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At least 19 recordsLinked to original sources

PubMed2004

Effects of oxygen tension in the gas atmosphere during in vitro maturation, in vitro fertilization and in vitro culture on the efficiency of in vitro production of mouse embryos.

Effects of oxygen (O2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O2 and then subjected to IVF and IVC under 5 or 20% O2 tension. Lowering the O2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O2 is superior to 20% O2 for IVM and IVC, and suggest that 20% O2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes.

Animals↗

PubMed2005

The first dromedary (Camelus dromedarius) offspring obtained from in vitro matured, in vitro fertilized and in vitro cultured abattoir-derived oocytes.

Dromedary offspring have never been produced fully in vitro. We have previously demonstrated that embryos obtained by culture in semi-defined medium (mKSOMaa) have better in vitro development ability than those cultured with oviductal epithelial cells. The aim of the present experiment was to study the pregnancy rate after embryo transfer of in vitro-produced (IVP) dromedary embryos cultured in semi-defined modified medium (mKSOMaa). IVM/IVF procedures were conducted on six hundred and sixty four (664) cumulus oocytes complexes (COCs) aspirated from ovaries collected at a local slaughterhouse and cultured in vitro (38.5 degrees C; 5% CO2, and maximum humidity >95%). Maturation was completed by incubation in TCM-199 medium supplemented with 10% heat-treated Fetal Calf Serum (FCS), 10 ng/mL EGF, 1 microg/mL FSH, 1 microg/mL E2 and 500 microM cysteamine for 30 h. In vitro fertilization was performed using fresh semen (0.5 x 10(6) spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 38.5 degrees C, 5% CO2 and 90% N2 with maximum humidity (>95%). All IVC steps were done in seven replicates. The cleavage rate (two cells to blastocyst stage) was 64% (425/664) and the percentage of oocytes reaching the blastocyst stage was 23% (155/664). The hatching rate of blastocyst obtained after culture was 46% (71/155). Good quality hatched blastocysts (n = 66) were transferred individually to synchronized recipients. Pregnancy rates, determined by ultrasonography at 15, 60 and 90 days after embryo transfer (ET), were 38%, 32% and 27%, respectively. Out of 18 pregnant females 5 aborted between the fifth and seventh month of pregnancy and 13 females (20%) remained pregnant. After 385 days of pregnancy, the first healthy and normal male-dromedary offspring produced fully in vitro was born at a birth weight of 38 kg. More dromedary calves (n = 4) were born later on. The remaining pregnant females (n = 8) are due to calf within the next months. In conclusion, this is the first reported offspring in camelids obtained by transfer of embryos produced by IVM, IVF and IVC using abattoir-derived oocytes, fresh semen and culture in a semi-defined medium.

Animals↗

PubMed1996

Embryo viability, duration of gestation and birth weight in sheep after transfer of in vitro matured and in vitro fertilized zygotes cultured in vitro or in vivo.

The influence of various in vitro procedures on embryo survival and the production of normal offspring was investigated in sheep. Zygotes produced from in vitro matured (IVM) and fertilized (IVF) oocytes derived from slaughtered Merino ewes were allocated to three culture treatments for 6.5 days. Two groups were cultured in vitro in the absence or presence of oviduct epithelial cells while the third group was cultured in vivo in the oviducts of synchronized ewes. A fourth group of zygotes obtained from superovulated Merino ewes was also cultured in vivo and served as controls. After culture, IVM-IVF morulae and blastocysts, and control embryos were transferred to final recipient ewes. Pregnancy was diagnosed at day 50 of gestation by ultrasonography and pregnancies were allowed to go to term. The survival to term of IVM-IVF zygotes cultured in vitro was reduced compared with both in vivo cultured IVM-IVF zygotes and control zygotes (25-35% versus 51-60%, respectively, P < 0.05). Day 6.5 IVM-IVF morulae had a lower survival rate than did control morulae regardless of culture treatment (P < 0.05), while survival rates of day 6.5 IVM-IVF blastocysts cultured in vivo did not differ from those of control blastocysts (P > 0.1). Both the gestation period and birth weight of IVM-IVF lambs were increased when compared with controls, the former significantly in all groups (154.0-154.9 days versus 150.6 days; P < 0.01), while the latter increase was on the borderline of significance (4.5-4.8 kg versus 4.0 kg; 0.01 < or = P < or = 0.1, respectively) and dependent on the prolongation of the gestation period. It is concluded that in vitro maturation and fertilization compromise subsequent embryonic and fetal development in sheep irrespective of the subsequent in vivo or in vitro culture treatment. Subjecting IVM-IVF zygotes to in vivo culture for 6.5 days minimizes only some of these effects, thus leading to the aberrant production of some offspring.

Animals↗

PubMed1998

Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM Working Group on the in vitro micronucleus test. Gesellschaft für Umwelt-Mutations-forschung.

In order to license a pharmaceutical or chemical, a compound has to be tested for several genotoxicity endpoints, including the induction of chromosomal aberrations in vitro. A working group within the GUM has evaluated published data on the in vitro micronucleus test with the aim of judging its suitability as a replacement for the in vitro chromosomal aberration test. After strict rejection criteria were applied, a database including 96 publications and 34 compounds was obtained. For 30 of these compounds, data on both tests were available. For 24 of the 30, concordant results in both test systems were obtained (80% correlation). The discordant results in 6 compounds can be explained by a known or suspected aneugenic potential of these compounds. Considering that cell types and test protocols were extremely heterogeneous, this correlation is rather encouraging. Comparison of the different protocols, and experience established within the working group yielded several recommendations for the routine use of the in vitro micronucleus test. Although many cell lines are suitable, those most often used in genotoxicity testing (e.g. CHL, CHO, V79, human lymphocytes, L5178Y mouse lymphoma cells) are recommended. Cytochalasin B may be used in the case of human lymphocytes; however, the possibility of its interaction with aneugenic test compounds should be considered. For continuously dividing cell lines, cytochalasin B is not recommended by the working group. Although, there seems to be flexibility in the choice of treatment and sampling times, the average generation time of the chosen cell line of choice should be taken into account when determining sampling time, and treatment of cells for at least one cell cycle duration is recommended. The use of appropriate cytotoxicity tests is strongly recommended. Although studies on some parameters of the test protocol may be useful, the introduction of the in vitro micronucleus test into genotoxicity testing and guidelines should not be delayed. Even in its present state, the in vitro micronucleus is a reliable genotoxicity test. Compared with the chromosomal aberration test, it detects aneugens more reliably, it is faster and easier to perform, and it has more statistical power and the possibility of automation.

Animals↗

PubMed2005

In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes.

The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P < 0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P < 0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.

Animals↗

PubMed2002

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes.

In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4-12h on COEM or for 4h in TALP medium supplemented with 20 microg of heparin.COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm (P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4h and no motile sperm were observed by 8h. Overall, the penetration rates of in vitro red deer oocytes were low (5-28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin (P<0.001). Penetration rates were similar for 4-12h pre-incubation of sperm on COEM (P>0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24h compared to 12h (P<0.001). There were no differences in penetration rates among the four donor stags used in the study. It was concluded that COEM sustains red deer sperm motility in vitro during the 24h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.

Animals↗

PubMed1993

Effect of glucose levels during the in vitro culture in synthetic oviduct fluid medium on in vitro development of bovine oocytes matured and fertilized in vitro.

The present study was conducted to determine the optimal glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM-199 + 10% FCS + hormones and granulosa cells were fertilized in vitro in a TALP medium with frozen-thawed, swim-up separated, and heparin-treated spermatozoa. After insemination, 1199 oocytes were cultured for 3 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% human serum (HS) and with 10 different glucose levels (0 to 5 mM), and further cultured for 5 days in SOFM + 10% HS containing 1.5 mM glucose (Experiment 1). In Experiment 2, 739 oocytes were cultured for 3 days following insemination in either SOFM + human serum albumin or SOFM + 10% HS containing 0.188 mM glucose. From Days 4 to 8, the oocytes were cultured in SOFM containing 4 different glucose levels. A high level of glucose (3.0 and 5.0 mM) at Days 0 to 3 significantly reduced the rate of blastocyst development (3.0 to 4.2%), and a yet higher (5.0 mM) glucose level at Days 4 to 8 also significantly lowered the rate of blastocyst development as compared with 1.5 mM glucose (19.5% vs 29.3%). The present results indicate that a lower level (0.188 mM: 28.8% in blastocyst development) of glucose is preferable in SOFM for the in vitro development to blastocysts at Days 0 to 3 after insemination. At Days 4 to 8, the original level (1.5 mM) of glucose contained in SOFM appears to be the most effective treatment.

Journal Article↗

PubMed1994

In vitro development of in vitro-matured porcine oocytes following chemical activation or in vitro fertilization.

Porcine cumulus-oocyte complexes were cultured in BSA-free Whitten's medium or modified Medium 199, each supplemented with porcine follicular fluid (PFF) and hormonal supplements (OMWM and OMM199, respectively) for 20 h; they then were cultured without hormonal supplements for an additional 20 (experiments 1 and 3) or 24 h (experiment 2). At the end of culture (experiment 1), the intracellular glutathione concentration was higher (p < 0.05) in oocytes matured in OMWM vs. OMM199. After activation by Ca2+ ionophore (experiment 2), the incidence of activation in the OMWM group was lower (p < 0.01) than in the OMM199 group. However, the incidence of pronuclear formation was higher (p < 0.01) in the OMWM group than in the OMM199 group at 8 h after activation. The percentage of embryos that developed to the morula stage was higher (p < 0.01) in the group matured in OMWM vs. OMM199 after 5 days of culture. After in vitro fertilization (experiment 3), the incidence of male pronuclear formation and the percentage of monospermic oocytes that formed one male and one female pronuclei were higher (p < 0.05) after maturation in OMWM vs. OMM199. The percentage of cleaved embryos that developed to the 8-cell and morula stages was higher (p < 0.05) in the OMWM group as compared to the OMM199 group. These results indicate that culture in modified Whitten's medium as compared with a standard medium (modified Medium 199) improves cytoplasmic maturation of porcine oocytes as evaluated by intracellular glutathione content, pronuclear formation, and development in vitro after artificial activation or fertilization in vitro.

Animals↗

PubMed1997

In vitro and in vivo development of embryos produced by in vitro maturation and in vitro fertilization of cat oocytes.

Cumulus-oocyte complexes of domestic cats were classified by morphology of ooplasm (A = good, B = fair, C = poor) and cultured for 24 h in TCM 199 with gonadotrophins (eCG, FSH, hCG or FSH/hCG). More of type A oocytes (52%) underwent in vitro maturation (IVM) than of type B (41%) or type C (17%). The gonadotrophin source did not affect frequency of IVM of type A (50-53%) or type B (38-44%) oocytes, but IVM of type C oocytes in hCG or FSH/hCG (27%/19%) was about double that in eCG or FSH alone (13%/10%). After IVF, frequency of cleavage for type A (54%), B (41%) and C (26%) oocytes was similar to the IVM frequency of the equivalent type. After 7 days, development to the morula (M) stage in vitro was similar among types (47-58%); however, higher percentages of type A and B oocytes developed to blastocysts (Bl), 31% and 29%, respectively, than of type C (15%). After transfer of day 5 (n = 70) or 6 (n = 32) M and Bl to day 4 or 5 recipients in trial 1 (n = 4) and 2 (n = 3), respectively, the three recipients in trial 2 gave birth to four live kittens. Development in vitro to M of IVM/IVF embryos frozen in propanediol plus sucrose during early cleavage was similar (64-69%) to that of cohort controls (64%), but Bl formation was reduced (13-17% versus 32%). Damage to the zona pellucida after plunging into liquid nitrogen at -30 degrees C was higher (11%) than that of the embryos cooled at 10 degrees C min-1 from -30 degrees C to -150 degrees C before storage (2%).

Animals↗

PubMed1990

Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos.

To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages.

Journal Article↗

PubMed2006

Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization.

OBJECTIVE: To determine an optimum condition for vitrification of various animal species and to examine the developing capacity into pups of mouse oocytes in preantral follicles originating from cryopreserved ovaries. DESIGN: Experimental animal study. SETTING: Laboratory environment. ANIMAL(S): Normal (C57BL/6 x DBA2) F1 mice. INTERVENTION(S): Vitrification of animal ovaries using polyester sheets as a storage device; collection of oocyte-granulosa cell complexes by enzymatic treatment; and in vitro growth (IVG), in vitro maturation (IVM), and IVF, with embryo transplantation to pseudopregnant mice. MAIN OUTCOME MEASURE(S): Histological analysis of vitrified and warmed ovaries from several animal species; measurement of successful rates in IVG, IVM, and IVF of mouse oocytes in oocyte-granulosa cell complexes collected from the vitrified and warmed ovaries; and achievement of pup birth. RESULT(S): The vitrification method used was effective for storage of various animal ovaries. The oocytes enclosed in preantral follicles that were yielded from the vitrified-warmed ovaries preserved the capacity for developing into pups after IVG, IVM, and IVF. CONCLUSION(S): The findings indicated that ovarian cryopreservation by vitrification and subsequent IVG, IVM, and IVF are promising methods for restoring young cancer patients' fertility. The practical information presented here is applicable to human ovarian tissues.

Animals↗

PubMed2005

Peroxidases, lignin and anatomy during in vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots.

In vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots with or without indolic-3-butyric acid (IBA) was studied in order to improve acclimatization of microplants after root formation and transplantation. Peroxidase (POD) activity and isoforms, lignin content and anatomical observations were evaluated in the course of the three interdependent phases (induction, initiation and expression) of microshoot rooting. Microshoots treated or not treated with IBA achieved high rooting percentages both in vitro and ex vitro. At the end of the 2-week acclimatization period, the percentage of surviving microplants ranged from 80% to 100%, for in vitro and ex vitro rooted microshoots, respectively. Microshoots rooted in vitro and ex vitro showed a relationship between rooting and POD activity but in a different time course. It appeared that root formation occurred after the microshoots had reached and passed a peak of maximum enzyme activity. In all treatments, electrophoretic analysis (native PAGE) of PODs revealed the appearance of one anionic and three cationic POD isoforms (C(1), C(3) and C(4)). An additional cationic POD isoform (C(2)) appeared only in the ex vitro rooting. The lignin content was similar in microshoots rooted both in vitro and ex vitro. The sequential anatomical changes during the rooting process were similar in both in vitro and ex vitro rooting treatments. In the case of in vitro rooting, pith cells had vacuoles entirely filled with a dark substance, while in the case of ex vitro rooting, pith cells contained many amyloplasts. The origin of the adventitious roots, in both rooting conditions, was located in the cambial ring. Roots with organized tissue systems emerged from the microshoot stem 10-14 days after the root induction treatments; on day 10 for rooting in vitro, while a 4-day delay was noted in microshoots rooted ex vitro.

Acclimatization↗

PubMed1985

Testing of 24 food, drug, cosmetic, and fabric dyes in the in vitro and the in vivo/in vitro rat hepatocyte primary culture/DNA repair assays.

Twenty-four dyes currently or previously used in the food, drug, cosmetic, and textile industries were tested in the in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assay and, to a limited extent, in the in vivo/in vitro HPC/DR assay. The positive control, Solvent Yellow 3 (o-aminoazotoluene), and five other dyes (4-dimethylaminobenzeneazo-1-naphthalene, 4-dimethylaminobenzeneazo-2-naphthalene, Direct Blue 53, Acid Blue 9, and 4-dimethylaminostilbene) induced DNA repair in rat hepatocytes both in vitro and in vivo, while 13 of the dyes (Food Red 1, Food Red 5, Food Orange 4, Food Red 7, Acid Red 14, Acid Red 27, Pigment Red 53, Acid Yellow 23, Food Black 1, Food Green 3, Acid Red 51, Acid Blue 74, and Natural Red 4) did not produce any detectable DNA repair in either the in vitro or in vivo/in vitro assays. Direct Blue 14 had weak activity in vitro but none was detected in vivo. In contrast, Solvent Yellow 5 was not active in vitro, but produced a weak positive response in vivo. Negative responses were also obtained for Solvent Yellow 14 and Acid Green 5 in the in vitro assay, whereas the responses produced by these dyes in the in vivo/in vitro assay were judged to be equivocal. An equivocal response was also obtained for Direct Red 28 in the in vivo/in vitro assay as well as in the in vitro assay. These findings provide information about the potential genotoxicity of a number of dyes for which previous genotoxicity data has been inconsistent or inadequate. For some dyes (eg, Solvent Yellow 5), discrepancies between the results obtained in the in vitro and in vivo/in vitro assays may implicate a role for intestinal microflora in their metabolic activation.

Animals↗

PubMed1994

Pregnancy and birth rates of live infants after in vitro fertilization in women with an without previous in vitro fertilization pregnancies: a study of eight thousand cycles at one center.

OBJECTIVE: Our aim was to compare the cumulative conception and live-birth rates after in vitro fertilization in women undergoing their first course of in vitro fertilization treatment with those in women undergoing their second course of treatment, having previously achieved an in vitro fertilization pregnancy. This study occurred in a tertiary referral-assisted conception unit. STUDY DESIGN: The cumulative conception rates obtained by life-table analysis in 4115 women having their first course of in vitro fertilization therapy (7327 treatment cycles leading to 1123 pregnancies) were compared by means of the log-rank test with those of 331 women in their second course of treatment, having previously achieved an in vitro fertilization pregnancy (561 treatment cycles leading to 138 second in vitro fertilization pregnancies). Similarly, the cumulative live birth rates of 3824 women in their first course of treatment (7136 treatment cycles leading to 732 live births) were compared with those of 105 women in their second course of treatment, having previously achieved an in vitro fertilization live birth (205 treatment cycles leading to 33 second in vitro fertilization live births). RESULTS: The cumulative conception rates and cumulative live birth rates were significantly higher in women having their second course of in vitro fertilization treatment than in those having their first course (cumulative conception rate: p = 0.0001; cumulative live birth rate, p = 0.007). After five cycles of in vitro fertilization, the cumulative conception rates and cumulative live birth rates were 49.8% (95% confidence interval, 46.3% to 53.5%) and 39.0% (95% confidence interval, 35.4% to 42.9%), respectively, in those having their first course of treatment compared with 69.9% (95% confidence interval, 57.6% to 81.3%) and 68.6% (95% confidence interval, 46.1% to 88.5%), respectively, in those having their second course. The estimated median numbers of cycles taken to achieve a pregnancy and live birth (assuming all women could potentially undergo the same number of cycles) were six and eight, respectively, in the first course of treatment compared with only three and five in the second course. CONCLUSION: Women who have achieved a previous in vitro fertilization pregnancy have significantly higher cumulative conception rate and cumulative live birth rates compared with those of women having their first course of treatment.

Adult↗

PubMed1994

Differences in pronucleus formation and first cleavage following in vitro fertilization between pig oocytes matured in vivo and in vitro.

To elucidate the developmental differences occurring after in vitro fertilization (IVF) of pig oocytes matured either in vitro (n = 1934) or in vivo (n = 1128), the present experiment investigated the morphological changes from penetration to the two-cell stage. Oocytes were examined every 2-4 h from 2 to 32 h after in vitro insemination to study sperm penetration, male and female pronucleus formation, synkaryosis and first cleavage. The penetration rate was significantly higher (P < 0.05) for in vivo matured oocytes (69.8%) than for in vitro matured oocytes (35.0%). Penetration of spermatozoa into the ooplasm was first recorded 6 h (in vitro matured oocytes) and 4 h (in vivo matured oocytes) after addition of the spermatozoa to the oocytes. For both in vivo and in vitro matured oocytes, 2 h were required for sperm head decondensation. However, maximum sperm head decondensation occurred 2 h later in in vitro matured oocytes. Within 6 h, 41.7 +/- 5.6% of the in vivo matured oocytes had completed second meiotic division, whereas only 20.8 +/- 6.5% of the in vitro matured oocytes reached this developmental stage (P < 0.01). For in vitro matured oocytes, male pronucleus formation was retarded 2-4 h after onset of insemination and development of the female pronucleus was enhanced compared with in vivo matured oocytes. Synchronized opposing pronuclei were observed 14 h after insemination in in vitro matured oocytes and after 8 h in in vivo matured oocytes. Synkaryosis was first observed at 16 and 18 h in in vivo and in vitro matured oocytes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

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.

Journal Article↗

PubMed1987

Effect of in vitro irradiation and cell cycle-inhibitory drugs on the spontaneous human IgE synthesis in vitro.

The in vitro effects of radiation, diterpine forskolin (FK), and hydrocortisone (HC) on the in vitro spontaneous IgE synthesis by peripheral blood B-lymphocytes from atopic patients were investigated. Without affecting cell viability, in vitro irradiation inhibited in a dose-dependent fashion de novo IgE synthesis in vitro by B cells from all patients examined with a mean 40% reduction of in vitro IgE product after treatment with 100 rads. In contrast, the in vitro IgE production by the U266 myeloma cell line was unaffected, even by irradiation with 1600 rads. The addition to B cell cultures from atopic patients of FK consistently resulted in a dose-dependent inhibition of the spontaneous IgE production in vitro. The addition to cultures of 10(-5) and 10(-6) molar concentrations of HC was also usually inhibitory, whereas lower HC concentrations were uneffective or even enhanced the spontaneous in vitro IgE synthesis. When 10(-6) molar concentrations of both HC and FK were combined in culture, a summation inhibitory effect on the spontaneous IgE synthesis was observed. In contrast, neither FK nor HC had inhibitory effect on the in vitro spontaneous IgE synthesis by the U266 myeloma cell line. The spontaneous in vitro IgE synthesis by B cells from patients with Hodgkin's disease, demonstrating high levels of serum IgE, was strongly reduced or virtually abolished after patients underwent total nodal irradiation to prevent the spread of the disease. In addition, the in vitro spontaneous IgE synthesis by B cells from atopic patients was markedly decreased or abolished by in vivo administration of betamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1998

Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization.

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo-matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro-matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro-matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro-matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro-matured oocytes were dissolved within 131.7 +/- 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro-matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for beta-D-Gal(1-3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro-matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro-matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro-matured and in vivo-matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional black to polyspermy in pig oocytes.

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