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RESEARCH |
University of York, Department of Biology, PO Box 373, York, Yorkshire YO10 5DD, UK
Correspondence should be addressed to P J Booth; Email: pjb11{at}york.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The identification of the amino acid requirements of the developing mammalian embryo has also suffered from these problems. When added individually to hamster embryos, amino acids can be classified into those that stimulate or inhibit development, or have no effect (Bavister & Arlotto 2002). Alternatively, amino acids can be classified according to whether they are essential or non-essential for the growth of somatic cells (Eagle 1958), and can be added to embryo culture media as mixtures of the two types. Use of these supplements in mouse (for reviews, see Gardner & Lane 1993, Summers & Biggers 2003) and pig embryo culture media (Van Thuan et al. 2002) has indicated that the presence of essential amino acids during cleavage impairs development, but the addition of both essential and non-essential amino acids after this stage is stimulatory. However, this approach is unlikely to be representative of the physiological situation in vivo where a complete set of amino acids will be present in the reproductive tract throughout preimplantation development (Engle et al. 1968, Iritani et al. 1974, Leese 2003, Summers & Biggers 2003). Under similar conditions in vitro, using a low concentration mixture of amino acids (Tay et al. 1997), the net rates of depletion or appearance of amino acids by individual human embryos varied between amino acids and the stage of development; no difference in turnover patterns was apparent between non-essential and essential amino acids (Houghton et al. 2002). These data suggest that allowing the embryo to determine its metabolism by presenting it with a mixture of substrates, rather than by imposing a subset of presumed substrate preferences, may help elucidate embryo nutritional requirements. In the present study, we have adopted this approach to determine the net uptake and release of amino acids by pig embryos from the zygote to the blastocyst stage in medium containing a mixture of 20 amino acids at low concentrations. The latter formulation was derived from the analysis of human Fallopian tubal fluid produced under vascular perfusion (Tay et al. 1997). The experiments were designed to determine the temporal pattern of amino acid turnover during preimplantation pig development, thereby allowing comparison with other species, and to assist in the formulation of improved culture media for pig embryos.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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NCSU-23aa medium contained amino acids including a reduced concentration of glutamine compared with normal NCSU (see Table 1
). The concentrations of amino acids were based on those measured in tubal fluid, produced during vascular perfusion of the human Fallopian tube, using medium 199 supplemented with 4% BSA as the perfusate (Tay et al. 1997). Consequently, low concentrations of amino acids were presented to the oocytes/embryos with the intention of allowing them to determine the rates of uptake or release of amino acids, thereby identifying their amino acid preferences.
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Experimental groups
Groups of embryos for amino acid profiling were selected at specific stages of development. On Day 0 at 6 h after insemination, groups of 20 presumptive zygotes were selected for amino acid profiling having been inseminated with (i) a normal concentration of sperm that had been previously optimised to generate maximal blastocyst yields, and (ii) a higher concentration sperm intended to produce highly polyspermic embryos. A third group comprised oocytes that were treated identically but were not inseminated.
On Day 1 at 32 h after insemination, groups of 6–20 embryos at the two-cell and four-cell stage were selected to represent slow and fast cleaving embryos, respectively. A third group of embryos, still at the one-cell stage, were assumed by default to comprise unfertilized oocytes, arrested zygotes and very slow cleavers. A fourth group, comprising five- to eight-cell embryos (and thereby, in comparison to the normal rate of pig embryonic development, were fragmenting) was also selected at the time. Compact morulae were selected on Day 4 and cultured in the amino acid profiling drops in groups of 7–8. The percentage of morulae developing to the blastocyst stage during the 24 h incubation period was recorded. Subsequently, on the basis of morulae producing 
25% or 
14.3% blastocyst rates (no group of morulae produced a blastocyst rate that fell between these two values) the amino acid depletion and appearance rate data were divided into two groups. Finally on Day 6, blastocysts were divided into two classes: (i) early unexpanded embryos and (ii) those that were at various stages of expansion. The depletion or appearance of amino acids from these embryos was performed in group sizes of 2–9. A blastocyst was defined as an embryo possessing a definite blastocoel cavity.
Polyspermy analysis
An estimate of the degree of polyspermia was carried out 12 h after the beginning of IVF in ~ 35 zygotes/replicate. Zygotes were fixed in acetic acid:ethanol (1:3) under coverslips for 5 days, stained with 1% (w/v) orcein and observed under a phase-contrast microscope.
Analysis of amino acid depletion and appearance
Each group of oocytes/embryos destined for amino acid profiling was washed five times in 100 µl drops of NCSU-23aa, before being transferred by a narrow-bore glass capillary tube (approximately equivalent to the embryos’ diameter) in a minimal volume to 4 µl drops of NCSU-23aa. After 24 h, the medium within the drop was gently mixed and the embryos were removed, in a similar manner as described for the addition of embryos to the medium, in order to minimize volume changes. The stages of development attained by the recovered embryos were recorded. Four µl drops of NCSU-23aa were located alongside those containing embryos and were used to control for non-specific changes in amino acid depletion and appearance during the incubation and sample storage periods (i.e. the breakdown of amino acids to ammonium). From each 4 µl drop, 2 µl was removed and diluted 1:11.5 in purified water (ELGA Purelab; Elga, UK) in HPLC tubes. Any variations in pipetting during sample recovery were corrected for by the previous addition of D-
-amino-n-butyric acid, an amino acid that cannot be metabolised, to the NCSU-23aa (Table 1
). All samples were stored at –80 °C before assay.
A reverse-phase HPLC analytical technique was employed, as previously described (Lamb & Leese 1994), with minor changes. Briefly, the amino acids were derivatised to fluorescent products by automated reaction of the 25 µl sample with an equal volume of o-phthaldialdehyde (OPA) containing 2 µl/ml 2-mercaptoethanol. The HPLC system was a Kontron 500 (Milan, Italy) series linked to a Jasco F920 fluorescence detector (Great Dunmow, UK). The flow rate through the column, a Zorbax Eclipse AAA 3.5 µm (4.6 x 75 mm) (Agilent Technologies, Cheshire, UK) was 1.4 ml/min with column temperature controlled at 25 °C. The two solvents required to generate the elution gradient were a 1:4 and 4:1 (v/v) ratio of methanol:sodium acetate (83 mM, pH 5.9).
The chemistry of the HPLC method did not permit the detection of cysteine and proline and, consequently, only 18 out of the 20 amino acids contained in the NCSU-23aa were measured. Furthermore, due to their very high concentrations in NCSU-23, taurine (7 mM) and hypotaurine (5 mM) exceeded the upper detectable limit of the assay. Due to the magnitude of the chromatographic peak created by hypotaurine, the separation of alanine was incompletely resolved. Consequently, a greater coefficient of variation of detection was recorded for alanine compared with the other amino acids.
Statistical analyses
The net rates of amino acid or nitrogen appearance and depletion were calculated as pmol/embryo per h. It should be appreciated that net rates of amino acid depletion and appearance represent the difference in absolute rates of uptake and release which, theoretically, could greatly exceed the net rates of depletion and appearance being observed.
Net rates of nitrogen appearance and depletion were determined by multiplying amino acid net rates of appearance or depletion by the number of nitrogen atoms contained in the respective amino acids (see Table 2).
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In those treatments containing embryo groups in which the complete set of 18 amino acids could not be measured, replicate sizes of treatments were reduced during the calculation of total net amino acid and nitrogen appearance, depletion, balance and turnover.
Data sets were tested for normal distribution by the Anderson–Darling test. Sets conforming to the null hypothesis were analysed by one-way ANOVA. Significance (P < 0.05) was investigated by the Fisher least squares difference test. Non-parametric data sets were analysed by the Mann–Whitney U test or the Kruskal–Wallis test according to the number of groups examined. Significant results generated by the Kruskal–Wallis test were examined post hoc by the Mann–Whitney U test.
Differences from zero in the net rates of appearance, depletion, balance and turnover of amino acids or nitrogen were determined in those data sets possessing a minimum of six pairs by the Wilcoxon signed ranks test.
Adjustments to the level of significance by the Benferroni method in cases of multiple statistical tests were not applied (Perneger 1998). All data are presented as mean ± S.E.M.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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, the net rates of appearance and release of the 18 amino acids examined are listed in an order corresponding to their molecular characteristics (Lehninger 2000).
The pattern of net amino acid depletion or appearance during preimplantation development is shown in Fig. 1, in which normally inseminated oocytes (Day 0), two-cell embryos (Day 1), morulae (Day 4) and blastocysts (Day 6) are compared. The net rates varied according to amino acid (P < 0.001) and, apart from serine and histidine, stage of development. Differences in net rates between stages of development were observed for: glycine, alanine, leucine, methionine, threonine, asparagine, glutamine, lysine, arginine, glutamate, aspartate (all P < 0.001), phenylalanine (P < 0.002), isoleucine (P < 0.003), tyrosine (P < 0.007), tryptophan (P < 0.01) and valine (P < 0.014).
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Glycine, isoleucine, valine, phenylalanine, tryptophan, methionine, asparagine, lysine, glutamate and aspartate consistently appeared whereas threonine, glutamine and arginine were consistently depleted at all stages of development. Only serine showed net rates of appearance/depletion that were not different from zero at any of these stages of development.
Over these same developmental stages, total net amino acid and nitrogen appearance, depletion, balance and turnover were all different (P < 0.01) from zero (Fig. 2). Total net amino acid and total net nitrogen appearance, depletion and turnover were all reduced quantitatively from Day 0 until Day 4 and then rebounded by Day 6 such that total net nitrogen depletion and turnover were greater on Day 6 than Day 0. Total net amino acid balance did not differ between Days 0 and 6. Total net nitrogen balance also did not differ between Days 0 to 4 but was significantly greater (P < 0.05) on Day 6 compared with the three preceding days, this was largely accounted for by the greater net depletion rate of arginine at this stage. Detailed analysis of each stage of development is presented below.
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The three oocyte/embryo types did not differ in total net amino acid appearance or turnover (Fig. 4). However, presumptive polyspermic oocytes exhibited a reduced (P < 0.05) total net depletion rate and a slightly positive value for total net amino acid balance (i.e. total net appearance exceeded total net amino acid depletion indicating that the embryos were in negative amino acid balance) in comparision with the other two oocyte/embryo types. No differences in total net nitrogen appearance (2.13 ± 0.10; 2.33 ± 0.12; 2.46 ± 0.17), depletion (–4.53 ± 0.32; –5.03 ± 0.20; –4.27 ± 0.07), balance (–2.40 ± 0.35; –2.70 ± 0.22; –1.80 ± 0.18) and turnover (6.67 ± 0.32; 7.35 ± 0.24; 6.73 ± 0.19) were observed between normally inseminated presumptive zygotes, uninseminated oocytes and presumptive polyspermic zygotes, respectively.
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The net amino acid depletion and appearance rates between uncleaved and unfertilized oocytes, two-cell, four-cell and fragmenting five- to eight-cell embryos (Fig. 5) were different in terms of glycine, valine, methionine, threonine, glutamine (all P < 0.001), arginine (P < 0.003), phenylalanine (P < 0.006), alanine (P < 0.02) and isoleucine (P < 0.036)
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Although total net amino acid appearance did not differ between any embryo types, total net depletion was greater in one-cell embryos causing total net amino acid balance and turnover to reflect these same differences (Fig. 6). Similar patterns and statistical differences were observed between total net nitrogen appearance (1.63 ± 0.06; 1.57 ± 0.08; 1.44 ± 0.07; 1.57 ± 0.11), depletion (–4.89 ± 0.31; –3.30 ± 0.15; –3.17 ± 0.38; (–2.6 ± 0.21), balance (–3.23 ± 0.32; –1.73 ± 0.22; –1.74 ± 0.40; –1.04 ± 0.29) and turnover (6.49 ± 0.30; 4.87 ± 0.11; 4.61 ± 0.37; 4.17 ± 0.18) for uncleaved and unfertilized oocytes, two-cell embryos, four-cell embryos and fragmenting five- to eight-cell embryos, respectively.
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25%) blastocyst rates had greater net rates of threonine (P < 0.015) and arginine (P < 0.001) depletion and higher net rates of glutamate (P < 0.029) appearance than those morulae that produced 14.3% blastocysts (Table 3). Total net amino acid appearance (0.97 ± 0.16 vs 0.78 ± 0.24), depletion (–1.05 ± 0.16 vs –1.24 ± 0.30), balance (–0.08 ± 0.28 vs –0.46 ± 0.52) and turnover (2.01 ± 0.17 vs 2.02 ± 0.14) were not influenced by the percentage of blastocysts generated, but total net nitrogen depletion (P < 0.006) and turnover (P < 0.001) were greater for those generating the high blastocyst yields. (Fig. 7).
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). Although the patterns of total net amino acid appearance, depletion, balance and turnover in the two blastocyst groups were similar to those of total net nitrogen, only total net amino acid depletion (–2.07 ± 0.23 vs –2.90 ± 0.32; P < 0.047) and turnover (3.67 ± 0.20 vs 4.70 ± 0.42; P < 0.016) were significantly greater in expanded blastocysts, whereas appearance (1.60 ± 0.34 vs 1.80 ± 0.29; NS) and balance (–0.47 ± 0.55 vs –1.11 ± 0.44; NS) were unaffected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The changes in the rates of net depletion and appearance of individual amino acids as development proceeded permitted classification into five distinct patterns (vide supra). The amino acid data presented by Partridge and Leese (1996) suggested that only aspartic acid gave a Type III response (a continuous fall in net appearance from Day 0 to Day 6) in the bovine. By contrast, alanine exhibited a Type I response (a pattern in which net amino acid appearance is high on Day 0, falls to a nadir on Day 1 or 4 but increases again by Day 6), glutamine was suggestive of a Type II pattern (one in which amino acid net appearance is low on Day 0, rises on Days 1 and 4, and falls again by Day 6), and threonine, glutamate and aspartate were indicative of a Type III response (comprising a continuous fall in net appearance from Day 0 to Day 6) (Partridge and Leese 1996). An increasing rate of net amino acid appearance (Type IV classification) was not apparent in the cow (Partridge and Leese 1996). A further difference between the bovine data and our own concerned the magnitude of net depletion or appearance rates of individual amino acids. For example, for those amino acids (with the exception of glutamine) that either consistently appeared or were depleted at all stages of development in the cow and pig, the mean net rates over the whole in vitro period in the cow were approximately 8–10 times higher than those recorded in the pig. This contrasts with the human (Houghton et al. 2002) in which the net rates were considerably lower. This again suggests, but does not prove, that a greater similarity exists in net amino acid appearance and depletion rates between the pig and the human compared with the cow. Finally, a notable difference in net amino acid rates between the pig and cow was that (with the exception of alanine, threonine and glutamine) those amino acids that appeared in the cow were often depleted in the pig. It remains to be established if in vivo derived pig embryos that possess greater developmental competence than their in vitro fertilised counterparts, present contrasting patterns of net amino acid depletion and appearance.
Explanations for these contrasting patterns of net amino acid appearance or depletion between the cow and pig could be due to differences in embryo metabolism or amino acid transporter systems (Van Winkle 2001) between ruminant and monogastric animals, although knowledge of amino acid transporter systems in domestic animal embryos is lacking. However, it should be emphasised that the concentrations of amino acids in the minimal essential medium (containing essential and non-essential amino acids) used by Partridge and Leese (1996) were up to 20 times higher than in the present study. As it is known that the pattern of amino acids consumed or produced is dependent on the concentrations of amino acids to which embryos are exposed (Epstein 1975), any such differences between studies may obscure the true response patterns shown between species. Alterations in the net rates of amino acid appearance and depletion that are dependent on the medium concentrations of amino acids have also been described for the mouse (Lamb & Leese 1994) and cow (Orsi & Leese 2004). On this basis, the similarity in pattern of amino acid net rates observed between human (Houghton et al. 2002) and pig embryos might be partially explained by the identical concentrations of amino acids (both based on those derived by Tay et al. (1997) from perfusion of Fallopian tube) added to their culture media. Consequently, we cannot exclude the intriguing possibility that similarities in net rates between pig and human embryos were due to the adjustment of the amino acid metabolism of the pig embryo towards that of the human under these conditions. The availability of a medium containing an amino acid formulation identical to that of porcine oviductal fluid would have resolved this uncertainty. Unfortunately, the only published data in the pig indicate 2–7 fold differences in amino acid concentration between studies (Engle et al. 1968, Iritani et al. 1974) (Table 1). Furthermore, the oviductal fluid in these studies was derived by sutured cannulation, a surgical procedure that could traumatise the tissue allowing pathophysiological alterations in secretory process to occur. In the absence of consistent information on the physiological concentrations of amino acids in porcine oviductal fluid that could, for example, be best determined by microsampling techniques (Nichol et al. 1992), we are of the opinion that the most appropriate approach to determine requirements is to present the developing embryo with a mixture of amino acids at relatively low concentrations and to measure net rates of uptake and release. This approach conforms to neither the ‘back to nature’ route nor the ‘let the embryo choose’ principle described by Summers and Biggers (2003) but represents an alternative valid method to discover empirically the amino acid preferences of early embryos from which data, more informed improvements in the constituents in culture media can be made.
Protein synthetic patterns could underlie the developmental changes in net rates of amino acid depletion and appearance. Prior to activation of the embryonic genome, the incorporation of amino acid substrates into protein steadily declines from the oocyte or zygote stage in the pig (Jarrell et al. 1991) and cow (Frei et al. 1989). During this period, maternal RNA transcripts are the only templates for protein synthesis until the late four-cell stage when transcription of the pig embryonic genome begins (Jarrell et al. 1991, Schoenbeck et al. 1992). At this point, protein synthesis gradually accelerates so that, by the blastocyst stage, it exceeds that recorded in the pig oocyte or one-cell zygote (Jarrell et al. 1991, Schoenbeck et al. 1992). These variations in protein synthesis may be reflected in the present study by the changes in the net rates of depletion or appearance of isoleucine, valine, phenylalanine, methionine and arginine and, to a lesser extent, leucine and tyrosine which all follow a pattern parallel to that of protein synthesis in that the net uptake of these amino acids appears to be lower on Days 1 and 4 compared with Days 0 and 6. In addition, the net appearance rate of glutamate, which is the end product of the majority of transamination reactions and a marker for deamination (Lehninger 2000), largely corresponds to the profile of protein synthetic changes (Jarrell et al. 1991). Analysis of total net amino acid and nitrogen turnover rates (Fig. 2) also appears to reflect the changes in protein synthesis documented in developing pig embryos (Jarrell et al. 1991, Schoenbeck et al. 1992).
In contrast to the above temporal changes, other amino acids exhibit alternate patterns of net amino acid depletion and appearance during development. These contrasting profiles probably reflect the complexity of metabolism that includes transamination and the various functions of amino acids in addition to their roles as protein monomers or intermediates for oxidation (see Gardner et al. 2000). Of those amino acids being depleted, arginine can function as a precursor of nitric oxide, a signalling molecule involved in mouse embryonic development (Manser et al. 2004) whereas glutamine is utilised not only as a significant energy source but also as a precursor of glycosylated products (Rieger 1992). The utilsation of glutamine declines between the two-cell stage and the early blastocyst in the cow (Rieger et al. 1992) as is the case for glutamine depletion in the pig (Fig. 1).
Of those amino acids appearing in the medium during the incubation period, alanine may be formed from pyruvate (Quinn & Wales 1973, Donnay & Leese 1999) and function to sequester ammonium (Van Winkle & Dickinson 1995) that is potentially toxic to embryos in vitro (Gardner & Lane 1993). The release of the remaining amino acids are more difficult to rationalise especially during the first 24 h of IVC (Day 0) when presumptive zygotes were transferred from fertilization medium which lacks amino acids (but is supplemented with BSA) to NCSU-23aa which is supplemented with both amino acids and BSA. In such a scenario, one might have anticipated a compensatory influx of amino acids from the culture medium as a response to the potential reduction of internal amino acid concentrations during the incubation in the fertilisation medium. In support of this suggestion, intracellular concentrations of amino acids in mouse embryos fall in response to transfer into a medium devoid of amino acids (Van Winkle & Campione 1996), a response that can occur within minutes (Eagle 1958, Schultz et al. 1981, White & Christensen 1983) and, even if only temporary, can exert lasting detrimental effects (Gardner & Lane 1996). Consequently, the net appearance on Day 0 of aspartate, glutamate, alanine and glycine, which represent a group of amino acids found at the highest endogenous concentrations in the oocyte and early cleaving embryo of the mouse and rabbit (Schultz et al. 1981, Miller & Schultz 1987), might be considered somewhat paradoxical. One interpretation is that the net release rather than uptake of these amino acids suggests that they are not limited at this stage of development, perhaps being made available by protein degradation, transamination or biosynthesis or liberation in exchange for other amino acids (see Bavister 1995). The accumulation of endogenous embryonic glutamate and aspartate during culture in the presence of glutamine (Van Winkle & Dickinson 1995) and the production of glutamate, aspartate and alanine from pyruvate (Quinn & Wales 1973) are some examples of metabolic interconversions in the mouse. The net release of glycine, on the other hand, especially on Day 0, might be explained by the estimated 90% loss of this amino acid during early embryonic development from the endogenous pool, in which it originally constitutes 60% of total amino acids in the unfertilized mouse egg, perhaps in exchange for other amino acids (Schultz et al. 1981).
It is also of interest that the aforementioned amino acids (glycine, alanine, aspartate and glutamate) plus asparagine, which appear during the incubation periods, are all classified as non-essential amino acids (Eagle 1958) and are major players in amino acid metabolism (Lehninger 2000). The net appearance rates of the remaining amino acids, which are all defined as essential, were lower than these non-essential amino acids. It is therefore possible that mechanisms exist to conserve the release of the essential amino acids; a system which might facilitate the ability of mouse embryos to develop in the absence of fixed nitrogen (Caro & Trounson 1984). Of the amino acids appearing during the culture of human embryos (that did not arrest prior to the blastocyst stage), those classed as non-essential were released at higher net rates compared with the essential amino acids (Houghton et al. 2002). However, it needs to be emphasised that the classification of amino acids into essential and non-essential is based on the nutritional requirements of somatic cells in vitro (Eagle 1958), and it may be inappropriate for the early embryo (see Houghton et al. 2002, Summers & Biggers 2003).
The classification of amino acids according to their molecular characteristics (Table 2) (Lehninger 2000) reveals obvious homogeneity in amino acid profiles within the triplets comprising the branched chain (leucine, isoleucine and valine) and benzenoid (phenylalanine, tryptophan and tyrosine) amino acids, while somewhat weaker similarities were observed between glutamate and aspartate (both negatively charged polar amino acids). However, interpretation of these inter-relationships is difficult owing to the scant knowledge available of amino acid metabolism by the porcine embryo plus the known complexity of metabolism and amino acid transporter systems (Van Winkle et al. 1990a,b).
The fertilisation-initiated increase in protein synthesis observed in lower organisms (reviewed by Epstein & Smith 1973) is not apparent in mammalian zygotes, at least as measured by amino acid uptake and incorporation in the mouse (Biggers & Stern 1973, Holmberg & Johnson 1979). This lack of change in protein synthetic parameters during the immediate post-fertilisation period parallels the similarity in amino acid net depletion and appearance rates observed in the present investigation between oocytes that were either uninseminated or were normally fertilized. By contrast, polyspermic mouse embryos have been shown to have increased rates of glucose metabolism, proportional to the number of pronuclei generated (Pantaleon et al. 2001). Although neither glutamine (nor pyruvate) metabolism is affected by polyspermia (Pantaleon et al. 2001), it is possible that elevated glucose utilisation is symptomatic of alterations in other metabolic pathways including those of other amino acids. This could explain the differences in net rates of amino acid depletion and appearance in the present study between polyspermic zygotes and normally fertilized or non-fertilised oocytes. It is notable that, of all the treatments investigated in this study, polyspermic zygotes were the only group that exhibited a total net appearance rate of amino acids that exceeded that of depletion (Table 4), perhaps suggestive of an imbalance in metabolism. This phenomenon could allow discrimination between mono- and polyspermia and thereby be a tool for the selection of embryos that are normally fertilised. Since the incidence of polyspermia (33% in this study) is an unavoidable consequence of pig IVF, further development of such afflicted oocytes (Han et al. 1999) could have influenced the amino acid patterns observed at later stages of development.
Blastocoel expansion is a highly energy demanding process such that, in the pig, ATP consumption triples between the morula and early blastocyst stages (Sturmey & Leese 2003), most likely to supply the Na+/K+ ATPase pump (Donnay & Leese 1999). These increased energy requirements are met by changes in the utilisation of substrates, partly by elevated rates of oxygen consumption, glucose utilisation and amino acid metabolism (Rieger et al. 1992, Donnay & Leese 1999, Sturmey & Leese 2003), and could explain the increased net rates of nitrogen depletion and turnover observed in the present study in those morulae producing higher blastocyst rates (Fig. 7) and those blastocysts that were already expanding at the time of selection (Fig. 8).
In conclusion, these data indicate that the net rates of appearance or depletion of amino acids by pig embryos vary between amino acids and the stage of development. Common features between the pig, cow and human are the net depletion of glutamine and arginine and the net appearance of alanine (Partridge & Leese 1996, Houghton et al. 2002). The net depletion of amino acids at all stages of preimplantation development suggests that a physiological mixture of all amino acids should be considered a routine supplement to pig culture media. At present, amino acids (apart from glutamine, taurine and hypotaurine) are usually absent from standard pig embryo culture media (i.e. NCSU; Petters & Reed 1991) or, if added, are supplemented in groups as non-essential or non-essential mixtures at high concentrations (Koo et al. 1997, Long et al. 1999, Van Thuan et al. 2002, Yoskioka et al. 2002). Further investigations should be undertaken to examine the interrelationship between amino acid patterns of net uptake and release at specific stages of development and subsequent embryo viability (Houghton et al. 2002, Brison et al. 2004). Such data could be combined with quantitative descriptors of cleavage times and morphological parameters in order to derive a viability index of individual embryos (Boise 2002).
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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