Freezability of porcine blastocysts at different peri-hatching stages

The freezability of porcine peri-hatching stage blastocysts was investigated by the cryopreservation of embryos at -196 degrees C with 1.5 M glycerol and by thawing, followed by in vitro culture. Of 66 expanded blastocysts frozen, 34 (51.5%) developed in vitro after thawing, while only 2 (6.7%, P<0.05) of 30 earlier stage blastocysts survived freezing. After freezing of 85 hatched blastocysts with an embryonic diameter of 150 to 300 mum, 59 (69.4%) surviving embryos were obtained, whereas none of the 78 advanced staged hatched blastocysts (>300 mum) survived the cryopreservation. High post-thaw survival (32 39 , 82..1%) was obtained with in vitro-hatched blastocysts precultured in Whittingham's M-16 medium containing 12mg/ml bovine serum albumin (BSA). In contrast, none of the 14 in vitro-hatched blastocysts precultured in the M-16 medium supplemented with 15% fetal calf serum (FCS) survived freezing. Similarly 51 of 56 hatced blastocysts (diameter = 150 to 300 mum) precultured in the M-16 medium supplemented with BSA survived cryopreservation, compared with 3 of 26 embryos precultured in the medium supplemented with FCS (P<0.001). Because both groups of the embryos precultured with BSA or FCS possessed normal ability to develop after transfer (developmental rate = 61.1 and 93.3%), the improved freezability of the embryos precultured with BSA may relate to a favorable change of embryonic cell membranes during the culture period. It was concluded that in vitro-hatched blastocysts precultured in medium containing BSA and in vivo-hatched blastocysts at the appropriate stage of development could both tolerate deep freezing to -196 degrees C; however, a differece in the freezability of embryos between breeds of pig was suggested from a further experiment performed with German Landrace embryos.     

Use of a protein-free medium for the in vitro development of sheep embryos]

The employment of protein-free medium for the culture of ovine embryos collected at the 1-2 cell stage from superovulated ewes was investigated. For this purpose sheep zygotes were randomly allocated in four treatment groups: T1) CZB medium + bovine serum albumin (BSA) on sheep oviductal monolayer (SOM), T2) CZB + polyvinyl alcohol (PVA) + SOM, T3) CZB + PVA + SOM supplemented with inositol (I) and serine (S), T4) TCM 199 + 10% fetal calf serum + SOM. Standard culture conditions were 2 ml of medium in 35 mm Petri dishes, under 5% CO2 in air at 39 degrees C. The percentages of morulae and blastocysts were recorded after 4 and 7 days of culture. After 4 days of culture there was no significant difference (P > 0.05) in the percentage of morulae between embryos cultured in T1 (86%), T2 (85%), T3 (88.8%), and T4 (87.5%). After 7 days the percentages of blastocysts were T1 (70%), T2 (50%), T3 (55.5%) and T4 (46.8%). These data suggest that a protein-free medium, CZB + PVA and CZB + PVA + I + S, can support ovine preimplantation embryo development in vitro; however CZB medium supplemented with BSA enhances development to blastocyst.     

Culture of ovine embryos in the absence of bovine serum albumin

Bovine serum albumin (BSA), a relatively impure protein, is routinely used as a component of embryo culture media. Since media containing BSA are chemically undefined, it would be desirable to replace BSA with substitutes of similar activity which are either chemically better defined and/or better standardized than BSA. Two commercial products, Ultroser((R)) G (USG) and Solcoseryl((R)) (SOL), were evaluated as replacements for BSA in culture with respect to the development of ovine embryos in vitro. A total of 126 late 8-cell and early 16-cell embryos were distributed among modified Brinster's medium for ovum culture (BMOC-2) containing either 1.5% BSA, 2.0% USG or 2.0% SOL. All three culture media supported development of ovine embryos. Results indicate that 8- and 16-cell embryos will develop into blastocysts in a BSA-free medium containing either USG or SOL. A higher number of embryos developed into blastocysts in media containing BSA than in media containing USG or SOL, and more blastocysts hatched in media containing BSA. Although the overall degree of embryonic development was more advanced in BSA-supplemented media, the concentrations of USG and SOL that were used in this study may not have been optimal for ovine embryo culture.     

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.     

Beneficial effects of taurine on mouse zygotes developing in protein-free culture medium

The objectives of this study were to determine if mouse zygotes from outbred mice can develop in simple culture medium in the absence of bovine serum albumin (BSA), and if taurine can be used as a medium supplement to improve development. Zygotes from 2 stocks of outbred mice (CD-1 and CF-1) were cultured in simple embryo culture medium (TE medium) lacking BSA and with or without taurine (24 mM), or in regular TE medium with BSA. The presence of BSA had little or no effect on development, but development to post-blastocyst endpoints was enhanced when CD-1 zygotes were cultured in medium containing taurine. In addition, when CD-1 blastocysts were transferred to pseudopregnant animals, embryos cultured in the presence of taurine developed into fetuses more often than those cultured in medium without taurine, and their weights were higher than those of embryos cultured in regular TE medium with BSA. These beneficial effects of taurine do not appear to be the nonspecific effects of a fixed nitrogen source, because the addition of glycine to BSA-free TE medium did not have similar beneficial effects. It was concluded that mouse zygotes from outbred mice do not require BSA for their preimplantation development in culture and that the presence of taurine in preimplantation culture medium is beneficial not only for preimplantation development of the zygotes, but also for their post-blastocyst development.     

The effects of bovine serum albumin and fetal bovine serum on the development of pre- and postcleavage-stage bovine embryos cultured in modified CR2 and M199 media

The effects of protein supplementation on bovine embryo development in vitro was evaluated using a 4 × 2 factorial arrangement with ten replications. A total of 6438 oocytes collected from abattoir ovaries were used. Bovine serum albumin (BSA) and fetal bovine serum (FBS) were added in various combinations to simple (modified CR2) and complex (M199) media during culture of precleavage-stage IVM/IVF-derived ova from 18 h after insemination to 72 h and postcleavage-stage embryos after 72 h of culture. Cleavage rates did not differ (p > 0.05) between media supplemented with FBS or with BSA. However, the postcleavage development to the blastocyst stage of in vitro-derived bovine embryos is better in media supplemented with FBS than BSA. A greater (p < 0.05) proportion of cleaved occytes developed to blastocysts and hatched blastocysts in media supplemented with FBS during postcleavage culture. The percentage of embryos that stopped development at the morula stage was significantly (p < 0.05) greater in media supplemented with BSA during postcleavage culture. Viability of blastocysts produced in CR2 and M199 supplemented with FBS were further assessed by transfer to recipients. In CR2, 25 transferred blastocysts resulted in seven pregnancies and the birth of three normal calves. In M199, 24 transferred blastocysts resulted in five pregnancies and the birth of two normal calves. There was no difference (p > 0.05) in rate of embryo development between CR2 and M199.     

Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan

Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage. Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media. Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion. Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation. The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation. Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions. For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable.     

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).     

In vitro culture of sheep oocytes matured and fertilized in vitro

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.     

Developmental responses of 2-cell embryos to oxygen tension and bovine serum albumin in Wistar rats.

To improve rat embryo culture conditions, responses of Wistar 2-cell embryos from 2 breeders to oxygen tension (5 vs 20%) and bovine serum albumin (BSA) (0 vs 3 mg/ml) were examined using rat 1-cell embryo culture medium (mR1ECM). Supplementation of 3 mg/ml BSA significantly stimulated and accelerated development to the blastocyst and expanded blastocyst stages during 72 and 96 h culture, while reduced oxygen tension stimulated cell division. Fetus development after transfer of blastocysts obtained from 72 h culture under 5% O2 with BSA was significantly higher than those cultured under atmospheric oxygen without BSA. However, the nuclear numbers of in vitro cultured blastocysts and fetus development after embryo transfer were still significantly lower than in vivo developed blastocysts, indicating the current culture condition is still suboptimal.     

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.     

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.     

Effects of glucose and protein sources on bovine embryo development in vitro

Oviductal factors may be obtained by ultrafiltration of conditioned medium, added to a simple media and used in bovine embryo culture. In this study, we aimed to analyze the development of bovine embryos produced with oviductal factors compared to those cultured in the presence of BSA or serum, the effects of glucose in presence of these protein supplements, and the ability of oviductal factors to support embryo development during the entire culture period. In vitro produced bovine zygotes from slaughterhouse ovaries were cultured in modified-synthetic oviduct fluid (mSOF) alone or supplemented with (1) oviductal factors, (2) BSA and (3) FCS. Oviductal factors showed embryotrophic activity, although with blastocyst rates lower than those in BSA and FCS. Glucose (1.5 mM) added at Day 2 of culture did not affect development in the presence of oviductal factors. The number of cells in expanded blastocysts was unaffected by the presence of glucose or any of the protein supplements used. Both BSA and FCS, respectively, improved blastocyst rates of Day 6 embryos produced with oviductal factors. The effect of oviductal factors was masked by the presence of BSA during the entire culture. FCS promoted an earlier appearance of blastocysts. It is concluded that the effect of glucose on in vitro embryo development depends upon the source of protein. Oviductal factors are not an appropriate supplement for embryos beyond Day 6 of culture in SOF, although blastocyst rates of such embryos may be increased by culturing them in the presence of FCS or BSA.     

Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media.

Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids [HECM-6] followed by tissue culture medium 199 + 10% bovine calf serum). Modifications were made to reduce or eliminate protein. Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured. There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively). Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%). There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%). Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium. Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium. This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis.     

Protein-free culture medium containing polyvinylalcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts

In vitro development of eight-cell hamster embryos to hatching blastocysts requires the presence of amino acids and a group of water-soluble vitamins in the culture medium. The present studies investigated the effect of type of macromolecule on blastocyst hatching and on the requirement for vitamins. Embryos were cultured for 3 days in the presence of the synthetic macromolecule polyvinylalcohol (PVA) and of different types of bovine serum albumin (BSA), both with and without vitamins. The results showed th at eight-cell embryos develop to hatching blastocysts in the presence of vitamins and amino acids with PVA as the only macromolecule in the medium. The presence of certain types of BSA reduced but did not eliminate the need for vitamins. Glutamine alone was as efficient as a complete amino acid supplement in supporting blastocyst hatching. These results demonstrate for the first time that eight-cell hamster embryos can be cultured to hatching blastocysts in a chemically defined medium.     

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

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

The 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.     

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.     

Improved in vitro development of porcine embryos with different energy substrates and serum

The effect of replacing 5.5 mM glucose in North Carolina State University (NCSU)-23 medium with 0.5 mM pyruvate/5.0 mM lactate on porcine IVF embryo development was investigated in Experiment 1. Culturing embryos with pyruvate/lactate for 7 days or with pyruvate/lactate from Days 0 to 2, and then glucose from Days 2 to 7 improved cleavage rates. In Experiment 2, embryos were cultured for 7 days in pyruvate/lactate containing NCSU-23 medium supplemented with 0.05% PVA, 0.4% BSA or 10% fetal bovine serum (FBS). The BSA supplement increased the rates of cleavage, blastocyst formation, and the number of total cells in blastocysts. In Experiment 3, embryos were cultured in pyruvate/lactate containing NCSU-23 medium supplemented with 0.4% BSA for 7 days (BSA-PL), 0.4% BSA from Days 0 to 4 and then 10% FBS from Days 4 to 7 (BSA-PL-->F ) or 0.4% BSA from Days 0 to 7 with addition of 10% FBS (BSA-PL + F ) at Day 4. More blastocysts in BSA-PL--> F and hatching or hatched blastocysts in BSA-PL-->F and BSA-PL+F were obtained. Total cell number in blastocysts derived from BSA-PL-->F and BSA-PL+F were increased. Our results demonstrated that supplementing pyruvate/lactate containing NCSU-23 medium with 0.4% BSA for 4 days and replacing it with 10% FBS for another 3 days improved porcine IVF embryo development.