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

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

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.     

Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition

Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.     

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

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

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

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

Successful piglet production after transfer of blastocysts produced by a modified in vitro system

Porcine in vitro production (IVP) systems, including in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes and their subsequent in vitro culture (IVC), have been modified by many researchers, but are still at a low level because of a low developmental rate of embryos to the blastocyst stage and their poor qualities. Our objectives were to establish reliable IVP procedures for porcine blastocysts and to examine the ability of the blastocysts to develop to term after transfer to recipients. Porcine cumulus-oocyte complexes were matured in vitro under 5% O(2) or 20% O(2), fertilized in vitro under 5% O(2), and subsequently cultured under 5% O(2) in 1) IVC medium supplemented with glucose (IVC-Glu) from Day 0 (the day of IVF) to Day 6; 2) IVC-Glu from Days 0 to 2, then IVC medium supplemented with pyruvate and lactate (IVC-PyrLac) from Days 2 to 6; 3) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6; and 4) IVC-PyrLac from Days 0 to 6. There were no significant differences in blastocyst formation rates on Day 6 between the 5% O(2) and 20% O(2) conditions (19.9% and 14.0%, respectively). However, the quality of blastocysts, as evaluated by the total cell number, was better after IVM under 5% O(2) than under 20% O(2) (mean cell number, 43.5 and 37.8, respectively). When IVP embryos were cultured in IVC-PyrLac from Days 0 to 2 and subsequently in IVC-Glu from Days 2 to 6, the rate of blastocyst formation (25.3%) and cell number (48.7) were higher than the rates (5.8% to 18.1%) and numbers (35.4 to 37.1) with the IVC-Glu then IVC-Glu, the IVC-Glu then IVC-PyrLac, and the IVC-PyrLac then IVC-PyrLac regimens, respectively. We then prepared conditioned medium (CM) from culture of porcine oviductal epithelial cells for 2 days in IVC-PyrLac and evaluated its effect on development to the blastocyst stage. Cultivation in CM for the first 2 days, followed by IVC-Glu for a further 4 days, had a significantly greater effect in increasing the number of cells in the blastocyst (58.3) than did in IVC-PyrLac (48.4). Finally, we evaluated the ability of blastocysts, generated by IVM under 5% O(2) and IVC in CM, to develop to term. When Day 5 expanding blastocysts (mean cell number, 49.7) were transferred to an estrus-synchronized recipient (50 blastocysts per recipient), the recipient remained pregnant and farrowed eight normal piglets. Furthermore, when Day 6 expanded blastocysts (mean cell number, 80.2) were transferred to two estrus-synchronized recipients, both gilts remained pregnant and farrowed a total of 11 piglets. These results suggest that an excellent piglet production system can be established by using this modified IVP system, which produces high-quality porcine blastocysts. This system has advantages for the generation of cloned and transgenic pigs.     

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.     

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.     

Comparison of sex ratio and cell number of IVM-IVF bovine blastocysts co-cultured with bovine oviduct epithelial cells or with Vero cells

The influence of 2 co-culture systems (BOEC and Vero cells) on the development rates, quality grades and sex ratios of IVM-IVF bovine embryos were studied. Zygotes obtained after IVF were co-cultured in each co-culture system for 7 and 8 d (Day 0 = day of insemination) in B2 medium. No effect of the co-culture system was observed on development rates measured on Days 7 and 8. However, Vero cell co-culture had a positive influence on embryo quality. Irrespective of their sex, embryos produced on Vero cells showed higher cells number than those co-cultured on BOEC (103.4 +/- 3.8 and 97 +/- 8.12 for BOEC vs 113.7 +/- 3.5 and 114 +/- 5.9 for Vero cells at Days 7 and 8, respectively; P < 0.05). The percentage of male embryos was increased in the two co-culture systems (60.7% males for BOEC; P < 0.05 vs 63% males for Vero cells; P < 0.01) on Day 7. In both co-culture systems the increase in the percentage of males was more obvious for embryos reaching the most advanced stage (expanded blastocysts). The results show that Vero cells improved the quality grade of bovine embryos produced in vitro, and thus are recommended for use as a safe co-culture system that does not contain pathogens.     

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

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

Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing

Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing.     

Oviductal and uterine influence on the development of Day-2 equine embryos in vivo and in vitro

The objective of this experiment was to contrast the influence of the oviductal and uterine environments on development of Day-2 embryos. Embryos were transferred to oviducts or uteri of synchronous recipient mares, or were incubated in oviductal co-culture, in uterine co-culture or in defined culture medium. Significantly more (P < 0.02) embryos transferred to the oviduct versus the uterus survived until Day 11 after ovulation (5 7 vs 0 7 , respectively). Significantly more (P < 0.001) embryos developed to expanded and hatched blastocysts in uterine co-culture than in culture medium (6 7 vs 0 7 , respectively). The rate of embryo development to expanded blastocysts was not significantly different (P > 0.1) in oviductal co-culture versus uterine co-culture (3 7 vs 6 7 , respectively), or in oviductal co-culture versus culture in medium (3 7 vs 0 7 , respectively). Three of 7 and 6 of 7 embryos developed to hatched blastocysts greater than 2000 mum in diameter during oviductal and uterine co-culture, respectively, while 0 of 7 embryos cultured in medium expanded to greater than 500 mum in diameter. Proportions of embryos that developed for at least 9 days.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20–22°C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4°C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22°C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zona dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS + albumin, TCM199 and TCM199 + calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20–22°C, the embryo survival rate decreased (PBS + albumin) or no embryo survived (TCM199 + calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Evaluation of different culture systems on the in vitro production of bovine embryos

The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified "well of the well" (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar (P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower (P < 0.05) in embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar (P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates (P < 0.05) were observed in the T3 group. In conclusion, embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system.     

Effects of cervical dilation and intrauterine infusions on the timing of oviductal transport of equine embryos

Equine embryos spend 5 to 6 d in the oviduct before entering the uterus as expanded blastocysts, and cannot be consistently collected nonsurgically until Day 7. Technologies such as cryopreservation and embryo splitting, which are most successful with embryos at the morula or early blastocyst stage, have not been used in mares because equine morulae and early blastocysts are located in the oviduct and cannot be recovered nonsurgically. These experiments test the hypothesis that transport of equine embryos through the oviduct can be hastened by cervical dilation or by acute, sterile endometritis induced by intrauterine oyster glycogen treatment. Cervical dilation with or without intrauterine infusion of 0.5 ml PBS on Day 4 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for mares with cervical dilation or cervical dilation plus PBS infusion vs mares receiving no treatments (0 of 5 and 0 of 5 vs 0 of 10, respectively). Intrauterine infusions of 40 ml of 1% oyster glycogen or 40 ml of PBS on Day 3 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for oyster glycogen- or PBS-treated vs untreated mares (2 of 12 and 3 of 11 vs 0 of 10, respectively). Cervical and uterine treatments on Day 3 or Day 4 and uterine lavages on Day 5 decreased (P < 0.05) Days 11 to Day 15 pregnancy rates compared with that of untreated mares. Day 11 to Day 15 pregnancy rates were 1 of 5 for mares with Day 4 cervical dilation and Day 5 uterine lavage, 1 of 5 for mares with Day 4 PBS infusion and Day 5 uterine lavage, 2 of 12 for mares with Day 3 oyster glycogen infusion and Day 5 uterine lavage, and 3 of 11 for mares with Day 3 PBS infusion and Day 5 uterine lavage vs 7 of 10 for mares that received no treatment or lavage. Cervical and uterine manipulations on Day 3 or 4 and uterine lavage on Day 5 appeared to decrease pregnancy rates by Days 11 to 15. The results of these experiments do not support the hypothesis that cervical dilation or uterine infusion hasten oviductal transport, since neither cervical manipulation nor transcervical infusion of oyster glycogen or PBS into the uterus significantly hastened the rate of embryo transport into the uterus.     

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.     

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

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