Transfer of cultured rabbit embryos

Embryo transfer experiments were carried out to study the developmental capacity of cultured rabbit embryos when transferred to recipients of variable postovulatory maturity. Rabbit embryos were flushed from the oviduct at 26 hours postcoitum (pc) and cultured in a modified Ham's F-10 medium supplemented with bovine serum albumin (BSA) for a period of 70 hours. At 96 hours pc the cultured embryos, which ranged from the early morula to the expanding blastocyst stage, were transferred to pseudopregnant recipients mated to vasectomized males 36 to 96 hours prior to the transfer procedure. Greatest embryo survival occurred when transfers were made to either the oviducts or uterine horns of recipients at 48 hours pc. Intermediate results for both implantation rates and number of young born were obtained with recipients at 36, 60, 72, and 84 hours pc. Transferred embryos consistently failed to survive the uterine environment of recipients 96 hours pc at transfer although this group was synchronous with embryonic chronological age. Oviductal transfers were generally more successful than uterine transfers. Markedly higher rates of embryo survival resulted from embryos that were collected 60 and 72 hours pc and transferred directly to synchronous recipients without an interim period of culture. Dissimilarity of development for in vivo grown rabbit embryos and those cultured in synthetic medium is demonstrated.     

Development of preimplantation rabbit embryos in uterine flushing-supplemented culture media

The development of cultured rabbit preimplantation embryos grown in standard media (Ham's F-10 or BSM II supplemented with bovine serum albumin (BSA) or homologous serum) or in Ham's medium supplemented with uterine flushings was compared. The uterine flushings derived from donors of 0.5-6 years of age. Uterine flushing supplemented media were used natively or after treatments like sterilization by filtration, lyophilization, three times freezing/thawing, heat denaturation, dialysis, or ultrafiltration. Compared with in vivo controls, embryonic growth was substantially reduced during in vitro culture, demonstrably by smaller diameters and impaired cell proliferation (measured by thymidine incorporation). The growth retardation was more pronounced in blastocysts (recovered at day 4 post coitum [p.c.]) than in morulae (recovered at day 3 p.c.). Development in uterine flushing media was notably better than in standard media but did not comply with in vivo development. Highest thymidine incorporation was observed in media with increased concentrations of uterine secretions and after sequential supplementation of flushings from subsequent progestational stages. Advanced donor ages, heating up to 80 degrees C, freezing, and lyophilizing did not affect incorporation data statistically significantly, whereas sterilization by filtration, ultrafiltration, and dialysis led to a significantly reduced thymidine incorporation in the cultured embryos. The positive effects of uterine flushing supplementation are attributed to the supply of components more adjusted to the needs of the cultured embryos and/or to a reduction of pathological effects in vitro like washing out of nutritive and regulatory components from the embryo into the surrounding culture medium.     

DNA ploidy abnormalities in rabbit preimplantation embryos are not increased by conditions associated with in vitro culture

Possible adverse effects of in vitro culture-associated physical factors were studied in 3- and 4-day-old rabbit embryos. Laboratory conditions were mimicked by exposure to visible light (320–740 nm, 1600 lx) or decreased temperature (22 ± 1°C). Embryos were exposed for a 24-hr period followed by either immediate evaluation or an additional 24 hr of standard in vitro culture (darkness, 37°C) and evaluation thereafter. Effects were assayed by cytophotometric measurement of the DNA content in Feulgen-stained cell nuclei and by cell number. The incidence of DNA aneuploid embryos and DNA aneuploid cell nuclei per embryo, as well as the average nuclear DNA content, was not significantly different between exposed embryos and controls. Both in vitro culture and reduced temperature caused a decrease in cell number. The temperature-induced cell number decrease was reversible within 24 hr after return to 37°C. These results demonstrate that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in cultured preimplantation embryos. Mol. Reprod. Dev. 50:30–34, 1998. © 1998 Wiley-Liss, Inc.     

Recombinant human activin A stimulates development of bovine one-cell embryos matured and fertilized in vitro

The effects of recombinant human activin A on the development of bovine one-cell embryos matured and fertilized in vitro were investigated. In experiment 1, one-cell embryos were cultured in a chemically-defined medium, of modified synthetic oviduct fluid supplemented with 1 mg/ml polyvinyl alcohol (mSOF-PVA), containing different concentrations of activin (0, 0.1, 1, 10, and 100 ng/ml) until 240 hr after in vitro fertilization. The addition of -1 ng/m activin to mSOF-PVA improved development to the blastocyst stage (14.5–17.1%), compared with no addition of activin (5.6%). However, there was no significant difference in hatching rate of embryos among treatments. In experiments 2 and 3, the embryos were also cultured in MSOF-PVA at various periods of exposure to 10 ng/ml activin to evaluate (development to the morula and blastocyst stages, respectively. The proportion of morulae was significantly higher in culture with activin at 20–120 hr postinsemination (37.2%) than with control (25.7%). Total number of cells in morulae at 120 hr postinsemination significantly increased by the addition of activin at 20–72 hr (26.1 cells) and 20–120 hr (24.2 cells) postinsemination, compared with control (20.1 cells). When activin was added to the medium during 20–120 hr and 20–192 hr postinsemination, the percentages of blastocysts (18.0% and 18.7%, respectively) were significantly higher than in the control (9.6%). However, the total number of cells in blastocysts was not significantly different. These results demonstrate that activin stimulates the development of bovine one-cell embryos to the morula and blastocyst stages in vitro. © 1996 Wiley-Liss, Inc.     

Differential development of rabbit embryos derived from parthenogenesis and nuclear transfer

Parthenogenetic development (PA) is often used as a model to investigate activation protocols for nuclear transfer (NT) embryos. The objective of this study was to compare the development, as well as the dynamics of the nuclear materials and microtubules of PA and NT embryos following similar activation treatment. Our results demonstrate that, during parthenogenesis, activation through either electrical pulses or chemical stimulation alone resulted in low cleavage rates and compromised development. A combination of two sets of electrical pulses and a 2-h-exposure to chemical activation medium (5 microg/ml cycloheximide (CHX) and 2 mM 6-dimethylaminopurine (6-DMAP) in KSOM+0.1% BSA) could effectively activate rabbit oocytes, and resulted in a 99% (n = 73) cleavage rate with greater than 60% (n = 73) developing to blastocysts at day 4. However, the same activation protocol following NT resulted in only 65-72% of oocytes cleaved (depending on donor cell type), with less than 20% developing to the blastocyst stage. The differences observed between NT and PA embryos subjected to the same activation protocol were also evident in terms of the time required for their development to the blastocyst stage, as well as the cell numbers present in blastocysts at day 6. Furthermore, laser confocal microscopy revealed that pronuclear formation in the NT embryos was delayed by comparison to that in the parthenotes. In conclusion, our study suggests that an effective protocol for parthenogenesis cannot promise a comparable outcome for NT embryos.     

Sequential analysis of zona thickness during in vitro culture of human zygotes: Correlation with embryo quality,age, and implantation

Zona pellucida thickness was measured daily in zygotes and cleavage stage embryos. Measurements were performed on a Nikon inverted microscope equipped with Hoffman modulation optics, using an ocular micrometer. Zona thickness of each zygote/embryo was measured four times, the zygote/embryo was then “rolled over,” and four more measurements were repeated for a total of eight. The zygotes/embryos were photographed daily and the measurements repeated on the prints. Subsequently, the mean zona thickness for each stage was calculated. A total of 81 patients (mean age 33.8 ± 4.2) participated in the study. A total of 427 embryos were evaluated. Categorical data differences between groups were evaluated by ANOVA and multiple linear regression. For nominal data, the Kruskal-Wallis test was applied; when P < 0.05 the differences were considered to be significant. We found that the average zona thickness on day 1 of in vitro culture was 17.7 ± 0.14 μm; 16.3 ± 0.14 μm on day 2 and 14.9 ± 0.14 μm on day 3 (P < .0001). When the zona thickness was analyzed in relation to the number of blastomeres on day 3 of culture, there was a highly significant correlation with blastomere number (P < .0001). Similarly, there was a highly significant correlation with embryo grade (P < .005) and fragmentation (P < .001). The data were also analyzed for embryos transferred that resulted in a successful pregnancy, revealing that embryos in a pregnancy cycle had significantly thinner zonae pellucidae (P < .0001), when compared to embryos that were not transferred or from nonconceptual cycles. The average zona thickness also decreased with age, and was most apparent after 35 years. Changes in zona thickness correlated with the number of blastomeres, grade, fragmentation, age and were more evident in embryos transferred from cycles resulting in successful pregnancies. Therefore, zona pellucida measurements should be included in the overall assessment of embryo quality, since this information may be useful in the selection of optimal embryos for transfer. Mol. Reprod. Dev. 47:99–104, 1997. © 1997 Wiley-Liss, Inc.     

Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UFs) on day 3 (D3) and day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 nonprotein amino acids (beta-alanine, taurine, ornithine, and citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06 +/- 3.63 mmol/L), D3UF (10.54 +/- 5.16 mmol/L), or D5UF (10.23 +/- 6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89 +/- 1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected and from which day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3 +/- 7.9%) or D5UF (PZM3-D5UF, 14.3 +/- 10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5 +/- 15.1), 72 (25.6 +/- 10.4), and 96 (18.7 +/- 10.0) hr and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8 +/- 9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6 +/- 8.3) was increased compared to PZM3-SAA (40.5 +/- 3.5). The osmolalities in D3OF, D3UF, D5OF, and D5UF were 318 +/- 8, 320 +/- 32, 321, and 293 +/- 8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360 mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hr and then transferred to 250 mOsM (33.3 +/- 3.4%), the blastocyst rate was higher than original PZM3 (21.2 +/- 2.2%) (288 mOsM).     

Two-compartment model for rabbit skin organ culture

Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d. The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid.     

A preliminary report on the establishment of pregnancies in an in vitro fertilization (IVF) programme at the University of British Columbia (UBC)

Between July 1982 and November 1983, two pregnancies were established using in vitro fertilization and embryo transfer (IVF and ET) procedures with three different schedules to induce follicular maturation. All women were cycling normally and had inoperable or absent fallopian tubes. Of 83 oocytes aspirated from 24 patients (31 cycles), 75% were considered mature and 25% immature by the morphological characteristics of the oocytes and cumulus cells. Oocytes were preincubated for 6–24 hours, and after insemination, 60% cleaved to the two-to-four-cell stage. The superovulation induction schedule employing hMG administered according to the individually adjusted treatment scheme established two pregnancies. This schedule was considered the superior regimen, as it gave the highest proportion of mature oocytes (89%) which cleaved (78%). The pregnancy-attaining follicle showed a high progesterone:estradiol-17β ratio (P₄/E₂) in its microenvironment of aspirated follicular fluid, culture media of granulosa cells, and oocyte-cumulus complex. Our observations indicate a high P₄/E₂ ratio in the pregnancy-attaining follicle, and thereby reflect a further parameter in influencing maturation of the oocytes most likely to implant.     

Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosa cells

Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture. Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin.     

冻融后小鼠休眠胚胎超微结构的变化

目的从亚细胞超微结构的角度揭示其抗冻能力优于正常孵化胚胎的原因。方法利用透射电子显微镜观察小鼠休眠胚胎与正常孵化期胚胎在细胞连接和各细胞器形态与分布上的差异,以及冻融培养后的变化,并进行相关比较分析。结果通过亚细胞结构对比分析发现:冷冻前小鼠休眠胚胎为紧缩状,处于能量代谢较低的"基态",通过冻融后培养,细胞器结构恢复与正常孵化胚胎冷冻前相似;而正常孵化胚胎经过冻融后,线粒体数量减少,细胞核松散,异染色质增多。结论小鼠休眠胚胎与正常孵化胚胎冻融后相比,其细胞状态更有利于物质储存及能量代谢,表明小鼠休眠胚胎从亚细胞超微结构的角度比其正常孵化胚胎更具抗冻性。     

The characterization of bovine embryos obtained from prepubertal calf oocytes and their viability after non surgical embryo transfer

We have previously shown that the addition of epidermal growth factor (EGF) during in vitro maturation was capable of stimulating the cytoplasmic maturation of cow and calf oocytes. The aim of the present study was to compare calf and cow blastocysts produced in the presence of EGF in terms of total cell number and cell distribution between trophectoderm (TE) and inner cell mass (ICM), pattern of protein synthesis, and ability to establish pregnancy after embryo transfer to recipients. For all experiment, embryos at Day 7 were obtained from IVM/IVF/IVC oocytes. No significant differences were noted in total cell number (cow= 138±46 vs CALF= 142±59; mean±SD) or ICM and TE cell number between calf (ICM= 35 ± 19, TE= 107± 52) and cow (ICM= 38± 21, TE= 99 ± 32) blastocysts, nor in the ICM/total cell number ratio (cow= 0.27± 11, CALF= 0.25 ± 12). No differences were noted in the constitutive and the neosynthetic protein profiles between cow and calf embryos obtained in vitro. The results of embryo transfer, showed that there was higher pregnancy loss following transfer of calf compared with cow embryos. After Day 35, the rate of pregnancy decreases, with only 22% of calf embryos maintaining pregnancy until calving compared with 39% for cow embryos. In conclusion, it would seem that embryos originating from calf oocytes are less capable of establishing pregnancies than embryos obtained from adult oocytes, althrough this difference was not significant. This low viability cannot be explained by differences in cell number or by the protein profiles identifed between these 2 groups of embryos.     

A review on disease transmission studies in relationship to production of embryos by in vitro fertilization and to related new reproductive technologies

This paper addresses the circumstances of germplasm contamination and updates on transmission of pathogenic agents by embryos produced in vitro and by associated techniques. It has been shown that some pathogenic agents might have been associated with the follicular oocytes and oviductal cells, collected for in vitro fertilization (IVF), resulting in infected embryos. Experimental introduction of pathogenic agent with oocytes or infected semen into the IVF system allows, in most cases, for the fertilization of eggs and for the production of some transferable quality embryos. Rendering of oocytes and embryos free of infectious pathogens, using the standard sequential washing or enzymatic treatment, is inconsistent and more difficult in the presently used in vitro fertilization system as compared to in vivo produced embryos.