Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro

Employing a total of 3465 bovine oocytes this study was aimed at improving the efficiency of bovine embryo production under defined and undefined conditions. Following in vitro maturation (IVM) and in vitro fertilization (IVF), oocytes were allocated to various culture treatments using synthetic oviduct fluid (SOF). In our 3 experiments we showed that: 1) the addition of fetal calf serum (FCS 10% v/v) to SOF droplets after 20 to 24 h significantly improved blastocyst yields on Day 6 (21 vs 12%; P < 0.01), but not at later stages and resulted in significantly higher Day-8 blastocyst cell numbers (148 +/- 61 vs 92 +/- 35; P < 0.05); 2) the removal of bovine serum albumin (BSA) from the standard SOF medium resulted in significantly reduced blastocyst yields on Days 6, 7 and 8, respectively (17 vs 8%; 28 vs 18%; 31 vs 21%; P < 0.05); 3) the presence or absence of cumulus cells surrounding the presumptive zygote in culture in SOF had no effect on cleavage rate, percentage of 5-8 cell embryos or blastocyst yields (Day 6,7 or 8); 4) the culture of presumptive zygotes in SOF in an atmosphere of 5% CO2 in air (20% O2) resulted in significantly reduced development compared with culture in 5% CO2, 5% O2, 90% N2 in terms of blastocyst yield on Days 6, 7 and 8 and on Day 8 hatching rate, respectively (5 vs 22%; 9 vs 33%; 13 vs 48%; 50 vs 8%; P < 0.001) and 5) embryo density (1 embryo per 1 or 3 microl SOF) or replacing the culture medium every 48 h had no effect when SOF was supplemented with serum; however, under serum-free conditions, changing of the media resulted in a slightly improved Day-6 blastocyst yield such that renewal of serum-free medium mimicked the effect of serum addition.     

Effects of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro

This study was conducted to evaluate the effect of bovine follicular fluid (BFF) on fertilizability and developmental capacity of bovine oocytes matured in vitro. Oocytes were collected from slaughterhouse ovaries, and matured in TCM199 supplemented with 5% superovulated cow serum (SCS), 2 mM pyruvate and 1 IU/mL PMSG. BFF was aspirated from small follicles (1 to 5 mm in diameter). In Experiment 1, BFF was added to the Brackett and Oliphant (BO) fertilization medium at concentrations of 0, 1, 5, 10 and 20%. After insemination with frozen-thawed and heparin-treated (10 micrograms/mL, 15 min) bull spermatozoa for 18 h, some of the oocytes were fixed and stained to evaluate the fertilization rate. The rest of the oocytes were co-cultured in serum-free embryo culture medium (ECM; TCM199 supplemented with 5% SCS, 2 mM pyruvate and 5 micrograms/mL insulin) with bovine oviductal epithelial cells (BOEC) at 38.5 degrees C under 5% CO2 in air, and the developmental capacity of embryos was examined at 2, 7 and 9 d. In Experiment 2, BFF was added to the serum-free ECM with BOEC at 0, 5, 10 and 20% concentrations, and embryos were cultured for 9 d. Fertilization rates and blastocyst rates in low (1 and 5%) BFF in fertilization medium were not significantly different from the control (without BFF). However, high concentrations of BFF (10 and 20%) in the fertilization medium suppressed both fertilization rates and development. Large vesicles with fast monolayer formation were observed at all concentrations of BFF added to ECM with BOEC. There were no significant differences in cleavage or development to blastocyst in different concentrations of BFF added to ECM. However, the rate of development to hatched blastocysts in 20% BFF was significantly lower than that of the control (P < 0.05). The results of the present study indicate that BFF addition to fertilization medium and ECM with BOEC does not improve fertilizability or developmental capacity and that high concentrations of BFF reduce the rate of both fertilization and development.     

The effect of co-culture system on developmental capacity of bovine IVM/IVF oocytes

Two experiments were conducted to compare the influence of different culture systems and the oviduct donor's cycle phase on the developmental potential of co-cultured bovine embryos derived from IVM/IVF oocytes and to establish an efficient freezing method for oviduct epithelial cells. In the first experiment, the effects of media (Menezo B2, synthetic oviduct fluid SOF); sera (no serum, fetal calf serum FCS, human serum HS); and the presence or absence of monolayer of bovine oviduct epithelial cells (BOEC) on developmental capacity of bovine embryos were investigated. In the second experiment, the influence of oviduct donor's hormonal status (superovulated versus unstimulated) and the cryopreservation of oviductal tissue on the support of developmental competence of bovine IVM/IVF-derived zygotes were examined. Oviduct epithelial cells were cryopreserved according to the modified two-step method previously applied to rabbit embryos. For zygotes co-cultured with a monolayer of BOEC the following blastocyst development rates were obtained: 40.1% (63/157); 34.5% (60/174); 13.0% (7/54); and 19.2% (14/73), respectively, in B2 serum-free medium, B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS medium. In the absence of BOEC the rates were 12.3% (10/81); 41.4% (36/87); and 8.9% (6/67), respectively, in B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS. It was shown that the source of oviduct epithelial cells and previous freezing had no influence on the proportion of cleaved zygotes (approximately 70%) or on the percentage of blastocysts (approximately 20%).     

Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification

The aim of this study was to assess the effect of a bovine in vitro culture system on blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were cultured in either synthetic oviduct fluid (SOF) in 5% CO2, 5% O2, 90% N2; or TCM199-granulosa cells (TCM199-GCM) in 5% CO2 in air. In vivo blastocysts were used as a control. Culture in SOF resulted in a significantly higher blastocyst yield on both Day 7 (31.3 vs 13.2%, P < 0.001) and 8 (36.8 vs 23.7%, P < 0.001) than did culture in TCM199-GCM. After vitrification, survival at 72 h of in vivo blastocysts was significantly higher than both in vitro groups, while significantly more blastocysts produced in TCM199-GCM survived compared to those produced in SOF (0, 43.5, 78.3% for SOF, TCM199-GCM and in vivo, respectively P < 0.01). In Experiment 2, SOF-GCM proved to be the best post-warming culture system of those tested and was adopted as the post-warming medium for all subsequent experiments. In Experiment 3, zygotes were cultured in SOF or SOF-GCM, in either 5% CO2 in air, or 5% CO2, 5% O2, 90% N2. In agreement with Experiment 1, culture in SOF in 5% O2 resulted in significantly more blastocysts at Day 7 (26.4 vs 17.3%, P < 0.01) and Day 8 (31.5 vs 23.2%, P < 0.01) than did culture in SOF-GCM. However, survival at 72 h post vitrification was significantly higher for SOF-GCM (44 vs 8.3%, P < 0.001). Increasing the O2 concentration to 20% significantly reduced the blastocyst eld from SOF (31.5 vs 17.3%, P < 0.001). In addition, the quality of blastocyst produced was reduced in terms of survival post vitrification (8.3 vs 0%, P < 0.05). In contrast, there was no difference in blastocyst yield (23.2 vs 25.2%) or survival (44.0 vs 36.9%) in SOF-GCM, irrespective of O2 concentration. Experiment 4 examined the duration of exposure to GCM necessary to acquire improved blastocyst quality. Zygotes were cultured in SOF; SOF until Day 3, followed by SOF-GCM for the remainder of the culture; SOF until Day 5, followed by SOF-GCM for the remainder of the culture; or SOF-GCM for the entire culture. Survival at 72 h post vitrification was significantly higher (P < 0.05) in Groups 2 (50.0%, 13/26) and 4 (55.3%, 26/47) than in Groups 1 (21.7%, 10/46) and 3 (10.8%, 4/37). In conclusion, culture system can affect blastocyst yield and quality and crytolerance is a useful indicator of blastocyst quality.     

In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.     

Evaluation of culture systems containing bovine oviduct epithelial cells or granulosa cells to mature and maintain the developmental competence of bovine oocytes in vitro

The effects of estrous cow serum (ECS), bovine oviduct epithelial cells (BOEC), and bovine granulosa cells (GC) on in vitro maturation (IVM) of immature oocyte-cumulus complexes (OCCs) were evaluated. Selected OCCs were cultured for 24 to 26 h in microdroplets of culture medium (CM; TCM 199 + 25 mM HEPES + 100 mug gentamicin sulfate/ml) or in CM medium supplemented or conditioned with 20% ECS, BOEC +/- 20% ECS or GC + 20% ECS. Supplemented media were incubated for 2 h before addition of OCCs, whereas media were conditioned by incubation with 20% ECS or BOEC +/- 20% ECS for 6 d, or with 20% ECS +/- GC for 24 or 48 h before addition of OCCs. The developmental competence of oocytes after TVM was assessed by insemination with glass wool separated, frozen-thawed bovine spermatozoa in microdroplets of modified medium (TALP) containing heparin (5 mug/ml) and BOEC for 18 h. The presumptive zygotes were cultured in microdroplets of CM medium + 20% ECS + BOEC for 7 to 9 d to assess embryo development to morula and blastocyst stages. The percentages of OCCs undergoing IVM (85 to 94%) and in vitro fertilization (IVF) (66 to 80%) were high, irrespective of the IVM conditions. Only after the IVM of OCCs in CM medium alone was the percentage of oocytes undergoing IVF significantly lower (66%; P<0.05). The proportion of IVF oocytes developing to blastocysts with a normal complement of cells (126 to 138) increased significantly (P<0.05) when the OCCs were matured in supplemented or conditioned CM medium containing ECS and/or somatic cells (18 to 28%) compared with those in CM medium alone (9%). When the CM medium was supplemented or conditioned with GC + 20% ECS, the proportion of fertilized oocytes developing to blastocysts increased significantly (28%; P<0.05). These results indicate that the potential of immature OCCs to be fertilized and to complete embryonic development to the blastocyst stage in vitro is enhanced by maturation in CM medium containing 20% ECS and/or BOEC or GC.     

Development of in vitro matured and fertilized bovine embryos, cultured from days 1-5 post insemination in either Menezo-B2 medium or in HECM-6 medium

Bovine embryos were produced by in vitro maturation and fertilization of abattoir oocytes. The embryos were randomly allocated either to coculture with bovine oviduct cells in Menezo-B2 medium (control group), or to culture in the defined HECM-6 medium. At Day 5 after insemination the HECM-6 embryos were transferred to Menezo-B2 medium with (HECM-B2/BOEC) or without (HECM-B2) oviduct cells for further culture. The proportion of cleaved embryos and blastocysts, the morphology and the speed of development were compared for the control and HECM groups. Significantly more HECM-6 embryos than control embryos cleaved (88 +/- 3% vs 76 +/- 5% (+/- SD)). Significantly fewer blastocysts developed in the HECM-B2 than in the control group (28 +/- 2% vs 35 +/- 3%), in addition the speed of development was delayed and the morphology was impaired. In the HECM-B2/BOEC group no differences in neither morphology, blastocyst rates (31 +/- 8%) nor speed of development could be demonstrated, when compared with the control group. A portion of the control and HECM-B2 embryos were vitrified at Days 7-8, but no differences were noted in survival or morphology at 48 and 72 h post thawing. It can be concluded, that the defined medium HECM-6 can support bovine embryonic development through the 8-16 cell in vitro block stage without the use of coculture in a reliable way. In our system it was however necessary to transfer the embryos at Day 5 to coculture in Menezo-B2 medium to ensure optimal continuation of development.     

Fertilizability and developmental capacity of individually cultured bovine oocytes

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.     

Development of in vitro matured and fertilized bovine oocytes co-cultured with Buffalo Rat Liver cells

This study was designed to evaluate the efficacy of Buffalo Rat Liver cells (BRLC) monolayers in supporting the development of in vitro matured and fertilized (IVM/IVF) bovine oocytes through to the hatched blastocyst stage compared to the commonly used co-culture system of bovine oviduct epithelial cells (BOEC). Cumulus oocyte complexes (COCs) obtained from 2- to 6-mm ovarian follicles at slaughter were matured for 24 h in TCM-199 supplemented with FBS and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed using 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium containing 18 to 20 matured oocytes. After 20 to 22 h of sperm exposure, 584 presumptive zygotes in 2 separate trials were randomly assigned to 3 treatment groups (BRLC co-culture, BOEC co-culture and control, consisting of medium alone). Zygotes were cultured in CZB media, a simple semi-defined medium, without glucose for the first 2 d, transferred to M199/FBS (TCM-199-HEPES supplemented with 20% HTFBS, 1 mM Sodium pyruvate), and cultured for an additional 8 days. Cleavage and development to morula and various blastocyst stages were recorded between d 3 and 11 after the start of IVF. Overall average cleavage rate was 75% (440 584 ) and did not vary across the treatments or trials. The proportion of embryos that reached the morula stage in both co-culture systems did not differ (P > 0.05) and was significantly higher (P > 0.05) compared to the control group. However, the percentage of the number of blastocysts, expanded blastocysts and hatched blastocysts varied across the treatment groups (P < 0.05), with the highest results obtained in the BRLC co-culture system. The production of blastocysts in BOEC co-culture was inconsistent between the 2 trials where a significant difference (40.6 vs 53.0%; P > 0.05) was observed. Rate of development to the blastocyst stage was similar between the 2 co-culture systems, with most of the embryos reaching the blastocyst stage by d 8 post insemination. The results of this study show that BRLC from a commercially available established cell line offer a more reliable alternative to a BOEC co-culture system for in vitro maturation, fertilization and culture of bovine embryos.     

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.     

The effect of glutamine on the development of sheep embryos in vitro

The effects of glutamine (Gln) on the in vitro development of sheep embryos cocultured with sheep oviduct epithelial cells (SOEC) or cultured in medium alone were investigated. The in vitro development was evaluated after culture in synthetic oviduct fluid (SOF) medium to Day 6, and then the viability of some of the morula/blastocyst stage embryos was assessed by transfer into recipient ewes. In Experiment 1, sheep embryos were cultured from Day 2 to Day 6 in SOF containing 0 or 1 mM Gln with or without (SOEC) support in a 2 x 2 factorial design. An interaction was found between the effects of Gln and SOEC (P<0.001). The addition of Gln increased blastocyst formation (6%, 2 36 vs 35%, 11 31 ) and the rate of pregnancy (50%, 4 8 vs 100%, 5 5 ) when the embryos were cultured in medium alone, but had no beneficial effect in the presence of SOEC. In Experiments 2 and 3, sheep embryos were cultured from Day 1 to Day 6 in SOF supplemented with 1 mM Gln, with 1 mM alpha-ketoglutarate or without supplementation (control). In Experiment 2, no other amino acids were added, but in Experiment 3 SOF was supplemented with 19 other amino acids. In Experiment 2, when Gln was the only amino acid, the rate of blastocyst formation was increased by the addition of Gln (24%, 8 35 ), but alpha-ketoglutarate caused no increase in blastocyst formation (3%, 1 34 ) compared to the control group (6%, 2 34 ). In Experiment 3, when 19 other amino acids were added, neither Gln nor alpha-ketoglutarate affected the rate of blastocyst formation or the subsequent development of embryos in recipient ewes. These results showed that Gln, when used as a single amino acid, has a beneficial effect on the development of sheep embryos in serum free culture without somatic cells. The data suggest that Gln is used as a source of amino groups rather than as a source of energy since no beneficial effects were found when its deaminated carbon skeleton (alpha-ketoglutarate) was used or when other amino acids were present.     

Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.     

Effect of growth factors on morula and blastocyst development of in vitro matured and in vitro fertilized bovine oocytes

Growth factors were studied as a means of increasing the development of in vitro matured (IVM) and in vitro fertilized (IVF) oocytes into morulae or blastocysts. Cell numbers of blastocysts were also counted. In Experiment 1, 2- to 8-cell embryos derived from bovine IVM/IVF oocytes were randomly allotted to one of 3 culture groups: a) synthetic oviduct fluid (SOF); b) SOF + 10 ng/ml epidermal growth factor (EGF); or c) SOF + 100 ng/ml EGF; all 3 culture media contained 10% fetal bovine serum. Culture resulted in 12%, 23% and 14% (P>0.05), respectively, developing into morulae and blastocysts. In Experiment 2, 5 ng/ml of transforming growth factor B (1) (TGFB (1)) added to CR(1aa) medium containing BSA increased the percentage of blastocysts to 56% vs 40% for the control (P<0.05). In Experiment 3, EGF and TGFB(1), added singly and in combination to CR(1aa) did not produce a synergistic effect. More embryos developed into morulae and blastocysts (45%) in a bovine oviduct epithelial co-culture than in any other treatment except in CR(1aa) + EGF (34%; P>0.05). In Experiment 4, 0, 1 and 5 ng/ml of platelet derived growth factor (PDGF) added to CR(1aa) yielded 39%, 70% and 52% morulae and blastocysts, respectively (P<0.05). Cell number was not increased, indicating that growth factors can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.     

In vitro deveropment of porcine oocytes fertilized in vitro with spermatozoa preincubated in two different media

This study was conducted to clarify the effects of sperm concentration and media during preincubation on fertilization and development of porcine oocytes fertilized in vitro (IVF). The effect of porcine oviduct epithelial cell aggregates (POECA) on in vitro development of IVF embryos was also examined. Oocytes matured in vitro for 48 to 50 h were inseminated with epididymal spermatozoa preincubated at 2 sperm concentrations (1 - 2 x 10(8)/ ml vs 4 - 5 x 10(8)/ ml) for 3 h in either Dulbecco's phosphate buffered saline (PBS) or Brackett and Oliphant medium (BO). For capacitation, spermatozoa were treated with heparin (100microg / ml) for 15 min at 38.5 degrees C under 5% CO(2) in air. Cleavage and development to the blastocyst stage were evaluated on Day 3 and Day 8 after culture with or without POECA. The effect of sperm concentration on preincubation did not affect the fertilization rate, but preincubation in PBS medium did result in a higher fertilization rate (P < 0.05) than did the BO medium. The proportion of embryos undergoing cleavage and development to the blastocyst stage was significantly higher (P < 0.05) in the POECA co-culture group than in the group without POECA co-culture. The present results indicate that PBS medium can be utilized as a simple preincubation medium for porcine spermatozoa and that the presence of POECA during in vitro culture improved the development of IVF oocytes to the blastocyst stage.     

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.