Fundamental radiological and geometric performance of two types of proton beam modulated discrete scanning systems.

PURPOSE: The purpose of this investigation was to compare and contrast the measured fundamental properties of two new types of modulated proton scanning systems. This provides a basis for clinical expectations based on the scanned beam quality and a benchmark for computational models. Because the relatively small beam and fast scanning gave challenges to the characterization, a secondary purpose was to develop and apply new approaches where necessary to do so. METHODS: The following performances of the proton scanning systems were investigated: beamlet alignment, static in-air beamlet size and shape, scanned in-air penumbra, scanned fluence map accuracy, geometric alignment of scanning system to isocenter, maximum field size, lateral and longitudinal field uniformity of a 1 l cubic uniform field, output stability over time, gantry angle invariance, monitoring system linearity, and reproducibility. A range of detectors was used: film, ionization chambers, lateral multielement and longitudinal multilayer ionization chambers, and a scintillation screen combined with a digital video camera. Characterization of the scanned fluence maps was performed with a software analysis tool. RESULTS: The resulting measurements and analysis indicated that the two types of delivery systems performed within specification for those aspects investigated. The significant differences were observed between the two types of scanning systems where one type exhibits a smaller spot size and associated penumbra than the other. The differential is minimum at maximum energy and increases inversely with decreasing energy. Additionally, the large spot system showed an increase in dose precision to a static target with layer rescanning whereas the small spot system did not. CONCLUSIONS: The measured results from the two types of modulated scanning types of system were consistent with their designs under the conditions tested. The most significant difference between the types of system was their proton spot size and associated resolution, factors of magnetic optics, and vacuum length. The need and benefit of mutielement detectors and high-resolution sensors was also shown. The use of a fluence map analytical software tool was particularly effective in characterizing the dynamic proton energy-layer scanning.

GATE as a GEANT4-based Monte Carlo platform for the evaluation of proton pencil beam scanning treatment plans.

Active scanning delivery systems take full advantage of ion beams to best conform to the tumor and to spare surrounding healthy tissues; however, it is also a challenging technique for quality assurance. In this perspective, we upgraded the GATE/GEANT4 Monte Carlo platform in order to recalculate the treatment planning system (TPS) dose distributions for active scanning systems. A method that allows evaluating the TPS dose distributions with the GATE Monte Carlo platform has been developed and applied to the XiO TPS (Elekta), for the IBA proton pencil beam scanning (PBS) system. First, we evaluated the specificities of each dose engine. A dose-conversion scheme that allows one to convert dose to medium into dose to water was implemented within GATE. Specific test cases in homogeneous and heterogeneous configurations allowed for the estimation of the differences between the beam models implemented in XiO and GATE. Finally, dose distributions of a prostate treatment plan were compared. In homogeneous media, a satisfactory agreement was generally obtained between XiO and GATE. The maximum stopping power difference of 3% occurred in a human tissue of 0.9 g cm(-3) density and led to a significant range shift. Comparisons in heterogeneous configurations pointed out the limits of the TPS dose calculation accuracy and the superiority of Monte Carlo simulations. The necessity of computing dose to water in our Monte Carlo code for comparisons with TPSs is also presented. Finally, the new capabilities of the platform are applied to a prostate treatment plan and dose differences between both dose engines are analyzed in detail. This work presents a generic method to compare TPS dose distributions with the GATE Monte Carlo platform. It is noteworthy that GATE is also a convenient tool for imaging applications, therefore opening new research possibilities for the PBS modality.

SU-E-T-299: Proton Pencil Beam Spot Scanning Phase Space in the IBA System and the Clinical Implications for Superficial Targets.

PURPOSE: In the treatment of superficial lesions with proton Pencil Beam Scanning (PBS), spot size is dominated by the nozzle contribution. Accuracy of phase-space modeling is therefore paramount. IBA's Dedicated (DN) and Universal Nozzles (UN) have different designs and, consequently, characteristics. Here we report the phase spaces of these two nozzles, without and with a range shifter (RS). METHODS: In-air spot fluence measurements were made for five proton energies: 225, 210, 180, 150 and 115 MeV and at five distances from isocenter pertinent to SAD-type treatments: +33, +20, +10, 0 and -10 cm ('+' implies upstream), without and with a 7.5 cm water-equivalently-thick RS (sufficient to pull back the lowest energy Bragg peak to patient surface), fixed with its upstream side 41 cm from isocenter. Data collected on a fixed horizontal beam-line with a DN and a gantry-mounted UN were compared. The full-width-at-half-maximum (FWHM) of a Gaussian fit to each spot fluence profile was extracted along the two principal axes. RESULTS: With no RS, the proton spots are ∼20-70% larger at isocenter in the UN than in the DN. Spots are less asymmetric, and eccentricity increases more slowly with energy, in the UN than in the DN. Over the 33 cm in-air travel upstream of isocenter, the spot FWHM varies by less than ∼2 mm. However, spot asymmetry becomes more severe upstream (for 115 MeV spots, 30-40% compared to <20% at isocenter for DN, but similar and <10% for UN). With an RS, spot FWHM at isocenter increases by 12.7 mm from 8.3 mm (DN) and 10.7 mm from 13 mm (UN) for 150 MeV protons (typical for brain treatments). With no RS, relatively distance-independent spot size facilitates SAD-type treatments. For patients with superficial lesions, where an RS is required and the phase space varies rapidly with distance, the RS should be permitted at two additional locations. US Army Medical Research and Materiel Command under Contact Agreement No. DAMD17-W81XWH-04-2-0022.

A Monte Carlo pencil beam scanning model for proton treatment plan simulation using GATE/GEANT4.

This work proposes a generic method for modeling scanned ion beam delivery systems, without simulation of the treatment nozzle and based exclusively on beam data library (BDL) measurements required for treatment planning systems (TPS). To this aim, new tools dedicated to treatment plan simulation were implemented in the Gate Monte Carlo platform. The method was applied to a dedicated nozzle from IBA for proton pencil beam scanning delivery. Optical and energy parameters of the system were modeled using a set of proton depth-dose profiles and spot sizes measured at 27 therapeutic energies. For further validation of the beam model, specific 2D and 3D plans were produced and then measured with appropriate dosimetric tools. Dose contributions from secondary particles produced by nuclear interactions were also investigated using field size factor experiments. Pristine Bragg peaks were reproduced with 0.7 mm range and 0.2 mm spot size accuracy. A 32 cm range spread-out Bragg peak with 10 cm modulation was reproduced with 0.8 mm range accuracy and a maximum point-to-point dose difference of less than 2%. A 2D test pattern consisting of a combination of homogeneous and high-gradient dose regions passed a 2%/2 mm gamma index comparison for 97% of the points. In conclusion, the generic modeling method proposed for scanned ion beam delivery systems was applicable to an IBA proton therapy system. The key advantage of the method is that it only requires BDL measurements of the system. The validation tests performed so far demonstrated that the beam model achieves clinical performance, paving the way for further studies toward TPS benchmarking. The method involves new sources that are available in the new Gate release V6.1 and could be further applied to other particle therapy systems delivering protons or other types of ions like carbon.

Ovum pick up and in vitro embryo production in cows superstimulated with an individually adapted superstimulation protocol.

The aim of this experiment was to apply an ovarian superstimulation protocol prior to ovum pick up (OPU), tailored to the individual donor response, to evaluate its advantages and disadvantages in terms of follicle numbers and diameters, the numbers of retrieved oocytes and day 7 cultured blastocysts. Ten adult non-lactating dairy cows were superstimulated with pFSH and subjected to ovum pick up-in vitro fertilisation (OPU-IVF) 6 times at 2-week intervals. On day 0 of each 2-week period, all follicles >8mm were ablated and an ear implant (Crestar, Intervet, Belgium) was inserted. On day 2, 48 h after follicle ablation the animals were administered six equal doses of pFSH, divided into morning and evening doses for 3 days. On day 7, 48 h following the last pFSH injection, follicle diameters were measured by ultrasound and all follicles were subjected to OPU. All cumulus-oocyte complexes (COC), regardless of their quality, were subjected to in vitro maturation-in vitro fertilisation-in vitro culture (IVM-IVF-IVC). The total dose of pFSH prior to the first OPU session was 300 microg per animal. During the following OPU sessions, the total pFSH dose was either kept unchanged, increased or reduced (+/-50 microg), according to the percentage of follicles of more than 11 mm in diameter, present in the previous session of that particular donor. The mean number of punctured follicles per session was 11.9 +/- 7.7 (mean +/- S.D.), with 16% of follicles exceeding 11 mm. These follicles yielded a mean of 5.6 +/- 4.1 cumulus oocyte complexes (COC), 32% of which had >/=3 layers of cumulus cells (quality 1 and 2). The recovery rate was 47%. Finally, all COC were subjected to IVM-IVF-IVC, which resulted in a mean of 2.0 +/- 2.3 blastocysts on day 7 postinsemination. The subtle changes in pFSH dose influenced the sizes but not the numbers of follicles, the latter parameter was influenced by the individual donor and the OPU session.

Impact of adding 5.5 mM glucose to SOF medium on the development, metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage.

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.

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.

Expression of Cu/Zn and Mn superoxide dismutases during bovine embryo development: influence of in vitro culture.

Temporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase-polymerase chain reaction (RT-PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5- to 8-cell embryos while no transcript was found at the 9-to 16-cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo-derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro-derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions.

Pulsatile activity and hatching of in vitro produced cow blastocysts: effects of serum supplementation.

The effect of serum added to a modified SOF medium on pulsatile activity and hatching of in vitro produced cow blastocysts was investigated by time-lapse cinematography. Embryos were generated from abattoir material and cultured in mSOF without serum or with 10% FCS added at 42h pi. Addition of serum significantly increases pulsatile activity before zona rupture and reduces the time of hatching. Pulsatile activity does not seem to be involved in the hatching process.

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.

High mortality rate associated with bovine respiratory syncytial virus (BRSV) infection in Belgian white blue calves previously vaccinated with an inactivated BRSV vaccine.

In a group of 60 Belgian White Blue calves less than 8 months old still housed in barns, a bovine respiratory syncytial virus (BRSV) outbreak was revealed on the basis of a direct diagnosis (immunofluorescence and virus isolation) performed on the lungs of dead animals, and the kinetics of BRSV neutralizing antibodies. Clinical signs, macroscopical and microscopical pulmonary lesions were also compatible with a BRSV infection. This outbreak is peculiar because the 35 oldest calves (204 +/- 29 days old) had been vaccinated 3-4 months before with an inactivated BRSV vaccine and 30% of these animals had died of respiratory distress. While they experienced a mild respiratory symptomatology, no death was recorded among the 25 youngest calves (69 +/- 29 days old) which had been left unvaccinated. Another peculiarity was found at the histological level where a massive infiltration of eosinophils was demonstrated in the pulmonary tissues of the dead animals. Together these data parallel the dramatic story described 30 years ago in children previously vaccinated with a formalin-inactivated human RSV (HRSV) vaccine upon a natural HRSV challenge. This illustrates that an immunopathological phenomenon also takes place after BRSV vaccination in cattle.

Comparative immunohistochemical distribution of connexin 37 and connexin 43 throughout folliculogenesis in the bovine ovary.

Among gap junctional proteins previously identified in the mouse ovary, connexins (Cx) Cx37 and Cx43 appeared to be essential for normal follicular growth. The aim of this work was to detect Cx37 expression in the bovine ovary, then to quantify and compare its follicular distribution pattern with that of Cx43 using quantitative analysis of immunofluorescently labeled ovary sections viewed with a confocal laser scanning microscope. Cx37 immunoreactivity was detected in bovine ovarian follicles and was predominantly localized at preantral stages. Unlike follicular Cx43 expression which was restricted to granulosa cells, Cx37 staining was observed in both oocyte and granulosa cell compartments. While no changes were seen during early follicular growth, the level of Cx37 expression decreased significantly at the onset of antral cavity formation (P

Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos.

The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H2O2 as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H2O2 degrading ability. Essential and nonessential amino acids also significantly reduced the H2O2 concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9-16-cell stage embryos and blastocysts to 0 M; 10(-7) M ; 10(-6) M, and 10(-5) M H2O2 in pyruvate-free mSOF was evaluated. Developmental rates of the H2O2-treated zygotes to the 5-8-cell or blastocyst stages and survival of H2O2-treated blastocysts were reduced in a dose-dependent manner whereas the 9-16-cell embryos were unaffected by those treatments. Blastocysts treated with H2O2 also tended to have lower numbers of bisbenzimide-stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10(-5) M H2O2 pulse highly reduced the H2O2 concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H2O2, the 9-16-cell embryos being more resistant than zygotes and blastocysts and that H2O2 and its toxic effects can be attenuated by including pyruvate in the medium.

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.

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.

Glucose metabolism during bovine preimplantation development: analysis of gene expression in single oocytes and embryos.

Glucose metabolism of the bovine embryo is low during the first cleavages and increases sharply after the major resumption of the genome (8-16 cells). The mRNA level for genes involved in glucose metabolism was tested by RT-PCR on individual oocytes and embryos at different stages of development. These genes were: glucose transport GLUT-1, hexokinase (HK), glucose-6-phosphatase-dehydrogenase (G6PDH), and glucose-phosphate-isomerase (GPI); actin was used as a reference transcript. RT-PCR results revealed three types of oocytes or embryos: positive with a PCR signal for each transcript considered, nul with no signal for any transcript, and heterogeneous with a PCR signal for some transcripts and none for others. The number of nul and heterogeneous samples was higher for slow than for fast-cleaving embryos (81% vs. 36%), and the proportion of positive embryos increased significantly at the 16-cell and morula stages (P < 0.002), suggesting a correlation between mRNA content and developmental capacity. In positive embryos, GLUT-1 level was reduced by half during maturation and fertilization. Actin and hexokinase mRNA levels decreased during the first cleavages, but significantly increased at the 16-cell and morula stages, respectively. GPI transcript remained stable throughout development, whereas there was a significant rise for G6PDH at the 4-cell stage, perhaps due to a polyadenylation process. Finally, the absence or decrease in intensity of several transcripts at the blastocyst stage suggests suboptimal culture conditions.

Effects of co-culture and embryo number on the in vitro development of bovine embryos.

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.

Effect of estrus-associated glycoprotein and tissue inhibitor of metalloproteinase-1 secreted by oviduct cells on in vitro bovine embryo development.

The effect of two glycoproteins (estrus-associated glycoprotein [EGP] and tissue inhibitor of metalloproteinase [TIMP-1]) secreted by bovine oviduct cells on in vitro bovine embryo development was assessed. A first set of experiments was conducted to determine whether the embryotrophic activity of the bovine oviduct-conditioned medium (BOCM) was correlated with the presence of EGP or TIMP-1. EGP and TIMP-1 were detected in BOCM, supporting the development of 22% zygotes to the blastocyst stage, as well as in BOCM yielding a low blastocyst rate (3-4% blastocysts). These glycoproteins do not seem to be necessary for bovine embryo development up to the blastocyst stage in our BOCM. In a second experiment, zygotes were cultured in modified synthetic oviduct fluid (mSOF) supplemented with different concentrations (0.5, 5, 50, and 500 micrograms/ml) of purified bovine EGP. In the third experiment, since purified bovine TIMP-1 was not available, zygotes were cultured in BOCM depleted of TIMP-1 by immunoprecipitation treatment. Adding EGP to mSOF, or removing TIMP-1 from BOCM, did not affect bovine embryo development up to the blastocyst stage, or mean number of cells per blastocyst after 8 days of culture. The results indicate that, under our culture conditions, EGP and TIMP-1 do not play an important role in sustaining bovine embryo development, and do not influence blastocyst quality, assessed in terms of total number of cells per embryo.