Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium

Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 +/- 4.3 and 47.1 +/- 4.3, respectively), similar glucose use (10.81 +/- 1.39 and 10.12 +/- 1.72 pmol/embryo/3 h, respectively) and pyruvate use (1.08 +/- 0.056 and 0.88 +/- 0.048 pmol/embryo/3 h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.     

Comparison of in vitro development and gene expression of in vivo- and IVM/IVF-derived porcine embryos after microinjection of foreign DNA

We compared the developmental ability and gene expression of in vivo- and IVM/IVF-derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF-derived and 129 in vivo-collected zygotes was used to examine developmental ability and gene expression following DNA microinjection. When either DNA injected or noninjected zygotes were cultured for 4 d in NCSU 23 followed by 5 d in Eagle's minimal essential medium (EMEM), the percentages of zygotes developing to blastocysts and hatched blastocysts were higher (P < 0.05) compared with groups cultured in NCSU 23 alone. The percentages of injected embryos reaching the morula and blastocyst stages were significantly lower (P < 0.05) than that of noninjected control embryos whether in vivo or IVM/IVF derived. The percentages of morula and blastocyst stage embryos expressing the gene were higher in the in vivo-derived embryos than in IVM/IVF-derived embryos. A lower proportion of (67 to 77%) mosaicism was observed in the in vivo-derived embryos than in IVM/IVF (90 to 100%) derived embryos. The total cell number of blastocysts cultured in both NCSU 23 and EMEM media was significantly higher than that of blastocysts cultured continuously in NCSU 23. Our results suggest that this dual culture system enhanced embryo viability following microinjection of foreign DNA.     

Energy status of nonmatured and in vitro-matured domestic cat oocytes and of different stages of in vitro-produced embryos: enzymatic removal of the zona pellucida increases adenosine triphosphate content and total cell number of blastocysts

In this study, we evaluated the adenosine triphosphate (ATP) content of individual domestic cat oocytes before and after in vitro maturation and of different stages of in vitro-produced embryos. To investigate the effects of assisted-hatching technique on the ATP content and total cell number, the zona pellucida of in vitro-produced blastocysts and expanded blastocysts (recovered 144 h postinsemination [hpi]) was completely removed by pronase treatment. The average (mean +/- SEM) ATP content of nonmatured oocytes (3.47 +/- 0.18 pmol) was significantly (P < 0.01) higher than that of in vitro-matured oocytes (2.17 +/- 0.10 pmol). After in vitro fertilization and culture, the ATP content of two-cell stages (24 hpi) was 1.17 +/- 0.08 pmol, which increased to 1.47 +/- 0.19 and 1.88 +/- 0.32 pmol at the four- (40 hpi) and eight-cell (48 hpi) stages, respectively. The ATP content then decreased to 1.48 +/- 0.10 pmol in 16-cell embryos (64 hpi), reaching a minimum of 0.49 +/- 0.04 pmol at the morula stage (120 hpi). Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 +/- 0.09, 1.79 +/- 0.01, and 4.17 +/- 0.21 pmol, respectively. After enzymatic removal of the zona pellucida (ERZP) at 144 hpi, ATP content and total cell numbers of blastocysts (4.15 +/- 0.37 pmol of ATP, 328.3 +/- 48.5 cells) and expanded blastocysts (5.81 +/- 0.54 pmol of ATP, 430.1 +/- 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 +/- 0.09 pmol of ATP, 65.3 +/- 4.6 cells; expanded blastocysts: 1.79 +/- 0.11 pmol of ATP, 121.4 +/- 6.5 cells). Our study describes, to our knowledge for the first time, changes in the energy status of domestic cat oocytes before and after maturation and during in vitro development after fertilization. The ERZP markedly increased the ATP content and total cell number of blastocyst stages, suggesting that this technique may improve the quality and viability of in vitro-produced domestic cat embryos.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

Improved in vitro development of porcine embryos with different energy substrates and serum

The effect of replacing 5.5 mM glucose in North Carolina State University (NCSU)-23 medium with 0.5 mM pyruvate/5.0 mM lactate on porcine IVF embryo development was investigated in Experiment 1. Culturing embryos with pyruvate/lactate for 7 days or with pyruvate/lactate from Days 0 to 2, and then glucose from Days 2 to 7 improved cleavage rates. In Experiment 2, embryos were cultured for 7 days in pyruvate/lactate containing NCSU-23 medium supplemented with 0.05% PVA, 0.4% BSA or 10% fetal bovine serum (FBS). The BSA supplement increased the rates of cleavage, blastocyst formation, and the number of total cells in blastocysts. In Experiment 3, embryos were cultured in pyruvate/lactate containing NCSU-23 medium supplemented with 0.4% BSA for 7 days (BSA-PL), 0.4% BSA from Days 0 to 4 and then 10% FBS from Days 4 to 7 (BSA-PL-->F ) or 0.4% BSA from Days 0 to 7 with addition of 10% FBS (BSA-PL + F ) at Day 4. More blastocysts in BSA-PL--> F and hatching or hatched blastocysts in BSA-PL-->F and BSA-PL+F were obtained. Total cell number in blastocysts derived from BSA-PL-->F and BSA-PL+F were increased. Our results demonstrated that supplementing pyruvate/lactate containing NCSU-23 medium with 0.4% BSA for 4 days and replacing it with 10% FBS for another 3 days improved porcine IVF embryo development.     

Factors influencing the in vitro hatching of mouse blastocysts

The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J × DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 μ1 of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.     

Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro

The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.     

Effects of fetal calf serum, amino acids, vitamins and insulin on blastocoel formation and hatching of in vivo and IVM/IVF-derived porcine embryos developing in vitro

The objective of this study was to determine the effects of fetal calf serum (FCS), non-essential MEM amino acids, MEM vitamins and insulin on blastocoel formation, expansion and hatching in porcine embryos developing in vitro. Addition of 20% FCS to the NCSU 23 medium significantly (P < 0.05) decreased by the compaction and blastocoel formation of 1- to 2-cell embryos developing in vitro. In contrast, more 1- to 2-cell embryos commenced hatching in the media containing amino acids than in control medium (25.7 vs 2.6%, P < 0.01). Amino acids and insulin synergistically enhanced the incidence of blastocoel formation and hatching of porcine embryos developing in vitro (P < 0.05). When early compacted embryos which developed in vitro in NCSU 23 medium were cultured in BSA-free NCSU 23 medium supplemented with 20% FCS, the incidence of hatching was significantly increased compared with that of the control groups (35.7 vs 4.1%, P < 0.01). However, addition of amino acids, vitamins or insulin to the NCSU 23 medium did not enhance the development of early morulae to the hatched embryos (P > 0.1). When either in vivo or IVM/IVF-derived 1- to 2-cell stage embryos were cultured 4 d in the modified NCSU 23 and an additional 4 days in the modified NCSU 23 supplemented in the FCS, the percentages (61.8 and 17.8%, in vivo- and IVM/TVF-derived, respectively) of hatched blastocysts were significantly higher (P < 0.01) than in the control groups (2.9 and 0%, in vivo and IVM/IVF-derived, respectively). These results suggested that dual culture conditions are required to optimize an in vitro culture system for the development of the porcine embryo in vitro.     

Development and quality of porcine embryos in different culture system and embryo-producing methods

In this study, the developmental ability and cellular composition of porcine IVF, parthenote and somatic cell nuclear transfer (SCNT) embryos were evaluated following different in vitro culture systems. Group 1, embryos were cultured in NCSU-23 with 5.55 mM D-glucose (NCSU+) until day 6 on 20% O(2) or 5% O(2) (Group 2). Group 3, embryos were cultured in D-glucose-free NCSU-23 (NCSU-) with 0.17 mM Na pyruvate/2.73 mM Na lactate for 58 h and subsequently cultured in NCSU+ until day 6 (NCSU -/+) on 20% O2 or 5% O(2) (Group 4). IVF blastocysts did not differ significantly with O(2) concentrations, but differed significantly with major energy source (glucose and pyruvate/lactate). In Group 3 and 4 IVF blastocysts, the total cell number and apoptosis rates were not significantly different with different O(2) concentrations. Blastocyst rate, total cell number and apoptosis rate in Groups 3 and 4 parthenote embryos also were not significantly different. Parthenote and SCNT, under the same culture treatment, exhibited significant differences in blastocyst and apoptosis rates (47.5 +/- 16.1 vs. 24.0 +/- 4.0 and 4.9 +/- 9.0 vs. 22.8 +/- 23.3). Apoptosis-generating rate increased in the order parthenote, IVF and then SCNT. In conclusion, in vitro development of porcine embryos was not affected by O(2) concentrations but was affected by major energy source. Even so, the concentration of each major energy source and the timing of its inclusion in culture could accomplish relatively high embryonic development, the apoptosis rate stressed that more work still needs to be done in developing a better defined culture system that could support SCNT embryos equivalent to in vivo preimplantation porcine embryos.     

Differential growth, cell proliferation, and apoptosis of mouse embryo in various culture media and in coculture

Sequential culture and coculture are two methods of improving the development of preimplantation embryos in vitro. Direct comparison of the efficiency of these methods is limited. Proliferation and apoptosis determine the total number of blastomere in preimplantation embryo, which is a sensitive parameter for evaluation of the development of embryo in vitro. In this study, we compared the proliferation and apoptosis of mouse embryo in different culture media, including CZB, KSOM, MTF, G1.2/G2.2 sequential culture media, and in human oviductal cell coculture. Sequential culture using G1.2/G2.2 was superior to KSOM, MTF, and CZB/CZB + G with respect to the formation of 3-4 cell embryos, morula, and blastocyst. G1.2/G2.2 cultured blastocyst had significantly more proliferating blastomeres and higher total cell number per blastocyst than those cultured in KSOM or CZB/CZB + G. Compared to embryos cultured in G1.2/G2.2, embryos cocultured in G1.2/G2.2 hatched more frequently. Cocultured blastocysts also had significantly higher percentage of proliferating cell and lower percentage of apoptotic cell per blastocyst than those cultured in G1.2/G2.2. It was concluded that G1.2/G2.2 facilitated the proliferation of blastomere whilst human oviductal cell coculture suppressed apoptosis in addition to stimulating proliferation of blastomere.     

Development of porcine embryos produced by IVM/IVF in a medium with or without protein supplementation: effects of extracellular glutathione

This study was designed to examine the effects of extracellular reduced glutathione on development of pig embryos, produced by in vitro maturation (IVM) and in vitro fertilisation (IVF), in a chemically defined North Carolina State University (NCSU) 23 medium or in NCSU 23 medium with bovine serum albumin (BSA). Microfilament distribution, as a marker of embryo quality, was also examined by immunocytochemical staining and confocal microscopy. When the inseminated oocytes were cultured in the defined medium containing 0-0.5 mM glutathione, blastocyst formation as observed only in the media with glutathione (8.5-16.0%). Increased numbers of blastomeres were observed in the blastocysts as the concentration of glutathione was increased (18.8 +/- 7.2 to 31.0 +/- 8.6). In NCSU 23 medium with 4 mg BSA/ml, addition of glutathione at concentrations of 0.125-0.5 mM significantly increased the proportions of oocytes that developed to blastocysts (39.2-52.5%) compared with the control (29.5%). However, no difference was observed in the average cell number in the blastocysts (41.9 +/- 15.6 to 49.1 +/- 15.5). There were no significant differences in the microfilament distribution in the embryos produced in the defined medium and in the BSA-containing medium. These results indicate that pig embryos produced by IVM/IVF can develop to the blastocyst stage in a defined medium. BSA and glutathione have a synergistic effect on pig embryo development.     

Role of glucose in mouse preimplantation embryo development

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.     

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.     

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.     

Actin filament distribution in blocked and developing pig embryos

Actin filaments play an important role in cell division. The present study was designed to examine the relationship between actin filament distribution and pig embryo development. When in vivo matured and fertilised pig oocytes were cultured in TCM 199 or NCSU 23, in various proportions, 45-65% of inseminated oocytes developed to the 2- to 4-cell stages but blastocyst development was observed only in NCSU 23 (34%) or NCSU 23 containing 10% TCM 199 (7%). Supplementation of NCSU 23 medium with 20% or more TCM 199 resulted in no blastocyst formation. Examination of actin filaments indicated that microfilaments were distributed in the cortex, at the junction of blastomeres and in the perinuclear area in the embryos cultured in NCSU 23, but perinuclear actin filaments were not observed in embryos cultured in TCM 199. When 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23 medium at 36 h after in vivo fertilisation, 57% of the cleaved embryos developed to blastocysts, which was no different from the proportion obtained after culture in NCSU 23 alone (56%). In addition, when 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23, most embryos showed perinuclear actin filaments within 6h. The results indicate that the composition of the culture medium plays an important role in the polymerisation of actin filaments, which in turn influences embryo development. It is possible that pig embryo development was blocked by some components in TCM 199 which prevented actin filament polymerisation.     

Amino acid metabolism of the porcine blastocyst

The pattern of depletion and appearance of a mixture of amino acids by single porcine blastocysts incubated in two different media has been determined non-invasively using high performance liquid chromatography. Zygotes were produced by the in vitro fertilisation of in vitro-matured, abattoir-derived immature oocytes and cultured in medium NCSU 23 with or without amino acids. Embryos grown in the absence of amino acids up to the blastocyst stage were transferred to amino acid-containing culture medium for measurement of turnover (Experiment 1). Blastocysts grown in NCSU 23+amino acids were transferred into fresh droplets of the same medium (Experiment 2). Although the specific pattern of amino acid production and depletion varied between experiments, a general pattern emerged, with arginine being significantly depleted (p<0.001) and alanine consistently appearing in the media, in quantities that varied depending with culture conditions. The data suggest that arginine is important during porcine blastocyst development, most likely contributing to the formation of nitric oxide and polyamines and that alanine is produced as a means of disposing of excess amino groups. A model for the interactions of amino acids during porcine early embryo development is proposed. The profile of amino acid metabolism by porcine blastocysts is qualitatively and quantitatively similar to that given by human embryos during the morula:blastocyst transition suggesting that the porcine blastocyst is a good model for the human.     

The effect of energy substrate concentration and amino acids on the in vitro development of preimplantation porcine embryos

As the pig becomes increasingly used for biomedical research, an effective and efficient in vitro culture system is essential. This study aimed to improve the commonly used porcine embryo culture medium, NCSU23, by altering the energy substrates and adding amino acids, using electrically activated diploid parthenotes from oocytes obtained from the ovaries of prepubertal and adult animals. Morphological development to day 6 and blastocyst cell number were examined. Glucose (5.56 mM) was replaced by pyruvate and lactate (0.2 mM and 5.7 mM, respectively) for either the entire culture period or for the first 48 h only. Blastocyst rates were not different between any of the treatments, and were similar for prepubertal and adult oocytes. When the embryos were cultured with pyruvate and lactate for the first 48 h and then glucose, there was a significant increase in blastocyst cell number compared to glucose only. Blastocysts produced using pyruvate and lactate for the entire time tended to have more cells than those exposed to glucose only and less than those who were cultured in pyruvate and lactate for the first 48 h and then glucose. Nonessential amino acids added for the first 48 h and nonessential and essential amino acids added for the remaining time significantly increased blastocyst cell number only when the embryos were grown in pyruvate and lactate followed by glucose. Blastocyst rates were not different between any of the treatments, and this result was the same when using sow or gilt oocytes. The modified medium was then tested using in vitro matured and fertilized embryos from sow oocytes. Blastocyst rates and cell number were significantly increased in the modified medium compared to those grown in unmodified NCSU23. This shows that altering energy substrates and adding amino acids can increase the quantity and cell number of IVP blastocysts compared with NCSU23.     

Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation

The present study investigated the effect of lactate/pyruvate supplement in culture medium and of chemical activation after electric stimulus on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were enucleated, reconstructed with fetal fibroblasts, and simultaneously fused/activated using a single pulse of 2.0 kV/cm for 30 microsec. In Experiment 1, reconstructed embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with either 5.5 mM glucose (Group A) or lactate (5.0 mM)/pyruvate (0.5 mM) (Group B). Compared to Group A, cleavage rate (64% vs. 47%) was higher and more blastocysts developed in Group B (17% vs. 6% at Day 6, 21% vs.11% at Day 7). Experiment 2, embryos reconstructed by electric stimulus (2.0 kV/cm for 30 microsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 microg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 microg/ml 6-dimethylaminopurine (Group E) treatment at 2 hr after electric stimulus. The reconstructed embryos were cultured for 7 days in NCSU-23 medium supplemented with lactate (5.0 mM)/pyruvate (0.5 mM). The rates of blastocyst formation on Day 6 and Day 7 in Group C (17 and 20%, respectively) or Group D (15, 20%, respectively) were higher than in Group E (9 and 12%, respectively). The percentage of two pseudo-pronucleus (PPN) formations in Group D (88%) was significantly higher than in Group C (71%) and Group E (72%). Mean cell numbers of blastocysts in Group D (63.4 +/- 15.8) were higher than in Group C (43.9 +/- 16.5) and Group E (32.9 +/- 17.9), due to increased trophectoderm (TE) cell numbers. Our results indicate that supplementing NCSU-23 medium with lactate/pyruvate and exposure of cytochalasin B after electrical stimulus can improve in vitro developmental competence of porcine SCNT embryos.     

3H-uridine incorporation in early porcine embryos.

The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two-cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 microM 3H-uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold (P less than 0.001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pellucida-intact blastocyst and amounted to 0.29-0.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased (P less than 0.01 compared with younger stages) and amounted to 0.86 fmol 3H-uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.