Theriogenology 81 (2014) 49–55

Contents lists available at ScienceDirect

Theriogenology journal homepage:

40th Anniversary Special Issue

The ART of studying early embryo development: Progress and challenges in ruminant embryo culture Pat Lonergan*, Trudee Fair School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 July 2013 Received in revised form 17 September 2013 Accepted 17 September 2013

The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and postnatal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Development In vitro culture In vivo culture Oviduct Embryo

1. Introduction The study of early mammalian embryo development is challenging for a number of reasons. First, the very early stages of development have historically been difficult to access in vivo because the embryo is located in the oviduct for the initial 3 to 4 days, from where it cannot be nonsurgically recovered using standard techniques of uterine flushing. This challenge can be overcome to some extent by producing embryos using IVF, although such embryos might be inferior to those derived in vivo. Furthermore, recent developments with endoscopy allow the collection of very early stage tubal embryos [1]. Second, the post-blastocyst hatching stages of embryo development, including the crucial period of maternal recognition of pregnancy, are particularly challenging to study in vitro because conceptus elongation is a maternally-driven process and does not occur in vitro (see later in text [Section 3]). That said, the development of * Corresponding author. Tel.: þ353 1 6012147; fax: þ353 1 6288421. E-mail address: [email protected] (P. Lonergan). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.

methods to culture embryos in vitro to the blastocyst stage has opened up a vista of possibilities to explore how embryos developdfrom basic morphological studies to highly sophisticated studies investigating underlying transcriptomic and proteomic regulatory networks that control development. In this review, we focus on specific aspects of early embryo development and, although an exhaustive review of the literature is not possible, we reference other recent reviews to which the reader can refer. 2. The effect of Theriogenology on the field of embryo culture Considering that this special issue commemorates the 40th year of Theriogenology, it is interesting to summarize the contribution that the Journal has made to the study of embryo culture. In some way, one can chart the development of technologies, and indeed personalities, in various areas of animal reproduction, including embryo culture, by flicking sequentially through the pages and issues of key journals in the area. Theriogenology is one such journal. At


P. Lonergan, T. Fair / Theriogenology 81 (2014) 49–55

the time of writing, a search on the PubMed database with the terms ‘embryo’ and ‘theriogenology’ reveals 2420 hits dating back to 1974. The first embryo-related article appeared in issue 2 of the Journal, entitled ‘Embryo transfer in cattle - experience of twenty-four completed cases’ by Betteridge and Mitchell [2]. A PubMed search for the terms ‘embryo culture’ and ‘Theriogenology’ reveals 888 hits, beginning in the early- to mid-80s (e.g., [3–5]). It is important to note that these early reports involved in vivoderived embryos recovered approximately 7 days after estrus. A search for ‘IVF’ and ‘Theriogenology’ yields 474 hits including some of the seminal papers that launched IVF in cattle [6,7]. It is probably fair to say that the effect of Theriogenology on the field of embryo technologies was facilitated by the publication of the proceedings of the International Embryo Transfer Society (IETS) as the first issue of Theriogenology from 1978 (the fourth annual meeting of what was then still a new society) to 2003 inclusive. To an extent, these two ‘institutions’ grew up together in the early years and their association guaranteed the publication of highly citable up-to-date reviews on various aspects of embryos and embryo technology. As noted by Betteridge [8] in his ‘A history of farm animal embryo transfer and some associated techniques’, these [IETS] proceedings ‘serve as a yardstick with which to measure changes in emphasis and intensity of activity in embryo transfer as time progresses’. 3. Autonomy and interdependency of the embryo and reproductive tract Up to the blastocyst stage, the ruminant embryo is relatively autonomous (i.e., does not need contact with the maternal reproductive tract) as evidenced by the fact that blastocysts can be successfully developed in vitro in large numbers using IVF technology. Furthermore, based on the fact that such in vitro-derived embryos, and those recovered from superovulated donors, can establish pregnancy after transfer to the uterus of synchronized recipients, the reproductive tract does not need exposure to the embryo before Day 7 (and even up to Day 16) [9] for a pregnancy to be established. In contrast, the post-hatching and preimplantation conceptus is dependent on substances in the uterine lumen, termed histotroph, that are derived from the endometrium, particularly the uterine glands, for growth and development. This is demonstrated by the fact that: (1) post-hatching elongation does not occur in vitro [10,11]; and (2) the absence of uterine glands in vivo results in a failure of blastocysts to elongate [12,13]. On the maternal side, preparation of the uterine luminal epithelium for attachment of trophectoderm and implantation in all studied mammals, including ruminants, involves carefully orchestrated spatiotemporal alterations in gene expression within the endometrium. In cyclic and pregnant animals, similar changes occur in endometrial gene expression up to initiation of conceptus elongation (approximately Day 13), suggesting that the default mechanism in the uterus is to prepare for, and expect, pregnancy [14]. Indeed, as already mentioned, it is possible to transfer an embryo to a synchronous uterus 7 days after

estrus and establish a pregnancy, as is routine in commercial bovine embryo transfer. It is only in association with maternal recognition of pregnancy, which occurs on approximately Day 16 in cattle, that significant changes in the transcriptomic profile are detectable between cyclic and pregnant endometria [14,15], when the endometrium responds to increasing interferon-tau secreted by the filamentous conceptus. 4. Interaction between the developing embryo and the oviductdtwo-way traffic or a one-way street? Successful culture of the early embryo requires recapitulation of the conditions experienced during development in vivo, initially in the oviduct and then in the uterus. The oviduct is a fascinating structure. It is the site of fertilization in all domestic mammals, and has the unique ability to facilitate transport of gametes in opposite directions and in the case of the mare, apparently has the ability to distinguish between fertilized and unfertilized oocytes [16]. During transit through the oviduct, the embryo morphologically undergoes the first mitotic cell divisions and transcriptionally undergoes embryonic genome activation (at the eight- to 16-cell stage). Despite clear evidence of an interaction between the developing conceptus and the uterine endometrium in early pregnancy [14], and evidence that temporal changes occur in oviduct epithelium gene expression during the estrous cycle [17] reflecting the changing requirements of the embryo, the evidence for reciprocal cross-talk during the transit of the embryo through the oviduct is less clear. There is very convincing evidence for a positive effect of the oviduct on the quality of the early embryo. For example, short-term culture of in vitro-produced zygotes in the oviducts of sheep [18–20], cattle [21], rabbits [22], and even mice [23,24] has been shown to improve embryo quality measured in a variety of ways including morphology, gene expression, cryotolerance, and pregnancy rate after transfer. In contrast, relatively little evidence exists demonstrating an effect of the embryo on the oviduct. The limited data reporting an effect of gametes on the oviduct come from litter-bearing species, in which any effect is likely to be amplified [25–27]. We have recently characterized the transcriptome of the bovine oviduct epithelium at the initiation of embryonic genomic activation on Day 3 after estrus in pregnant and cyclic heifers (Maillo et al., unpublished). The isthmic regions, from which all eight-cell embryos and unfertilized oocytes were collected, were compared. Although large differences in gene expression were observed between the isthmus and ampulla, preliminary data suggest that the presence of an eight-cell embryo had no effect on the transcriptome of the isthmus, although a local effect at the precise position of the embryo cannot be ruled out. 5. In vitro embryo production As mentioned in the Introduction, the ability to generate large numbers of embryos in vitro at a relatively low cost has been an enormous benefit to the study of early

P. Lonergan, T. Fair / Theriogenology 81 (2014) 49–55

development. There are, however, significant challenges associated with the technology revolving around issues of oocyte quality at one end of the process and embryo quality at the other end [28]. Highlighting the importance of this area, and in recognition of some of the inherent problems facing users of this technology, a symposium was held in Orlando, Florida in conjunction with the 32nd Annual Conference of the IETS and published in Theriogenology, entitled, ‘Realizing the promise of IVF in cattle: optimizing embryonic and fetal survival’, at which many related topics were addressed including procedures for transferring a better embryo, errors in embryonic and fetal development, recipient management, and cryopreservation [29]. Evidence of the potential of this technology in commercial practice is reflected in a follow-on IETS workshop, also in Florida, in October 2012, entitled: ‘Bringing the in vitroproduced embryo to the commercial cattle producer’. In 2011, the last year for which data are available, close to 400,000 in vitro-produced bovine embryos were transferred worldwide; of those, Brazil accounted for approximately 85%, reflecting an enormous increase in the use of IVF technology in commercial practice there in recent years [30]. The in vitro production of ruminant embryos is a threestep process involving in vitro oocyte maturation, in vitro fertilization, and in vitro culture. In terms of efficiency, in cattle, approximately 90% of immature oocytes, generally recovered from follicles at unknown stages of the estrous cycle, undergo nuclear maturation in vitro from prophase I to metaphase II (the stage at which they would be ovulated in vivo); approximately 80% undergo fertilization after insemination and cleave at least once, to the two-cell stage. However, only 30% to 40% of such oocytes reach the blastocyst stage, at which they can be transferred to a recipient or frozen for future use. Thus, the major fall-off in development occurs during the last part of the process (in vitro culture), between the two-cell and blastocyst stages, suggesting that postfertilization embryo culture is the most critical period of the process in terms of determining the blastocyst yield. However, evidence demonstrates that the quality of the oocyte is crucial in determining the proportion of immature oocytes that form blastocysts and that the postfertilization culture environment, within certain limits, does not have a major influence on the capacity of the immature oocyte to form a blastocyst [20]. It would appear that when the oocyte has been removed from the follicle, its ability to develop to the blastocyst stage is more or less sealed and despite attempts at temporarily inhibiting resumption of meiosis to allow cytoplasmic maturation to proceed in vitro, thereby improving development or modifications of maturation media, blastocyst yields in vitro using oocytes recovered from slaughtered animals rarely exceed 40% on a consistent basis [28,31]. In contrast, there is considerable evidence that the postfertilization culture environment is critical in determining the quality of the blastocyst. For example, by culturing in vitro-produced bovine zygotes in vivo, in the ewe oviduct, it is possible to dramatically increase the quality of the resulting blastocysts, measured in terms of cryotolerance, to a level similar to that of totally in vivoproduced embryos [18–20]. Furthermore, in the reciprocal


experiment, the culture in vitro of in vivo-produced bovine zygotes resulted in blastocysts of low cryotolerance [20]. In other words, the culture of ‘poor quality’ zygotes, produced by in vitro maturation and fertilization, in vivo leads to the production of high-quality blastocysts (albeit at low frequency) and, conversely, the culture of ‘high quality’ zygotes, produced by in vivo maturation and fertilization, in vitro leads to the production of poor quality blastocysts (albeit at high frequency). Other evidence of the effect of culture conditions on embryo quality come from an examination of the trancriptome. Many articles have reported differences in gene expression between bovine blastocysts produced in vitro or derived in vivo. Other elaborate studies have demonstrated that the developing embryos exhibit a clear temporal sensitivity to their culture environment [32,33]. This has been achieved by culturing bovine zygotes produced using IVM/IVF either in vitro in synthetic oviduct fluid (SOF) or in vivo in the sheep oviduct and recovering embryos at various stages up to the blastosyst stage to pinpoint at which stage the divergence in gene expression seen at the blastocyst stage originates [32]. An extension of this study examined the effect of culturing in vitro-produced embryos in vitro in SOF for the first 2 to 4 days followed by culture in the sheep oviduct to the blastocyst stage and vice-versa on blastocyst cryotolerance and gene expression [33]. Interestingly, blastocysts derived from embryos that spent the first 2 days in vivo and the last 4 in vitro had the lowest survival rates after cryopreservation; those which spent the last 2 days only in SOF had intermediate rates of survival, and those which spent the last 4 days of culture in vivo had high rates of survival, compared with those cultured entirely in vivo; this difference was reflected in the expression of a small number of candidate genes [33]. These data suggest that the period around the time of embryonic genome activation (EGA) is crucial to the establishment of a good quality embryo. Following a similar line of investigation, but using a whole transcriptome approach rather than a candidate gene approach, and transfer of early-stage embryos to homologous bovine oviducts, Gad et al. [34] recently examined the influence of alternative culture conditions (in vivo or in vitro) before and during the EGA event on bovine embryonic developmental rate and gene expression pattern. In agreement with previous studies [20], although alternative culture conditions (in vivo or in vitro) either before or after the time of EGA had no effect on the developmental rates, the origin of the oocyte seemed to determine the embryo development. Total developmental rates until Day 7 for in vitro-generated embryos, which were transferred to in vivo conditions at the four-cell or 16cell stages were 24.1% and 27.5%, respectively. In contrast, total developmental rates until Day 7 in vitro for embryos flushed at the four-cell or 16-cell stage were 64.4% and 68.2%, respectively [34]. The study provides detailed information on molecular mechanisms and pathways that are influenced by altered culture conditions during a specific embryonic developmental time point and highlight the significant effect of in vitro culture conditions during EGA on the transcriptome profile of the resulting blastocyst. Such data contribute greatly to our knowledge of the


P. Lonergan, T. Fair / Theriogenology 81 (2014) 49–55

regulation of embryonic development, and could be used to advance new strategies to modify the in vitro culture conditions at this critical stage of development to enhance the growth of good quality embryos.

global transcriptome using microarray or RNA sequencing technology. Metabolomic profiling of embryo culture media might provide a useful adjunct to more traditional methods of embryo assessment including aspects of morphology, timing of cleavage, etc.

6. Embryo metabolism Our understanding of preimplantation embryo metabolism has progressed dramatically since the pioneering work of the late 1960s and 1970s and has been summarized in a recent review by Leese [35]. The mammalian embryo undergoes dramatic changes in morphology and energy requirements as it develops from a unicellular zygote through the early cleavage divisions to form a multicellular blastocyst. Like most mammalian cells, preimplantation embryos derive their ATP predominantly by oxidative phosphorylation, initially from pyruvate, lactate, and amino acids. After morula compaction, glucose becomes an important substrate but in quantitative terms makes only a modest contribution to ATP generation. In general, embryos throughout pre-elongation development are reliant on oxidative phosphorylation via oxidation of pyruvate and amino acids for the generation of ATP for embryo development. However, there is a switch to an increased contribution of glycolysis during compaction and blastulation (for review, see [35,36]). The composition of bovine oviduct fluid, in which the first few cell divisions occur, has been relatively well characterized in recent years in terms of energy substrates, ions, and amino acids [37–41]. Glycine, alanine, and glutamate are the predominant amino acids; glycine was the most abundant at two- to three-fold higher than alanine, threeto six-fold higher than glutamate, and up to 30-fold higher than amino acids present at lowest concentrations such as asparagine, methionine, and tryptophan [37]. Furthermore, oviductal amino acid concentrations are modulated by progesterone; nine of 20 amino acids increased after supplementation with progesterone, with glycine showing the largest increase of approximately two-fold [40]. Knowledge on how embryos modify their culture environment in vitro can provide information on the embryo needs. Partridge and Leese [42] reported that individual amino acids are depleted at different rates by bovine preimplantation embryos in vitro, suggesting that amino acid requirements change during development. Amino acid depletion was higher at later developmental stages implying an increase in amino acid requirement as development progresses. Subsequently, amino acid turnover has been used to predict developmental competence of embryos from a variety of species (human [43–45]; pigs [46]; and cattle [47]). Until quite recently, approaches to studying the metabolism of preimplantation embryos were based primarily on the targeted analysis of single or selected metabolites. More recently, there has been a move toward a more comprehensive analysis of the metabolome (metabolomics), describing changes in all low molecular weight molecules present in cells or biological fluids at a particular developmental stage or physiological state [48,49]. This is somewhat analogous to the study of individual candidate genes using reverse transcription polymerase chain reaction and the

7. Systems of embryo culturedfrom microdrops to microfluidics Gandolfi and Moor [50] began their landmark article describing the successful coculture of in vivo-derived sheep embryos with oviduct epithelial cells with the sentence, ‘The culture of mammalian embryos from one-cell to the expanded blastocyst stage is an uncertain process’. It is probably fair to say that the process is a little more certain these days, especially using in vivo-derived embryos; in contrast, most IVF-derived embryos typically fail to reach the blastocyst stage, but this is likely because of poor oocyte quality rather than inadequacies of embryo culture media. Tervit and colleagues [51,52] were among the first to report successful culture of ruminant zygotes to the blastocyst stage in vitro using SOF medium, which was based on the composition of ovine oviduct fluid. It was later reported that supplementation of SOF with serum improved development of sheep embryos to the blastocyst stage [53]. Around the same time many other groups were using coculture techniques, such as those described by Gandolfi and Moor [50] and Eyestone and First [54], and at the beginning of the 90s, coculture of cattle and sheep embryos was the most favored method to support embryo development in vitro [36]. At the time, very little was known about the metabolic requirements of ruminant embryos. Coculture advanced the application of in vitro embryo production because it was now possible to produce large numbers of blastocysts for transfer or other studies. However, as pointed out by Bavister [55], such undefined systems did not facilitate the understanding of factors that influence embryo development. The development of serum-free and somatic cell-free culture systems for ruminant embryosdso-called defined and semidefined mediadcame from the desire to define the cellular requirements of the embryo during early development. One such medium was an adjustment of the original SOF formulation with the inclusion of nonessential and essential amino acids [56]. Compared with other species, especially the mouse, progress in the development of systems for the culture of early embryos of domestic ruminants has mostly occurred since the 90s [57]. Over the past 25 years, the most well studied variables aimed at improving embryo development have revolved around the chemical composition of the media used. Since the first successful coculture of ruminant embryos (in vivo-derived sheep zygotes) to the blastocyst stage in the presence of oviduct epithelial cells [50], we have seen developments in the use of defined media, of which the exact composition is known, sequential media, which, in theory, take into account the changing metabolic needs of the embryo, and methods of culturing embryos individually, or in groups, but in an individually identifiable manner using wells-in-wells [58], meshes [46,59], or on adhesive substances [59,60]. More recently, innovative

P. Lonergan, T. Fair / Theriogenology 81 (2014) 49–55

ways of bar-coding individual embryos have even been reported [61,62]. New developments in culture platforms and the potential to improve assisted reproductive technologies have been comprehensively reviewed by Smith et al. [63]. Automation of embryo assessment (using time-lapse) and production (using microfluidics) is achievable. As pointed out by Thompson [64], uptake of such technologies is slow because of cost and the conservative nature of embryologists. Microfluidics represents a very promising technology although it seems to have been promising for some time now and has not been taken up on any large scale [65–67]. 8. In vivo culturedaccessing the oviduct Culture of embryos in the oviducts of intermediate hosts was a crucial milestone in the development of methods to culture embryos in vitro. Although to go back to such in vivo culture methods might seem like a retrograde step, the application of technologies such as endoscopy to allow access to the oviducts of living animals has facilitated the study of early development in vivo in an unrivalled manner [1,68–70]. Eyestone et al. [71] were among the first to describe the culture of one- and two-cell bovine embryos, collected from superovulated heifers, to the blastocyst stage in the ovine oviduct. As mentioned in Section 4, culture of in vitroproduced bovine embryos in the oviduct of the ewe has been shown by several authors to be a suitable environment for the development of embryos from the zygote to blastocyst stage and even through the early stages of elongation [72]. Though not perfect, one advantage of this in vivo culture system is the ability to culture large numbers of embryos in a ‘near in vivo’ environment and in a costeffective manner. Although the yield of blastocysts after such in vivo culture is not superior to that after culture in vitro, the quality of the blastocysts has been shown to be significantly improved [18,23]. Culture of bovine embryos in intermediate host oviducts of a variety of species including sheep, rabbit, and mouse, has been reviewed recently by Rizos et al. [23]. However, heterologous transfer and culture of embryos (i.e., bovine embryos in the ovine oviduct) is never totally satisfactory from an experimental design viewpoint. Recently, endoscopy has been successfully used to access the oviducts of cattle for the in vivo culture of in vitro-matured or -fertilized embryos in the homologous oviduct [1,68–70]. Although this technique requires a significant level of skill and experience (currently only practiced routinely by one group worldwide for the tubal transfer of embryos), it offers much promise for comparative studies of embryo development in different conditions (e.g., [34,73]). 9. Outcome measures Typical outcome measures in studies involving ruminant embryos include fertilization rate, cleavage rate (the proportion of inseminated oocytes undergoing at least one mitotic division), and blastocyst development (the proportion of inseminated oocytes reaching the blastocyst stage). Blastocyst quality is typically assessed using


noninvasive morphological criteria. Time-lapse monitoring has been used to study the kinetic characteristics of early embryo development in vitro in a variety of animal species. In cattle, the group at Louvain Le-Neuve in Belgium used time-lapse to study the development of normal and frozenthawed cow blastocysts [74,75], and later, the development of early bovine embryos [76], describing a lag-phase at the eight-cell stage. Holm et al. [77] used time-lapse to show that faster cleaving embryos are more likely to reach the blastocyst stage. Since then, other groups have used more sophisticated imaging approaches and mathematical algorithms to predict developmental outcome [78–80]. Others have used more invasive measures including staining and counting cell number, assessing cryotolerance, and examining gene expression patterns. The problem with many of these techniques is that they involve destroying the embryo. In a novel approach, El-Sayed et al. [81] addressed the relationship between the transcriptional profile of embryos and pregnancy success based on gene expression analysis of blastocyst biopsies taken before transfer to recipients. This allowed the identification of differentially regulated genes between embryos resulting in a calf delivery versus those not resulting in a pregnancy. This approach was subsequently applied to human embryo assessment [82]. Information on live birth rate and offspring health is mostly restricted to humans because of the difficulty in capturing posttransfer data in domestic species. Such data are nonetheless critical, especially considering the evidence of long-term consequences of embryo culture on offspring health [53]. 10. Concluding remarks Despite the challenges involved in successfully culturing embryos in vitro to a stage at which they can be transferred to a recipient with the realistic expectation of acceptable pregnancy rates, we have come a long way in our understanding of the factors regulating early embryo development and the interaction (or lack of interaction at the very early stages) between the embryo and the maternal reproductive tract. With the number of bovine embryos transferred worldwide increasing annually, there is a greater need than ever to optimize conditions of embryo culture in vitro to maximize embryo quality, cryotolerance, and ultimately, pregnancy rate after transfer. References [1] Besenfelder U, Havlicek V, Kuzmany A, Brem G. Endoscopic approaches to manage in vitro and in vivo embryo development: use of the bovine oviduct. Theriogenology 2010;73:768–76. [2] Betteridge KJ, Mitchell D. Embryo transfer in cattle - experience of twenty-four completed cases. Theriogenology 1974;1:69–82. [3] Allen RL, Bondioli KR, Wright Jr RW. The ability of fetal calf serum, new-born calf serum and normal steer serum to promote the in vitro development of bovine morulae. Theriogenology 1982;18:185–9. [4] Betterbed B, Wright Jr RW. Development of one-cell ovine embryos in two culture media under two gas atmospheres. Theriogenology 1985;23:547–53. [5] Voelkel SA, Amborski GF, Hill KG, Godke RA. Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos. Theriogenology 1985;24:271–81. [6] Greve T, Bousquet D, King WA, Betteridge KJ. In vitro fertilization and cleavage of in vivo matured bovine oocytes. Theriogenology 1984;22:151–65.


P. Lonergan, T. Fair / Theriogenology 81 (2014) 49–55

[7] Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozenthawed semen. Theriogenology 1986;25:591–600. [8] Betteridge KJ. A history of farm animal embryo transfer and some associated techniques. Anim Reprod Sci 2003;79:203–44. [9] Betteridge KJ, Eaglesome MD, Randall GC, Mitchell D. Collection, description and transfer of embryos from cattle 10–16 days after oestrus. J Reprod Fertil 1980;59:205–16. [10] Alexopoulos NI, Vajta G, Maddox-Hyttel P, French AJ, Trounson AO. Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture. Reprod Fertil Dev 2005;17: 799–808. [11] Brandao DO, Maddox-Hyttel P, Lovendahl P, Rumpf R, Stringfellow D, Callesen H. Post hatching development: a novel system for extended in vitro culture of bovine embryos. Biol Reprod 2004;71:2048–55. [12] Spencer TE, Gray CA. Sheep uterine gland knockout (UGKO) model. Methods Mol Med 2006;121:85–94. [13] Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE. Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation. Reproduction 2002;124:289–300. [14] Forde N, Carter F, Spencer TE, Bazer FW, Sandra O, Mansouri-Attia N, et al. Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant? Biol Reprod 2011; 85:144–56. [15] Bauersachs S, Ulbrich SE, Reichenbach HD, Reichenbach M, Buttner M, Meyer HH, et al. Comparison of the effects of early pregnancy with human interferon, alpha 2 (IFNA2), on gene expression in bovine endometrium. Biol Reprod 2012;86:46. [16] Hunter RH. Components of oviduct physiology in eutherian mammals. Biol Rev 2012;87:244–55. [17] Bauersachs S, Rehfeld S, Ulbrich SE, Mallok S, Prelle K, Wenigerkind H, et al. Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle. J Mol Endocrinol 2004;32:449–66. [18] Lazzari G, Colleoni S, Lagutina I, Crotti G, Turini P, Tessaro I, et al. Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species. Theriogenology 2010;73:748–57. [19] Enright BP, Lonergan P, Dinnyes A, Fair T, Ward FA, Yang X, et al. Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality. Theriogenology 2000;54:659–73. [20] Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev 2002;61:234–48. [21] Tesfaye D, Lonergan P, Hoelker M, Rings F, Nganvongpanit K, Havlicek V, et al. Suppression of connexin 43 and E-cadherin transcripts in in vitro derived bovine embryos following culture in vitro or in vivo in the homologous bovine oviduct. Mol Reprod Dev 2007; 74:978–88. [22] Boland MP. Use of the rabbit oviduct as a screening tool for the viability of mammalian eggs. Theriogenology 1984;21:126–37. [23] Rizos D, Ramirez MA, Pintado B, Lonergan P, Gutierrez-Adan A. Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct. Theriogenology 2010;73: 777–85. [24] Rizos D, Pintado B, de la Fuente J, Lonergan P, Gutierrez-Adan A. Development and pattern of mRNA relative abundance of bovine embryos cultured in the isolated mouse oviduct in organ culture. Mol Reprod Dev 2007;74:716–23. [25] Lee KF, Yao YQ, Kwok KL, Xu JS, Yeung WS. Early developing embryos affect the gene expression patterns in the mouse oviduct. Biochem Biophys Res Commun 2002;292:564–70. [26] Alminana C, Heath PR, Wilkinson S, Sanchez-Osorio J, Cuello C, Parrilla I, et al. Early developing pig embryos mediate their own environment in the maternal tract. PloS One 2012;7:e33625. [27] Fazeli A, Affara NA, Hubank M, Holt WV. Sperm-induced modification of the oviductal gene expression profile after natural insemination in mice. Biol Reprod 2004;71:60–5. [28] Lonergan P, Fair T. In vitro-produced bovine embryos - dealing with the warts. Theriogenology 2008;69:17–22. [29] Hansen PJ. Realizing the promise of IVF in cattle–an overview. Theriogenology 2006;65:119–25. [30] Stroud B. IETS 2012 Statistics and Data Retrieval Committee Report. Embryo Transfer Newsletter 2012;30:15–26.

[31] Albuz FK, Sasseville M, Lane M, Armstrong DT, Thompson JG, Gilchrist RB. Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes. Hum Reprod 2010;25: 2999–3011. [32] Lonergan P, Rizos D, Gutierrez-Adan A, Moreira PM, Pintado B, de la Fuente J, et al. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod 2003;69:1424–31. [33] Lonergan P, Rizos D, Kanka J, Nemcova L, Mbaye AM, Kingston M, et al. Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality. Reproduction 2003;126:337–46. [34] Gad A, Hoelker M, Besenfelder U, Havlicek V, Cinar U, Rings F, et al. Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions. Biol Reprod 2012;87:100. [35] Leese HJ. Metabolism of the preimplantation embryo: 40 years on. Reproduction 2012;143:417–27. [36] Thompson JG. In vitro culture and embryo metabolism of cattle and sheep embryos - a decade of achievement. Anim Reprod Sci 2000; 60–61:263–75. [37] Hugentobler SA, Diskin MG, Leese HJ, Humpherson PG, Watson T, Sreenan JM, et al. Amino acids in oviduct and uterine fluid and blood plasma during the estrous cycle in the bovine. Mol Reprod Dev 2007;74:445–54. [38] Hugentobler SA, Humpherson PG, Leese HJ, Sreenan JM, Morris DG. Energy substrates in bovine oviduct and uterine fluid and blood plasma during the oestrous cycle. Mol Reprod Dev 2008;75: 496–503. [39] Hugentobler SA, Morris DG, Sreenan JM, Diskin MG. Ion concentrations in oviduct and uterine fluid and blood serum during the estrous cycle in the bovine. Theriogenology 2007;68:538–48. [40] Hugentobler SA, Sreenan JM, Humpherson PG, Leese HJ, Diskin MG, Morris DG. Effects of changes in the concentration of systemic progesterone on ions, amino acids and energy substrates in cattle oviduct and uterine fluid and blood. Reprod Fertil Dev 2010;22: 684–94. [41] Leese HJ, Hugentobler SA, Gray SM, Morris DG, Sturmey RG, Whitear SL, et al. Female reproductive tract fluids: composition, mechanism of formation and potential role in the developmental origins of health and disease. Reprod Fertil Dev 2008;20:1–8. [42] Partridge RJ, Leese HJ. Consumption of amino acids by bovine preimplantation embryos. Reprod Fertil Dev 1996;8:945–50. [43] Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod 2002;17: 999–1005. [44] Brison DR, Houghton FD, Falconer D, Roberts SA, Hawkhead J, Humpherson PG, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod 2004;19:2319–24. [45] Sturmey RG, Brison DR, Leese HJ. Symposium: innovative techniques in human embryo viability assessment. Assessing embryo viability by measurement of amino acid turnover. Reprod Biomed Online 2008;17:486–96. [46] Booth PJ, Watson TJ, Leese HJ. Prediction of porcine blastocyst formation using morphological, kinetic, and amino acid depletion and appearance criteria determined during the early cleavage of in vitroproduced embryos. Biol Reprod 2007;77:765–79. [47] Hemmings KE, Leese HJ, Picton HM. Amino acid turnover by bovine oocytes provides an index of oocyte developmental competence in vitro. Biol Reprod 2012;86:1–12. [48] Singh R, Sinclair KD. Metabolomics: approaches to assessing oocyte and embryo quality. Theriogenology 2007;68(Suppl. 1): S56–62. [49] Botros L, Sakkas D, Seli E. Metabolomics and its application for non-invasive embryo assessment in IVF. Mol Hum Reprod 2008;14: 679–90. [50] Gandolfi F, Moor RM. Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells. J Reprod Fertil 1987;81:23–8. [51] Tervit HR, Rowson LE. Birth of lambs after culture of sheep ova in vitro for up to 6 days. J Reprod Fertil 1974;38:177–9. [52] Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972;30:493–7. [53] Thompson JG, Mitchell M, Kind KL. Embryo culture and long-term consequences. Reprod Fertil Dev 2007;19:43–52.

P. Lonergan, T. Fair / Theriogenology 81 (2014) 49–55 [54] Eyestone WH, First NL. Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium. J Reprod Fertil 1989;85:715–20. [55] Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995;1:91–148. [56] Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development. Biol Reprod 1994;50:390–400. [57] Leese HJ, Donnay I, Thompson JG. Human assisted conception: a cautionary tale. Lessons from domestic animals. Hum Reprod 1998; 13(Suppl. 4):184–202. [58] Vajta G, Korosi T, Du Y, Nakata K, Ieda S, Kuwayama M, et al. The Well-of-the-Well system: an efficient approach to improve embryo development. Reprod Biomed Online 2008;17:73–81. [59] Matoba S, Fair T, Lonergan P. Maturation, fertilisation and culture of bovine oocytes and embryos in an individually identifiable manner: a tool for studying oocyte developmental competence. Reprod Fertil Dev 2010;22:839–51. [60] Gopichandran N, Leese HJ. The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos. Reproduction 2006;131:269–77. [61] Penon O, Novo S, Duran S, Ibanez E, Nogues C, Samitier J, et al. Efficient biofunctionalization of polysilicon barcodes for adhesion to the zona pellucida of mouse embryos. Bioconj Chem 2012;23: 2392–402. [62] Novo S, Penon O, Barrios L, Nogues C, Santalo J, Duran S, et al. Direct embryo tagging and identification system by attachment of biofunctionalized polysilicon barcodes to the zona pellucida of mouse embryos. Hum Reprod 2013;28:1519–27. [63] Smith GD, Takayama S, Swain JE. Rethinking in vitro embryo culture: new developments in culture platforms and potential to improve assisted reproductive technologies. Biol Reprod 2012;86:62. [64] Thompson JG. Culture without the petri-dish. Theriogenology 2007; 67:16–20. [65] Swain JE, Lai D, Takayama S, Smith GD. Thinking big by thinking small: application of microfluidic technology to improve ART. Lab Chip 2013;13:1213–24. [66] Krisher RL, Wheeler MB. Towards the use of microfluidics for individual embryo culture. Reprod Fertil Dev 2010;22:32–9. [67] Beebe D, Wheeler M, Zeringue H, Walters E, Raty S. Microfluidic technology for assisted reproduction. Theriogenology 2002;57: 125–35. [68] Havlicek V, Kuzmany A, Cseh S, Brem G, Besenfelder U. The effect of long-term in vivo culture in bovine oviduct and uterus on the




[72] [73]






[79] [80]




development and cryo-tolerance of in vitro produced bovine embryos. Reprod Domest Anim 2010;45:832–7. Besenfelder U, Havlicek V, Mosslacher G, Brem G. Collection of tubal stage bovine embryos by means of endoscopy. A technique report. Theriogenology 2001;55:837–45. Besenfelder U, Brem G. Tubal transfer of bovine embryos: a simple endoscopic method reducing long-term exposure of in vitro produced embryos. Theriogenology 1998;50:739–45. Eyestone WH, Leibfried-Rutledge ML, Northey DL, Gilligan BG, First NL. Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct. Theriogenology 1987;28:1–7. Rexroad Jr CE, Powell AM. The ovine uterus as a host for in vitroproduced bovine embryos. Theriogenology 1999;52:351–64. Maillo V, Rizos D, Besenfelder U, Havlicek V, Kelly AK, Garrett M, et al. Influence of lactation on metabolic characteristics and embryo development in postpartum Holstein dairy cows. J Dairy Sci 2012; 95:3865–76. Massip A, Mulnard J. Time-lapse cinematographic analysis of hatching of normal and frozen-thawed cow blastocysts. J Reprod Fertil 1980;58:475–8. Massip A, Mulnard J, Vanderzwalmen P, Hanzen C, Ectors F. The behaviour of cow blastocyst in vitro: cinematographic and morphometric analysis. J Anat 1982;134:399–405. Grisart B, Massip A, Dessy F. Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium. J Reprod Fertil 1994;101:257–64. Holm P, Shukri NN, Vajta G, Booth P, Bendixen C, Callesen H. Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex. Theriogenology 1998;50:1285–99. Sugimura S, Akai T, Hashiyada Y, Somfai T, Inaba Y, Hirayama M, et al. Promising system for selecting healthy in vitro-fertilized embryos in cattle. PloS One 2012;7:e36627. Kirkegaard K, Agerholm IE, Ingerslev HJ. Time-lapse monitoring as a tool for clinical embryo assessment. Hum Reprod 2012;27:1277–85. Wong CC, Loewke KE, Bossert NL, Behr B, De Jonge CJ, Baer TM, et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat Biotechnol 2010;28:1115–21. El-Sayed A, Hoelker M, Rings F, Salilew D, Jennen D, Tholen E, et al. Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients. Physiol Genomics 2006;28:84–96. Jones GM, Cram DS, Song B, Kokkali G, Pantos K, Trounson AO. Novel strategy with potential to identify developmentally competent IVF blastocysts. Hum Reprod 2008;23:1748–59.

The ART of studying early embryo development: progress and challenges in ruminant embryo culture.

The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at ...
190KB Sizes 0 Downloads 0 Views