 days post-surgery, while most of the AA-implanted mice lost the signal pressure within 14-49 days post-surgery. In conclusion, we believe that LC implantation in mice is superior to the AA technique and is more appropriate for long-term telemetry studies, especially for smaller (transgenic) animals.

Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro.

This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism.

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.

Osmotic behavior of in vitro produced bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival.

The osmotic behavior of bovine blastocysts produced in vitro was filmed during exposure to and dilution of cryoprotectant solutions used for vitrification. The relationship between the changes in the diameter of embryos and their subsequent survival was assessed. Embryos collected on Day 6 and Day 7 postinsemination were exposed to 10% glycerol (GLY) for 5 min, 10% GLY + 20% ethylene glycol (EG) for 5 min, and 25% Gly + 25% EG for 30 s, before dilution in 0.85 M galactose and finally in embryo transfer freezing medium (ETF). Embryos that had a higher probability of survival behaved as perfect osmometers, shrinking, reexpanding, or swelling according to an identical pattern, whereas embryos that deviated from this standard usually did not survive. The initial embryo diameter, duration of shrinkage and expansion in 10% glycerol, duration of reexpansion in ETF, and final embryo diameter were clearly predictive of the ability to hatch after culture in vitro. On a given day postinsemination, larger blastocysts were more likely than smaller blastocysts to survive and hatch after exposure to cryoprotectants with or without vitrification.

Embryo production by ovum pick up in unstimulated calves before and after puberty.

The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty.

Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos.

Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide--BIS) and a nonpermeant one (propidium iodide--PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2% vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dilution step in 0.42 M galactose (Experiment 4) before dilution in ETF decreased the proportion of cells permeant to PI and increased the hatching rate after 72 h of co-culture. In conclusion, double staining with BIS and PI allowed for discrimination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful tool for adjusting equilibration and dilution conditions during a cryopreservation procedure.

Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity.

Methodological studies were undertaken to test the validity of a three-step vitrification procedure for bovine in vitro produced embryos using glycerol and ethylene glycol as cryoprotectants. Embryos were produced in a low-phosphate culture system (medium VT1 + 10% foetal calf serum) and vitrified at day 7 post-insemination either in a mixture of 25% glycerol--25% ethylene glycol or a mixture of 10% glycerol--40% ethylene glycol. In the first mixture 67% (n = 283) of blastocysts were re-expanded after 72 h of culture and 53% were hatched while in the second one (n = 65) only 5% survived. The mean number of cells of the surviving blastocysts was correlated with the rate of survival (R2 = 0.47; P = 0.0024). Embryo size (diameter < or > to 180 microm) did not influence blastocyst survival or cell number, but hatching rate was higher for embryos > 180 microm. Embryo survival, hatching rate and cell number 72 h post-warming were not affected by the mode of vitrification (direct plunging into nitrogen liquid or vitrification into nitrogen liquid vapour), the mode of preparation of the vitrification solutions (molar or molal basis) or by the concentration of galactose used as a diluent (0 to 0.85 M). Only one calf was born after transfer of 22 vitrified blastocysts. These results confirm the apparent lack of correlation for cryopreserved embryos between in vitro survival or hatching and viability after transfer.

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification.

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n = 181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O2. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O2: 62% vs. 5% O2: 25%, P < 0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O2 than under 5% O2 as indicated by lower total and trophectoderm cell numbers (respectively 79 +/- 6 and 56 +/- 6 at 20% O2 vs. 100 +/- 10 and 74 +/- 10 at 5% O2, P < 0.05), by an altered ICM/trophectoderm ratio (20% O2: 28% vs. 5% O2: 23%, P < 0.05), by a higher total nuclear fragmentation (20% O2: 3.7% vs. 5% O2: 1.5%, P < 0.05) and a trend to decreased hatching (20% O2: 32% vs. 5% O2: 81%, P = 0.07). Whereas, for BRL co-culture, 20% O2 yielded higher quality blastocysts than 5% O2 as evaluated by higher ICM and trophectoderm cell numbers (19 +/- 1 and 71 +/- 5 at 20% O2 vs. 15 +/- 2 and 48 +/- 9 at 5% O2, respectively, P < 0.05), by lower nuclear fragmentation in the ICM (20% O2: 2.2% vs. 5% O2: 6.7%, P < 0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O2 seems to be the best combination to evaluate blastocyst survival and quality after vitrification.