[Scaffold hopping computational approach for searching novel ß-lactamase inhibitors]. / Virtual'nyi skrining s ispol'zovaniem izmeneniia strukturnogo ostova ligandov dlia poiska novykh ingibitorov ß-laktamaz.

We present a novel computational ligand-based virtual screening approach with scaffold hopping capabilities for the identification of novel inhibitors of ß-lactamases which confer bacterial resistance to ß-lactam antibiotics. The structures of known ß-lactamase inhibitors were used as query ligands, and a virtual in silico screening a database of 8 million drug-like compounds was performed in order to select the ligands with similar shape and charge distribution. A set of numerical descriptors was used such as chirality, eigen spectrum of matrices of interatomic distances and connectivity together with higher order moment invariants that showed their efficiency in the field of pattern recognition but have not yet been employed in drug discovery. The developed scaffold-hopping approach was applied for the discovery of analogues of four allosteric inhibitors of serine ß-lactamases. After a virtual in silico screening, the effect of two selected ligands on the activity of TEM type ß-lactamase was studied experimentally. New non-ß-lactam inhibitors were found that showed more effective inhibition of ß-lactamases compared to query ligands.

Seeking, accepting and declining help for emotional distress in cancer: A systematic review and thematic synthesis of qualitative evidence.

Many individuals affected by cancer who experience emotional distress report not wanting help. This review aims to understand why individuals affected by cancer seek, accept or decline help for emotional distress and what influences these actions. A systematic review and thematic synthesis of the qualitative literature was conducted. Using pre-defined search terms, four electronic databases were searched from January 2000 to May 2016. Pre-determined inclusion and exclusion criteria were then applied. Identified papers were quality appraised. In total, 32 papers were included in the synthesis. Four themes emerged from data synthesis: attaining normality-the normality paradox; being emotionally literate; perceptions of help; needs-support gap. Attaining normality is ideographic, context dependent and temporally situated; some individuals maintain normality by not seeking/declining help whereas others seek/accept help to achieve a new normality. Thus, attaining normality paradoxically functions to explain both why individuals sought/accepted help or did not seek/declined help. Data indicate that a context dependent, systems thinking approach is merited to enhance psychosocial care. In particular, clinicians must actively explore the personal context of an individual's distress to ensure that help desired and help offered are mutually understood. Further research must address the limitations of the current evidence base to advance theoretical understanding.

General practitioners' 'lived experience' of assessing psychological distress in cancer patients: an exploratory qualitative study.

While psychological distress in cancer patients is common, little is known about how general practitioners (GPs) assess distress. Using semi-structured interviews, a phenomenological study of seven GPs was conducted to explore GPs' experiences of assessing distress. Findings revealed five themes: (1) Being in the Relay Team - receiving and passing the baton: where the assessment of distress was conceptualised as a relay baton passed between a team of health care professionals, with GPs most involved at diagnosis and in the palliative phase. (2) Being in a Relationship: where the doctor-patient relationship was described as a powerful facilitator to assessment. (3) Being Skilled: where GPs perceive they are skilled at assessment adopting a patient-centred approach. (4) Being Challenged - encountering barriers: challenges with assessment were identified regarding the GPs' own emotions, patient related factors and time; the duality of family as both barrier and facilitator was voiced. (5) The Intruder in the Room: where GPs did not use validated screening tools which were viewed as an intruder in the doctor-patient relationship. Further research to objectively assess GPs' skills in distress assessment and attitudes towards the use of screening tools within the cancer care context are merited.

Automated identification of crystallographic ligands using sparse-density representations.

A novel procedure for the automatic identification of ligands in macromolecular crystallographic electron-density maps is introduced. It is based on the sparse parameterization of density clusters and the matching of the pseudo-atomic grids thus created to conformationally variant ligands using mathematical descriptors of molecular shape, size and topology. In large-scale tests on experimental data derived from the Protein Data Bank, the procedure could quickly identify the deposited ligand within the top-ranked compounds from a database of candidates. This indicates the suitability of the method for the identification of binding entities in fragment-based drug screening and in model completion in macromolecular structure determination.

IgG N-glycans as potential biomarkers for determining galactose tolerance in Classical Galactosaemia.

N-glycan processing and assembly defects have been demonstrated in untreated and partially treated patients with Classical Galactosaemia. These defects may contribute to the ongoing pathophysiology of this disease. The aim of this study was to develop an informative method of studying differential galactose tolerance levels and diet control in individuals with Galactosaemia, compared to the standard biochemical markers. Ten Galactosaemia adults with normal intellectual outcomes were analyzed in the study. Five subjects followed galactose liberalization, increments of 300 mg to 4000 mg/day over 16 weeks, and were compared to five adult Galactosaemia controls on a galactose restricted diet. All study subjects underwent clinical and biochemical monitoring of red blood cell galactose-1-phosphate (RBC Gal-1-P) and urinary galactitol levels. Serum N-glycans were isolated and analyzed by normal phase high-performance liquid chromatography (NP-HPLC) with galactosylation of IgG used as a specific biomarker of galactose tolerance. IgG N-glycan profiles showed consistent individual alterations in response to diet liberalization. The individual profiles were improved for all, but one study subject, at a galactose intake of 1000 mg/day, with decreases in agalactosylated (G0) and increases in digalactosylated (G2) N-glycans. We conclude that IgG N-glycan profiling is an improved method of monitoring variable galactosylation and determining individual galactose tolerance in Galactosaemia compared to the standard methods.

Further analysis of the population history of ancient Egyptians.

The origins of state formation in ancient Egypt have been the focus of recent research utilizing biological data to test hypotheses regarding in situ development of local groups, or large-scale in-migration, possibly by an invading army. The primary goal of the present research is to further test these hypotheses. Our secondary goal is to compare different distance measures and assess how they might affect interpretation of population history. We analyze craniodental nonmetric data using several different measures of biological distance, as well as a method for estimating group diversity using multidimensional scaling of distance estimates. Patterns of biological variation and population relationships were interpreted in temporal and geographic contexts. The results of our analyses suggest that the formation of the ancient Egyptian state likely included a substantial in situ process, with some level of contribution by outside migrants probable. The higher level of population structure in Lower Egypt, relative to Upper Egypt, suggests that such influence and migration by outsiders may not have been widespread geographically. These findings support, but serve to refine further those obtained by the second author in a previous study. Moreover, our comparison of distance measures indicates that the choice of measure can influence identification and interpretation of the microevolutionary processes shaping population history, despite being strongly correlated with one another.

Pharmacological effects of a novel isosorbide-based butyrylcholinesterase inhibitor.

Isosorbide-2-benzylcarbamate-5-benzoate, a novel butyrylcholinesterase inhibitor, shows interspecies variation in its inhibitory activity (IC(50) of 4.3 nM for human plasma butyrylcholinesterase, but 1.09 microM for mouse plasma butyrylcholinesterase). Stability studies revealed that this drug is resistant to hydrolysis by human plasma (no degradation in 1 h). However, it was found to undergo rapid degradation when incubated with mouse plasma or mouse liver homogenate, yielding benzyl carbamate and benzoic acid. The addition of the carboxylesterase inhibitor bis-(4-nitrophenyl) phosphate (BNPP) inhibited the degradation of the novel drug, indicating that it may be a substrate for both butyrylcholinesterase and carboxylesterase. The absence of carboxylesterase from human plasma explains the drug's stability in this medium. In vivo, pharmacodynamic studies on single doses of 1 mg/kg to naïve male C57BL/6 mice revealed maximal plasma butyrylcholinesterase inhibition 20 min after intraperitoneal administration (approximately 60% inhibition) and 1 h after administration by gavage (approximately 45% inhibition). While this plasma butyrylcholinesterase inhibition was short-lived, the drug also penetrated the blood-brain barrier resulting in a slight (10-15%) but persistent (> or =72 h) reduction in brain butyrylcholinesterase activity.

Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture.

Manipulation or non-physiological embryo culture environments can lead to defective fetal programming in livestock. Our demonstration of reduced fetal methylation and expression of ovine IGF2R suggests pre-implantation embryo procedures may be vulnerable to epigenetic alterations in imprinted genes. This highlights the potential benefits of epigenetic diagnostic screening in developing embryo procedures.

Prediction of mandibular growth rotation: assessment of the Skieller, Björk, and Linde-Hansen method.

The purpose of this investigation was to assess the method proposed by Skieller, Björk, and Linde-Hansen in 1984 to predict mandibular growth rotation. Our sample consisted of 40 randomly selected, untreated, adolescent subjects representative of the patient population generally encountered in orthodontic practice. The four independent variables identified in the Skieller study as having the highest predictive value (mandibular inclination, intermolar angle, shape of the lower border of the mandible, and inclination of the symphysis) were identified on initial lateral cephalograms. The proposed regression equations were applied and predicted mandibular rotations obtained. Final lateral cephalograms made 6 years after the initial profile radiographs were superimposed and actual mandibular rotation recorded. The observed and predicted rotations were compared and regression analyses performed to determine the amount of variability in observed values accounted for by the four variables individually and in combination. Only 5.6% of the variability in mandibular growth rotation could be accounted for using the four variables individually. Only 9% of the variability could be accounted for with a combination of the variables. In addition, we performed a Monte Carlo analysis, which mirrored the Skieller analysis but used random numbers instead of actual cephalometric data, to determine if the Skieller results may simply have capitalized on chance. Using the same forward stepwise selection procedure with a rejection level of P >.1, we found after 5000 simulations that a mean of 84% and a median of 94% of mandibular growth rotation variability could be accounted for using meaningless data in the Skieller analysis. This result was comparable to the Skieller value of 86%. In conclusion, information derived from pretreatment lateral cephalograms using the Skieller, Björk, and Linde-Hansen method does not permit clinically useful predictions to be made in a general population relative to the direction of future mandibular growth rotation.

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.

Bovine blastocyst production in vitro after inhibition of oocyte meiotic resumption for 24 h.

The aims of the present study were to assess the effect of various substances on meiotic resumption and subsequent development to the blastocyst stage of bovine oocytes. Immature cumulus-oocyte complexes were cultured for 24 h in (a) Medium 199 (M199) alone, or M199 supplemented with (b) 10% fetal calf serum (FCS), (c) 1 micrograms cycloheximide ml-1, (d) 2 mmol 6-dimethylaminopurine (6-DMAP) l-1, or (e) 0.1 mmol vanadate l-1. After 24 h, groups (a) and (b) were inseminated with frozen-thawed spermatozoa and subsequently cultured, while groups (c-e) were washed and cultured for a second 24 h in M199 + FCS, after which they were inseminated and cultured. At all time points a representative sample of oocytes were fixed and stained with orcein to observe the nuclear status, while others were labelled with [35S]methionine to study protein biosynthesis. Incubation with 6-DMAP, cycloheximide or vanadate completely blocked germinal vesicle breakdown with most oocytes remaining at the germinal vesicle stage after 24 h culture (89%, 100% and 85%, respectively). This inhibitory effect was fully reversible in the case of 6-DMAP and cycloheximide; after a second period of incubation, germinal vesicle breakdown occurred in almost all cases (99% and 100%, respectively), and most reached metaphase II (85% and 83%, respectively). In contrast, inhibition with vanadate was only reversible in 56% of oocytes, with only 6% reaching metaphase II. Cleavage rates at 72 h after insemination and blastocyst yields on day 8 of culture were, respectively: (i) M199, 72% and 34%; (ii) M199 + FCS, 80% and 45%; (iii) M199 + cycloheximide, 81% and 19%; (iv) M199 + 6-DMAP, 77% and 14%. 6-DMAP did not modify methionine incorporation. However, cycloheximide completely blocked protein synthesis when present during the period of labelling. Addition of epidermal growth factor to cycloheximide-inhibited oocytes was without effect. In contrast, epidermal growth factor overcame the effect of 6-DMAP in about 50% of oocytes, resulting in lower developmental rates after IVF. These results give an indication of the feasibility of in vitro meiotic inhibition as a tool in the study of the mechanisms involved in acquisition of competence.

Effects of supplementation with fetal calf serum on development of bovine embryos in synthetic oviduct fluid medium.

The effects of fetal calf serum (FCS) or serum fractions on the development of bovine embryos was investigated. Bovine zygotes were produced in vitro and were cultured in a semi-defined culture medium (mSOF). In the first experiment, blastocysts produced in mSOF supplemented with 10% whole heat-treated FCS or desalted FCS appeared about 1 day earlier, their proportion was significantly (P < 0.05) higher (whole: 30%, desalted: 29%) and they had significantly (P < 0.05) more cells at day 8 (119 cells, 127 cells) than did blastocysts produced in mSOF without any supplement (16%, 98 cells) or mSOF supplemented with a glucose concentration equivalent to that of serum (15%, 88 cells). Our results indicate that high molecular mass components (> 5 kDa) of serum are responsible for the effects of FCS on the kinetics of development, on the percentage of blastocysts obtained and the total number of cells in blastocysts. A further analysis using time-lapse microcinematography showed that the acceleration of development induced by serum occurred between the 9-16-cell and morula stages. Finally, in an experiment designed to analyse by microcinematography the effect of the addition of FCS using semen from a different bull to inseminate the oocytes, a different batch of serum and adding mSOF at a different time (42 h after insemination), acceleration was similarly observed between these two stages. Our microcinematographic studies demonstrate that the addition of FCS at two developmental stages (three-four-cell and five-eight-cell) before the 8-16-cell stage accelerates development just after this critical blocking stage.

Characterization of calf follicular fluid and its ability to support cytoplasmic maturation of cow and calf oocytes.

The aims of the present study were to characterize the follicular fluid from prepubertal calf follicles of known size and quality and to study the ability of follicular fluid to support cytoplasmic maturation of calf and cow oocytes. Follicular fluid was obtained from 67 calf follicles classified according to size (S: small < 6 mm, M: medium 6-8 mm and L: large > 8 mm in diameter) and quality (HY: healthy, EA: early atretic and A: atretic). Quality was first determined by mitosis:pycnosis ratios in granulosa cell smears and confirmed by insulin-like growth factor binding protein (IGFBP) patterns. There was approximately 90% agreement between the two methods of follicle classification and on this basis the calf follicular fluid was pooled into nine groups. The accuracy of this pooling was confirmed by evaluation of oestradiol concentrations in the nine pools of follicular fluid using radio-immunoassay. Increases in follicle size were characterized by a decreased intensity of bands for IGFBP-2, IGFBP-5 and IGFBP-4, an increase in the proportion of healthy follicles and a decrease in the proportion of follicles in the early stages of atresia. This finding is in agreement with previously published results in cows. All classes of calf follicular fluid contained lower concentrations of oestradiol than previously reported for corresponding classes of cow follicular fluid. Cow oocytes were matured in M199 alone, or supplemented with 10% fetal calf serum (FCS), or 10% calf follicular fluid from one of three pools (LHY, LEA, LA), fertilized, and cultured for 8 days in synthetic oviduct fluid. Addition of FCS or calf follicular fluid to cow oocytes during in vitro maturation increased the yield of blastocysts on day 8 over the control (23%, 21/91), FCS (39%, 37/96, P < 0.05), LA (41% 21/52, P < 0.05), LEA (32%, 28/88), LHY (36%, 32/88), although not significantly in all cases. The rate of hatching of blastocysts was also improved: control (38%, 8/21), FCS (54%, 20/37), LA (62%, 13/21), LEA (75%, 21/28, P < 0.02), LHY (59% 19/32). In contrast, the addition of either FCS, calf follicular fluid or cow follicular fluid did not improve development of calf oocytes compared with the unsupplemented control. In conclusion, it is probable that serum and follicular fluid contain factors that stimulate the acquisition by oocytes, during maturation, of developmental competence and to which prepubertal oocytes are unable to respond. Specific receptors for these factors may develop only around puberty.

In vitro production of bovine embryos using individual oocytes.

The development of a bovine in vitro embryo production system where individual oocytes could be followed through to the morula or blastocyst stage would be of interest to several fields of study and would allow us to characterise developmentally competent oocytes and their corresponding follicular environment. Several studies have, however, reported significantly reduced embryo development when oocytes or embryos were cultured individually compared to in groups. The aim of this study was to establish such an embryo production system, with embryo development rates similar to that observed under control (grouped) conditions. This study showed that conservation of the oocyte/embryo medium densities generally employed for grouped culture does not facilitate embryo development if oocytes/embryos are cultured individually. However, individual oocytes could effectively undergo IVM/IVF/IVC to the expanded blastocyst stage with some small modifications to the standard protocol. Individual IVF was effective if carried out in either 100 microliters of medium in wells or in 50 microliters droplets. Individual IVC, if carried out in 10 or 20 microliters droplets of SOF with FCS added at either 0 or 24 hr, was effective in terms of blastocyst yields but 20 microliters droplets did yield significantly fewer hatched blastocysts compared to grouped controls (p < 0.05). An entirely individual embryo production system was effective when it included individual IVM in 10 microliters droplets of M199 + 10 ng/ml EGF resulting in day 8 blastocyst yields not significantly different from controls (38% vs. 35% respectively). The use of 10% FCS during individual IVM appeared, at least under our experimental conditions, to be detrimental to subsequent development. The uses of an individual system for embryo production are many and varied. The results of this study show clearly that a large proportion of bovine oocytes can develop to the blastocyst stage when matured, fertilized, and cultured individually. This opens the way for studies regarding the quality of specific oocytes in such a way as will greatly improve our understanding of the events of late folliculogenesis.

Prepubertal bovine oocyte: a negative model for studying oocyte developmental competence.

To identify potential markers of maturation quality, differences in developmental capacity between cow and calf oocytes were compared in parallel with their constitutive and neosynthetic protein profiles before and after in vitro maturation (IVM). A comparison was also made between the protein profiles of follicular fluid (FF) from calf and cow ovaries. The effect of epidermal growth factor (EGF) during IVM on the subsequent development of prepubertal calf oocytes was examined. The effect of the presence of fetal calf serum (FCS) during development of embryos originating from calf oocytes was also examined. No differences were noted between the constitutive proteins of cow and calf oocytes and only a minor modification was observed before IVM in the pattern of neosynthesized proteins (presence of a band of 37 kD and a slight increase in the intensity of band of 78 kD in cow as compared to calf oocytes). However, the comparison of constitutive protein profiles from calf and cow FF demonstrated quantitative (the bands of 34 and 45 kD were more intense for cow than for calf) differences. EGF receptors (EGF-R) were demonstrated on cumulus-oocytes complexes (COCs) by immunofluorescence. There was no difference in intensity between cow and calf COCs. Furthermore, the addition of EGF during IVM of calf oocytes dramatically stimulated cumulus expansion and significantly increased the cleavage rate at 72 h post-insemination (82% vs 67%), as well as the proportion of embryos at the 5- to 8-cell stage at this time (54% vs 43%). Also, blastocyst yields at day 6 (11% vs 5%) and at day 8 (17% vs 10%) were significantly higher in the presence of EGF P < 0.05). The addition of FCS to synthetic oviduct fluid droplets at day 2 of culture (48 hpi) had no effect on cleavage, blastocyst yield, or blastocyst cell number. In conclusion, differences in developmental ability between calf and cow oocytes would appear to be not solely linked to differences in oocyte protein patterns. It is likely that the FF, which constitutes the microenvironment in which the oocyte develops, plays a major modulating role in determining the fate of the oocyte/follicle.

[In vitro oocyte maturation in domestic ruminants]. / Maturation ovocytaire in vitro chez les ruminants domestiques.

In vitro maturation represents the first step towards the in vitro production of embryos in domestic species. This production is of great interest from both zootechnic and basic points of view. Beyond nuclear aspects of maturation (progression of meiosis to metaphase II) cytoplasmic aspects confer to oocytes their potential to be fertilized and to develop normally. The culture medium used for maturation could influence this competence. Furthermore, the origin of oocytes (physiologic and genetic status of the oocyte donor, characteristics of the follicle) could also be determinant. The improvement of in vitro maturation techniques will certainly require a better understanding of these different aspects. Additionally, the maintenance of the oocytes in meiotic block during a pre-maturation in vitro culture will allow them to complete the storage of molecules necessary for fertilization and development.

Role of epidermal growth factor in bovine oocyte maturation and preimplantation embryo development in vitro.

Epidermal growth factor (EGF) has been shown to have a positive effect during in vitro maturation (IVM) and has been reported in follicular fluid at levels capable of stimulating meiosis in a variety of species. The aim of the present work was to study the effect on subsequent development of EGF present in defined medium during bovine 1) oocyte maturation or 2) embryo culture. The presence of EGF during IVM, irrespective of concentration (1, 10, 100 ng/mg), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphase II, the rate of cleavage, and the proportion of embryos reaching the 5- to 8-cell stage at 72 h postinsemination. Blastocyst rates on Days 7 and 9 were also significantly improved for oocytes matured in the presence of EGF (10% vs. 18-24% on Day 7 and 21% vs. 31-32% on Day 9, for Tissue Culture Medium 199 [M199] and M199 + EGF, respectively). The presence of fetal calf serum (FCS) during IVM resulted in similarly elevated rates of development. There was no cumulative effect when EGF and FCS were present together during IVM. The presence of EGF also altered the pattern of proteins neosynthesized during maturation. The maturation-promoting effect of EGF was evident for denuded oocytes also, suggesting that EGF may act, at least in part, directly on the oocyte. Immunofluorescence studies revealed the EGF receptor on immature cumulus-oocyte complexes. When present during postfertilization culture in defined medium (synthetic oviduct fluid), EGF stimulated development in comparison to that of the control but could not replace serum. The results suggest a physiological role for EGF in the regulation of bovine oocyte maturation and development.

Effect of follicle size and quality on the ability of follicular fluid to support cytoplasmic maturation of bovine oocytes.

Recent studies have shown that the developmental capacity of in vitro-matured oocytes is affected by the origin of follicular fluid (FF) supplemented to the maturation medium. The aims of this study were (1) to determine if follicle size and quality would influence the capacity of FF to support bovine oocyte maturation and (2) to determine if fetal calf serum (FCS) and FF had an additive effect when added together to the maturation medium. Follicular fluid collected from 108 follicles was classified according to size ( < 6, 6-8, > 8 mm in diameter) and quality (healthy, early atretic, and atretic). Quality, first determined by mitosis/pycnosis ratios in granulosa cell smears, was subsequently confirmed by insulin-like growth factor binding protein (IGFBP) patterns and estradiol concentrations. While most small- or medium-sized follicles showed some atresia (88% and 67%, respectively), fewer of the large follicles were atretic (30%). In experiment 1 bovine oocytes (n = 2,152) were matured either in TCM199 alone, with 10% FCS, or with 10% FF from the following follicle types: small healthy (SH); small early atretic (SEA); small atretic (SA); medium healthy (MH); medium early atretic (MEA); medium atretic (MA); large healthy (LH); large early atretic (LEA); and large atretic (LA). Following IVM, oocytes were fertilized and subsequently cultured in synthetic oviduct fluid (SOF). Day 8 blastocyst yields were 23% in TCM199 alone; 37% in TCM199 plus FCS; and, in medium supplemented with FF, SH, 36%; MH, 32%; LH, 30%; SEA, 21%; MEA, 26%; LEA, 28%; SA, 32%; MA, 33%; and LA, 38%. All FF from healthy or atretic follicles resulted in significantly improved blastocyst yields compared to M199 alone (P < 0.05). However, FF from early atretic follicles irrespective of size did not yield a significant improvement. In experiment 2 we examined the effect of addition of FF-LH and serum together to the maturation medium. In terms of blastocyst yield, no additional benefit was observed when TCM199 was supplemented with 10% FCS + 10% FF (33%) compared to 10% FCS or FF alone (35% and 30%, respectively). The efficacy of FF as a supplement to the maturation medium to improve cytoplasmic maturation appears to vary with follicle quality but not size. However, in general, the addition of 10% FF or FCS to the maturation media resulted in a similar blastocyst yield with no additional improvement when media was supplemented with both FCS and FF.

Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro.

Employing a total of 3465 bovine oocytes this study was aimed at improving the efficiency of bovine embryo production under defined and undefined conditions. Following in vitro maturation (IVM) and in vitro fertilization (IVF), oocytes were allocated to various culture treatments using synthetic oviduct fluid (SOF). In our 3 experiments we showed that: 1) the addition of fetal calf serum (FCS 10% v/v) to SOF droplets after 20 to 24 h significantly improved blastocyst yields on Day 6 (21 vs 12%; P < 0.01), but not at later stages and resulted in significantly higher Day-8 blastocyst cell numbers (148 +/- 61 vs 92 +/- 35; P < 0.05); 2) the removal of bovine serum albumin (BSA) from the standard SOF medium resulted in significantly reduced blastocyst yields on Days 6, 7 and 8, respectively (17 vs 8%; 28 vs 18%; 31 vs 21%; P < 0.05); 3) the presence or absence of cumulus cells surrounding the presumptive zygote in culture in SOF had no effect on cleavage rate, percentage of 5-8 cell embryos or blastocyst yields (Day 6,7 or 8); 4) the culture of presumptive zygotes in SOF in an atmosphere of 5% CO2 in air (20% O2) resulted in significantly reduced development compared with culture in 5% CO2, 5% O2, 90% N2 in terms of blastocyst yield on Days 6, 7 and 8 and on Day 8 hatching rate, respectively (5 vs 22%; 9 vs 33%; 13 vs 48%; 50 vs 8%; P < 0.001) and 5) embryo density (1 embryo per 1 or 3 microl SOF) or replacing the culture medium every 48 h had no effect when SOF was supplemented with serum; however, under serum-free conditions, changing of the media resulted in a slightly improved Day-6 blastocyst yield such that renewal of serum-free medium mimicked the effect of serum addition.

Effect of recovery method on yield of bovine oocytes per ovary and their developmental competence after maturation, fertilization and culture in vitro.

An important aim of an oocyte recovery method is to maximize the number of oocytes per ovary which can be employed for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). In this study, primary bovine oocytes were collected by 2 methods: aspiration of visible follicles (2 to 6mm in diameter) or surface dissection in which the ovary surface is finely dissected. The oocytes were classified on the basis of cumulus cover and cytoplasmic appearance. The total number of oocytes and the yield of good-quality oocytes recovered per ovary by surface dissection and aspiration were 44.2 and 13.9 and 13.5 and 4.6 (P<0.05), respectively. When a sample group of selected oocytes recovered by each method was measured, no significant difference was found in the mean diameter (144.11m vs 142.54m). A representative sample of good-quality oocytes recovered by each method was put through the IVM/IVF/IVC procedure: no significant difference in cleavage rate, cleavage index or blastocyst yield was found. However, when the blastocyst yield was compared on a per ovary basis, a significant difference was observed in favor of surface dissection (3.30+/-0.46 vs 0.96+/-0.16;P<0.05). When unselected oocytes recovered by surface dissection of the ovaries were put through the standard embryo production system, an average of 15.4 blastocysts per dam was obtained.