The quality after culture in vitro or in vivo of porcine oocytes matured and fertilized in vitro and their ability to develop to term

The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro‐matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) (‘ET‐vivo’ embryos), or cultured in vitro for 5 or 6 days (‘IVC’ embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET‐vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term.     

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.     

Effect of varying glucose concentrations during in vitro maturation and embryo culture on efficiency of in vitro embryo production in buffalo

This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm ) along with a control (0 mm ) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC‐I) (12–72 hpi) and IVC‐II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC‐I and IVC‐II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.     

Improvement of the developmental ability of nuclear transfer embryos by using blastomeres from in vitro fertilized embryos selected according to the early developmental stage and cell division status as donor cells in cattle

This study was conducted to improve the developmental ability of nuclear transfer (NT) embryos by using blastomeres from in vitro fertilized (IVF) embryos with high quality as donor cells. The IVF embryos selected at the 2-cell stage at 24-h postinsemination (hpi) and again at the ≥8-cell stage at 48 hpi (Selected-IVF-embryos) showed the highest blastocyst formation rate among embryos. When blastomeres from the Selected-IVF-embryos (Selected-NT group) or Nonselected-IVF-embryos (Non-selected-NT group) were used as donor cells for NT, the blastocyst formation rate in the Selected-NT group (25.6%) was significantly higher than that in the Non-selected-NT group (13.5%). When blastomeres from the Selected-IVF-embryos at 108 (contained many cells before cell division) and 126 hpi (contained many cells immediately after cell division) were used as donor cells for NT (108- and 126-NT groups, respectively), the 126-NT group showed a significantly higher blastocyst formation rate (32.1%) than the 108-NT group (16.8%). Embryo transfer of blastocysts in the 126-NT group showed that 11 of 23 recipients became pregnant; nine calves were obtained. For the NT embryos reconstructed using in vivo derived embryos, 9 of 20 recipients became pregnant; seven calves were obtained. These results indicate that the blastocyst formation rate of NT embryos can be improved by using blastomeres from IVF embryos selected at the early developmental stage, especially immediately after cell division, and that the resultant NT embryos have a high developmental ability to progress to term that is comparable to NT embryos reconstructed using in vivo derived embryos.     

Telomerase Activity in Swamp Buffalo (Bubalus bubalis) Oocytes and Embryos Derived from In Vitro Fertilization, Somatic Cell Nuclear Transfer and Parthenogenetic Activation

The objective of this study was to examine the telomerase activity in swamp buffalo oocytes and pre-implantation stage embryos derived from in vitro fertilization (IVF), somatic cell nuclear transfer (NT) and parthenogenetic activation (PA). Immature and mature oocytes, and embryos at the 2-4 cell, 8-16 cell, morula and blastocyst stages produced by IVF, NT and PA were collected and the telomerase activity was assayed by using a Telomerase PCR ELISA kit. Telomerase activity was detected in all developmental stages evaluated from immature oocytes to blastocyst stage embryos. Telomerase activity was detected in higher amounts in immature as compared with mature oocytes (p < 0.05). Embryos derived from NT showed a profile of telomerase activity similar to that of IVF. In IVF and NT embryos, telomerase activity was low in the 2-4 cell and 8-16 cell stages, but the activity significantly increased (p < 0.05) at the morula stage, reaching its highest level at the blastocyst stage. In PA embryos, low levels of telomerase activity were detected from the 2-4 cell to the morula stage, and the highest level of telomerase activity was found at the blastocyst stage. Telomerase activity in NT blastocysts is higher than that derived from IVF and the activity is highest in PA blastocysts. These results suggest that the successful reprogramming of telomerase activity in buffalo NT embryos follow a pattern similar to that in embryos derived from IVF and PA.     

Effects of the Reproductive Status on Morphological Oocyte Quality and Developmental Competence of Oocytes after In Vitro Fertilization and Somatic Cell Nuclear Transfer in Cat

This study was conducted to examine the effects of the reproductive cycle of donor cat on the quality of oocytes at recovery and developmental competence of oocytes after in vitro fertilization (IVF) and somatic cell nuclear transfer (NT). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular or luteal stages. After collection of oocytes, the oocytes were classified into four grades according to the morphological condition of oocyte cytoplasm and cumulus cells. A total of 16 558 oocytes were obtained from 198 ovarian pairs. The total mean numbers of oocytes and the mean numbers of oocytes with high quality (grade I) were significantly higher in ovarian pairs at the inactive stage (111.1 and 19.0 oocytes, respectively) than in ovarian pairs at the follicular stage (67.1 and 11.4 oocytes, respectively). A significant difference in the proportions of oocytes with grade I out of the total examined oocytes was observed between the follicular and luteal stages of ovaries (14.9% vs 20.2%, p < 0.05). The proportions of IVF embryos cleaved and developed to blastocysts significantly decreased with decreased quality of oocytes at recovery, irrespective of the reproductive status of ovaries. Moreover, there were no significant differences in the proportions of cleavage and development to the blastocyst stage of IVF and NT embryos among three oestrous stages of ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries has no apparent effects on the in vitro development of oocytes after IVF and NT, but the quality of oocytes at recovery influences the development of IVF embryos.     

Optimizing injection time of GFP plasmid into sheep zygote

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.     

Effects of glutathione treatments during sperm washing and in vitro fertilization on the in vitro early development of embryos of Japanese Black cattle

This study was conducted to examine the effects of adding glutathione (1 mM) to media used for sperm washing and in vitro fertilization (IVF) on the improvement of early development of embryos produced using cryopreserved spermatozoa of the less IVF-competent bull (the one considered unqualified as spermatozoa supplier for the production of bovine blastocysts using IVF). The cryopreserved spermatozoa of this bull were characterized by normal motility and lower ATP content and blastocyst productivity than those of IVF-competent bulls. The addition of glutathione to the sperm washing medium was more effective in improving the productivity of blastocysts and ATP content than the addition of glutathione to the IVF medium or no glutathione addition at all (control). These results suggest that this simple method may be used to improve the potential of cryopreserved spermatozoa of less IVF-competent bulls to fertilize oocytes in vitro and to induce normal embryonic development after fertilization.     

Effects of purple sweet potato anthocyanins on development and intracellular redox status of bovine preimplantation embryos exposed to heat shock

The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0=day of IVF) with 0, 0.1, 1 and 10 microg/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 microg/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P<0.05) and an increase in intracellular ROS (P<0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 microg/ml anthocyanins significantly (P<0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P<0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 microg/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.     

Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo

The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.     

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.     

Medium without ammonium accumulation supports the developmental competence of human embryos

L-Glutamine has been shown to play an important role during in vitro culture of mammalian embryos. However, it is easily decomposed into ammonium, which is believed to have deleterious effects on preimplantation embryos. In this study, we assessed prospectively the developmental competence of human embryos cultured in medium containing L-glutamine or a novel stable glutamine derivative and vitamins. The subjects of this study were 41 women who underwent IVF/ET treatment from September to November 2006 and from whom 6 or more oocytes were retrieved. Sibling oocytes were randomly divided into EA/BA (EmbryoAssyst and BlastAssyst containing a novel stable glutamine derivative and vitamins), and BAS groups (BlastAssyst system containing L-glutamine). There was no difference in pronuclear formation rate between EA/BA and BAS (74 vs. 69%). The blastulation rates of embryos based on the number of zygotes cultured in EA/BA on Days 5 (Day 0=insemination, 54%) and 6 (63%) were significantly higher (P<0.05) than those cultured in BAS (Day 5: 33% and Day 6: 45%, respectively). The present data indicate that a medium containing a novel stable glutamine derivative and vitamins supports the developmental competence of human embryos.     

Birth of piglets through the non-surgical transfer of blastocysts produced in vitro

In this study, we attempted to produce piglets by non-surgically transferring blastocysts produced in vitro, using a flexible catheter as the transfer instrument. Cumulus-oocyte complexes (COCs) were aspirated from the follicles of ovaries obtained at a local slaughterhouse. They were then matured in modified North Carolina State University (NCSU)-37 medium for 44-46 h and fertilized in porcine gamete medium (PGM). Ten hours after in vitro fertilization (IVF), presumptive zygotes were removed from the cumulus cells and cultured in porcine zygote medium (PZM)-5. Blastocysts were cultured for five days after IVF and, using a catheter for deep intrauterine insemination without sedation, they were transcervically transferred into the uterine horn of six recipients (45-50 blastocysts/recipients) whose estrous cycles were synchronized, at 5 days after human chorionic gonadotropin (hCG) injection. Of the six recipients, one sow became pregnant and farrowed seven piglets (four live piglets) 119 days after hCG injection. The body weight at birth of the newborns ranged from 0.8 to 1.4 kg. These results indicate that it is possible to obtain piglets by transcervically transferring blastocysts produced by IVF and in vitro cultures in chemically defined media.     

Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or G1 phase after intracytoplasmic nuclear injection in cattle

We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.     

Effect of replacement of pyruvate/lactate in culture medium with glucose on preimplantation development of porcine embryos in vitro

The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.     

Blastocyst production from in vitro-produced day-2 bovine embryos classified by cleavage stage, and cytogenetical evaluation of the resultant day-8 blastocysts

The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8+/-42.4, P<0.05), largest number of metaphases per blastocyst (9.5+/-4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6+/-33.4, P<0.05), a large number of metaphases per blastocyst (11.9+/-5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.     

Effect of Growth Factors on Bovine Blastocyst Development in a Serum-Free Medium

Shamsuddin, M.: Effect of growth factors on bovine blastocyst development in a serum-free medium. Acta vet. scand. 1994, 35, 141-147. - To investigate the effect of growth factors on pre-implantation development, bovine zygotes, produced by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes, were cultured in a serum-free medium to which the following growth factors were added one at a time: epidermal growth factor (EGF), acidic fibroblast growth factor (a-FGF), insulin-like growth factor-II (IGF-II), platelet-derived growth factor from human platelets (PDGF), and platelet-derived growth factor-AB, human, recombinant (PDGF-AB). All growth factors were added at a dose of either 10 or 50 ng/ml, except PDGF which was added at a dose of either 5 or 15 ng/ml. The control medium was TCM 199 supplemented with sodium pyruvate (0.25 mmol/1), BSA (10 mg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), and sodium selenite (5 ng/ml). Embryos were cultured for 8 days (day of insemination = Day 0). The mean percentages of first cleavage on Day 2 varied from 67% to 86% and the differences between the 2 doses, or between the control and growth factor- treated groups were not significant (p≥0.13). The effects of the two doses on subsequent development up to the blastocyst stage did not differ either (p≥0.12). There was no stimulatory effect of any of the used exogenous growth factors on embryo development up to the morula or blastocyst stage on Day 7, or blastocyst stage on Day 8. Moreover, medium supplemented with PDGF had fewer blastocysts than the control (p≤0.03). The results indicate that growth factor supplementation may not necessarily increase the yield of blastocysts from bovine IVM-IVF oocytes in a serum-free medium.     

Different Culture Media Requirements of IVF and Nuclear Transfer Bovine Embryos

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.