Determinants of thermal homeostasis in the preimplantation embryo: a role for the embryo's central heating system?

A number of factors may impinge on thermal homeostasis in the early embryo. The most obvious is the ambient temperature in which development occurs. Physiologically, the temperature in the lumen of the female tract is typically lower than the core body temperature, yet rises at ovulation in the human, while in an IVF setting, embryos are usually maintained at core body temperature. However, internal cellular developmental processes may modulate thermal control within the embryo itself, especially those occurring in the mitochondria which generate intracellular heat through proton leak and provide the embryo with its own 'central heating system'. Moreover, mitochondrial movements may serve to buffer high local intracellular temperatures. It is also notable that the preimplantation stages of development would generate proportionally little heat within their mitochondria until the blastocyst stage as mitochondrial metabolism is comparatively low during the cleavage stages. Despite these data, the specific notion of thermal control of preimplantation development has received remarkably scant consideration. This opinion paper illustrates the lack of reliable quantitative data on these markers and identifies a major research agenda which needs to be addressed with urgency in view of laboratory conditions in which embryos are maintained as well as climate change-derived heat stress which has a negative effect on numerous clinical markers of early human embryo development.

The Quiet Embryo Hypothesis: 20 years on.

This article revisits the hypothesis, proposed in 2002, that the successful development of oocytes and preimplantation mammalian embryos is associated with a metabolism which is "quiet" rather than "active", within limits which had yet to be defined. A distinction was drawn between Functional Quietness, Loss of quietness in response to stress and Inter-individual differences in embryo metabolism and here we document applications of the hypothesis to other areas of reproductive biology. In order to encompass the requirement for "limits" and replace the simple distinction between "quiet" and "active", evidence is presented which led to a re-working of the hypothesis by proposing the existence of an optimal range of metabolic activity, termed a "Goldilocks zone", within which oocytes and embryos with maximum developmental potential will be located. General and specific mechanisms which may underlie the Goldilocks phenomenon are proposed and the added value that may be derived by expressing data on individual embryos as distributions rather than mean values is emphasised especially in the context of the response of early embryos to stress and to the concept of the Developmental Origins of Health and Disease. The article concludes with a cautionary note that being "quietly efficient" may not always ensure optimal embryo survival.

Amino Acids and the Early Mammalian Embryo: Origin, Fate, Function and Life-Long Legacy.

Amino acids are now recognised as having multiple cellular functions in addition to their traditional role as constituents of proteins. This is well-illustrated in the early mammalian embryo where amino acids are now known to be involved in intermediary metabolism, as energy substrates, in signal transduction, osmoregulation and as intermediaries in numerous pathways which involve nitrogen metabolism, e.g., the biosynthesis of purines, pyrimidines, creatine and glutathione. The amino acid derivative S-adenosylmethionine has emerged as a universal methylating agent with a fundamental role in epigenetic regulation. Amino acids are now added routinely to preimplantation embryo culture media. This review examines the routes by which amino acids are supplied to the early embryo, focusing on the role of the oviduct epithelium, followed by an outline of their general fate and function within the embryo. Functions specific to individual amino acids are then considered. The importance of amino acids during the preimplantation period for maternal health and that of the conceptus long term, which has come from the developmental origins of health and disease concept of David Barker, is discussed and the review concludes by considering the potential utility of amino acid profiles as diagnostic of embryo health.

Intraovarian injection of platelet-rich plasma in assisted reproduction: too much too soon?

The prospect of ovarian rejuvenation offers the tantalising prospect of treating age-related declines in fertility or in pathological conditions such as premature ovarian failure. The concept of ovarian rejuvenation was invigorated by the indication of the existence of oogonial stem cells (OSCs), which have been shown experimentally to have the ability to differentiate into functional follicles and generate oocytes; however, their clinical potential remains unknown. Furthermore, there is now growing interest in performing ovarian rejuvenation in situ. One proposed approach involves injecting the ovary with platelet rich plasma (PRP). PRP is a component of blood that remains after the in vitro removal of red and white blood cells. It contains blood platelets, tiny anucleate cells of the blood, which are responsible for forming athrombus to prevent bleeding. In addition, PRP contains an array of cytokines and growth factors, as well as a number of small molecules.The utility ofPRP has been investigatedin a range of regenerative medicine approaches and has been shown to induce differentiation of a range of cell types, presumably through the action of cytokines. A handful ofcasereports have described the use of PRP injections into the ovaryin the human, and while these clinical data report promising results, knowledge on the mechanisms and safety of PRP injections into the ovary remain limited.In this article, we summarise some of the physiological detail of platelets and PRP, before reviewing the existing emerging literature in this area. We then propose potential mechanisms by which PRP may be eliciting any effects before reflecting on some considerations for future studies in the area. Importantly, on the basis of our existing knowledge, we suggest that immediate use of PRP in clinical applications is perhaps premature and further fundamental and clinical research on the nature of ovarian insufficiency, as well as the mechanism by which PRP may act on the ovary, is needed to fully understand this promising development.

Glucose concentration during equine in vitro maturation alters mitochondrial function.

The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturation medium containing either 5.6 or 17 mM glucose, as well as expression of key genes in oocytes and cumulus cells. Oocytes were fertilised by ICSI and cultured. Analysis of spent medium revealed that COC glucose consumption and production of lactate and pyruvate were similar between treatments. However, the glycolytic index was decreased at 17 mM and analysis of mitochondrial function of COCs revealed that IVM in 17 mM glucose was associated with decreased ATP-coupled respiration and increased non-mitochondrial respiration compared to that for 5.6 mM glucose. We also found that the metabolic enzyme lactate dehydrogenase-A (LDHA) was downregulated in cumulus cells of oocytes that completed IVM in 17 mM glucose. There was no difference in maturation or blastocyst rates. These data indicate that COC mitochondrial function and gene expression are altered by high glucose concentration during IVM. Further work is needed to determine if these changes are associated with developmental changes in the resulting offspring.

Metabolic profile of in vitro derived human embryos is not affected by the mode of fertilization.

The pattern of metabolism by early embryos in vitro has been linked to a range of phenotypes, including viability. However, the extent to which metabolic function of embryos is modified by specific methods used during ART has yet to be fully described. This study has sought to determine if the mode of fertilization used to create embryos affects subsequent embryo metabolism of substrates. A metabolic profile, including consumption of key substrates and the endogenous triglyceride content of individual IVF and ICSI supernumerary embryos, was assessed and compared. Embryo development and quality was also recorded. All embryos were donated at a single clinical IVF center, on Day 5, from 36 patients aged 18-38 years, The data revealed that consumption of glucose and pyruvate, and production of lactate, did not differ between embryos created by IVF or ICSI. Similarly, the mode of insemination did not impact on the triglyceride content of embryos. However, ICSI-derived embryos displayed a more active turnover of amino acids (P = 0.023), compared to IVF embryos. The specific amino acids produced in higher quantities from ICSI compared to IVF embryos were aspartate (P = 0.016), asparagine (P = 0.04), histidine (P = 0.021) and threonine (P = 0.009) while leucine consumption was significantly lower (P = 0.04). However, importantly neither individual nor collective differences in amino acid metabolism were apparent for sibling oocytes subjected to either mode of fertilization. Embryo morphology (the number of top grade embryos) and development (proportion reaching the blastocyst stage) were comparable in patients undergoing IVF and ICSI. In conclusion, the microinjection of spermatozoa into oocytes does not appear to have an impact on subsequent metabolism and viability. Observed differences in amino acid metabolism may be attributed to male factor infertility of the patients rather than the ICSI procedure per se.

Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos.

Mitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Oxygen Consumption Rate (OCR) is an established measure of mitochondrial function. OCR by mammalian oocytes and embryos has generally been restricted to overall uptake and detailed understanding of the components of OCR dedicated to specific molecular events remains lacking. Here, extracellular flux analysis (EFA) was applied to small groups of bovine, equine, mouse and human oocytes and bovine early embryos to measure OCR and its components. Using EFA, we report the changes in mitochondrial activity during the processes of oocyte maturation, fertilisation, and pre-implantation development to blastocyst stage in response to physiological demands in mammalian embryos. Crucially, we describe the real time partitioning of overall OCR to spare capacity, proton leak, non-mitochondrial and coupled respiration - showing that while activity changes over the course of development in response to physiological demand, the overall efficiency is unchanged. EFA is shown to be able to measure mitochondrial function in small groups of mammalian oocytes and embryos in a manner which is robust, rapid and easy to use. EFA is non-invasive and allows real-time determination of the impact of compounds on OCR, facilitating an assessment of the components of mitochondrial activity. This provides proof-of-concept for EFA as an accessible system with which to study mammalian oocyte and embryo metabolism.

Effect of lactation on conceptus-maternal interactions at the initiation of implantation in cattle: I. Effects on the conceptus transcriptome and amino acid composition of the uterine luminal fluid.

Approximately 65-75 days postpartum (dpp), the estrous cycles of nonlactating (dried off immediately postpartum: n = 12) and lactating (n = 13) Holstein Friesian cows were synchronized and on day 7 a single blastocyst derived from superovulated nulliparous Holstein Friesian heifers was transferred to each cow. A control group of nulliparous heifers (n = 8) were synchronized, inseminated to a standing heat, and slaughtered on the same day as nonlactating and lactating recipients (day 19; estrus = day 0). The uterine horn ipsilateral to the corpus luteum was flushed with 10 ml phosphate-buffered saline and the conceptus, and uterine luminal fluid (ULF) was snap-frozen in liquid nitrogen. Gene expression analysis of the conceptus was performed by RNA sequencing, while amino acid composition of ULF was determined by high-performance liquid chromatography. No differentially expressed genes (DEGs) were observed between conceptuses recovered from nonlactating and lactating cows. Eight DEGs were identified between conceptuses recovered from nonlactating cows and heifers. A total of 269 DEGs (100 up- and 169 downregulated) were identified between conceptuses recovered from lactating cows compared to heifers. Alanine, glycine, serine, threonine, arginine, leucine, and valine were significantly lower in abundance in ULF recovered from heifers compared to nonlactating or lactating cows. This study demonstrates that the environment in which the embryo develops post the blastocyst stage can have an effect on the conceptus transcriptome and amino acid composition of the ULF but this was mainly observed between the two extreme groups in terms of metabolic status (nulliparous heifers vs postpartum lactating cows).

Effect of metabolic status on conceptus-maternal interactions on day 19 in dairy cattle: II. Effects on the endometrial transcriptome.

The aim of this study was to test the hypothesis that the metabolic stresses associated with lactation alter the ability of the endometrium to respond appropriately to the conceptus by examining endometrial gene expression on day 19 of pregnancy. Immediately after calving, primiparous Holstein cows with similar production and fertility estimated breeding values were randomly divided into two groups and either dried off (i.e. never milked) immediately or milked twice daily. Approximately 65-75 days postpartum, grade 1 blastocysts recovered from superovulated Holstein heifer donors (n = 5) were transferred (1 per recipient) into lactating (n = 11) and nonlactating (n = 11) recipients. Control nulliparous Holstein heifers (n = 6) were artificially inseminated. RNA-sequencing was performed on intercaruncular endometrial samples recovered at slaughter from confirmed pregnant animals on day 19 (n = 5 lactating and nonlactating cows; n = 4 heifers). Differentially expressed genes (DEGs) were identified between both postpartum groups compared to heifers and between lactating and nonlactating cows. Functional annotation of DEGs between cows and heifers revealed over-representation of categories, including endosome, cytoplasmic vesicle, endocytosis, regulation of exocytosis, and cytokine receptor activity. Functional categories including transcription factor binding sites, cell motility, and cell migration were enriched for DEGs between endometria from lactating and nonlactating cows. In conclusion, while the evidence for a major effect of lactation on the endometrial transcriptome is relatively weak, these data suggest that the metabolic status of the animal (heifer vs cow) modulates the response of the endometrium to the developing conceptus.

Genistein crosses the bioartificial oviduct and alters secretion composition.

The dietary derived isoflavone and oestrogen analogue, genistein, is known to perturb fundamental reproductive events such as implantation and embryo cleavage. However the question of whether genistein is able to traverse the oviduct epithelial monolayer and impact oviduct fluid secretion remains unclear. This study tests these research questions using a bioartificial oviduct to show that genistein permeates the oviduct lumen in vitro with a biphasic (burst and plateau) kinetic profile, faster than spontaneous diffusion, and alters the amino acid composition of in vitro derived oviduct fluid (ivDOF) but not as an oestrogen analogue. In addition to offering insights into the potential mechanisms of these findings, this manuscript demonstrates the potential to use the bioartificial oviduct model to characterise the transport or barrier properties of the oviduct towards a range of circulating xenobiotics.

Modelling oviduct fluid formation in vitro.

Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage, and genome activation. However, the composition and regulation of this critical environment remains rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium, to investigate the formation and composition of in vitro derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes, and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility, and optimising embryo culture media.

Sexually Dimorphic Gene Expression in Bovine Conceptuses at the Initiation of Implantation.

In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst-specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium.

Biological optimization, the Goldilocks principle, and how much is lagom in the preimplantation embryo.

The quiet embryo hypothesis postulates that early embryo viability is associated with a relatively low metabolism (Leese, 2002 BioEssays 24: 845-849). This proposal is re-visited here using retrospective and prospective data on the metabolic activity and kinetics of preimplantation development alongside the concept that an optimal range of such indices and of energetic efficiency influences embryogenesis. It is concluded that these considerations may be rationalized by proposing the existence of a "Goldilocks zone," or as it is known in Sweden, of lagom-meaning "just the right amount"-within which embryos with maximum developmental potential can be categorized. Mol. Reprod. Dev. 83: 748-754, 2016 © 2016 Wiley Periodicals, Inc.

Expression and function of transient receptor potential channels in the female bovine reproductive tract.

The epithelium lining the oviduct is critical for early reproductive events, many of which are mediated via intracellular calcium ions. Despite this, little is known about the regulation of calcium homeostasis in the oviductal epithelium. Epithelial transient receptor potential channels (TRPCs) modulate calcium flux in other tissues, and their expression and functional regulation have therefore been examined using the bovine oviduct as a model for the human. The effects of FSH, LH, 17ß-estradiol, and progesterone on TRPCs expression and intracellular calcium flux were determined. Transient receptor potential channels 1, 2, 3, 4, and 6 were expressed in the bovine reproductive tract, and their gene expression varied throughout the estrous cycle. In more detailed studies undertaken on TRPC1 and 6, we show that protein expression varied through the estrus cycle; specifically, 17ß-estradiol, FSH, and LH individually and in combination upregulated TRPC1 and 6 expression in cultured bovine oviduct epithelial cells although progesterone antagonized these effects. Functional studies showed changes in calcium mobilization in bovine oviduct epithelial cells were dependent on TRPCs. In conclusion, TRPC1, 2, 3, 4, and 6 are present in the epithelium lining the bovine oviduct, and TRPC1 and 6 vary through the estrous cycle suggesting an important role in early reproductive function.

Applying metabolomic analyses to the practice of embryology: physiology, development and assisted reproductive technology.

The advent of metabolomics technology and its application to small samples has allowed us to non-invasively monitor the metabolic activity of embryos in a complex culture environment. The aim of this study was to apply metabolomics technology to the analysis of individual embryos from several species during in vitro development to gain an insight into the metabolomics pathways used by embryos and their relationship with embryo quality. Alanine is produced by both in vivo- and in vitro-derived human, murine, bovine and porcine embryos. Glutamine is also produced by the embryos of these four species, but only those produced in vitro. Across species, blastocysts significantly consumed amino acids from the culture medium, whereas glucose was not significantly taken up. There are significant differences in the metabolic profile of in vivo- compared with in vitro-produced embryos at the blastocyst stage. For example, in vitro-produced murine embryos consume arginine, asparagine, glutamate and proline, whereas in vivo-produced embryos do not. Human embryos produce more alanine, glutamate and glutamine, and consume less pyruvate, at the blastocyst compared with cleavage stages. Glucose was consumed by human blastocysts, but not at a high enough level to reach significance. Consumption of tyrosine by cleavage stage human embryos is indicative of blastocyst development, although tyrosine consumption is not predictive of blastocyst quality. Similarly, although in vivo-produced murine blastocysts consumed less aspartate, lactate, taurine and tyrosine than those produced in vitro, consumption of these four amino acids by in vitro-derived embryos with high octamer-binding transcription factor 4 (Oct4) expression, indicative of high quality, did not differ from those with low Oct4 expression. Further application of metabolomic technologies to studies of the consumption and/or production of metabolites from individual embryos in a complete culture medium could transform our understanding of embryo physiology and improve our ability to produce developmentally competent embryos in vitro.

Human embryos from overweight and obese women display phenotypic and metabolic abnormalities.

STUDY QUESTION: Is the developmental timing and metabolic regulation disrupted in embryos from overweight or obese women? SUMMARY ANSWER: Oocytes from overweight or obese women are smaller than those from women of healthy weight, yet post-fertilization they reach the morula stage faster and, as blastocysts, show reduced glucose consumption and elevated endogenous triglyceride levels. WHAT IS KNOWN ALREADY: Female overweight and obesity is associated with infertility. Moreover, being overweight or obese around conception may have significant consequences for the unborn child, since there are widely acknowledged links between events occurring during early development and the incidence of a number of adult disorders. STUDY DESIGN, SIZE, DURATION: We have performed a retrospective, observational analysis of oocyte size and the subsequent developmental kinetics of 218 oocytes from 29 consecutive women attending for ICSI treatment and have related time to reach key developmental stages to maternal bodyweight. In addition, we have measured non-invasively the metabolic activity of 150 IVF/ICSI embryos from a further 29 consecutive women who donated their surplus embryos to research, and have related the data retrospectively to their body mass index (BMI). PARTICIPANTS/MATERIALS, SETTING, METHODS: In a clinical IVF setting, we compared oocyte morphology and developmental kinetics of supernumerary embryos collected from overweight and obese women, with a BMI in excess of 25 kg/m(2) to those from women of healthy weight. A Primovision Time-Lapse system was used to measure developmental kinetics and the non-invasive COnsumption/RElese of glucose, pyruvate, amino acids and lactate were measured on spent droplets of culture medium. Total triglyceride levels within individual embryos were also determined. MAIN RESULTS AND THE ROLE OF CHANCE: Human oocytes from women presenting for fertility treatment with a BMI exceeding 25 kg/m(2) are smaller (R(2) = -0.45; P = 0.001) and therefore less likely to complete development post-fertilization (P < 0.001). Those embryos that do develop reach the morula stage faster than embryos from women of a BMI < 25 kg/m(2) (<0.001) and the resulting blastocysts contain fewer cells notably in the trophectoderm (P = 0.01). The resulting blastocysts also have reduced glucose consumption (R(2) = -0.61; P = 0.001), modified amino acid metabolism and increased levels of endogenous triglyceride (t = 4.11, P < 0.001). Our data further indicate that these differences are independent of male BMI. LIMITATIONS, REASONS FOR CAUTION: Although statistical power has been achieved, this is a retrospective study and relatively small due to the scarcity of human embryos available for research. Consequently, subanalysis of overweight and obese was not possible based on the sample size. The analysis has been performed on supernumerary embryos, originating from a single IVF unit and not selected for use in treatment. Thus, it was not possible to speculate how representative the findings would be of the better quality embryos transferred or frozen for each patient. WIDER IMPLICATIONS OF THE FINDINGS: The data indicate that a high BMI of women at conception is associated with distinct phenotypic changes in the embryo during the preimplantation period, highlighting the importance of prepregnancy body weight in optimizing the chances of fertility and safeguarding maternal and offspring health. These changes to the metabolic fingerprint of human embryos which are most likely a legacy of the ovarian conditions under which the oocyte has matured may reduce the chances of conception for overweight women and provide good evidence that the metabolic profile of the early embryo is set by sub-optimal conditions around the time of conception. The observed changes could indicate long-term implications for the health of the offspring of overweight and obese women. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Hull IVF Unit Charitable Trust and the Hull York Medical School. There are no conflict of interests.

Metabolic heterogeneity during preimplantation development: the missing link?

BACKGROUND: Most tissues in the body rely on the presence of gap junctions in order to couple their component cells electrically and metabolically via intercellular transport of ions, metabolites and signalling agents. As a result, cells within tissues achieve a high degree of, 'metabolic homogeneity' which enables them to develop in an integrated way and co-ordinate their response to physiological signals and environmental cues. Unusually, the developing mammalian preimplantation embryo does not form functional gap junctions until it has divided into 8 or more cells. We discuss the implications of this 'missing link' during the first few days of development for the maintenance of homogeneity between embryonic cells and for the co-ordination of the embryonic response to intrinsic genetic damage and external environmental signals. METHODS: No systematic review has been carried out. The physiology of preimplantation development and the general nature of gap junctions have been reviewed briefly before examining experimental evidence which addresses the following points: (i) whether there are functional differences between early blastomeres; (ii) when during preimplantation development the embryo is most sensitive to environmental perturbation and (iii) the consequences for early embryos of ablating gap junction formation and function. RESULTS AND CONCLUSIONS: General conclusions are confounded by species differences, especially in the timing of embryonic genome activation (EGA) and the extent of intrinsic genotypic and phenotypic variation (low in embryos from inbred mice; high in human embryos). Nevertheless, we propose that the absence of gap junctions requires cleavage stage mammalian embryos to behave cell autonomously in a metabolic sense, contributes to their heightened sensitivity to environmental perturbation compared with the later stages of preimplantation development and poses more problems in the early human embryo, where there is a high degree of heterogeneity between the blastomeres. We argue that the legacy of metabolic heterogeneity, in part generated by the absence of gap junctions, is 'rescued' by the onset of apoptosis following EGA. In the context of human-assisted conception, since early embryos lacking gap junctions are more sensitive to environmental stress during cleavage, this would support transfer to the natural environment as early as possible after fertilization.

A simple approach for COnsumption and RElease (CORE) analysis of metabolic activity in single mammalian embryos.

Non-invasive assay of the consumption and release of metabolites by individual human embryos could allow selection at the cleavage stage of development and facilitate Single Embryo Transfer in clinical IVF but will require simple, high throughput, sensitive methods applicable to small volume samples. A rapid, simple, non-invasive method has therefore been devised using a standard fluorescence plate reader, and used to measure the consumption of pyruvate and glucose, and release of lactate by single bovine embryos at all stages of preimplantation development in culture; amino acid profiles have been determined using HPLC. Early embryos with an 'intermediate' level (6.14±0.27 pmol/embryo/h) of pyruvate uptake were associated with the highest rate (68.3%) of blastocyst development indicating that a mid "optimum" range of pyruvate consumption correlates with high viability in this bovine model.