Bovine embryo culture in vitro - Human Reproduction

Human Reproduction Vol. 15, (Suppl. 5) pp. 59-67, 2000

Bovine embryo culture in vitro: new developments and post-transfer consequences Jeremy G.Thompson1'2 and AJim Peterson Reproductive Technologies Group, AgResearch Ruakura Research Centre, PB 3123, Hamilton, New Zealand Present address: Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville Rd., Woodville S.A. 5011, Australia 2

To whom correspondence should be addressed at Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville Rd., Woodville S.A. 5011, Australia. E-mail: [email protected]

The past decade has seen a significant shift away from co-culture systems for cattle blastocyst production. In particular, recent adoption of sequential media systems has increased performance. However, wholly defined systems, such as the replacement of albumin with nonbiological macromolecules, fail to reproduce the nutritive role that this molecule has during development. Cattle blastocysts developed in protein-free medium are metabolically compromised. A further new concept is the use of metabolic inhibitors to stimulate embryo development in vitro. Non-toxic levels of NaN3, 2,4-dinitrophenol or very low oxygen atmospheres (—2%) significantly increase both the yield (by -10-20%) and the quality of blastocysts when these treatments are applied during the peri-compaction period in vitro. Nevertheless, there are also negative consequences of cattle embryo culture, such as fetal oversize and/or significant post-day 35 fetal loss. We have recently found that much of this loss is due to failure of normal allantoic development within the conceptus. Early fetal development is supported by vascularization within the yolk sac, but from day 35 to day 110, loss occurs through poor nutrient supply and an inability to remove nitrogenous wastes, leading to fetal death around day 35. The cause of disrupted allantois development has not been identified as yet, but may share a common 'cause-effect' mechanism with the fetal oversize syndrome. Key words: cattle/embryo culture/fetal loss

© European Society of Human Reproduction and Embryology

Introduction Following the birth of Louise Brown in 1978 (Steptoe and Edwards, 1978), the clinical application of mammalian embryo production in vitro was successfully demonstrated and has grown extensively to meet the demand that exists from infertile couples. The basic research and technical development that led to human embryo production had been performed using rodents. Somewhat in contrast, the application of cattle in-vitro embryo production (IVP) has been slow compared to that seen in the treatment of human infertility. This slow development was due to two reasons. First, techniques for the collection and maturation in vitro of immature cattle oocytes needed to be developed (e.g. Staigmiller and Moor, 1984; Sirard and Lambert, 1985). Secondly, there was no reliable invitro culture system that supported in-vitro matured and fertilized ova to the blastocyst stage. The latter was an absolute necessity, as it had been shown that cattle (and sheep) embryos require to be transferred to a synchronized recipient at the appropriate site to obtain acceptable post-transfer rates of development (Moore and Shelton, 1964; Rowson et al., 1969, 1972). Furthermore, this required a uterine transfer to take place (either surgically or non-surgically), as uterine transfer procedures are easier and more cost-effective compared to oviductal transfers. The advent of co-culture technology, developed initially in sheep (Gandolfi and Moor, 1987) and then applied to cattle (Eyestone and First, 1989), overcame this problem, despite an earlier report 59

J.G.Thompson and A.J.Peterson

that a relatively simple, semi-defined medium also supported cattle and sheep embryo development following IVF (Tervit et al, 1972). Nevertheless, despite the development of these techniques, the application of IVP in cattle breeding remains limited within the commercial sector. There are several reasons for this, including low oocyte recovery rates and poor oocyte quality from oocyte retrieval procedures, low embryo survival to term following transfer (e.g. 30-35%) and poor survival following storage in liquid nitrogen. Furthermore, for much of this decade, little progress has been achieved in improving the proportion of bovine embryos developing in vitro (Thompson and Duganzich, 1996), with few studies reporting results of >50% development to blastocysts from cleaved embryos. In this review, we will focus on progress in two areas: that of in-vitro development of cattle embryos and the pathology of posttransfer loss of early fetuses. Both have received considerable attention in our laboratories. Sequential medium development The development of more defined embryo culture systems has been one of the key achievements in cattle embryology over the last decade. It is now generally accepted that although it is an efficient system for embryo production, co-culture is difficult to improve upon, due to the different interactions between medium components and the two cell types in culture. Furthermore, there is the increased risk associated with transfer of viral and other sub-cellular pathogens between somatic cells and the embryo (reviewed by Bavister, 1995; Leese et al, 1995, 1998; Thompson, 1997). Over the past decade, knowledge of the basic cell biology of early embryo development and the in-vivo environment has substantially increased. Initially this led to revised formulations, such as SOFaaBSA (Gardner et al 1994), or new formulations, such as CR1 (Rosenkrans and First, 1994). However, even these formulations failed to produce consistent results and still require the addition of serum (as a growth factor 'cocktail') to achieve consistent and reliable results (Pinyopummintr and Bavister, 1994; Carolan et al, 1995; Thompson et al, 1998a). In particular, addition of serum following early cleavage, but prior to compaction and blas60

tulation, led to reliable, and in some cases improved, embryo development (Carolan et al, 1995, Thompson et al, 1998a). Nevertheless, there has been a continuous effort to understand early embryo cell biology, coupled with an understanding of the temporal relationship between the reproductive tract fluid milieu and embryo development (e.g. Gardner et al, 1996). This has led to the concept that media components and physical conditions should be altered during development to the blastocyst stage (reviewed by Thompson, 1996; Gardner and Lane, 1997). This concept has subsequently been termed 'sequential' media systems (Gardner and Lane, 1997). In particular, human IVF laboratories seeking more robust systems for human blastocyst culture have championed the application of sequential systems (Gardner and Lane, 1997; Gardner et al, 1998), to reduce the need to transfer more than one or two embryos. Indeed, several human IVF medium manufacturers now market 'sequential' medium systems. For cattle embryos, our laboratory has developed a sequential medium system for fertilization and culture of IVP embryos, known as 'SOF-98' (AgResearch, Hamilton, New Zealand). The performance of this new system, based on the development of synthetic oviduct fluid (SOF) medium (Tervit et al, 1972; Gardner et al, 1994) yields improved fertilization and embryo development rates compared to the SOFaaBSA formulation (Thompson et al, 2000). A further development of this concept is the use of perfusion culture as the vehicle to introduce changes in media composition (Bavister, 1995; Thompson, 1996; Gardner, 1998). Although not a new technology, there has been little requirement until now for the application of perfusion equipment to in-vitro embryo development. The general principle of perfusion culture is that embryos are held in a chamber through which a medium can flow either continuously or intermittently. The benefit of this is that the composition of the medium can be altered with minimal disturbance to the embryos. With the advent of sequential medium, the benefits of a perfusion system become evident. However, the availability of the components necessary for microperfusion culture is extremely limited and led us to 'adapt' commer-

Bovine embryo culture in vitro

cially available equipment to embryo culture, although such adaptions continue to yield less than optimal results (Lim et al., 1996; McGowan and Thompson, 1997). This has necessitated the development of specialist embryo perfusion componentry, which has been surprisingly difficult (J.Thompson, personal observation). Cattle blastocysts require albumin Biological media components, such as serum albumin, have been widely used in embryo culture systems. However, due to their undefined nature and variability not only in constituents, but also in biological effects on cells and embryos, plus the potential risk of cross-infection from contaminated biological fluids, much research has been conducted into embryo production using 'defined' media (Bavister, 1995) in particular, the use of nonproteinaceous macromolecules. Poly vinyl alcohol (PVA) has been mooted as a preferred additive, especially as this provides surfactant activity similar to albumin. More recently, this list has been extended to include glycosaminoglycans, in particular hyaluronic acid (Gardner et al., 1999), already known to stimulate cattle embryo development in vitro (Furnus et al., 1998). However, evidence from the mouse suggests that albumin may have intracellular roles in addition to exogenous support roles (e.g. metal ion chelation) (Dunglison and Kaye, 1993; Dunglison et al., 1995). Although albumin is not an absolute requirement for bovine embryo growth in vitro (Keskintepe et al, 1995; Eckert et al., 1998), development results are generally poorer following in-vitro development in PVA than for albumin-supplemented medium (e.g. Eckert et al., 1998; Krisher et al., 1999). Furthermore, few calves have been derived from blastocysts produced in protein-free IVP conditions. We have investigated if the total protein content of IVP cattle blastocysts is affected by the presence of protein in the culture medium. Our initial examination of total protein content in embryos revealed that during early cleavage, protein content decreases, followed by an increase during compaction and blastulation (Thompson et al., 1998b). Hence, protein degradation exceeds protein synthesis during early cleavage. Furthermore, the total protein content was

lower in blastocysts resulting from culture in PVAsupplemented medium, compared to those in vivo or cultured in vitro in media supplemented with either BSA or fetal calf serum (Figure 1; Thompson et al., 1998b). This was not attributed to a difference in protein synthesis rates, but rather the action of protein uptake via pinocytosis (Thompson et al., 1998b). This suggested a nutritive role for exogenous albumin, which we examined by measuring energy substrate utilization. We observed that blastocyst stage embryos cultured in PVA medium compared to BSA medium had considerably higher uptake of the key Krebs cycle substrate, pyruvate, yet a reduced oxygen uptake (Eckert et al., 1998). This result would normally be difficult to explain, as oxygen uptake and pyruvate oxidation are usually closely correlated. However, in a separate study, Lee et al. (1998a) found that oxidation of pyruvate was significantly lower in blastocysts derived from culture in PVA medium compared to BSA medium. We believe that this demonstrates the importance of a further role for pyruvate. Intracellular pyruvate is utilized to detoxify NH3 produced by endogenous protein degradation and amino acid metabolism; the resulting alanine may then be transported from the cell. Such a role has been previously reported for bovine embryos (Partridge and Leese, 1996). Kaye and colleagues at the University of Queensland have demonstrated that albumin has a significant nutritive role to play during mouse embryo development during post-compaction stages (Dunglison and Kaye, 1993; Dunglison et al., 1995). It would appear from our data that cattle post-compacting embryos also show a similar requirement for protein. Whether albumin can be replaced by a more inert protein molecule, such as casein, will require further experimentation. Above all, however, despite the need for further research, we believe that the move towards more 'defined' systems, especially protein-free systems, must be tempered by the physiological requirements of the embryo during development. Metabolic regulation using inhibitors Energy substrate preference during early cleavage of mammalian embryos developing in vitro has long been recognized. For example, Brinster 61

J.G.Thompson and A.J.Peterson 1000-|

b

o £. .a

b

b

100-

10-

b -r

Bu)

E

tein content

a

2

a.

•\

.

PVA

BSA

PCS- D1

PCS- D5

n vivo

Figure 1. Log10 plot histograms of protein content (± SEM) of day 7 blastocysts produced in vitro and incubated in medium supplemented with either: polyvinyl alcohol (PVA); bovine serum albumin (BSA); fetal calf serum (FCS-D1), BSA from day 1-5 of development and FCS for days 5-7 of development. A further group was also included (in vivo): day 7 blastocysts derived in vivo following superovulation and embryo collection. Different superscripts signify significant differences (P < 0.001). (Previously published in Molecular Reproduction and Development. J.G.Thompson et al., 1998b. Reprinted with permission from John Wiley and Sons, Inc., New York.)

(1965a,b) demonstrated that glucose and lactate as sole energy substrates were ineffective to support early cleavage in the early mouse embryo. However, cleavage in vitro will occur in the presence of pyruvate. Energy substrate preferences of embryos from sheep and cattle during in-vitro culture have been well researched over the past decade (e.g. Javed and Wright, 1991; Rieger et al, 1992a,b; Gardner et al, 1993; Eckert et al, 1998). Briefly, early cleavage is dependent on ATP generation almost entirely from oxidative phosphorylation. However, as compaction proceeds, there is a shift towards a greater contribution to ATP production via glycolysis, which coincides with the transition from oviduct to uterus, where oxygen availability may be a rate-limiting factor (Thompson, 1997). There is also evidence that a difference exists in both the level of ATP generated and the proportion generated via glycolysis between in-vivo derived and IVP expanded blastocysts in cattle. In-vivo derived embryos appear to produce less ATP overall, of which a greater proportion is generated by glycolysis than IVP expanded blastocysts (Thompson, 1997). From such observations, we proposed the hypothesis that facilitating the shift in ATP production from oxidative phosphorylation to glycolysis during compaction and blastulation favours cattle embryo development in vitro. We tested this hypothesis in three ways. First, day 5-7 (i.e. peri-compaction/blastulation) cattle 62

embryos were cultured in vitro in oxygen levels ranging from 0 to 7% O2. Second, peri-compaction embryos were cultured in non-toxic levels of the oxidative phosphorylation inhibitor, sodium azide. Third, embryos were incubated in the uncoupler of oxidative phosphorylation, 2,4-dinitrophenol (DNP) (Thompson et al, 2000). All three experiments yielded significant improvements in embryo development and quality as measured on day 7 of development. Either low O2 levels (—2%) or 5-10 |imol/l NaN3 (Figure 2) or DNP were found to be stimulatory. A similar strategy involving EDTA, but targeted at the inhibition of glycolysis during early development, has also been described (Lane and Gardner, 1997). EDTA was found to assist the development of 2-cell embryos from mouse strains that would normally suffer a culture-induced developmental ('2-cell') block (Abrumzuk et al, 1977; Mehta and Kiessling, 1990; Nasr-Esfahini et al, 1992). For many years it was believed that EDTA sequestered the toxic effects of contaminating heavy metal cations, perhaps by inhibiting the production of reactive oxygen species, catalysed by ions such as Fe 2+ and Cu 2+ (e.g. Johnson and Nasr-Esfahini, 1994). Lane and Gardner (1997) demonstrated that, at a cellular level, EDTA depressed glycolytic rates within pre-compaction mouse embryos, a result also demonstrated for bovine embryos (Gardner et al, 1997). These authors believe that in the


10 0

5

10

15 1

NaN3(umoll" )

Figure 2. Development of cleaved bovine embryos to compact morula and blastocyst stages ( • ) and grade 1 and 2 compact morula and blastocyst (O) during incubation in SOFaaBSA medium supplemented with NaN3 (0, 5, 10 and 20 uM) from day 5 to day 7 of development. Different letters indicate significant differences (P < 0.05). (Previously published in Journal of Reproduction and Fertility. J.G.Thompson et al., 2000. Reprinted with permission.)

mouse, at least, the mechanism of EDTA is via the intracellular chelation of Mg 2+ , a necessary co-factor for several glycolytic enzymes (Lane and Gardner, 1997). The beneficial effect of EDTA is optimal only when introduced during early development, when abnormal rates of glycolysis induced by the glucose concentration may compromise development (Lane and Gardner, 1997). The use of such direct and indirect inhibitors of metabolism to regulate embryo development opens a wide spectrum of compounds that can be used strategically to enhance development in vitro. Perhaps more importantly, such data demonstrate that the way in which embryonic cells produce ATP is a key regulator of embryonic development. Post-transfer embryonic loss Pre-elongation culture of ruminant embryos prior to transfer can yield a number of abnormalities manifesting themselves later in development after transfer. The best known of these is the so-called 'large lamb/calf syndrome, first described in sheep by Walker et al. (1992). Other abnormalities include: high neonatal mortality (Walker et al, 1992; Behboodi et al., 1995; Massip et al, 1996), hydro-allantois (van Wagtendonk-de Leeuw et al., 1998) and abnormal limb and organ development (Walker et al, 1992; Sinclair et al, 1997). However, the phenomenon is not universal, there being

high variability over the incidence reported internationally (Kruip and den Daas, 1997). One reason for this is that fetal oversize can partly be attributed to the culture conditions used (Thompson et al, 1995). Perhaps of greater concern is the high incidence of early fetal loss following transfer of cattle IVP embryos also reported by many laboratories. We have recently identified, using serial slaughter of recipients of IVP embryos produced in a SOF-based system, that a major cause of fetal loss in the first trimester is malformation of allantoic development (Peterson and McMillan, 1998a,b). This malformation can range from aplasia through to apparently normal growth and vascularization but impaired haematopoesis and fetal placental development. Some 25% of IVP conceptuses are affected during allantoic emergence (days 22-34), reducing to 10% by day 70. This distribution of affected conceptuses is very close to the pattern of embryo and fetal loss during the first trimester. In contrast, allantoic growth and development was normal in all control conceptuses which resulted from artificial insemination. Growth and differentiation of the other extra-embryonic membranes, the amnion, yolk sac and trophoblast appeared normal, with the vascularized yolk sac maintaining fetal development to approximately day 35 of gestation. The trophoblast expands to 63

J.G.Thompson and A.J.Peterson

fill both uterine horns with syncytial burrs occurring over the caruncles. The phylogeny of the mammalian allantois has been well documented (Amoroso, 1953) and allantoic development in bovine conceptuses after AI (Melton et al., 1951) or natural mating (Greenstein et al., 1958) has been described. Neither reported any malformation in development, further confirmation that this pathology is confined to the IVP conceptus. There has been very little recent information on the growth of the bovine allantois since these two reports. The allantois is the last extra-embryonic membrane in ruminants to develop and those factors that control its emergence from the hindgut of the embryo are unknown. Ruminants differ from primates and rodents in having a large fluid filled lumen, whereas the lumen is vestigial in primates and absent in rodents. However, it is the allantoic mesoderm that contributes to the vascular component of the placenta in all mammals (Amoroso, 1953). Surprisingly little consideration has been given to the ruminant allantois as a haematopoetic organ. Indeed it has been completely overlooked as a possible contributor of haematopoetic stem cells (Al Salami et al., 1985). There is extensive vasculogenesis and haematopoesis in the allantoic mesoderm of both sheep and cattle, and in the former we have identified the early expression of genes associated with endothelial differentiation and P-globin synthesis (Ledgard and Peterson, 1998). The avian allantois acquires its endothelial network through vasculogenesis. It produces HSC which are capable of colonizing the bone marrow of the embryo, and the endothelial cells or endothelial cell precursors from the allantois have an important role in the development of bone marrow (Caprioli et al., 1998). This suggests that a consequence of allantoic malformation, especially of haematopoesis, may affect subsequent bone marrow and other fetal and neonatal haematopoetic organs. Interestingly there has been a recent report of the death of a neonatal nuclear cloned calf from severe anaemia, lymphoid aplasia and hypoplastic thymus, spleen and lymph nodes (Renard et al., 1999). In contrast to the variable pathologies in allantoic development, haematopoesis and vasculogenesis 64

appeared normal in the yolk sac with a patent vitelline-embryo circulation. This suggests that despite the same germ cell lineage (endoderm and mesoderm), there are differing factors influencing differentiation in the two membranes. Certainly there are marked differences in protein secretory patterns of the sheep yolk sac and allantois (Lee et al., 1998b) and recent information comparing haematopoesis in avian yolk sac and allantois suggests that there are specific haematopoetic programmes in each of these membranes (Caprioli et al., 1998). The results suggest that something associated with the in-vitro conditions causes the allantoic pathology but it is not readily apparent that either stage of development or grade of embryo after culture predicts the occurrence of this condition. Removing antibiotics and phenol red from the media has no effect on its occurrence, neither does shortening the time in culture from seven to five days. In fact, in the latter experiments the condition was exacerbated (Thompson et al., 1999). This suggests that a factor, or factors, is absent or reduced in the media compared to oviductal and uterine luminal fluid. Such a suggestion is supported by the observation that in-vivo cultured bovine embryos (i.e. in sheep oviduct) appear not to suffer the post-day 35 pattern of loss associated with allantoic malformation (Galli and Lazzari, 1996). Intriguingly, similar in-vitro culture conditions do not seem to affect subsequent development in either sheep or red deer conceptuses, for their survival remains constant between pregnancy detection and term (Thompson et al., 1995; Peterson et al, 1997). Allantoic malformation leading to placental insufficiency is a major cause of the fetal loss after the transfer of the in-vitro produced bovine embryo. Its cause and how it can be rectified remains to be elucidated. Until it is, the application of cattle IVP embryos will continue to produce poor calving results when compared to AI and natural mating. Furthermore, a link between allantois development and fetal oversize has not been made to date, although it is feasible that both phenomena are a result of the same 'cause-effect' mechanism. Further work is required to understand fully these intriguing perturbations to in-utero development.


References Abramczuk, J., Solter, D. and Koprowski, H. (1977) The beneficial effect of EDTA on development of mouse one cell embryos in chemically defined medium. Dev. Biol., 61, 378-383. Al Salami, M., Simpson-Morgan, M.W. and Morris, B. (1985) In Morris, B. and Miyasaka, M. (eds), Immunology of the Sheep, F.Hoffman-La Roche and Co., Basle, pp. 19-36. Amoroso, E.C. (1953) In Parkes, A.S. (ed.), Marshall's Physiology of Reproduction. Longmans, Green and Co., London, vol. II, pp. 17-311. Bavister, B.D. (1995) Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update, 1, 91-148. Behboodi, E., Anderson, G.B. and Bondurant, R.H. et al. (1995) Birth of large calves that developed from in vzYro-derived bovine embryos. Theriogenology, 44, 227-232. Brinster, R.L. (1965a) Studies on the development of mouse embryos in vitro, ii. The effect of energy source. J. Exp. Zool., 158, 59-68. Brinster, R.L. (1965b) Studies on the development of mouse embryos in vitro, iv. Interaction of energy sources. J. Reprod. Fertil., 10, 227-240. Caprioli, A., Jaffredo, T. and Gautier, R. et al. (1998) Blood-borne seeding by hematopoetic and endothelial precursors from the allantois. Proc. Natl. Acad. Sci. USA , 95, 1641-1646. Carolan, C , Lonergan, P. and Van Langendonckt, A. et al. (1995) Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilisation in vitro. Theriogenology, 43, 1115-1128. Dunglison, G.F. and Kaye, P.L. (1993) Insulin regulates protein metabolism in mouse blastocysts. Mol. Reprod. Dev., 36, 4 2 ^ 8 . Dunglison, G.F., Jane, S.D., McCaul, T.F. et al. (1995) Stimulation of endocytosis in mouse blastocysts by insulin: a quantitative morphological analysis. J. Reprod. Fertil, 105, 115-123. Eckert, J., Pugh, P.A., Thompson, J.G. et al. (1999) Exogenous protein affects developmental competence and metabolic activity of bovine preimplantation embryos in vitro. Reprod. Fertil. Dev., 10, 327-332. Eyestone, W.H. and First, N.L. (1989) Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned media. J. Reprod. Fertil, 85, 715-720. Furnus, C.C., deMatos, D.G. and Martinez, A.G. (1998) Effect of hyaluronic acid on development of in vitro produced bovine embryos. Theriogenology, 49, 1489-1499. Galli, C. and Lazzari, G. (1996) Practical aspects of IVM/IVF in cattle. Anim. Reprod. Sci., 42, 371-379. Gandolfi, F. and Moor, R.M. (1987) Stimulation of early

embryonic development in sheep by co-culture with oviduct epthelial cells. J. Reprod. Fertil, 81, 23-28. Gardner, D.K. (1998) Embryo development and culture techniques. In Clark, A.J. (ed.), Animal Breeding. Technology for the 21st Century. Harwood, Amsterdam, pp. 13^-6. Gardner, D.K. and Lane, M. (1997) Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum. Reprod. Update, 3, 367-382. Gardner, D.K., Lane, M. and Batt, P. (1993) Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo. Mol. Reprod. Dev., 36, 313-319. Gardner, D.K., Lane, M., Calderon, I. et al. (1996) Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil. Steril. 65, 349-353. Gardner, D.K., Lane, M.W. and Lane, M. (1997) Bovine blastocyst cell number is increased by culture with EDTA for the first 72 h of development from the zygote. Theriogenology, 47, 278. Gardner, D.K., Lane, M., Spitzer, A. et al. (1994) 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., 50, 390^00. Gardner, D. K., Rodriegez-Martinez, H. and Lane, M. (1999) Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer. Hum. Reprod., 14, 2575-2580. Gardner, D.K., Vella, P., Lane, M. et al. (1998) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril, 69, 84-88. Greenstein, J.S., Murray, W.G. and Foley, R.C. (1958) Observations on the morphogenesis and histochemistry of the bovine preattachment placenta between 16 and 33 days of gestation. Anat. Rec, 132, 321-341. Javed, M.H. and Wright, R.W. Jr (1991) Determination of pentose phosphate and Embden-Meyerhoff pathway activities in bovine embryos. Theriogenology, 35, 1029-1037. Johnson, M.H. and Nasr-Esfahini, M.H. (1994) Radical solutions to cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro! Bioessays, 16, 31-38. Keskintepe, L., Burnley, C.A. and Brackett, B.G. (1995) Production of viable bovine blastocysts in defined in vitro conditions. Biol. Reprod., 52, 1410-1417. Krisher, R.L., Lane, M. and Bavister, B.D. (1999) Developmental competence and metabolism of bovine

65

J.G.Thompson and AJ.Peterson

embryos cultured in semi-defined and defined culture media. Biol. Reprod., 60, 1345-1352. Kruip, T.A.M. and den Daas, J.H.G. (1997) In vitro produced and cloned embryos: Effects on pregnancy, parturition and offspring. Theriogenology, 47, 43-52. Lane, M. and Gardner, D.K. (1997) EDTA stimulates development of cleavage stage mouse embryos by inhibiting the glycolytic enzyme 3-phosphoglycerate kinase. Biol. Reprod., 56 (Suppl. 1), Abstr. 193. Ledgard, A.M. and Peterson, A.J. (1998) Expression of genes associated with allantoic emergence during early pregnancy in sheep. Proc. Aust. Soc. Reprod. Biol., 29, 52 (abstr.). Lee, E.S., Pugh, P.A., Allen, N. et al. (1998a) [5-3H]glucose and [l-14C]-pyruvate utilization by fresh and frozen-thawed day 7 IVP bovine blastocysts cultured in PVA- or BSA-supplemented medium. Theriogenology, 49, 233. (abstr). Lee, R.S.F., Wheeler, T.T. and Peterson, A.J. (1998b) Large-format, two-dimensional polyacrylamide gel electrophoresis of ovine periimplantation uterine luminal fluid proteins: Identification of aldose reductase, cytoplasmic actin, and transferrin as conceptus-synthesised proteins. Biol. Reprod., 59, 743-752. Leese, H.J., Alexiou, M., Comer, M.T. et al. (1995) Assessment of the embryo nutritionl requirements and role of co-culture techniques. In Shaw, R.W. (ed.), Assisted Reproduction. Progress in Research and Practice. Advances in Reproductive Endocrinology. Parthenon, Carnforth, UK, vol. 7, pp. 11-23. Leese, H.J., Donnay, I. and Thompson, J.G. (1998) Human assisted conception: A cautionary tale. Lessons from Domestic Animals. Hum. Reprod., 13 (Suppl. 4), 101-119. Lim, J.M., Reggio, B.C., Godke, R.A. et al. (1996) A continuous flow, perifusion culture system for 8- to 16-cell bovine embryos derived from in vitro culture. Theriogenology, 46, 1441-1450. McGowan, L.T. and Thompson, J.G. (1997) Perfusion culture of bovine in vitro produced embryos. Proc. Aust. Soc. Reprod. Biol, 29, 24 (abstr.). Massip, A., Mermillod, P., Van Langendonkt, A. et al. (1996) Calving outcome following transfer of embryos produced in vitro in different conditions. Anim. Reprod. Sci., 44, 1-10. Melita, T.S. and Kiessling, A.A. (1990) Development potential of mouse emryos conceived in vitro and cultured in ethylenediaminetetraacetic acid with or without amino acids or serum. Biol. Reprod., 43, 600-606. Melton, A.A., Berry, R.O. and Butler, O.D. (1951) The interval between the time of ovulation and attachment of the bovine embryo. J. Anim. Sci., 10, 993-1005. Moore, N.W. and Shelton, J. N. (1964). Egg transfer in the sheep. Effect of the degree of synchronization between donor and recipient, age of egg, and site of

66

transfer on the survival of transferred eggs. J. Reprod. Ferdl, 7, 145-152. Nasr-Esfahini, M.H., Winston, N.J. and Johnson, M.H. (1992) Effects of glucose, glutamine, ethylenediaminetetra acetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryos in-vitro. J. Reprod. Fertil, 96, 219-231. Partridge, R.J. and Leese, H.J. (1996) Consumption of amino acids by bovine preimplantation embryos. Reprod. Fertil. Dev., 8, 945-950. Peterson, A.J., Ledgard, A.M. and Berg, D. (1997) Conceptus development and associated fertility in red deer (Cervus Elephus) from days 11 to 25 after mating. Theriogenology, 47, 503 (abstr). Peterson, A.J. and McMillan, W.H. (1998a) Allantoic aplasiala consequence of in vitro production of bovine embryos and the major cause of late gestation embryo loss. Proc. Aust. Soc. Reprod. Biol, 29, 4 (abstr.). Peterson, A.J. and McMillan, W.H. (1998b) Variation in the rate of emergence of the bovine allantois. Proc. Aust. Soc. Reprod. Biol, 29, 63 (abstr.). Pinyopummintr, T. and Bavister, B.D. (1994) Development of bovine embryos in a cell-free culture medium: effects of type of serum, timing of its inclusion and heat inactivation. Theriogenology, 41, 1241-1249. Renard, J.-P, Chastant. S., Chesne, P. et al. (1999) Lymphoid hypoplasia and somatic cloning. Lancet, 353, 1489-1491. Rieger, D., Loskutoff, N.M. and Betteridge, K.J. (1992a) Developmentally related changes in the metabolism of glucose, glutamine by cattle embryos produced and co-cultured in-vitro. J. Reprod. Fertil, 95, 585-595. Rieger, D., Loskutoff, N.M. and Betteridge, K.J. (1992b) Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in-vitro. Reprod. Fertil. Dev., 4, 547-557. Rosenkrans, C.F. Jr and First, N.L. (1994) Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro. J. Anim. Sci., 72, 434^137. Rowson, L.E.A., Moor, R.M. and Lawson, R.A.S. (1969) Fertility following egg transfer in the cow; effect of method, medium and synchronization of oestrus. /. Reprod. Fertil, 18, 517-523. Rowson, L.E.A., Lawson, R.A.S., Moor, R.M. et al. (1972) Egg transfer in the cow: synchronization requirements. J. Reprod. Fertil, 28, 427^31. Sirard, M.A. and Lambert, R.D. (1985) In-vitro fertilisation of bovine follicular oocytes obtained by laparoscopy. Biol. Reprod., 33, 487-494. Sinclair, K.D., Maxfield, E.K., Robinson, J.J. et al (1997) Culture of sheep zygotes can alter fetal growth and development. Theriogenology, 47, 380. Staigmiller, R.B. and Moor, R.M. (1984) Effect of follicle cells on the maturation and developmental


competence of ovine oocytes matured outside the follicle. Gamete Res., 9, 221-229. Steptoe, P.C. and Edwards, R.G. (1978) Birth after the reimplantation of a human embryo [letter]. Lancet 2, 366. Tervit, H.R., Whittingham, D.G. and Rowson, L.E.A. (1972) Successful culture in-vitro of sheep and cattle ova. J. Reprod. Fertil., 30, 493^97. Thompson, J.G. (1996) Defining the requirements for bovine embryo culture. Theriogenology, 45, 27^40. Thompson, J.G. (1997) Comparison between in vivo derived and in vitro produced pre-elongation embryos from domestic ruminants. Reprod. Fertil. Dev., 9, 341-354. Thompson, J.G. and Duganzich, D. (1996) Analysis of culture systems for bovine in vitro embryo production. Proceedings of the International Embryo Transfer Society (1991-1995). Theriogenology, 45,195 (abstr.). Thompson, J.G., Gardner, D.K., Pugh, PA. et al. (1995) Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol. Reprod., 53, 1385-1391. Thompson, J.G., Allen, N.W., McGowan, L.T. et al. (1998a) The effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development and following transfer. Theriogenology, 49, 1239-1249. Thompson, J.G. Sherman, A., Allen, N. W. etal. (1998b) Total protein content and protein synthesis within preelongation stage bovine embryos. Mol. Reprod. Dev., 50, 139-145. Thompson, J.G., Cox, S., McMillan, W.H. et al. (1999) Fetal abnormality is more pronounced following transfer to synchronised recipients of day 5 bovine IVP embryos compared to day 7 embryos. Proc. Aust. Soc. Reprod. Biol., 30, 136 (abstr.). Thompson, J.G., McNaughton, C , Gasparinni, B. et al. (2000) Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos. /. Reprod. Fertil., 118, 47-55. Van Wagtendonk-de Leeuw, A.M., Aerts, B.J.G. and den Daas, J.H.G. (1998) Abnormal offspring following in vitro production of bovine preimplantation embryos: a field study. Theriorgenology, 49, 883-894. Walker, S.K., Heard, T.M. and Seamark, R.F. (1992) Invitro culture of sheep embryos without co-culture: successes and perspectives. Theriogenology, 37, 111-126.

67