Effect of antibiotics on development in vitro of hamster pronucleate ova

Antibiotics are commonly added to embryo culture media, but effects on embryo development have not been examined thoroughly. Hamster ova were used to investigate whether penicillin, streptomycin or gentamicin affect embryo development in vitro. Ova were collected 10 h post activation by spermatozoa in vivo and cultured in five treatments: 1) Control: chemically-defined medium HECM-9 with no antibiotics; 2) HECM-9 with 100 IU/mL penicillin; 3) HECM-9 with 50 microg/mL streptomycin; 4) HECM-9 with 10 microg/mL gentamicin and 5) HECM-9 with both 100 IU/mL penicillin and 50 microg/mL streptomycin. Individually, penicillin, streptomycin and gentamicin did not affect embryo development to the 8-cell stage at 58 h post oocyte activation, or morula/blastocyst stages, or blastocysts alone at 82 h post activation. However, when penicillin and streptomycin were both present in the culture medium the percentages of 8-cell embryos at 58 h and blastocysts at 82 h were significantly lower than the control. No antibiotic treatment improved hamster embryo development in vitro. We caution against the use of penicillin and streptomycin together for hamster embryo culture, and show that it is not necessary to include any antibiotics in embryo culture media for up to 72 h if proper sterile technique is used with an oil overlay.     

Succinate and malate improve development of hamster eight-cell embryos in vitro: Confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% ± 3.9 vs. 54.5% ± 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% ± 7.2), but not pyruvate (85.8% ± 6.2) or glutamine (84.1% ± 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%–58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% ± 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 ± 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20–24). The MCN was the highest (33.4 ± 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 ± 1.1) or malate (24.7 ± 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (±4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% ± 2.0) or blastocysts (28.9% ± 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium. Mol. Reprod. Dev. 47:440–447, 1997. © 1997 Wiley-Liss, Inc.     

Development of preimplantation embryos of the golden hamster in a defined culture medium

Eight-cell embryos were recovered from mated golden hamsters that had been superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Embryos were cultured for 24 or 32 h in a defined medium (modified Tyrode's solution) designed for fertilization of hamster oocytes in vitro. This medium was supplemented in some experiments with amino acids (glutamine, phenylalanine, methionine and isoleucine) and with vitamins (Eagle's Minimum Essential Medium vitamin supplement). At the end of the culture period, the numbers of embryos developing to the blastocyst stage were recorded. In other experiments, the effects of varying the osmotic pressure (225, 250, 275 and 300 m0smol/kg) and the pH (6.8 and 7.4) of the culture medium on blastocyst formation were examined. A difference was found between the ability of early 8-cell embryos (approx. 54 h post-egg activation) and late 8-cell embryos (approx. 62 h post-egg activation) to develop in culture. In the unsupplemented culture medium, only 2% of early 8-cell embryos developed to the blastocyst stage compared with 22% of late 8-cell embryos. A marked effect of the four amino acids on development was found. In the presence of amino acids 36% of early 8-cell embryos developed into blastocysts (18-fold increase). The amino acids also increased the percentage of late 8-cell embryos that developed into blastocysts from 22% to 66%. These data suggest that an important metabolic change may occur in hamster embryos during a critical period at the 8-cell stage of development. No additional effect on development was observed when vitamins were included in the culture medium. No significant effect of either osmotic pressure of pH of the culture medium on development was found. When blastocysts formed from cultured 8-cell embryos were transferred surgically to pseudopregnant hamsters, about 25% developed into normal-looking fetuses and 5 normal-looking young were born, 4 of which have survived. These results represent an approach towards achieving complete preimplantation development of hamster embryos in vitro.     

Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.     

Effects of volume, culture media and type of culture dish on in vitro development of hamster 1-cell embryos

We examined effects of medium volume and two different culture media (HECM-3 and HECM-4) on in vitro development of hamster embryos. Groups of 5 to 8 1-cell embryos were cultured for 72 h in either < or =100 or > or =100 microl volumes. In the first experiment, embryos were cultured in Petri dishes with 2, 5, 20, 50 or 100 microl of medium using the two media (2 x 5 factorial experiment). Optimal volumes for morula and blastocyst development were 100 microl of HECM-3 and > or =50 microl of HECM-4; in HECM-4, > or =20 microl volumes were suitable whereas in HECM-3 < or = 50 microl volumes were unsuitable. In the second experiment, embryos were cultured in 100, 200, 500 and 1000 microl of HECM-3 and HECM-4 using organ culture dishes. Controls were 100 microl drops in Petri dishes. In organ culture dishes, blastocyst development was < or =6% in HECM-3 and 33-41% in HECM-4, and suitable volumes for development to at least morulae were > or =200 microl of HECM-3, and > or =100 microl of HECM-4. In both experiments development to morula and blastocyst stages with 100 microl volume in Petri dishes was significantly higher with HECM-4 (96 and 85% in Experiment 1 and 2, respectively) than that with HECM-3 (52 and 40% in Experiment 1 and 2, respectively; P < 0.05). These results indicate that attention should be paid to both type and volume of medium and interaction with type of culture dish for optimizing development of embryos in vitro.     

Environmental variables influencing in vitro development of hamster 2-cell embryos to the blastocyst stage

This study is a systematic analysis of environmental variables influencing development of 2-cell hamster embryos to the blastocyst stage in vitro. Experiments were done using a chemically defined (protein-free) culture medium (HECM-2). Physicochemical variables examined were temperature, the concentrations of CO2, HCO3-, Ca2+, Mg2+, K+, and O2, the presence of a silicone oil overlay, and osmotic pressure. The optimal temperature for development in vitro was 37.5 degrees C; lower temperatures were inhibitory. There was no significant effect on blastocyst development of alterations in the concentrations of NaHCO3, CaCl2, MgCl2, and KCl, or in the ratios of Ca2+:Mg2+ and Na+:K+, over the ranges tested. Development to the blastocyst stage was significantly stimulated by increased CO2 concentrations (7.5% and 10%), by reduced O2 concentrations (10% and 5%), and by the presence of silicone oil overlying the culture medium. Reduction of blastocyst development in the absence of an oil overlay was not caused by increased osmotic pressure. Cleavage stage embryos were not affected by osmolalities ranging from 250 to 350 mOsmols, but blastocyst development was inhibited at greater than or equal to 325 mOsmols. Under optimized conditions (37.5 degrees C, 10% CO2, 25 mM HCO3-, 2.0 mM Ca2+, 0.5 mM Mg2+, 3.0 mM K+, 10% O2, 250-300 mOsmols, with silicone oil overlay), 51-57% of 2-cell hamster embryos developed to the blastocyst stage. This represents a significant improvement over previous "standard" culture conditions, which supported development of 26% blastocysts from 2-cell hamster embryos. The culture system described here provides an adequate baseline response to support detailed investigations into the regulation of embryo development in the hamster.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Glucose inhibits development of hamster 8-cell embryos in vitro

Relative preferences of energy substrates (glucose, pyruvate, and lactate) for in vitro development of hamster 8-cell embryos were investigated. Using protein-free modified Tyrode's medium (TLP-PVA) containing 10 mM lactate (L), 0.1 mM pyruvate (P), and amino acids (Phe, Ile, Met and Gln), we found that development of hamster 8-cell embryos to blastocysts was supported better in the absence of glucose than in medium containing (standard) 5 mM glucose (88.1% and 50%, respectively). Addition of even 0.25 mM glucose to the medium significantly inhibited blastocyst formation (54.1%). Medium T-PVA, containing 5 mM glucose as sole energy substrate (without pyruvate, lactate, and amino acids), very poorly supported embryo development (less than or equal to 7.9% blastocysts), but addition of 0.1 mM pyruvate enhanced blastocyst formation (52%). Elimination of pyruvate in TL-PVA medium containing 5 mM glucose and amino acids markedly reduced blastocyst formation by 4-fold (13.5%); the optimal pyruvate concentration was 0.2 mM. However, if the same medium was devoid of glucose, blastocyst formation was high both in the absence (71.1%) and presence (83.3%) of 0.1 mM pyruvate. Similarly, in glucose-free T-PVA medium, addition of either 10 mM lactate or amino acids supported 8-cell embryo development to blastocysts (61.7% and 60.5%, respectively) as opposed to 18.8% and 30.6%, respectively, in the presence of 5 mM glucose. This augmented development in the absence of glucose is suggested to the due to the efficient conversion of lactate to pyruvate and of amino acids to amphibolic intermediates and hence their utilization via the Krebs cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose

The failure of hamster 2-cell embryos to develop in vitro (2-cell block) was examined with experiments in which concentrations of glucose and phosphate in the culture medium were varied. Embryos were cultured in a protein-free modified Tyrode's solution that normally contains 5.0 mM glucose and 0.35 mM sodium dihydrogen phosphate. In the presence of 0.35 mM phosphate but without glucose, 23% of 2-cell embryos reached the 4-cell stage or further after culture for 1 day and 27% after 2 days. Glucose inhibited embryo development even at 0.1 mM (4% development to greater than or equal to 4-cells after culture for 2 days); there was no dose-related inhibition above this glucose concentration. In a second experiment, phosphate levels were varied in the absence of glucose. Phosphate was highly inhibitory to development, with 97% of 2-cell embryos reaching the 4-cell stage or further after culture for 1 day in the absence of phosphate compared to 9-21% in the presence of 0.1-1.05 mM phosphate. After culture for 2 days, 26% of embryos reached the 8-cell stage or further when phosphate was absent compared to 0% development to 8-cells with 0.1 mM phosphate or higher. In a factorial experiment, phosphate blocked development when glucose was present or absent, whereas glucose did not block embryo development in the absence of phosphate. However, 2-deoxyglucose (a non-metabolizable analogue of glucose) inhibited embryo development in the absence of phosphate. These data show that the in vitro block to development of hamster 2-cell embryos is caused at least in part by glucose and/or phosphate. Deletion of these compounds from the culture medium eliminates the 2-cell block to development in virtually all embryos, and approximately 25-75% of embryos develop to the 8-cell or morula stages in vitro. The observations provide a possible explanation for the 2-cell and 4-cell blocks that occur in conventional culture media: stimulation of glycolysis by glucose and/or phosphate may result in inefficient adenosine triphosphate (ATP) production. The data indicate marked dissimilarities in the regulation of in vitro development of early cleavage stage hamster embryos compared with embryos of inbred mice, since the latter have an inactive glycolytic pathway prior to the 8-cell stage of development and will grow from 1-cell to blastocyst with both phosphate and glucose in the culture medium.     

A comparative study on developmental capacity to blastocysts derived from 1-and 2(3)-cell bovine embryos after in vitro maturation and fertilization

The present study was conducted to compare the developmental capacity of 1-and 2(3)-cell embryos after 18 and 30 h of fertilization, and blastocyst cell number and in vitro survival after freezing and thawing of bovine blastocysts derived from the 1-and 2-cell embryos. Oocytes were matured and fertilized by conventional IVM/IVF methods. After 18 or 30 h of fertilization, 1-cell embryos (18 h-fertilization) or 1- and 2(3)-cell embryos (30 h-fertilization) were cultured for 8 or 10 d in synthetic oviduct fluid medium (SOFM) supplemented with 10% human serum (HS), minimum essential medium (MEM) essential or nonessential amino acids and glutamine. The separate culture of 1- and 2(3)-cell embryos after 30 h of fertilization showed higher (p < 0.01) cleavage, development to expanded and hatched blastocysts than culture of 1-cell embryos after 18 h of fertilization. Two-cell embryos of 30 h-fertilization group had higher developmental capacity to expanded and hatched blastocysts than 1-cell embryos at 18 or 30 h after insemination (Experiment I). However, there was no significant difference in the mean cell number of blastocysts derived from the culture of 1-cell and 2(3)-cell embryos, respectively (Experiment II). The in vitro survival or hatching after freezing and thawing of blastocysts was significantly affected by embryonic quality before freezing, but did not significantly differ with blastocysts derived from 1- and 2(3)-cell embryos after 18 or 30 h of fertilization. The results indicate that the culture of 2(3)-cell embryos after 30 h of fertilization is an effective method to produce more transferable embryos (blastocysts) in bovine IVM, IVF and IVC techniques.     

Effect of visual light on in vitro embryonic development in the hamster

The effect of light exposure during collection and culture of hamster embryos on their subsequent development in vitro was examined. When embryos were collected under dark conditions (70 lux) within 10 minutes and then cultured in a HECM-1 medium in 5% CO(2) in air, the developmental rates of 1-cell embryos to the 4- and 8-cell stages were 88.6% (93 105 ) and 66.7% (70 105 ), respectively. These rates were significantly higher than those under light conditions (1600 lux): 51.9% (56 108 ) and 34.3% (37 108 ). Light irradiation during the culture of 1-cell embryos suppressed subsequent development. The degree of suppression correlated inversely with duration of light irradiation, and light irradiation of 30 minutes or more completely blocked development to the 2-cell stage. When 1-cell embryos were irradiated through a yellow filter, cutting the light wavelengths to less than 500 nm, embryonic development was still suppressed. However, the degree of the suppression varied and 45.7% (53 116 ), 6.0% (7 116 ), and 0.9% (1 116 ) of the embryos developed to the 2-, 4-, and 8-cell stages, respectively, under 30 minute light irradiation. Inhibitory effects of light irradiation on the development of 2- and 8-cell embryos were also observed, showing an inverse correlation with duration; the developmental rates of 2-cell embryos to the 8-cell stage under 0, 10, and 30 minutes of irradiation were 85.6% (107 125 ), 1.6% (2 122 ), and 0% (0 129 ), respectively, and those of 8-cell embryos to the blastocyst stage were 79.8% (91 114 ), 74.8% (86 115 ), and 0% (0 110 ), respectively. These findings indicate that early-stage embryos are sensitive to light exposure; however, severe light exposure adversely affects the development of embryos at any stage. Thus, the protection of embryos from light exposure at all stages of embryo manipulation, from collection to culture, is essential.     

Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 microL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst.     

Relationship between development, metabolism, and mitochondrial organization in 2-cell hamster embryos in the presence of low levels of phosphate.

The effect of low concentrations of inorganic phosphate (P(i)) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 microM KH(2)PO(4). Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with P(i) did not alter TCA-cycle activity. However, culture with > or 2.5 microM P(i) significantly increased (P < 0.01) EMP activity compared to control. Mitochondrial organization was significantly (P < 0.01) disrupted by P(i) in a dose-dependent manner. Development to the 8-cell, morula/blastocyst, and blastocyst stages was significantly reduced (P < 0.05) in the presence of > or =2.5 microM P(i) compared to both control and 1.25 microM P(i). This study clearly demonstrates that, for hamster embryos, inclusion of even exceptionally low concentrations of P(i) in culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence.     

Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media.

Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids [HECM-6] followed by tissue culture medium 199 + 10% bovine calf serum). Modifications were made to reduce or eliminate protein. Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured. There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively). Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%). There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%). Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium. Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium. This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis.     

Developmental responses of 2-cell embryos to oxygen tension and bovine serum albumin in Wistar rats.

To improve rat embryo culture conditions, responses of Wistar 2-cell embryos from 2 breeders to oxygen tension (5 vs 20%) and bovine serum albumin (BSA) (0 vs 3 mg/ml) were examined using rat 1-cell embryo culture medium (mR1ECM). Supplementation of 3 mg/ml BSA significantly stimulated and accelerated development to the blastocyst and expanded blastocyst stages during 72 and 96 h culture, while reduced oxygen tension stimulated cell division. Fetus development after transfer of blastocysts obtained from 72 h culture under 5% O2 with BSA was significantly higher than those cultured under atmospheric oxygen without BSA. However, the nuclear numbers of in vitro cultured blastocysts and fetus development after embryo transfer were still significantly lower than in vivo developed blastocysts, indicating the current culture condition is still suboptimal.     

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, freezability and amino acid consumption of bovine embryos cultured in synthetic oviductal fluid (SOF) medium containing amino acids at oviductal or uterine-fluid concentrations

In this study, we examined the development, freezability and amino acid consumption of in vitro produced bovine embryos cultured in a chemically defined medium (SOF+polyvinyl alcohol), supplemented with 24 amino acids at concentrations measured in bovine oviductal or uterine fluid. Amino acids at concentrations in oviductal fluid tested by Elhanssan (EOAA) significantly improved development to the hatched blastocyst stage, compared to Sigma amino acid solutions BME and MEM (SAA). Amino acids at concentrations in uterine fluid tested by Li (LUAA) were not compared to SAA, and development in LUAA was not significantly different from development in EOAA. Amino acids at concentrations in uterine fluid tested by Elhanssan (EUAA) significantly reduced cleavage rate and blocked further embryo development. When the IVF embryos were cultured in EOAA for 48, 72, 96, or 120 h and then transferred to LUAA, blastocyst and hatched blastocyst rates were not significantly affected. The freezability of blastocysts cultured in EOAA for the first 72 h and then moved to LUAA was improved compared to that in SAA. During the 1-8-cell stages, embryos secreted all 23 amino acids (total, 6,368 pmol/embryo). During the 8-cell to morula stages, embryos continued to secrete 21 amino acids (total, 2,495 pmol/embryo), meanwhile embryos began to absorb Arg (70 pmol/embryo) and Gln (18 pmol/embryo). After the morula stage, embryos began to absorb 15 amino acids including Glu, Gly, Arg, and Gln (total, 2,742 pmol/embryo) and secreted eight amino acids (total, 1,616 pmol/embryo). Embryos absorbed only Arg (183 pmol/embryo) and secreted the other 22 amino acids (total, 3,697 pmol/embryo) when the culture medium was not changed during the entire culture period (zygote to blastocyst).     

Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.     

Oviduct Epithelial Cell Co-culture of early Porcine Embryos

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144–168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4-and 8-16-cell embryos formed blastocysts in culture at a rate of 80–90%.