Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

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.     

Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture)

The objective of this study was to test a new co-culture system of bovine embryos, which we call "mixed co-culture." This system consists of culturing embryos on cell monolayers composed of both Vero and BRL cells (Vero/BRL). Cumulus-oocyte complexes from ovaries of slaughtered cows were matured and fertilized in vitro. The presumptive zygotes were cultured with Vero/BRL (Group 1), BRL (Group 2) or Vero (Group 3) cell monolayers, in 40 microL drops of Menezo B2 medium supplemented with 10% FBS and antibiotics. The development of the presumptive zygotes was compared on Day 2 [48 h post insemination (pi)] and Day 7 (168 h pi). On Day 2, there was no difference between the groups. On Day 7, the highest percentage of compacted morulae/blastocysts was observed in mixed co-culture of Vero/BRL cells: 40% versus 36% on BRL versus 27% on Vero cell monolayers. The differences were statistically significant (P < or = 0.05). Among compacted morulae/blastocysts, blastocysts prevailed in mixed co-culture: 67% on Vero/BRL as compared with 55% on BRL and 27% on Vero cell monolayers. The differences were highly statistically significant (P < or = 0.01). The results suggest that Vero/BRL cells improve the development of bovine embryos.     

Effect of protein supplementation in potassium simplex optimization medium on preimplantation development of bovine non-transgenic and transgenic cloned embryos

The present study evaluated the effect of protein supplementation in potassium simplex optimization medium (KSOM) on bovine preimplantation embryo development. The in vitro fertilized (IVF) (Experiment 1), non-transgenic (Experiment 2) and transgenic cloned embryos (Experiment 3) were cultured for 192 h in KSOM supplemented with 0.8% BSA (KSOM-BSA), 10% FBS (KSOM-FBS) or 0.01% PVA (KSOM-PVA). Transfected cumulus cells with an expression plasmid for human alpha1-antitrypsin gene and a green fluorescent protein (GFP) marker were used to produce transgenic cloned embryos. Modified synthetic oviductal fluid (mSOF) supplemented with 0.8% BSA (mSOF-BSA) was used as a control medium. In Experiment 1, cleavage rate was significantly (P < 0.05) lower (69.1%) in IVF embryos cultured in KSOM-FBS than in KSOM-BSA (80.3%). The rate of hatching/hatched blastocyst formation was significantly (P < 0.05) lower in embryos cultured in KSOM-PVA than in KSOM-FBS (2.2% versus 10.8%). Blastocysts cultured in KSOM-FBS contained significantly (P < 0.06) higher numbers of inner cell mass cells (50.4 +/- 20.2) than those cultured in mSOF-BSA (36.9 +/- 19.2). In Experiment 2, the rate of blastocyst formation was significantly (P < 0.05) lower (20.5%) in embryos cultured in KSOM-PVA than in other culture media (33.3-38.5%). The rate of hatching/hatched blastocysts was significantly (P < 0.05) lower in KSOM-PVA (13.9%) and KSOM-FBS (17.1%) than in KSOM-BSA (30.8%) and mSOF-BSA (33.9%). The numbers of total and trophectoderm cells (104.6 +/- 32.2 and 71.7 +/- 25.5, respectively) were significantly (P < 0.05) lower in blastocysts cultured in KSOM-PVA than in KSOM-BSA (125.7 +/- 39.7 and 91.7 +/- 36.2, respectively). In Experiment 3, no significant differences in embryo development, GFP expression and blastocyst cell numbers were observed among the culture groups. In conclusion, the present study demonstrated that KSOM and mSOF supplemented with BSA were equally effective in supporting development of bovine non-transgenic and transgenic cloned embryos. Moreover, different developmental competence in response to protein supplementation of KSOM was observed between bovine non-transgenic and transgenic cloned embryos.     

A serum-free medium for use in a cumulus cell co-culture system for bovine embryos derived from in vitro maturation and in vitro fertilization

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the >or= 2-cell (51 to 60%), >or= 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.     

Optimization of culture medium for cloned bovine embryos and its influence on pregnancy and delivery outcome

This study was conducted to establish an effective culture system for supporting in vitro development of cloned bovine embryos and to evaluate whether improved development in the optimal culture system could contribute to enhancing pregnancy and delivery outcomes after transfer. Enucleated oocytes at the metaphase II stage were reconstructed with serum-starved ear fibroblasts and cloned embryos were subsequently cultured for 168 h in vitro. In Experiment 1, cloned embryos were cultured in either modified Charles Rosenkrans 2 amino acid medium (mCR2aa) or modified synthetic oviduct fluid medium (mSOF). More (P < 0.05) 2-cell embryos (78% versus 92%), morulae (51% versus 69%) and blastocysts (2% versus 39%) were obtained after culture in mSOF than after culture in mCR2aa. In Experiment 2, cloned embryos were successively cultured in mSOF supplemented with various macromolecules during different periods of culture. A successive culture of oocytes in BSA-containing medium for 72 h and then in FBS-containing medium for the next 96 h yielded a higher rate of blastocyst formation (49% versus 25-36%) than other combinations (BSA to BSA or PVA to PVA, BSA or FBS). This macromolecule supplementation also significantly increased the number of total blastomeres (117.3 cells/blastocyst) and inner cell mass cells (ICM, 49.7 cells/blastocyst), and the ratio of ICM cells to trophoblast cells (TB, 0.98). In Experiment 3, a total of 85 blastocysts obtained from each 2-step culture were transferred individually to recipient cows at the end of the culture period and 32 pregnancies (38%) were diagnosed on Day 60 after transfer. However, no (P > 0.05) significant differences due to culture were apparent in the pregnancy outcome. Although six calves were produced using the 2-step culture regime of either BSA-BSA or PVA-FBS, no calves were produced using the successive culture of BSA then FBS, which optimized preimplantation development. In conclusion, mSOF has more potential to support the development of clone embryos than mCR2aa, and successive supplementation of BSA and FBS to mSOF further promotes blastocyst formation. However, enhanced development in vitro might not directly contribute to improving pregnancy outcomes.     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

Culture of bovine embryos to the blastocyst stage using Buffalo rat liver (BRL) cells

A comparison was made between the development of in vitro matured and fertilized bovine oocytes in co-culture with bovine oviduct epithelial (BOE) cells or with Buffalo rat liver (BRL) cells. Both cell types supported development from the 1-cell to the blastocyst stage with equal efficiencies (4.4% for BRL cells, 4.0% for BOE cells). Medium conditioned by either cell type supported development to the blastocyst stage as efficiently as co-cultures (6.4 and 7.3% blastocysts for BOE and BRL conditioned medium, respectively). A higher percentage of blastocyst development was found when embryos were cultured closely apposed in small drops of BRL-conditioned medium compared with larger volumes (20.5 versus 7.0%). The ability of BRL-conditioned medium to support embryonic development was dependent on the duration of the conditioning period (optimum 24 to 48 h), and was not lost when the medium was stored at -20 degrees C for extended periods. The effects were independent of the conditions used to promote maturation in vitro and the procedure for fertilization. With 2 different methods to produce embryos in culture, both the BRL cell co-culture and BRL-conditioned medium in microdrops supported embryo development to the blastocyst stage. The use of the BRL cell line reduces the variability associated with primary BOE cell cultures.     

Development, chromosomal composition, and cell allocation of bovine cloned blastocyst derived from chemically assisted enucleation and cultured in conditioned media

Treatment of in vitro matured bovine oocytes with colcemid results in a membrane protrusion that contains maternal chromosomes, which can be easily removed by aspiration. Four experiments were designed to evaluate the overall and temporal effects of conditioned medium (CM) by bovine cumulus cells on development of nuclear transfer (NT) bovine embryos and to examine the chromosomal composition and allocation of inner cell mass (ICM) and trophectoderm (TE) of the subsequent blastocysts. The nuclear transfer embryos were cultured in various CR1aa media conditioned by preculture with bovine cumulus cells. Development to the blastocyst stage in BSA-containing CM (BCM) and serum-containing CM (SCM) were similar to co-culture group (24-30%). The 24 hr-conditioned BCM yielded higher blastocyst development than 48 and 72 hr-conditioned BCM. Temporary exposure of embryos to BCM and SCM followed by CR1aa was also studied. Morula and blastocyst development were not different among the groups cultured in BCM for 72, 96, and 168 hr, but were significantly higher (P < 0.01) than groups exposed to BCM for 24 and 48 hr, respectively. Blastocyst development in SCM for 24 hr (29%), 96 hr (25%), and 168 hr (27%) were much higher (P < 0.05) than those in SCM for 48 hr (12%) and 72 hr (10%). The analyses of chromosomal composition of the resulting blastocysts indicate approximately 80% of the blastocysts cultured in CR1aa with co-culture or groups initially exposed to BCM for 24 hr followed by culture in CR1aa were diploid. However, the incidence of diploidy were only 36-60% in SCM-cultured groups and groups cultured in BCM beyond 48 hr. Conditioned media did not affect the allocation of ICM and TE in the blastocyst. No difference was found in the ratio of inner cell mass to total cells in co-culture, BCM or SCM groups (0.424, 0.441, and 0.473, respectively). In conclusion, bovine cumulus cell-CM and CR1aa with co-culture supported comparable development and blastocyst ICM:total cell ratio of bovine NT embryos. However, CM affected the blastocyst chromosomal composition and induced higher mixploidy.     

Influence of protein supplements on the secretion of leukaemia inhibitory factor by mitomycin-pretreated Vero cells: possible application to the in vitro production of bovine blastocysts with high cryotolerance

A co-culture system for bovine embryos using mitomycin-treated Vero cells and serum-supplemented modified synthetic oviduct fluid (mSOF) supports the development of in vitro maturation and fertilization-derived oocytes to hatched blastocysts. In this system, it has been suggested that one contribution made by the co-culture cells to embryo development is production of the cytokine leukaemia inhibitory factor (LIF). However, there are concerns about exposure of early embryos to serum due to its incompatibility with embryo cryosurvival. In this study, the influence of two protein supplements (synthetic serum substitute (SSS), a lipid-free human serum-derived product) and oestrous cow serum (ECS)) on Vero cell LIF secretion was compared, with the aim of designing a co-culture system that is supportive of bovine embryo cryopreservation. Vero cells cultured for 72 h in medium 199 + 5% fetal bovine serum (FBS) (recommended maintenance medium for this cell line) secreted detectable amounts of LIF (13.1 +/- 0.9 pg LIF per 10(5) cells). Culture in mSOF, the medium routinely used in this laboratory for embryo culture, also supported LIF secretion in Vero cells. However, the amount of LIF was tenfold higher (24.7 +/- 6.2 pg LIF per 10(5) cells; P < 0.05) when mSOF was supplemented with 10% (v/v) ECS compared with supplementation with 2% (v/v) SSS. Results of a second series of experiments in which supplementation with each protein was normalized to 10% revealed similar differences in LIF secretion, indicating that LIF secretion was affected by the type, not the amount, of protein. Time course analysis revealed stepwise increases (P < 0.05) in cumulative LIF secretion with every 24 h of culture in mSOF + either SSS or ECS. In terms of embryo development and post-cryopreservation viability, medium supplementation with 2% (v/v) SSS alone versus the two-step system of 2% (v/v) SSS (days 1-4) + 10% (v/v) ECS (days 4-10) had no influence (P > 0.05) on the ability of bovine blastocysts to hatch, with or without intervening cryostorage. However, the rate of blastocyst formation (expressed as the percentage of cleaved embryos) was only 27% in the presence of 2% (v/v) SSS, and increased almost twofold (P < 0.05) when ECS was added beginning on day 4 of co-culture. In summary, Vero cell LIF secretion was increased markedly by ECS. A two-step system of medium supplementation, in which embryos are exposed to ECS beginning on day 4 of in vitro development combined high rates of blastocyst formation with cryotolerance. This effect may be a result of limiting embryo exposure to serum-derived lipid until after the eight-cell stage and providing an increase in LIF during the critical developmental stages of compaction and cavitation.     

Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media

This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos.     

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.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P = 0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P /= 0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P = 0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P < 0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P = 0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P >/= 0.535).     

Increasing the rate of blastocyst formation and hatching from in vitro-produced bovine zygotes

This study was designed to identify parameters that would facilitate early selection of superior embryos, as well as to define culture conditions that could increase the proportion of embryos proceeding to the blastocyst stage. In the first experiment, the developmental potential of bovine embryos that had reached different stages of development after 60 h of culture following insemination was assessed. No 2-cell embryos underwent further cleavage. Of the 4-cell embryos (n = 188) only 12.2% progressed to the blastocyst stage, while 62.5% of 8-cell embryos (n = 480) did so (P < 0.01). In a further experiment, the effects of conditioning the culture medium (TCM 199) either with Buffalo rat liver cells (BRLC) or bovine oviductal epithelial cells (BOEC) and the effects of co-culture with either of these 2 cell types were examined. The percentage of 8-cell embryos proceeding to the morula and blastocyst stages was independent of cell type and culture system. However, BOEC-conditioned medium supported significantly lower production of blastocysts than any of the other culture methods. Only 24.1% of the former proceeded to the blastocyst stage after the full 10 d of culture, and only 3% hatched, values that were significantly lower than in the other 3 groups (P < 0.01). Among the latter, 44% progressed to the blastocyst stage in BRLC-conditioned medium while 44 and 46% reached that endpoint after co-culture with BOEC or BRL cells, respectively. The percentages that hatched among these were 28.2, 31 and 28.5%, respectively.     

Improvement of in vitro co-culture systems for bovine embryos using a low concentration of carbon dioxide and medium supplemented with beta-mercaptoethanol

Two experiments were conducted to investigate the effect of carbon dioxide (CO2) gas atmosphere and beta-mercaptoethanol on the development of bovine embryos in an in vitro co-culture system. In Experiment 1, in vitro-matured bovine oocytes were inseminated and then co-cultured with cumulus cells in culture medium (CM; 25 mM HEPES buffered TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate). Oocytes matured and fertilized in 2 or 5% CO2 in air exhibited similar cleavage rates, but the proportion of embryos that developed to the blastocyst stage was higher for embryos co-cultured in 2 versus 5% CO2 in air. In Experiment two, 4- to 8-cell embryos produced under the condition of 2% CO2 in air were co-cultured with cumulus cells in CM supplemented with various levels of beta-mercaptoethanol (0, 5, 10, 50 microM). The percentage of embryos that developed to the blastocyst stage in CM with 10 microM beta-mercaptoethanol was higher (P<0.05) than that of embryos co-cultured with 0 or 50 microM beta-mercaptoethanol. These results indicate that cumulus cell co-culture in an atmosphere of 2% CO2 in air has a marked stimulatory effect on in vitro development of bovine embryos and that addition of beta-mercaptoethanol to the co-culture medium 2 d after insemination improved the in vitro development of bovine 4- to 8-cell embryos to the blastocyst stage.     

Intracytoplasmic glutathione concentration and the role of beta-mercaptoethanol in preimplantation development of bovine embryos

In vitro-matured/in vitro-fertilized bovine oocytes were cultured on cumulus cell layers in a serum-free medium (bovine embryo culture medium; BECM) supplemented with 3 mg/ml fatty acid-free BSA. The intracytoplasmic glutathione concentration of embryos was found to change significantly (P < 0.008) during the preimplantation stages, beginning to increase at the 9- to 16-cell stage (20.7 pM/embryo) and reaching the highest (P < 0.03) level at the hatched-blastocyst stage (36.7 pM/embryo). A significantly (P < 0.06) lower concentration of glutathione was obtained at the 2- to 8-cell stage (7.1 pM/embryo) than at any other stage. When inseminated oocytes were cultured in BECM supplemented with different concentrations of beta-mercaptoethanol (2-ME) to promote glutathione synthesis, higher (P < 0.05) percentages of embryos developed to the 9- to 16-cell, morula and blastocyst stages at 96, 144 and 192 h post insemination, following the addition of 6.25 and 12.5 microM than after no supplementation with 2-ME. However, when 16-cell embryos were cultured in BECM supplemented with 6.25 and 12.5 microM of 2-ME, blastocyst formation was not significantly (P > 0.9) increased. When the combined effects of 2-ME and/or cumulus cells were compared in a 2 x 2 factorial design, there was a significant (P < 0.03) effect of 2-ME on the development of oocytes to blastocysts. The presence of cumulus cells significantly (P < 0.001) affected development after the fourth cleavage (morula compaction and blastocyst formation), but there was no significant (P > 0.11) interaction between 2-ME and cumulus cells. In conclusion, intracytoplasmic glutathione concentration of bovine embryos derived from in vitro-culture increases during preimplantation development. The glutathione synthesis promoter 2-ME exerts its embryotropic role on the development before the fourth cleavage, thus yielding an improvement in blastocyst formation.     

The differential requirement of albumin and sodium citrate on the development of in vitro produced bovine embryos

In vitro culture for bovine embryos is largely not optimal. Our study was to determine the components necessary for early embryo development. In experiment 1, IVF embryos were cultured for two days in CR1aa medium containing sodium citrate and BSA from two sources (Sigma vs. ICPbio), subsequently for additional five days with cumulus monolayer in 10% FBS CR1aa. We found that supplementation with both Sigma-BSA and sodium citrate significantly increased total blastocyst (BL) development compared with the ICPbio-BSA groups (37% vs. 19-21%), and enhanced the total number of high quality (C1 BL, IETS standard) blastocysts (26% vs. 11-17%) (P < 0.05). In experiment 2 with serum free and/or somatic free culture, we found that CR1aa culture can support a comparable embryo development with a supplement of Sigma BSA. The addition of sodium citrate did not increase blastocyst development in either the Sigma-BSA or the ICPbio-BSA groups. An inferior blastocyst development occurring in ICPbio-BSA culture (1-3%) could be rescued by culture in CRlaa supplemented with 10% FBS (29%), more importantly, by culture in CR1aa with a replacement of Sigma BSA (24%) (P <0.05). C1 blastocysts rescued by FBS and Sigma BSA in ICPbio-BSA culture possessed indistinguishable morphology to embryos developed in a Sigma-BSA, FBS and somatic co-culture system, showing similar cell number/blastocyst (129-180, P > 0.05). Our study found a beneficial effect of sodium citrate and BSA on the in vitro development of bovine IVF embryos during co-culture. We also determined that differential embryotrophic factor(s) contained in BSA and serum, probably not sodium citrate, is necessary for promoting competent morula and blastocyst development in cattle.     

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos.