Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro

The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.     

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.     

Effect of protein supplementation and presence of an antioxidant on the development of bovine zygotes in synthetic oviduct fluid medium under high or low oxygen tension

The aim of this study was to investigate the effect of protein supplementation of culture medium and the presence of a putative antioxidant on bovine zygote development under 5% (low) and 20% (high) O2. In Experiment 1, presumptive zygotes (n=992) were cultured in synthetic oviduct fluid (SOF) alone or supplemented with 3 mg/mL PVP, 3 mg/mL BSA (SOFB), and/or 10% FCS (SOFBF) in 5% CO2, 5% O2, 90% N2. In Experiment 2, zygotes (n=1916) were cultured in SOF, SOFB or SOFBF with or without taurine under high and low O2. In Experiment 1, presence of BSA or BSA plus FCS significantly increased the speed of development compared to SOF or SOF+PVP. Blastocyst quality was also improved, as evidenced by increased hatching rate and cell numbers. In Experiments 2, taurine had no effect on development irrespective of oxygen concentration or protein supplementation. In conclusion, the presence of protein in the culture medium and culture under reduced O2 significantly improved embryo development. Taurine had no effect on development.     

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.     

Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival

Two experiments were conducted to examine the effect of membrane stabilization through the modification of in vitro culture medium or freezing medium on post-thaw survival of in vitro-produced bovine embryos. In Experiment 1, Day 7 (Day 0 = day of IVF) late morulae and blastocysts that developed following culture in SOF/aa/BSA (IVC medium) were frozen slowly to -35 degrees C in the presence of 1.5 M ethylene glycol prepared in ovum culture medium (OCM) or in OCM supplemented with 10, 25 or 50% fetal calf serum (FCS) or 5, 10 or 25 mg/mL BSA. Post-thaw survival was assessed by re-expansion and/or hatching following 48 h of culture in IVC medium + 10% FCS. Overall, survival was significantly (P < 0.01) affected by embryo stage, with more hatched blastocysts surviving (71%) than blastocysts (59%) or late morulae (51%). Addition of FCS significantly (P < 0.01) reduced survival compared with control embryos or those frozen in BSA-supplemented medium (50.48 vs 68.01 vs 63.53%, respectively). There was also a significant interaction between embryo stage and protein type (P < 0.05). The survival of late morulae/early blastocysts following freezing was improved in the presence of additional BSA but not FCS. In Experiment 2, the IVC medium was supplemented with liposomes containing lecithin, sphingomyelin and cholesterol. Sphingomyelin and cholesterol at ratios of 1:1, 1:4 and 4:1 were added to 50, 100 or 150 micrograms/mL lecithin to yield a final lipid concentration of 200 micrograms/mL. A further group contained 200 micrograms/mL lecithin only. Blastocysts were frozen in 1.5 M ethylene glycol in OCM, then thawed and assessed as in Experiment 1. The presence of liposomes during IVC did not affect the proportion of cleaved embryos that developed to blastocysts or survival following freezing. However, the survival of blastocysts that developed in the presence of 200 micrograms/mL lecithin only was significantly lower than in any other treatment (6%; P < 0.03). These studies demonstrate that the protein composition of the freezing medium can significantly affect survival after thawing and that the survival of late morulae can be improved with additional BSA. The presence of lecithin only in the liposome preparation did not affect embryo development, but significantly reduced survival after freezing, suggesting it can affect post-thaw embryo survival, perhaps by altering embryonic membrane composition.     

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.     

Effects of platelet-derived growth factor (PDGF) on the in vitro production of bovine embryos in protein-free media

The purpose of our experiments was to explore the effects of platelet-derived growth factor (PDGF)-supplementation at the various steps of in vitro production of bovine embryos using protein-free media. Cumulus-oocyte-complexes (COC) were collected by slicing abattoir ovaries and then dividing the COC into 2 morphological categories. After maturation for 24 h in TCM-199 supplemented with hormones and either 20% estrous cow serum (ECS) or 1 mg/ml polyvinyl-alcohol (PVA), oocytes were co-incubated for 19 h with frozen/thawed spermatozoa from bull of proven fertility. The semen was diluted in Fert-Talp supplemented with heparin, hypotaurine and epinephrine and either 6 mg/ml bovine serum albumin (BSA) or 1 mg/ml PVA. Presumptive zygotes were transferred into embryo culture medium containing either 20% ECS or 1 mg/ml PVA for a total of 10 d. The PDGF was added at concentrations of 1, 10 or 100 ng/ml to the maturation medium (Experiment 1), fertilization medium (Experiment 2) or culture medium from Day 1 on (Experiment 3), respectively, or at 1 ng/ml PDGF to both the fertilization and culture medium from Day 3 on (Experiment 4), with each medium supplemented with PVA. Oocytes/embryos incubated in the absence of PDGF in media supplemented with either ECS or PVA served as controls. An average of 20 COC was incubated in 1 droplet under silicone oil, and each experiment contained 4 to 6 replicates. No significant differences were found among the various concentrations of PDGF, nor did PDGF-supplementation during maturation (Experiment 1) or embryo culture on Day 1 (Experiment 3) significantly affect development of oocytes/embryos (34.7 +/- 3.5 to 40.4 +/- 2.5% morulae, 11.9 +/- 2.4 to 18.8 +/- 2.5% blastocysts; and 23.2 +/- 2.3 to 27.5 +/- 3.4% morulae, 11.5 +/- 2.6 to 12.7 +/- 2.3% blastocysts, respectively; x +/- SEM). In the presence of 10 ng/ml PDGF in the fertilization medium development to morulae and blastocysts was similar to that of the ECS-group, and was higher (P < 0.05) than that of the PVA-control (ECS: 32.1 +/- 4.6 and 13.8 +/- 2.7%; PVA: 17.5 +/- 0.8 and 6.1 +/- 1.3%; PDGF: 30.6 +/- 3.0 and 14.0 +/- 2.2%, respectively). Development to morulae/blastocysts was increased, and was at the same level as in the ECS-group when the fertilization and/or embryo culture medium on Day 3 contained PDGF compared with the PVA-control group (morulae: ECS 25.3 +/- 4.4%, PVA 13.9 +/- 2.2% [P < 0.05], PDGF 16.7 +/- 3.2 to 19.1 +/- 1.1%; blastocysts: ECS 5.3 +/- 2.1%, PVA 5.0 +/- 1.7%, PDGF 7.1 +/- 1.6 to 9.1 +/- 1.7%, respectively). These results indicate that under our laboratory conditions PDGF can elevate low rates of development and the addition of PDGF to the fertilization medium enhances bovine preimplantation embryonic development. Thus, PDGF can be potentially an important factor in a completely defined medium to substitute the effects of serum.     

Factors affecting cryosurvival of nuclear-transferred bovine and swamp buffalo blastocysts: effects of hatching stage, linoleic acid-albumin in IVC medium and Ficoll supplementation to vitrification solution

The objective was to determine whether the hatching stage of cattle and swamp buffalo somatic cell nuclear-transferred (SCNT) blastocysts affected cryosurvival after vitrification, and whether addition of linoleic acid-albumin (LAA) to the IVC medium and Ficoll to the vitrification solution improves cryosurvival. Fused couplets were activated with ethanol and cycloheximide-cytochalasin D (day 0), and were allowed to develop in the presence of 0.3% BSA or 0.1% LAA+0.2% BSA. Hatching blastocysts were harvested at day 7.0 (cattle) or day 6.5 (buffalo), and classified into one of three categories, according to the ratio of extruding embryonic diameter from zona to embryonic diameter inside the zona. The blastocysts were vitrified in 20% DMSO+20% ethylene glycol+0.5M sucrose, with or without 10% Ficoll in TCM199+20% FBS, using Cryotop as a cryodevice. The post-thaw survival of the blastocysts was assessed by in vitro culture for 24h. In cattle, when the LAA-supplemented IVC medium and the Ficoll-free vitrification solution were used, cryosurvival of the early-hatching blastocysts (77%) was not different from those of middle- and late-hatching blastocysts (74 and 80%, respectively). Inclusion of Ficoll in the vitrification solution did not improve the cryosurvival of SCNT blastocysts (54 to 68%). Early-hatching SCNT blastocysts produced in the absence of LAA were sensitive to the vitrification procedure (cryosurvival 56%; P<0.05 versus 80% in the late-hatching blastocysts). The full-term developmental potential of SCNT blastocysts was proven only in the non-vitrified control group. In buffalo, the mean cryosurvival of hatching SCNT blastocysts produced with LAA (89%) was not different from that of those produced without LAA (87%). In conclusion, bovine SCNT blastocysts, regardless of their hatching stage, were relatively resistant to vitrification by the ultra-rapid cooling procedure when the blastocysts were produced in the presence of LAA. Furthermore, swamp buffalo SCNT blastocysts were more tolerant of vitrification than bovine SCNT blastocysts.     

Effect of human leukemia inhibitory factor on in vitro development of IVF-derived bovine morulae and blastocysts

This study was conducted to investigate whether human leukemia inhibitory factor (hLIF) improves the subsequent development of IVF-derived bovine morulae and blastocysts. To obtain IVF-derived bovine morulae, ova were matured and fertilized in vitro and cultured in 0.5 ml of synthetic oviduct fluid (SOF) medium supplemented with 10% human serum (HS) for 5 d at 39 degrees C under a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2). Morulae and early blastocysts at Day 5 of culture were cultured in 0.5 ml of SOF medium with or without 5000 U/ml recombinant hLIF for 2 or 3 d (2 groups). To investigate the effect of addition of hLIF on the subsequent development of morulae, SOF medium was supplemented with 8 mg/ml BSA instead of HS. To test whether hLIF affects the subsequent development of IVF-derived bovine blastocysts, only good blastocysts that developed from SOF medium with or without hLIF at Days 7 and 8 of culture were frozen by a conventinal slow freezing method and again cultured in SOF medium with or without the addition of hLIF for 3 d after thawing (4 groups). Survival of frozen-thawed bovine embryos was evaluated for re-expansion and hatching of blastocysts during 3 d of culture. There was no significant difference in the developmental rate of Day 5 embryos to blastocysts between those cultured with (47.8%) and without (47.6%) addition of hLIF. However, the addition of hLIF before freezing significantly increased the hatching rate of IVF-derived bovine morulae (P < 0.05), whereas addition of hLIF after thawing did not increase the subsequent development of blastocysts. These results suggest that hLIF added at the Day 5 morula stage may contribute to bovine embryonic development through the hatching process.     

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.     

Effect of cooling and warming rates during cryopreservation on survival of in vitro-produced bovine embryos

The effect of cooling and warming rates during cryopreservation on subsequent embryo survival was studied in 607 bovine morulae and 595 blastocysts produced by in vitro maturation, fertilization and culture (IVM/IVF/IVC). Morulae and blastocysts were prepared by co-culturing presumptive zygotes with bovine oviductal epithelial cells (BOEC) in serum-free TCM199 medium for 6 and 7 d, respectively. The embryos in 1.5 M ethylene glycol in plastic straws were seeded at -7 degrees C, cooled to -35 degrees C at each of 5 rates (0.3 degrees, 0.6 degrees , 0.9 degrees, 1.2 degrees, or 1.5 degrees C/min) and then immediately plunged into liquid nitrogen. The frozen embryos were warmed either rapidly in a 35 degrees C water bath (warming rate > 1,000 degrees C/min) or slowly in 25 degrees to 28 degrees C air (< 250 degrees C/mm). With rapid warming, 42.1% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts. The proportions of rapidly wanned morulae that hatched decreased with increasing cooling rates (30.4, 19.0, 15.8 and 8.9% at 0.6 degrees , 0.9 degrees, 1.2 degrees and 1.5 degrees C/min, respectively). With slow warming 25.9% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts, while <10% of the morulae that had been cooled faster developed. The hatching rate of blastocysts cooled at 0.3 degrees C/min and warmed rapidly (96.3%) was higher than those cooled at 06 degrees and 0.9 degrees C/min (82.7 and 84.6%, respectively), and was also significantly higher than those warmed slowly after cooling at 0.3 degrees, 0.6 degrees or 0.9 degrees C/min (69.1, 56.6 and 51.8%, respectively). Cooling blastocysts at 1.2 degrees or 1.5 degrees C/min resulted in lowered hatching rates either with rapid (71.2 or 66 0%) or slow warming (38.2 or 38.9%). These results indicate that the survival of in vitro-produced bovine morulae and blastocysts is improved by very slow cooling during 2-step freezing, nevertheless, slow warming appears to cause injuries to morulae and blastocysts even after very slow cooling.     

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.     

Effects of microinjection-related treatments on the subsequent development of in vitro-produced bovine oocytes

The effects of gene injection-related handling on the subsequent development of in vitro-produced bovine oocytes were studied. In Experiment 1, centrifuged oocytes were stored in an injection chamber for 30 min on a warm (+39 degrees C) stage at 18, 22, 26 or 30 h post insemination. In Experiment 2, centrifuged oocytes were stored for 60, 90 or 120 min on a warm stage, while in Experiment 3 they were stored for 60 min on a warm or cool (+22 degrees C) stage. In Experiment 4, the centrifuged zygotes were injected with buffer either into the pronucleus or cytoplasm. Development to morulae and blastocysts at Day 7 was monitored. The results indicate that handling of oocytes either very early (18 h post insemination) or very late (30 h post insemination) significantly reduced development (P<0.05). The duration of the storage or the temperature during storage did not have any significant effect on the development of the embryos. Development (counted from the cleaved ova), however, was significantly lower (P<0.01) in pronucleus-injected than in cytoplasm-injected or control embryos (27.7, 45.5 and 44.0% morulae and blastocysts, respectively). The conclusion of this study is that the main reason for decreased development of pronucleus-injected, in vitro-produced bovine zygotes is the pronucleus-injection itself rather than injection-related handling or the overall damage caused by zygote piercing.     

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.     

Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.     

Effect of culture-medium supplementation with alpha-mannosidase and/or beta-N-acetyloglucosaminidase on in vitro bovine embryonic development

Glycosidases are enzymes with a potential role in embryonic development. The objectives of this study were to assess: (a) whether in vitro bovine embryonic development is affected by the addition of beta-N-acetyloglucosaminidase (beta-NAGASE) and/or alpha-mannosidase to the culture medium and (b) whether these enzymes are utilized by bovine embryos during their development in vitro. Bovine embryos were produced using standard methods of IVM, IVF and IVC. Presumptive zygotes were cultured in groups of 20 in 50 microl drops of SOF medium (plus 5% FBS after 24 h culture) incubated in 5% CO2, 5% O2 and 90% N2 at 38.5 degrees C. The groups of zygotes were allocated to four treatments in which the culture medium was supplemented with: (1) beta-NAGASE, (2) alpha-mannosidase, (3) beta-NAGASE plus alpha-mannosidase, and (4) control (no supplement). Embryos were evaluated and samples of culture medium collected and frozen prior to assay for glycosidases at day 7 of culture. The experimental design was a randomised block arrangement of 4 treatments x 7 replicates with 20 zygotes per plot (culture droplet). Data were analysed by ANOVA and presented as mean +/- S.E.M. The osmolarity of the control culture medium was 272 mOsm. This was increased to 279 mOsm by the addition of alpha-mannosidase, 424 mOsm by beta-NAGASE and 337 mOsm with a combination of the two enzymes. The beta-NAGASE supplemented medium and the combined supplement reduced (0%) the development of zygotes to morula or blastocyst stages (P < 0.002) relative to control medium (35.7 +/- 8.4%). Embryo development was also reduced to 21.9 +/- 3.2 (P< 0.002), relative to control, by alpha-mannosidase supplementation. The reduced embryo development in the beta-NAGASE-supplemented medium was attributed to increased osmolarity of the culture medium. Embryos appeared to utilize alpha-mannosidase because its concentration decreased from 600.95 +/- 174.03 IU/l in drops without zygotes/embryos to 211.01 +/- 71.59 IU/l in drops with zygotes/embryos. Other culture media supplementation showed no significant differences between droplets, with or without zygotes/embryos. It was concluded that beta-NAGASE increased medium osmolarity, embryos utilized alpha-mannosidase and both glycosidases (singly or in combination) inhibited the development of bovine zygotes to morulae/blastocysts.     

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.     

Effects of increased ambient temperature on the development of in vitro derived bovine zygotes

In this study, presumptive bovine zygotes were subjected to two consecutive 24-h cycles of heat treatment during the first 48 h (Experiment I) of in vitro culture (IVC) or 24h of heat treatment during the fourth day of IVC (Experiment II). In Experiment I, the percentage of heat treatment zygotes that developed to > or =8-cell stage embryos after 72 h IVC was 2.0% (n = 459) compared with 28.4% (n = 458) for the control zygotes (P<0.001). The subsequent yield of morulae or blastocysts after 144 h IVC for the heat treatment and control groups was 0.9% (n = 457) and 12.3% (n = 456) (P<0.001), respectively. These results demonstrate that heat treatment during the first 48 h of IVC significantly impaired embryo development. In Experiment II, the percentage of zygotes that developed into morulae and blastocysts following heat treatment during the fourth day of IVC was 4.5% (n = 468) compared to 10.5% (n = 456) for the control group (P<0.001). This study has demonstrated that in vitro heat stress during the critical stage of early embryo development significantly increases the incidence of early embryonic mortality.