Studies on a chemically defined medium for in vitro culture of in vitro matured and fertilized porcine oocytes

The present study evaluated the effects of various components in a chemically defined medium on the development of IVM/IVF porcine embryos. The investigated components included energy substrates (lactate, pyruvate or glucose, alone or in various combinations), amino acids (glutamine, glycine or alanine), PVP and HEPES buffer. The effects of each energy substrate were the same as the control. However, a mixture of lactate with either of the other energy substrates increased the development rate. Glutamine tended to decrease rate of the development more than other amino acids, and this inhibition was dose dependent. Both PVP and HEPES buffer did not affect development rate. However, more than 35 mM HEPES buffer induced fragmentation From the above results, a new culture medium was designed (supplemented with 0.276 mM glycine, 0.176 mM alanine, 15 mM HEPES buffer and 1% (wt/vol) PVP in BSA-free Whitten's medium with or without glucose). The new medium resulted in a higher embryo development rate (20.4 and 16.3%) than that obtained with the control medium (10.0%).     

Sperm penetration in vitro of pig follicular oocytes matured in culture.

Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4.5-5 h at 37 degrees C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17-20 h after insemination 60.6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10.0 and 16.7% with ejaculated spermatozoa, and 3.3, 19.6 and 26.4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27.8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.     

Developmental capability of rat oocytes matured in vitro in defined medium

The normality of in vitro matured oocytes was compared to that of in vivo matured (ovulated) oocytes at the following stages of development: germinal-vesicle breakdown, first polar body formation, fertilization (two polar bodies and two pronuclei with a sperm tail or first cleavage), and fetal development (day 20 fetuses). At all points, the in vitro oocytes exhibited a reduced ability, with oocytes matured cumulus-free having the poorest. The exposure of oocytes to human chorionic gonadotropin (hCG) for 2 hr before collection or during incubation improved their rates of maturation and development to day 20 fetuses but not their ability to undergo fertilization. While beneficial, the exposure to gonadotropins before or during maturation was not essential, as evidenced by the production of two day 20 fetuses matured and fertilized in vitro without any gonadotropin (luteinizing hormone or hCG) treatment in vivo or in vitro. These data demonstrate that in the population of in vitro matured oocytes there exist individuals wholly competent of complete normal development, albeit in a reduced proportion in comparison to normally matured and ovulated oocytes. That the in vitro handling, treatment, and culture of the oocytes may be responsible for some of the reduced developmental ability observed is suggested by the developmental abilities of ovulated oocytes under different conditions. Ovulated oocytes fertilized in the donor had the highest rates of development (46%), followed by those fertilized after transfer into mated recipients' oviducts (20%). The lowest rate was achieved with in vitro fertilized oocytes (7%), which represented the group subject to the greatest degree of manipulation and distinction from the normal in vivo process.     

The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined medium (modified Tyrode's solution) without glucose. When different concentrations of NaCl were added to the medium, the proportions of embryos developed to the >/=8-cell, morula and blastocyst stages 96, 144 and 192 h post insemination, respectively, were significantly higher at 89 to 114 mM than 64 to 76 and 126 to 139 mM NaCl. A high proportion (28%) of blastocyst-stage embryos 192 h post insemination was obtained at 89 mM NaCl. When calculated osmolarity in the medium with 64 mM NaCl was varied by adding D-sorbitol, significantly higher proportions of morula-stage embryos were obtained at 265 to 315 mOsm (27 to 38%) than 215 (9%) and 365 (2%) mOsm, but the development to the blastocyst stage was difficult at any osmolarities (215 to 365 mOsm) tested. In the medium with a fixed osmolarity (315 mOsm) but with different concentrations (64 to 114 mM) of NaCl, there were no differences in the proportions (29 to 33%) of morula-stage embryos among different NaCl concentrations. However, significantly higher proportions of embryos developed to the blastocyst stage at 89 to 101 mM (22 to 23%) than 64 to 76 (0 to 9%) and 114 (11%) mM NaCl. When Cl- concentration in the medium with 64 mM NaCl was adjusted by adding choline chloride, significantly higher proportions of embryos developed to the morula stage at 97 to 122 mM (32 to 40%) than 72 (6%) and 147 (2%) mM Cl-, but few embryos developed to the blastocyst stage at any Cl- concentrations (72 to 147 mM) tested. In the medium with 64 or 114 mM NaCl and each with 2 different Na (+)K (+) ratios, there were no differences in the proportions of morula- and blastocyst-stage embryos between different Na+ K+ ratios (31 and 39 at 64 mM NaCl, and 39 and 47 at 114 mM NaCl) at each NaCl concentration. When glucose was added to the medium with 89 mM NaCl 120 h postinsemination, there were no significant differences in the proportions (40 to 48%) of morula-stage embryos 144 h post insemination among different concentrations (0 to 6.95 mM) of glucose. The proportion (33%) of blastocysts 192 h post insemination at 2.78 mM glucose was significantly higher than the values at 0 (22%), 5.56 (19%) and 6.95 (15%) mM but not different compared with the values at 1.39 (23%) and 4.17 (28%) mM. In conclusion, NaCl concentration in a defined medium is one of the most important factors for the development of bovine embryo to the blastocyst stage, but the development of embryos up to the morula stage is also regulated by osmolarity and/or Cl-concentration.     

Developmental competence of porcine oocytes selected by brilliant cresyl blue and matured individually in a chemically defined culture medium

The objective of this study was to investigate the effects of oocyte selection using brilliant cresyl blue (BCB) and culture density during individual in vitro maturation (IVM) on porcine oocyte maturity and subsequent embryo development using a chemically defined medium. Cumulus-oocyte complexes (COCs) were classified as BCB-positive or BCB-negative after exposure to a BCB solution for 90 min. The classified COCs were matured in a group (15 COCs per 100-μL droplet) or individually (1 COC per 1-, 2.5-, 5-, or 10-μL droplet). Meiotic competence, intraoocyte glutathione concentration, and developmental competence after intracytoplasmic sperm injection were monitored. The BCB selected oocytes competent for nuclear and cytoplasmic maturation. Furthermore, meiotic competence for oocytes matured individually in a 5-μL droplet was superior (P < 0.05) to that of oocytes matured in a 1-μL droplet. Also, the culture density in a 5-μL droplet during IVM resulted in a higher (P < 0.05) rate of cleaved embryos than that in a 1-μL droplet and produced a similar rate of blastocysts compared with that of a group culture system. Conversely, BCB selection did not improve cleavage and blastocyst formation. In conclusion, it was possible to predict porcine oocytes competent for maturation using oocyte selection with BCB. Moreover, a 5-μL droplet during the individual IVM culture was most suitable for oocyte maturation and subsequent embryo development, although every culture density used in this study supported development up to the blastocyst stage.     

Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

The purpose of the present study was to investigate the optimal concentration of osmolarity, calcium and bicarbonate for sperm penetration and formation of pronuclei (PN), and to investigate the time required for capacitation, penetration across the zona pellucida and formation of PN in bovine cumulus-free oocytes matured in vitro. Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro. Bovine sperm penetrated the zona pellucida in medium containing 240 to 440 mOsm, whereas PN formation was observed in a narrow range of osmolarities, from 280 to 360 mOsm. Maximal penetration by spermatozoa and PN formation was obtained in the medium with 2.5 mM calcium. High rates of spermatozoa penetration were observed in the medium with 37 to 49 mM NaHCO3. However, PN were formed regardless of the concentration of NaHCO3. The times required for sperm capacitation and penetration through the zona pellucida were 260 and 50 min, respectively. The first development of PN was recorded at 120 min after sperm penetration. Therefore, our study suggests that fertilization ability of spermatozoa in vitro appears to be more stable in high concentrations of NaCI. Oocytes are more sensitive to osmotic stress than spermatozoa. Calcium is required for both sperm penetration and PN formation in cumulus-free oocytes, but bicarbonate may be needed mainly for the penetration of spermatozoa.     

Sperm factors related to in vitro penetration of porcine oocytes

This study was designed to evaluate the relationship between sperm factors and penetration capacity in an in vitro system with immature porcine oocytes. The sperm parameters evaluated in 145 ejaculates were volume, sperm concentration, total cells in the ejaculate, ATP content, morpho-anomalies, percentage of motile sperm cells, forward progressive motility (FPM), acrosome status (NAR), hypo-osmotic swelling test (HOS), osmotic resistance test (ORT), eosin-nigrosin viability stain and sperm membrane integrity (DCF). Porcine oocytes (a total of 8,736) were used to evaluate the capacity of the different sperm assays to predict penetration. Many parameters were found to be related to in vitro penetration ability; all conventional semen parameters, except sperm concentration and eosinnigrosin staining, were significantly better in high (>75%) than in low penetration rates (<75%). When the ejaculates were preselected the number of significantly related parameters was lower. When studying all conventional semen parameters through a stepwise multiple linear regression analysis of seminal measurements, up to 72.3% of total variance of the penetration rate could be predicted. However, as many as 4 parameters were needed (FPM in fresh semen, folded tail, NAR in post-treatment semen and DCF) for accurate prediction. On the other hand, the multiple logistic regression needed 7 parameters to discriminate 83.96% of the cases correctly. In summary, the results from the present study showed that almost all studied parameters were significantly different for predicting penetration process attained or failed, but most of them were correlated together. These findings emphasize the complexity of sperm functions and the difficulty of assessing the fertilizing ability.     

Effects of caffeine and/or heparin in a chemically defined medium with or without glucose on in vitro penetration of bovine oocytes and their subsequent development

Bovine cumulus-enclosed oocytes were matured in culture for 22 to 24 h, freed from cumulus cells and inseminated with frozen-thawed spermatozoa in a chemically defined medium containing 1 mg polyvinylalcohol/ml with or without 5 mM caffeine and/or 10 micrograms heparin/ml and 13.9 mM glucose. Penetration of oocytes was observed only in the medium containing caffeine and/or heparin. Regardless of the presence of glucose, similar proportions of oocytes were penetrated in the medium containing heparin with (73 and 83%) or without (36 and 41%) caffeine. However, when the medium was supplemented with caffeine only, a higher penetration rate was observed in the presence (41%) than in the absence (27%) of glucose. When oocytes inseminated in medium containing caffeine and heparin with or without glucose were cultured in a chemically defined, protein-free medium, 72 and 90% and 9 and 21% of inseminated oocytes developed to the > or = 2-cell and blastocyst stages 48 and 192 h post insemination, respectively. These results, obtained using chemically defined conditions, indicate that glucose is required for stimulating fertilization in vitro of bovine oocytes and that synergistic action of caffeine and heparin appears independently of the reversing activity of caffeine on the inhibition of heparin-induced sperm capacitation by glucose.     

Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium

This study was performed to establish an individual bovine oocyte-IVP system using a chemically defined simple medium (mSOFaa containing 1 mg/ml polyvinyl alcohol: PVA) and to investigate the effects of epidermal growth factor (EGF) during oocyte maturation on in vitro maturation, fertilization and embryonic development. Cumulus-oocyte complexes were collected from bovine ovaries and were matured in mSOFaa containing PVA (control medium) supplemented with 0, 1, 10 or 50 ng/ml of EGF. Two further groups (TCM199 and mSOFaa, supplemented with 10% fetal calf serum were also included. In this study, mSOFaa containing PVA were used as a basic medium for fertilization and embryo development in vitro. Experiments were conducted in both group- and individual-IVP systems. In the group-IVP system, the proportion of matured oocytes (MII) in the control medium (62.7% +/- 5.0%) was significantly (p < 0.05) lower than in all other treatments, and in the individual-IVP system, the addition of 1 ng/ml EGF significantly (p < 0.05) increased the maturation rate (1 ng/ml EGF vs control: 76.2% +/- 5.4% vs 57.1% +/- 14.4%). The addition of EGF did not affect the proportions of penetrated and normally fertilized oocytes in either individual- or group-culture systems. In the group-IVP system, no significant difference among treatments was found in the rate of blastocyst formation, whereas in the individual-IVP system the control medium supplemented with 10 ng/ml EGF resulted in a significantly (p < 0.05) higher the rate of blastocyst formation (20.0 +/- 5.2%) than that in the control medium (6.2% +/- 3.5%). These results indicate that bovine oocytes can successfully develop to blastocysts in an individual-IVP system using a single chemically defined medium, and that the group-IVP system also resulted in a similar level of blastocyst formation to that in a standard multiple-media system in our laboratory. The effect of EGF during oocyte maturation medium differed depending on whether embryos were cultured individually or in groups.     

Fertilizability and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium

This study investigated effects of adding hypotaurine (HT), beta-merocaptoethanol (beta-ME), or both into a chemically defined maturation medium (TCM-199 containing 0.1% polyvinyl alcohol: PVA) on maturation, fertilization and development of individually (single) cultured bovine oocytes. Mean GSH concentration in the oocytes cultured in the medium supplemented with either beta-ME (1.11 +/- 0.05 nM) or HT plus beta-ME (0.97 +/- 0.03 nM) was significantly (P < 0.05) higher than that in the medium containing PVA alone (0.75 +/- 0.03 nM). Adding beta-ME showed a significantly (P < 0.05) higher rate of the second metaphase stage (93.6 +/- 3.3%) than in the medium containing PVA alone (single-control) (65.2 +/- 7.9%). Adding both HT and beta-ME showed significantly (P < 0.05) higher rates (92.6 +/- 2.7%) of normal fertilization than did adding HT alone (63.5 +/- 4.6%). Also, adding both HT and beta-ME significantly (P < 0.05) lowered the polyspermy rate than did adding HT alone. Adding either beta-ME or both HT and beta-ME showed no significant difference in cleavage. Blastocyst development did not improve significantly adding either HT, beta-ME or both, although beta-ME alone or HT plus beta-ME tended to result in a higher rate of blastocysts (6.4 and 6.8%, respectively) than resulted without additives (1.6%). Our results show that adding beta-ME to a chemically defined maturation medium increased the intracellular GSH level of bovine oocytes cultured individually, and can improve the maturation rate leading to the blastocyst stage throughout in vitro production.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

Effects of different protein supplements in fertilization medium on in vitro penetration of cumulus-intact and cumulus-free bovine oocytes matured in culture

Bovine oocytes matured in culture were inseminated with frozen-thawed spermatozoa in BO medium containing 5 mM-caffeine, 10 mug/ml of heparin and different protein supplements at various concentrations. When cumulus-enclosed oocytes were inseminated, no significant differences were observed in the penetration rates (89 to 100%) between media with and without protein supplements and among the different concentrations of each protein supplement, except for 20% calf serum (CS), in which the penetration rate decreased drastically (43%). Notably higher incidences of polyspermy were obtained in medium with FCS (75 to 86%) than with either no supplement (25%) or with BSA (20 to 24%) and CS (13 to 49%). On the other hand, there was almost no penetration of cumulus-free oocytes in the nonsupplemented control medium. Concentration-dependent increases in penetration and polyspermy occurred with BSA, FCS and CS supplementation. A high concentration (5%) of FCS yielded a high incidence (97%) of polyspermy. A decrease in the penetration of cumulus-enclosed oocytes was observed when spermatozoa were capacitated with a high concentration (20%) of CS; difficulty of sperm penetration of cumulus-free oocytes occurred when the capacitation medium lacked protein supplementation; and an increased rate of polyspermy was observed following supplementation with FCS in both cumulus-enclosed and cumulus-free oocytes after insemination with spermatozoa from 5 different bulls.     

In vitro development of theDrosophila chorion in a chemically defined organ culture medium

Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.     

Studies on zona hardening in rat oocytes that are matured in vitro in a serum-free medium

The present study confirms that zona pellucidae of rat oocytes became resistant to chymotrypsin digestion (zona hardening) after undergoing in vitro maturation (IVM) in a serum-free medium. However, zona hardening did not occur when empty zonae without oocytes were cultured in the same IVM conditions, suggesting that oocyte-derived factor(s) is responsible for zona hardening in oocytes matured in the serum-free medium. Zona hardening occurred primarily after dictyate oocytes were cultured for 6-8 hours in the IVM medium without serum. Zona hardening could be prevented or alleviated if oocytes were cultured in the IVM medium containing bovine foetal calf serum, a soybean trypsin inhibitor, or beta-mercaptoethanol, and in vitro fertilization rates for such oocytes were normal. Possible mechanisms of the phenomenon of zona hardening in oocytes matured in serum-free conditions are discussed in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy.     

Cultivation of Pasteurella haemolytica in a chemically defined medium

A chemically defined medium was developed for the aerobic cultivation of Pasteurella haemolytica. Studies on the growth of strain H44L were conducted in a medium consisting of 15 amino acids, inorganic salts, citrate, nicotinamide, pantothenate, thiamine or thiamine monophosphate, and carbon sources. The amino acids were provided as l isomers, because racemic mixtures of some amino acids inhibited growth. The carbon source consisted of a mixture of 1.0% d-galactose and 0.1% d-glucose. Culture populations of strain H44L reached 2 x 10(10) cells per milliliter after 16 hr of incubation at 37.5 C. Other strains of P. haemolytica, from a wide variety of sources, were tested for growth in the medium, and 23 of 24 strains grew well. Five strains of P. haemolytica var. ureae failed to grow in the medium.     

In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.     

The effect of endotoxin-contaminated medium on in vitro fertilization and development of bovine oocytes matured in vitro

The purpose of this study was to determine whether the presence of bacterial endotoxin in the bovine serum albumin (BSA) used to supplement media utilized for sperm preparation and co-culture of bovine sperm and oocytes affects in vitro penetration and embryonic development of oocytes matured in vitro. The chromogenic limulus amoebocyte lysate (LAL) test was used for quantification of the content of endotoxin. The proportion of penetrated ova was significantly greater (P greater than 0.0005) for the endotoxin-contaminated group (89%) versus the non-contaminated group (61%), but this was probably not due to endotoxin contamination. The presence of endotoxin resulted in a high rate of polyspermy (27% versus 4%, respectively; P greater than 0.0005), while the occurrence of parthenogenetic activation was the same for each group (8%). The proportion of total embryos put into culture that developed to the blastocyst stage by day 8 was similar (30% and 26%) for the contaminated and non-contaminated group, respectively. Fifty-three and 69%, respectively, hatched on day 10. These results suggest that endotoxin induces polyspermy, but has no adverse effect on embryonic development.     

Embryonic stem cell development in a chemically defined medium

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cell in vitro differentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported in Xenopus, due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possibly in vivo.