B2 Medium Research Articles

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.

Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates.

We investigated whether further in-vitro culture of human multicellular embryos that survive cryopreservation can select the viable embryos for transfer. Embryos for cryopreservation were supernumerary multicellular embryos obtained after in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments, with <20% of their volume filled with anucleate fragments. These had been cryopreserved using a slow-freezing and slow-thawing protocol with 1.5 M dimethylsulphoxide as the cryoprotectant. From the start of our cryopreservation programme until September 12, 1994, the thawing strategy was to thaw frozen embryos up to the exact number needed for transfer. Embryos for transfer were selected on the basis of their morphological appearance and embryo transfer to the patient was done on the day of thawing. From September 12, 1994 onwards we used a more selective thawing strategy where a cohort of up to a maximum of 12 frozen embryos per patient is thawed from which embryos of the best morphological quality, and which are furthest advanced in terms of cleavage after a 24 h in-vitro culture period in Menezo B2 medium, are selected. We took delivery rates, embryo implantation rates and birth rates into account to see if there is any difference between the following three types of transfers used: 187 transfers exclusively of embryos having continued to cleave after thawing, 107 mixed transfers of embryos with and without further cleavage and 53 transfers exclusively of embryos with no further cleavage. The overall outcome in terms of delivery rate and embryo implantation and birth rates were not different between the new and the earlier thawing policies (6.6, 5.2 and 3.6% versus 6.0, 4.1 and 2.7% respectively). Only when a distinction was made between transfers on the basis of the presence of embryos with further cleavage, did the advantage of selection on the basis of cleavage capacity become evident. Significantly higher delivery and embryo implantation and birth rates (11.2, 7.7 and 6.5% respectively) were recorded with transfers exclusively of embryos with further cleavage versus mixed transfers of embryos with and without further cleavage (1.9, 2.9 and 0.6% respectively). Fifty-three transfers exclusively of embryos with no further cleavage did not lead to any delivery. Our results demonstrate that selection of human multicellular embryos which survive cryopreservation and continue to cleave in vitro can significantly improve the delivery rate per transfer and the implantation rate per transferred embryo.

Successful in vitro maturation of human oocytes not exposed to human chorionic gonadotropin during ovulation induction, resulting in pregnancy

To report a case of successful in vitro maturation of human oocytes not exposed to hCG during ovulation induction, resulting in pregnancy after transfer of a frozen-thawed embryo resulting from intracytoplasmic sperm injection (ICSI) of the in vitro-matured human oocytes. Clinical study. Hospital-based private IVF center. A couple with infertility due to severe oligospermia. Five immature oocytes were retrieved from a patient who failed to use hCG during ovarian stimulation and were cultured for 48 hours in B2 medium containing FSH and hCG. Five oocytes extruded the first polar body and underwent ICSI with frozen-thawed husband spermatozoa. In vitro maturation, fertilization after ICSI, embryo development, and pregnancy. All five oocytes extruded the first polar body and were injected using ICSI. Three oocytes were fertilized, but two showed three pronuclei. The remainder, a two-pronuclei embryo subsequently cleaved and was cryopreserved. An ongoing pregnancy was obtained after-the transfer of this frozen-thawed embryo. Immature human oocytes at the germinal-vesicle stage that have not been exposed to hCG during ovarian stimulation can be matured in vitro and a normal pregnancy can result from ICSI of the in vitro-matured oocytes.

Isthmic and ampullary fluid enhance fertilization and cleavage rate of cow oocytes in vitro

The aim of the work was to determine the susceptibility of non-cultured and cultured rabbit embryos at different stages of development to the adverse effects of vitrification. The experiment was carried out on non-cultured embryos at the 8- to 16-cell, morula and blastocyst stages, and on embryos collected at the 2-cell stage and cultured for 24, 48 or 72 h. The embryos were cultured in Menezo's B2 medium supplemented with 20% FCS (fetal calf serum), or were cocultured with oviductal epithelium cells. A mixture of 35% glycerol + 35% 1,2-propanediol in serum-supplemented, phosphatebuffered saline was used as a vitrification medium. The embryos were vitrified in glass ampoules or plastic straws. Dilution after rapid thawing was done in one step, in a 1 M sucrose solution.After vitrification of non-cultured embryos in glass ampoules, the survival rates of the 8- to 16-cell, morula and blastocyst stages were 55.9%, 88.4% and 83.1%, respectively.The survival rates of embryos derived from cultures of 2-cell embryos in Menezo's B2 medium for 24 h (8- to 16-cell stage), 48 h (morula stage) and 72 h (blastocyst stage) and then vitrified in glass ampoules were 46.7%, 62.4% and 36.2%, respectively. The percentage of live foetuses remaining 28 days after the transfer of 103 vitrified and 115 cultured and vitrified 8- to 16-cell embryos was 21.4% and 1.8%, respectively. After the transfer of 111 vitrified morulae and 112 cultured and vitrified morulae, the percentage of live foetuses 28 days later was 11.7% and 7.1%, respectively.After vitrification in plastic straws, the survival rates in vitro of non-cultured morulae, and of morulae resulting from the culture of 2-cell embryos for 48 h in Menezo's B2 medium and in the coculture system were 89.1%, 93.8% and 91.2%, respectively.

Culture of bovine embryos in buffalo rat liver cell-conditioned media or with leukemia inhibitory factor.

Experiments were designed to study development of bovine embryos in TCM-199 medium conditioned by preculture with buffalo rat liver (BRL) cells. Conditioned media were harvested after BRL cells were cultured until confluency (CON), or for an additional 2 d with the same cells but new medium (CON-N) or the same medium (CON-S). Glucose in TCM-199 was depleted by BRL cells to different concentrations depending on coculture procedures: CON = 3.94 mM, CON-N = 1.67 mM, and CON-S = 1.11 mM glucose. In Exp. 1, development of bovine zygotes in CON-S resulted in fewer blastocysts than development in CON (10 vs 28%, P < .05); CON-N was not different from CON (26% blastocysts). Experiment 2 examined effects of moving embryos to a fresh drop of different or identical conditioned medium after culture for 3 d. Initial culture in CON-N and final culture in CON resulted in a greater (P < .01) number of blastocysts compared with the control of CON followed by CON (32 vs 19% blastocysts). This was not entirely due to changing from low to high glucose because adding glucose to CON-N after 3 d yielded only 18% blastocysts. To test the hypothesis that beneficial effects of BRL cell-conditioned media may be due to secretion of leukemia inhibiting factor (LIF), LIF was added to B2, a more appropriate medium than Medium-199 for culturing bovine embryos without conditioning or coculture with BRL cells. In the absence of serum, the percentage of blastocysts per cleaved embryo (17 to 28%) was not improved with LIF; however, the mean number of cells per blastocyst was higher (P < .05) in treatments with LIF (65 to 74 cells) than without LIF (47 cells). In B2 medium + 10% fetal calf serum, LIF was of no benefit; development to blastocysts was good with or without LIF (43% of cleaved).

Protein phosphorylation in bovine oocytes following fertilization and parthenogenetic activation in vitro

This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast’s age and type of somatic cells used as donor nuclei.Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus–oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei.Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell–cytoplast complexes (SC–CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 μs. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 °C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24–35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level.

Association of [formula omitted] serover [formula omitted] type hardjobovis with bovine ova and embryos produced by in vitro fertilization

The association of Leptospira borgpetersenii serovar hardjo type hardjobovis with in vitro fertilized (IVF) embryos was investigated in in vivo and in vitro. The organism could not be isolated from IVF embryos that were generated from ova harvested from experimentally infected donor heifers and cultured in commercial INRA Henezo B2 medium containing low levels of penicillin and streptomycin. Recipient heifers (n = 14) to which embryos cultured in Menezo B2 medium were transferred, did not develop antibody titres to Leptospira , and this organism was not isolated from the resulting fetuses (n = 5). Similarly, the organism could not be isolated from IVF embryos that were produced from in vitro-exposed oocytes to Leptospira and cultured in Menezo B2 medium, or TCM-199 medium supplemented with penicillin and streptomycin. In contrast, leptospires were isolated from IVF embryos that were produced from in vitro- exposed oocytes and cultured in TCM-199 medium free of antibiotics. The sequential washing procedure that was used with media free of antibiotics did not render infected embryos free of the pathogenic microorganism. Oocytes exposed to Leptospira in vitro were not transferred into the recipients. The presence of serovar hardio in the IVF system had no detrimental effect on fertilization rates or on embryonic development to the blastocyst stage. This study shows that it is possible to obtain transferable stage embryos from oocytes recovered from infected donors and from oocytes exposed in vitro to Leptospira . Such embryos may be associated with the microorganism, and the use of culture media supplemented with antibiotics for processing IVF embryos would be advisable in order to prevent any risk of transmission.

Stimulation of Human Sperm during Capacitation in Vitro by an Adenosine Agonist with Specificity for A2 Receptors

The effects of an adenosine agonist with specificity for A2 receptors, on human sperm prepared for in vitro fertilization (IVF), were examined to verify physiological effects and possible pharmacological use. 5' -N-ethyl-carboxamidoadenosine (NECA), when added at 100 microM over 30 min in B2 medium, did not induce a spontaneous acrosome reaction after 0, 3, and 6 h capacitation in B2, nor did it modify sperm motility. However, NECA increased the number of capacitated spermatozoa able to respond (p < 0.05) to A23187 (10 microM) after 6 h preincubation in B2 medium. This effect was associated with an increase in cAMP production, which was measured by RIA after 10 and 20 min incubation with NECA in uncapacitated sperm, and with changes in the kinetics of protein tyrosine phosphorylation as revealed by Western blot. Phosphorylation of a 95-kDa protein was enhanced by NECA in uncapacitated sperm and inhibited in capacitating sperm (incubated 1 h in B2), whereas phosphorylation of a 50-kDa protein was systematically enhanced whatever the preincubation time. NECA can stimulate uncapacitated human sperm via cAMP production and protein phosphorylation/dephosphorylation without inducing an acrosome reaction or influencing motility. Cyclic AMP-dependent protein kinase A seems to positively control protein phosphorylation involved in human capacitation. Adenosine present in the tubal fluid or produced by the spermatozoon itself may influence capacitation in vivo through sperm A2 receptors. In cases of male infertility, use of NECA in sperm handling for IVF should be evaluated as a means to improve capacitation without increasing the possibility of a premature spontaneous acrosome reaction.

Surface localization of P34H an epididymal protein, during maturation, capacitation, and acrosome reaction of human spermatozoa.

During epididymal transit, spermatozoa acquire new surface antigens that are involved in the acquisition of their fertilizing ability. We have previously described a 34-kDa (P34H) human epididymal sperm protein that shows antigenic and functional homologies with the hamster P26h. P34H is localized on the acrosomal cap of human spermatozoa and has been proposed to be involved in the interaction with the zona pellucida. The aim of this study was to document the expression of P34H on the sperm surface during transit along the male and female genital tracts. Immunohistochemical techniques were performed on human testes and epididymides by means of an antiserum specific for P34H. No labelling was detected on those spermatozoa found within the seminiferous tubules or in the vasa efferentia. P34H first appeared in the caput epididymidis and was restricted to the acrosomal cap. Signal intensity then increased considerably from the proximal corpus to the cauda region of the epididymis. After ejaculation, the same pattern of P34H distribution was observed, but the intensity was much lower than that characterizing the cauda epididymal spermatozoa. Strong labeling was restored after incubation in B2 medium and was maximal after 5 h of capacitation. After acrosomal exocytosis induced by a Ca2+ ionophore, the percentage of P34H-labeled spermatozoa decreased proportionally to the number of acrosome-reacted spermatozoa as determined by Pisum sativum-fluorescein isothiocyanate (FITC) labeling. P34H appeared to be strongly anchored to the sperm plasma membrane during epididymal transit as indicated by the requirement for detergent to extract this surface antigen from ejaculated spermatozoa. This confirms the importance of P34H binding to the sperm plasma membrane during epididymal maturation. We have previously proposed that P34H is involved in sperm-zone pellucida interaction. The appearance and accumulation of P34H on the sperm plasma membrane during epididymal maturation, followed by its inaccessibility associated with ejaculation, its unmasking during capacitation, and finally its elimination after the acrosome reaction, are in agreement with te proposed function of this sperm antigen.

Role of endometrial factors in regulating secretion of components of the immunoreactive human embryonic interleukin-1 system during embryonic development.

In the study reported here, we localized at the protein level the major components of the interleukin (IL)-1 system in the human embryo, and we investigated the endometrial factors influencing their secretion during embryonic development. To localize these components, we performed immunohistochemical experiments in 44 oocytes and 78 embryos. The following primary antibodies were used: monoclonal mouse anti-human IL-1 receptor type I (IL-1R tl), monoclonal mouse anti-human IL-1 beta, and polyclonal rabbit anti-human IL-1 receptor antagonist (IL-1ra). For embryo culture, human embryos at different developmental stages were cultured in 100-microliters drops of Ham's F-10 medium + 4 mg/ml BSA (n = 33), in 100-microliters drops of Menezo B2 culture medium (n = 18), or in wells with 1 ml of Menezo B2 culture medium (n = 8). For embryo coculture, endometrial stromal cells (ESC) and endometrial epithelial cells (EEC) were isolated from human secretory endometrium and cultured until confluence in 75% Dulbecco's Modified Eagle's Medium and 25% MCDB-105 containing antibiotics and supplemented with 10% charcoal-Dextran-treated fetal bovine serum. Individual human embryos were cocultured with experimental EEC and ESC (n = 23 and n = 4, respectively) for 5 days in 600-microliters drops of Menezo B2 medium, and conditioned medium was removed every 24 h. Human embryos were also cultured with EEC-conditioned medium (n = 9). IL-1 alpha, IL-1 beta, and IL-1ra levels were determined by ELISA in the 24-h culture- or coculture-conditioned media. Immunostaining confirmed the presence of IL-1 beta, IL-1ra, and IL-1R tl in oocytes and embryos in all stages analyzed, with no statistical differences. IL-1 alpha, IL-1 beta, and IL-1ra were absent in conditioned media of cultured embryos and embryos cocultured with ESC. However, when human embryos were cocultured with EEC or with EEC-conditioned medium alone, two different populations of embryos were observed: IL-1 producers (57% and 56%) and IL-1 nonproducers (43% and 44%, respectively). Finally, the IL-1 profile of a single human embryo cocultured with maternal EEC which successfully implanted and developed is presented, this pattern being similar to that described in the IL-1 producer population. These results demonstrate the presence of the IL-1 system in the human embryo. However, the selective release of IL-1 only when embryos were cocultured with EEC or EEC-conditioned medium indicates an obligatory role of the endometrium in the regulation of the embryonic IL-1 system. Furthermore, the differential embryonic production of IL-1 may be related to the implantation capability of the embryos.

In vitro development of IVM/IVF bovine embryos in B2 medium supplemented with albumin (BSA) or polyvinyl alcohol(PVA)

In vitro development of IVM/IVF bovine embryos in B2 medium supplemented with albumin (BSA) or polyvinyl alcohol(PVA)

Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue.

Secretion of interferon tau (IFN tau) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Ménézo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-microliters drops of BRL cell-conditioned medium, mean IFN tau secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFN tau secretion by trophoblastic tissue increased to mean levels of > 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.

Andrology: Variations in spontaneous and induced acrosome reaction: correlations with semen parameters and in-vitro fertilization results

To determine whether variations in spontaneous and induced acrosome reactions are correlated with semen quality, and to identify the inducers of clinical interest, the acrosome reaction, sperm concentration, motility and morphology were recorded in 117 semen samples from patients undergoing an in-vitro fertilization (IVF) attempt. The spontaneous acrosome loss after 24 h incubation in Ménézo B2 medium and after induction by calcium ionophore A23187, progesterone, human follicular fluid, cyclic adenosine 3'-5'-phosphate (cAMP) analogue and phorbol ester (TPA) were measured using the fluorescein isothiocyanate-GB24 antibody. The mean (range) spontaneous acrosome reaction was 3.4 +/- 0.6% (0.0-30.0). Response to the tested inducers was 23.3 +/- 1.6% (0.0-72.0) for calcium ionophore, 5.4 +/- 0.8% (0.0-60.0) for progesterone, 1.0 +/- 0.7% (0.0-24.0) for human follicular fluid, 2.6 +/- 0.7% (0.0-39.0) for the cAMP analogue and 2.3 +/- 0.7% (0.0-31.0) for TPA. The response to calcium ionophore was correlated significantly to sperm concentration, motility and morphology, while the responses to progesterone and TPA were correlated significantly to motility and acrosome morphology. The responses to other inducers were not linked to classic semen parameters. When studying acrosome reaction as a function of IVF results, the responses to calcium ionophore and TPA were discriminant. The results of this study show that the spontaneous acrosome loss and the responses to acrosome reaction inducers are highly variable and partially linked to semen quality. The responses to calcium ionophore and TPA could be of interest in predicting the fertilizing ability in vitro.

DNA and chromatin structure: Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the accurate and objective evaluation of sperm chromatin condensation and chromatin stability of sperm nuclei. It was also possible to determine the influence of incubation on sperm chromatin. Different types of spermatozoa were studied: unprocessed spermatozoa at 1 and 45 min after ejaculation, after swim-up (migrated), spermatozoa incubated for 6 h in non-capacitating conditions (aged), or in B2 medium (capacitated) or B2 medium followed 1 h later with A23187 (reacted). All types of spermatozoa were analysed before and after treatment with various decondensation agents: sodium dodecyl sulphate (SDS), SDS plus EDTA and SDS plus disulphide-reducing agent [dithiotreitol (DTT)]. Sperm nuclei were enzymatically isolated and stained with propidium iodide. Three flow cytometric parameters were then measured: forward light scatter (cellular size), side light scatter (cellular complexity) and fluorescence (uptake of propidium iodide). Fluorescence was the most suitable parameter to study the degree of condensation and resistance to decondensation of DNA in the spermatozoa. Unprocessed spermatozoa 1 min after ejaculation underwent decondensation by all assessed treatments (anionic detergent, chelating or disulphide-reducing agents). Unprocessed spermatozoa 45 min after ejaculation and migrated spermatozoa did not undergo decondensation with SDS treatment, but decondensation occurred after treatment with SDS+EDTA or SDS+DTT. Spermatozoa incubated for 6 h under both non-capacitating (aged spermatozoa) and capacitating conditions (capacitated spermatozoa) and reacted spermatozoa were decondensed only after treatment with SDS+DTT.(ABSTRACT TRUNCATED AT 250 WORDS)

Relevance of acrosome function in the evaluation of semen in vitro fertilizing ability

To determine whether or not acrosome evaluation can enhance the prediction of IVF results when associated to conventional semen parameters. Acrosome reaction, sperm concentration, motility, and morphology were recorded in 131 semen samples from patients undergoing an IVF attempt. Spontaneous acrosome loss after a 24-hour incubation in B2 medium and after induction by calcium ionophore A23187 and phorbol ester, phorbol 12-myristate 13-acetate 4-O-methyl ether (TPA). Statistically significant differences between fertilization failures and successes were found for concentration, viability, spontaneous and induced acrosome reaction, and most parameters of motility and morphology. However, none of the parameters could predict > 64% of IVF results when studied alone. A progressive discriminant analysis allowed to predict up to 83% of IVF results, by classifying sperms through their normal forms, rapid motility, spontaneous acrosome loss, enlarged heads, multiflagellar forms, vitality, linear motility, and acrosome response to TPA. The other parameters, including concentration and response to calcium ionophore, had no additive value. The study of acrosome function, through spontaneous acrosome loss and response to TPA, is of great interest in clinical practice when associated to some parameters of motility and morphology. However, it appears that response to calcium ionophore, one of the most studied parameters, is of poor practical interest.

Fertilization and early embryology: Maturation of human cumulus-free germinal vesiclestage oocytes to metaphase II by coculture with monolayer Vero cells

The objective of this study was to evaluate and compare the efficacy of coculture with Vero cells (monkey kidney cells) and in-vitro culture in B2 Ménézo medium for the maturation of germinal vesicle (GV)-stage cumulus-free human oocytes to metaphase II. Cumulus-free human GV-stage oocytes obtained after ovulation induction and retrieval via vaginal ultrasound echo-guided puncture were put either in coculture with Vero cells or in 25 microliters droplets of B2 Ménézo medium. In a controlled randomized study, 145 GV-stage oocytes were matured in vitro: 72 were placed in the coculture system with Vero cells and 73 were cultured in B2 alone. After 30 h of in-vitro culture, 82.4% had dissolved the nuclear membrane and extruded the first polar body in the coculture system compared with 37% in B2 medium (P < 0.001). A significant difference was also noticed in the number of GV-stage oocytes which underwent GV breakdown; the proportion of oocytes which did not extrude the first polar body (metaphase I) was 1.6% in the coculture system compared to 44.0% in the B2 medium. It can be concluded that coculture with Vero cells improves the in-vitro maturation of cumulus-free human GV-stage oocytes.

Long-term culture of cloned bovine embryos for evaluation of their developmental potential

To evaluate the value of supplementing a standard culture medium with 10% heat-inactivated mature follicular fluid (FF).Prospective randomized study evaluating the in vitro development of nontransferred, nonfrozen human pre-embryos in three culture conditions from day 3 to day 8 postfertilization. Preliminary evaluation by RIA and electrophoresis of factors responsible for these results.Ten percent mature FF supplementation of Inra Menezo (B2 medium) was associated with a significantly higher proportion of human pre-embryos reaching the morula (95% versus 72% with 10% maternal serum, P = 0.04) and the blastocyst stage (50% versus 11% with B2 alone, P = 0.02). The concentration of insulin-like growth factors I and II did not differ significantly between serum and mature FF-supplemented medium. Protein electrophoresis showed a difference of two bands corresponding to a molecular weight of 17,000 present in the serum and not in the FF-supplemented medium.Culture medium supplementation with 10% mature FF is associated with a significantly higher proportion of pre-embryos reaching the morula and blastocyst stage. The observed difference could be explained by the presence of a low molecular weight (17,000) embryotoxic factor contained in the serum-supplemented medium.

Development of In Vitro Matured/In Vitro Fertilized Bovine Oocytes In A Simple Defined (KSOM) Medium.

The experiments reported here take advantage of the large number of in vitro matured and in vitro fertilized (IVM/IVF) bovine oocytes which can be produced, permitting the design of controlled experiments to establish a simple defined medium for the study of early embryo requirements and to further test this medium as a component of co-culture. A total of 1386 IVM/IVF oocytes were used to compare a simple medium (KSOM) with complex culture conditions used successfully for culture of bovine embryos. All experiments were extensively replicated factorials. In Experiment 1, KSOM was equivalent to the complex Menezo B2 medium in producing blastocysts from IVM/IVF produced embryos and was superior to Menezo B2 medium when both were used with buffalo rat liver cells (BRLC), yielding 25% vs 8% blastocysts, respectively (P<0.05). In Experiment 2 a, KSOM was tested with 0 or 1 ng/ml of platelet derived growth factor (PDGF) and in Experiment 2 b, 0, 1 and 5 ng/ml of PDGF were tested. In Experiment 2 a blastocyst formation was higher (P<0.05) when PDGF was added to the co-culture and also was higher (P<0.05) for morulae plus blastocysts in Experiment 2 b when PDGF treatments were combined and compared to no PDGF. The results with the simple KSOM medium are sufficiently promising to indicate that growth factors, amino acids and other specific requirements of the embryo may be examined in future studies with KSOM as a base. Additionally, KSOM appears to be superior to Menezo B2 medium often used commercially for co-culturing bovine embryos with BRLC.

Production, freezing and transfer of bovine IVF embryos and subsequent calving results

Ultrasound-guided oocyte aspirations were performed repeatedly, on a weekly basis, on 155 different cows. An average of 4.9 oocytes with 4.1 classified as usable were collected. Following in vitro maturation (IVM), fertilization (IVF) and culture (IVC), a few Day 7 morulae, and all Day 7 and most Day 8 blastocysts were either transferred or frozen. The transfer of 2268 fresh IVF embryos resulted in 1220 pregnancies (53 8%). Day 7 embryos resulted in a higher (56%) pregnancy rate than did Day 8 (43%) or Day 9 (41%). In addition, the transfer of embryos classified as grade 1 on either Day 7 or 8 resulted in a higher pregnancy rate than did grade 2 embryos. Although there was no difference in pregnancy rate, embryos resulting from co-culture with Buffalo Rat Liver Cells n Menezo's B2 medium developed faster than embryos co-cultured with TCM 199 culture medium. Pregnancy rate following transfer of IVF blastocysts frozen on Day 7 was 42% and dropped to 20% for blastocysts frozen on Day 8. Pregnancy rate was not influenced when synchrony between the IVF embryo and recipient was within ± 36 h. The sex ratio of the resulting calves did not deviate from normal but there was a higher than normal incidence of early abortions and dystocias. There appeared to be some increase in calf birth weights.

In vitro maturation, fertilisation and culture of bovine oocytes in a modified Ménézo B2 medium

In this study we investigated the influence of essential amino acid composition of the medium on in vitro maturation and culture of bovine oocytes (cleavage of oocytes and development capacity of embryos). Oocytes were matured and cultured in vitro in a modified Ménézo B2 medium (MMM). The medium was modified by adding amino acids, not as single compounds, but as amino acid supplements for MEM (Minimum Essential Medium) or BME (Basal Medium Eagle) tissue culture medium, which differ completely in their content of essential amino acids. In the medium with MEM supplements the content of all essential amino acids except glutamine is at least twice as high as in the BME-supplemented medium. Oocytes were randomly allocated to one of four treatments: (1) in vitro maturation (IVM) and in vitro culture (IVC) in MMM with MEM amino acids (MEM medium); (2) IVM in MEM medium, IVC in MMM with BME supplements (BME medium); (3) IVM in BME medium, IVC in MEM medium; (4) IVM and IVC in BME medium. After IVM in MEM medium, more cumulus-oocyte complexes displayed marked expansion of the cumulus cells and cleavage occurred more frequently than after IVM in BME medium. More oocytes underwent cleavage when cultured in MEM medium than in BME medium. A higher number of embryos entered the compact morula or blastocyst stage when cultured in BME medium. Development to the blastocyst stage occurred faster after IVM and IVC in BME medium than in the other groups. The results demonstrate that the content of essential amino acids of the maturation and culture medium affects the development of bovine embryos in vitro.