Digital Microfluidic Dynamic Culture of Mammalian Embryos on an Electrowetting on Dielectric (EWOD) Chip

Current human fertilization in vitro (IVF) bypasses the female oviduct and manually inseminates, fertilizes and cultivates embryos in a static microdrop containing appropriate chemical compounds. A microfluidic microchannel system for IVF is considered to provide an improved in-vivo-mimicking environment to enhance the development in a culture system for an embryo before implantation. We demonstrate a novel digitalized microfluidic device powered with electrowetting on a dielectric (EWOD) to culture an embryo in vitro in a single droplet in a microfluidic environment to mimic the environment in vivo for development of the embryo and to culture the embryos with good development and live births. Our results show that the dynamic culture powered with EWOD can manipulate a single droplet containing one mouse embryo and culture to the blastocyst stage. The rate of embryo cleavage to a hatching blastocyst with a dynamic culture is significantly greater than that with a traditional static culture (p<0.05). The EWOD chip enhances the culture of mouse embryos in a dynamic environment. To test the reproductive outcome of the embryos collected from an EWOD chip as a culture system, we transferred embryos to pseudo-pregnant female mice and produced live births. These results demonstrate that an EWOD-based microfluidic device is capable of culturing mammalian embryos in a microfluidic biological manner, presaging future clinical application.     

Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering

AIM:To investigate the interaction between mesenchymal stem cells(MSCs) and bone grafts using two different cultivation methods:static and dynamic.METHODS:MSCs were isolated from rat bone marrow.MSC culture was analyzed according to the morphology,cell differentiation potential,and surface molecular markers.Before cell culture,freeze-dried bone(FDB) was maintained in culture for 3 d in order to verify culture medium pH.MSCs were co-cultured with FDB using two different cultivation methods:static co-culture(two-dimensional) and dynamic co-culture(threedimensional).After 24 h of cultivation by dynamic or static methods,histological analysis of Cell adhesion on FDB was performed.Cell viability was assessed by the Trypan Blue exclusion method on days 0,3 and 6 after dynamic or static culture.Adherent cells were detached from FDB surface,stained with Trypan Blue,and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture.Statistical analyses were performed with SPSS and a P < 0.05 was considered significant.RESULTS:The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures.Rat MSCs were positive for CD44,CD90 and CD29 and negative for CD34,CD45 and CD11bc.FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH(P > 0.05).In histological analysis,there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods(P < 0.05).The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method.On day 0,the cell viability in the dynamic system was significantly higher than in the static system(P < 0.05).There was a statistical difference in cell viability between days 0,3 and 6 after dynamic culture(P < 0.05).In static culture,cell viability on day 6 was significantly lower than on day 3 and 0(P < 0.05).CONCLUSION:An alternative cultivation method was developed to improve the MSCs adhesion on FDB,demonstrating that dynamic co-culture provides a superior environment over static conditions.     

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.     

Effect of vitamin A supplementation at different gaseous environments on in vitro development of pre-implantation sheep embryos to the blastocyst stage

Vitamin A (all-trans retinol) is an important antioxidant whose role in embryo development in vitro and in vivo is well established. Oxidative stress is a major cause of defective embryo development. This study evaluated the effects of all-trans retinol supplementation to maturation and embryo culture media under different gaseous environments on the development of ovine oocytes and embryos in vitro. The percentages of cleavage, morula and blastocyst, total cell count and comet assay were taken as indicators of developmental competence of embryos. In experiments I and II, all-trans retinol at concentrations of 0, 2, 4, 6, 8 and 10 μM were supplemented to the oocyte maturation medium and cultured in an environment of 5% or 20% O2 respectively. All-trans retinol supplementation (6 μM) to the maturation medium at 5% O2 levels significantly increased blastocyst yield and total cell number (P < 0.05). Maturation of oocytes in a 20% O2 environment bettered cleavage rates in the 6 μM supplemented group compared with the control group (P < 0.05). In experiments III and IV, all-trans retinol, at the aforesaid concentrations was supplemented to embryo culture media under a 5% or 20% O2 environment, respectively. All-trans retinol supplementation to the embryo culture medium at 5% O2 levels did not yield any significant result whereas the culture at 20% O2 levels gave significantly higher blastocyst yield in the 6 μM supplemented group compared with the control group (P < 0.01).     

Survival of 4-cell mouse embryos derived from in vitro fertilization after ultrarapid freezing and thawing

Four-cell mouse embryos obtained by fertilization in vitro were frozen ultrarapidly, immediately after one-step exposure to a highly concentrated solution (modified VS1) at room temperature, and later thawed in a 37 degrees C waterbath. The proportion of morphologically normal embryos on thawing was 95.7% (198/207), and 83.8% (166/198) of these embryos developed to morulas and early blastocysts after 24 hr in culture. All frozen-thawed embryos that developed to morulas and early blastocysts were transferred to the uterine horns of recipients on day 3 of pseudopregnancy and 47.0% (78/166) of transferred embryos developed to normal young.     

Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity

Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72 h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72 h (P<0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P<0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72 h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences.     

Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development

The objective of the present study was to assess the in vitro viability of ovine embryos at different stages of development after combining cell sampling and vitrification. Precompacted morulae, compacted morulae and blastocysts were obtained from superovulated Sarda ewes at 4, 5 or 6 d following insemination. Embryo cell biopsy was carried out in a 100-microl drop of PBS + 10% fetal calf serum (FCS) with 10 micromol nocodazole and 7.5 microg/ml cytochalasin-b by aspiration (3-5 cells). Embryos were cryopreserved at room temperature after exposure of 2 solutions for 5 min, transferred into a vitrification solution, loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. In Experiment 1, the in vitro viability of manipulated or vitrified embryos after in vitro co-culture in TCM 199 medium with 10% FCS and sheep oviductal epithelial cells (SOEC) in 5% CO2 humidified atmosphere in air at 39 degrees C was significantly lower (P < 0.05 and P < 0.01, respectively) at precompacted morula (60 and 30%) and compacted morula (62 and 39%) stages than intact embryos at the same stages (87 and 88%). No differences were found at the blastocyst stage. In Experiment 2, the in vitro survival rate of precompacted morulae which were manipulated and immediately vitrified was lower (P < 0.05) than in those manipulated and, after a temporary period of culture, vitrified at blastocyst stage (21 vs 48%); while no differences were found at compacted morula and blastocyst stages. The results show that 1) the stage of development influences the subsequent in vitro viability of manipulated and vitrified ovine embryos, 2) temporary culture after manipulation and before vitrification improves the in vitro viability of embryos, and 3) the hole in the zona pellucida resulting from biopsy does not affect blastocyst survival after subsequent vitrification.     

Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage

The present series of experiments investigated the effect of a reducing environment created by addition of reduced glutathione (GSH) or thioredoxin (TRX) to in vitro culture medium on the developmental competence of in vitro produced porcine embryos, and their intracellular redox status. Porcine cumulus-oocyte complexes were collected from ovaries matured and fertilized in vitro. The putative zygotes were then cultured for 6 days in modified NCSU-37 medium with or without (control) GSH or TRX, and their developmental competence was evaluated. In addition, the intracellular redox status of the cultured embryos was compared quantitatively using an index based on the ratio of the intracellular GSH content relative to the intracellular H(2)O(2) level. The proportion of embryos that developed to the blastocyst stage was significantly increased when 0.5 or 1.0 microM GSH (29.6% or 30.4%, P < 0.05 or 0.01, respectively) or 1.0 mg/ml TRX (30.6%, P < 0.01) was added to the medium compared to that without any supplementation (control; 20.1%). The intracellular redox status of embryos at the 8- to 12-cell stage or the blastocyst stage in the group cultured in the presence of GSH or TRX was significantly reduced in comparison with the control (P < 0.05 to 0.001). Furthermore, administration of GSH or TRX enhanced the total cell number (from 48.3 to 49.2) and lowered the proportion of apoptotic cells (from 6.2% to 7.0%) in blastocysts compared with the control (cell number 39.3; apoptosis rate 11.1%, P < 0.05). These results suggest that GSH or TRX can improve the in vitro development of porcine embryos, while maintaining an intracellular reductive status.     

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.     

The protective action of polyvinyl alcohol during rapid-freezing of mouse embryos

Biological products like serum and BSA are routinely used in embryo freezing solutions. These products are undefined and can potentially expose the embryos to infectious agents. Therefore, this experiment was designed to evaluate in vitro and in vivo survival of mouse embryos frozen in solutions supplemented with a chemically defined macromolecule, polyvinyl alcohol (PVA). Morula-stage embryos from superovulated mice were collected, frozen by a rapid freezing procedure, and cryoprotectant diluted out (after thawing) in media supplemented with either 10% fetal calf serum (FCS), 0.1 mg/mL PVA, or a combination of 10% FCS and 0.1 mg/mL PVA. Frozen-thawed (good to excellent quality) and nonfrozen (control, collected in FCS supplemented medium) embryos were cultured in medium M16 (32) supplemented with either 4 mg/mL BSA or 0.1 mg/mL PVA for 72 h. Embryos frozen in solutions supplemented with FCS or PVA and nonfrozen embryos were transferred to pseudopregnant recipients. Recipients were humanly killed on Day 15 after transfer, and the rate of implantation and percentage of live fetuses were recorded. The supplementation of collection, freezing and cryoprotectant dilution solutions with FCS, PVA or FCS plus PVA did not influence (P > 0.05) the rate of survival and in vitro development of embryos to hatched/hatching blastocyst-stage. However, a higher (P < 0.01) in vitro development rate to hatching/hatched-stage was recorded when frozen-thawed embryos were cultured in medium supplemented with BSA than with PVA. There was no difference (P > 0.05) in the rate of implantation (68 vs 72%) or percentage of live fetuses (62 vs 60%) between pregnant recipients with embryos frozen in medium with FCS or PVA. The rate of implantation and development of embryos frozen in medium supplemented with PVA or FCS was comparable (P > 0.05) to that of nonfrozen embryos. It may be concluded that PVA can be substituted for FCS in medium for freezing mouse embryos; however, it can not be completely substituted for BSA in the in vitro culture of embryos to the hatched blastocyst stage.     

Effects of fetal calf serum, amino acids, vitamins and insulin on blastocoel formation and hatching of in vivo and IVM/IVF-derived porcine embryos developing in vitro

The objective of this study was to determine the effects of fetal calf serum (FCS), non-essential MEM amino acids, MEM vitamins and insulin on blastocoel formation, expansion and hatching in porcine embryos developing in vitro. Addition of 20% FCS to the NCSU 23 medium significantly (P < 0.05) decreased by the compaction and blastocoel formation of 1- to 2-cell embryos developing in vitro. In contrast, more 1- to 2-cell embryos commenced hatching in the media containing amino acids than in control medium (25.7 vs 2.6%, P < 0.01). Amino acids and insulin synergistically enhanced the incidence of blastocoel formation and hatching of porcine embryos developing in vitro (P < 0.05). When early compacted embryos which developed in vitro in NCSU 23 medium were cultured in BSA-free NCSU 23 medium supplemented with 20% FCS, the incidence of hatching was significantly increased compared with that of the control groups (35.7 vs 4.1%, P < 0.01). However, addition of amino acids, vitamins or insulin to the NCSU 23 medium did not enhance the development of early morulae to the hatched embryos (P > 0.1). When either in vivo or IVM/IVF-derived 1- to 2-cell stage embryos were cultured 4 d in the modified NCSU 23 and an additional 4 days in the modified NCSU 23 supplemented in the FCS, the percentages (61.8 and 17.8%, in vivo- and IVM/TVF-derived, respectively) of hatched blastocysts were significantly higher (P < 0.01) than in the control groups (2.9 and 0%, in vivo and IVM/IVF-derived, respectively). These results suggested that dual culture conditions are required to optimize an in vitro culture system for the development of the porcine embryo in vitro.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

α-Tocopherol and l-ascorbic acid increase the in vitro development of IVM/IVF swamp buffalo ( Bubalus bubalis) embryos

This study was conducted to investigate the effects of capacitating agents added at in vitro fertilization (IVF) and antioxidants supplemented during in vitro culture (IVC) on the development of buffalo embryos. In experiment I, in vitro embryo development of buffalo embryos was compared when the IVF medium was supplemented with heparin, caffeine and calcium ionophore A23187 either alone or in combination. There was no significant difference (P > 0.05) in the cleavage rates of oocytes among the treatment groups but the development rate to the blastocyst stage and the cell numbers of blastocyst in the heparin-treated group were significantly higher (P < 0.05) than that of other treatments. In experiment II, in vitro embryo development of buffalo embryos was compared when IVC medium was supplemented with either α-tocopherol (250 and 500 μM) or l-ascorbic acid (250 and 500 μM). The rate of development to the blastocyst stage of embryos cultured in medium supplemented with 250 μM α-tocopherol (33%, 41/123) and 250 μM l-ascorbic acid (31%, 38/123) was significantly higher (P < 0.05) than that of those cultured in medium alone (19%, 20/108) but not significantly different (P < 0.05) from medium supplemented with either 500 μM α-tocopherol (24%, 30/123) or 500 μM l-ascorbic acid (25%, 33/133). These results suggest that buffalo spermatozoa treated with heparin were suitable for IVF and that α-tocopherol and l-ascorbic acid added during IVC increased the rate of buffalo embryo development.     

Viability of bovine blastocysts obtained after 7, 8 or 9 days of culture in vitro following vitrification and one-step rehydration

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).     

Exposure of bovine oocytes to EGF during maturation allows them to develop to blastocysts in a chemically-defined medium

When cumulus-enclosed bovine oocytes were cultured for 24 h in serum-free medium containing 0 to 50 ng/ml EGF, the proportions of oocytes reaching metaphase II were higher (P < 0.05) in the presence of 30 ng/ml EGF (88.1 +/- 1.3%) than under control conditions (65.5 +/- 3.5%) or in the presence of 10 ng/ml (73.9 +/- 4.5%) and 50 ng/ml (73.6 +/- 4.0%) EGF. When oocytes matured under these conditions were inseminated in vitro, the proportions of oocytes penetrated were higher (P < 0.05) in 10 to 50 ng/ml EGF (96.7 +/- 3.3 to 100%) than in its absence (77.9 +/- 8.9%). However, the proportions of penetrated oocytes with male and female pronuclei did not differ among the different groups (96.7 +/- 3.3 to 100%). When oocytes were matured under the same conditions, fertilized in vitro, and cultured until 192 h post insemination in a chemically-defined medium, the proportion of embryos at the >/=2-cell stage was higher (P < 0.05) in the groups treated with 30 ng/ml (96.1 +/- 2.5%) and 50 ng/ml (90.6 +/- 3.5%) EGF than in the controls (71.8 +/- 3.1%) at 48 h post insemination. Although there were no differences in the proportions (37.3 +/- 5.3 to 47.2 +/- 5.8%) of >/=morulae at 144 h post insemination among treatments, the proportion of embryos developing to the blastocyst stage was higher (P < 0.05) in the presence of 10 to 50 ng/ml EGF (16.5 +/- 2.0 to 20.8 +/- 4.9%) than in control medium (3.4 +/- 2.1%). The mean blastocyst cell number at 192 h post insemination did not differ between culture media in the presence (91 to 107 cells) and the absence (116 cells) of EGF (10 to 50 ng/ml) during maturation. Thus, higher proportions of oocytes matured in serum-free medium with EGF than without EGF could develop to the blastocyst stage in a chemically-defined medium after in vitro fertilization. These results indicate that EGF can induce not only nuclear maturation but also cytoplasmic maturation of cumulus-enclosed bovine oocytes in vitro.     

Improved development of DNA-injected bovine embryos co-cultured with mouse embryonic fibroblast cells

The in vitro development of DNA-injected bovine zygotes, produced in vitro, was compared when cultured with or without mouse embryonic fibroblasts (MEF). The in vivo viability of the embryos produced in these in vitro culture systems was assessed by single or double transfer to recipients taken to term. For these experiments, in vitro fertilized oocytes were not injected (Experiment 1) or were injected with pBL1 gene (Experiment 2) and then cultured for 2 days in CR1aa medium supplemented with 3 mg/ml BSA at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air. Embryos that developed to the 4- to 8-cell stage at the end of this period were randomly assigned to the two cultured systems and cultured for a further 5 days in groups of 10 to 15 embryos in 0.75 ml medium. These two culture systems were CR1aa medium alone or co-culture with MEF in CR1aa medium supplemented with 10% fetal bovine serum (FBS). Every 48 h, 0.5 ml of the medium was replaced with fresh CR1aa medium and at Day 5 of culture, both media were supplemented by the addition of 5.56 mM glucose and 1x GMS-X supplement solutions. Results were assessed as morphological development of the embryos and data were analyzed by Chi-square test or Student's t-test.The development rate of in vitro fertilization (IVF)-derived embryos co-cultured with MEF (24.4%, 49/201) was significantly higher than those cultured alone (14.4%, 28/194; P<0.05) in Experiment 1. There was a similar difference between the treatments in the proportions of embryos which reached the hatching stage or hatched (10.9%, 22/201 vs. 4.1%, 8/194, respectively; P<0.05). DNA-injected embryos co-cultured with MEF (13.7%, 28/205) showed a higher developmental rate than that of the embryos cultured without MEF (6.7%, 13/193; P<0.05) in Experiment 2. Following the transfer to recipients of one or two DNA-injected blastocysts, the pregnancy rates for two culture systems were similar (MEF co-culture 27.4%, 23/84; CR1aa culture 24. 2%, 16/66). However, the numbers of calves born alive from these pregnancies were higher on the MEF co-culture group (82.6%, 19/23) than the CR1aa culture group (56.2%, 9/16). It was concluded that in vitro embryo development to the blastocyst stage and subsequent in vivo development to term of DNA-injected bovine embryos was improved in comparison to culture in CR1aa alone when the last 5 days of in vitro culture were in a MEF co-culture system.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure^? and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure^? and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure^?, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure^? method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure^? treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure^? method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure^? could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.     

In vitro evaluation of early embryo viability and development in summer heat-stressed, superovulated dairy cows

Our study evaluated the viability and the development in vitro of embryos flushed from superovulated heat-stressed (hot season) and unstressed (cool season) Holstein cows 6 to 8 d after artificial insemination (AI). Plasma progesterone (P(4)) levels were measured in all cows. The incidence of unfertilized ova was significantly increased in hot-season cows (P < 0.05). There was no seasonal effect on the highly variable P(4) levels 6 to 8 d after AI. Morulae and blastocysts flushed from hot-season cows were significantly less viable in culture than morulae or blastocysts flushed from cool-season cows (P < 0.001 and P < 0.0025, respectively). Furthermore, there was a significant seasonal effect both on blastulation (P < 0.0025) and hatching of blastocysts developed from morulae in culture (P < 0.005) and on expansion of blastocysts placed in culture at the blastocyst stage (P < 0.05). These data lead us to suggest that the reduced fertility of summer heat-stressed dairy cows may result from decreased viability and developmental capacity of Day-6 to Day-8 embryos. In addition, the results indicate that the efficiency of embryo transfer procedures may be significantly lowered by the reduced embryo viability associated with hot weather and that reduced embryo viability may be the cause of the well-documented seasonal reduction in the efficiency of AI.