Production of dromedary (Camelus dromedarius) embryos by IVM and IVF and co-culture with oviductal or granulosa cells

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 degrees C, 5% CO2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 microg/ml FSH, and 500 microM cysteamine. Matured COCs (n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 x 10(6)spermatozoa/ml in modified TALP-solution). Inseminated oocytes (n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes (n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively (P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.     

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 x 10(6) cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.     

Development of in vitro matured and fertilized bovine oocytes co-cultured with Buffalo Rat Liver cells

This study was designed to evaluate the efficacy of Buffalo Rat Liver cells (BRLC) monolayers in supporting the development of in vitro matured and fertilized (IVM/IVF) bovine oocytes through to the hatched blastocyst stage compared to the commonly used co-culture system of bovine oviduct epithelial cells (BOEC). Cumulus oocyte complexes (COCs) obtained from 2- to 6-mm ovarian follicles at slaughter were matured for 24 h in TCM-199 supplemented with FBS and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed using 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium containing 18 to 20 matured oocytes. After 20 to 22 h of sperm exposure, 584 presumptive zygotes in 2 separate trials were randomly assigned to 3 treatment groups (BRLC co-culture, BOEC co-culture and control, consisting of medium alone). Zygotes were cultured in CZB media, a simple semi-defined medium, without glucose for the first 2 d, transferred to M199/FBS (TCM-199-HEPES supplemented with 20% HTFBS, 1 mM Sodium pyruvate), and cultured for an additional 8 days. Cleavage and development to morula and various blastocyst stages were recorded between d 3 and 11 after the start of IVF. Overall average cleavage rate was 75% (440 584 ) and did not vary across the treatments or trials. The proportion of embryos that reached the morula stage in both co-culture systems did not differ (P > 0.05) and was significantly higher (P > 0.05) compared to the control group. However, the percentage of the number of blastocysts, expanded blastocysts and hatched blastocysts varied across the treatment groups (P < 0.05), with the highest results obtained in the BRLC co-culture system. The production of blastocysts in BOEC co-culture was inconsistent between the 2 trials where a significant difference (40.6 vs 53.0%; P > 0.05) was observed. Rate of development to the blastocyst stage was similar between the 2 co-culture systems, with most of the embryos reaching the blastocyst stage by d 8 post insemination. The results of this study show that BRLC from a commercially available established cell line offer a more reliable alternative to a BOEC co-culture system for in vitro maturation, fertilization and culture of bovine embryos.     

The vitrification of in vitro fertilized cow blastocysts by the open pulled straw method

In 6 replicates, a total of 450 immature oocytes recovered from 144 slaughterhouse-derived bovine ovaries were matured and fertilized in vitro, then cultured for 7 to 9 d on a granulosa cell monolayer in tissue culture medium 199 (TCM-199) supplemented with fetal calf serum. Of 126 blastocysts (28% of oocytes cultured), 117 (26% of oocytes cultured) were vitrified in Hepes/bicarbonate-buffered TCM-199 medium and 20% fetal calf serum, with ethylene glycol and dimethylsulfoxid as the cryoprotectants. After thawing in 1.2 mL holding medium with 0.25-M sucrose and after 1 min in holding medium with 0.15-M sucrose, blastocysts were cultivated in vitro for 24 h. The re-expansion rate of blastocysts was 69.2% (81 blastocysts), and 39.5% (32 blastocysts) were hatched. Re-expansion and hatching rates differed between the blastocysts vitrified on 7 and 8+9 days (74.6% and 46% vs. 62% and 29%, respectively). After transfer to recipient cows, 3 out of 6 were diagnosed by ultrasonography as pregnant. Three calves were born from 18 transferred embryos (16.7%). The open pulled straw (OPS) method seems to be a convenient, simple and effective method for cryopreservation of 7 to 9 d bovine embryos.     

Nonspecies-specific effects of mouse oviducts on the development of bovine IVM/IVF embryos by a serum free co-culture

In Experiment 1, development of bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes was examined under 4 culture conditions: 1) co-culture with mouse ampullae continuously for 8 d, 2) co-culture with mouse ampullae that were replaced with fresh ampullae at 48-h intervals, 3) co-culture with bovine granulosa cell monolayers, and 4) culture in medium alone. Culture medium consisted of tissue culture medium 199 (TCM-199) supplemented with 1% fetal calf serum (FCS). Inseminated oocytes were transferred to each of the culture treatment 24 h after insemination and were cultured for 8 d. The number of blastocysts per number of cleaved ova obtained after co-culture with mouse ampullae (42.9%) was significantly (P<0.05) higher than that obtained after co-culture with granulosa cell monolayers (28.3%) or culture without cells (4.2%). In Experiment 2, the developmental ability of bovine IVM/IVF embryos co-cultured with mouse ampullae supplemented with or without serum was examined. When serum was excluded from the culture medium, 26.4% (33 125 ) of the total number of embryos cultured were able to develop to the blastocysts stage using this co-culture system. This value was comparable to that obtained in a serum-supplemented co-culture system (30.7%; 39 125 ). In addition, the developmental ability of embryos that reached to the 4-cell stage or beyond at 46 to 48 h after insemination was not significantly different when the embryos were co-cultured with mouse ampullae with (38.5 vs 44.6%) or without (37.0 vs 33.8%) serum.     

The effect of co-culture system on developmental capacity of bovine IVM/IVF oocytes

Two experiments were conducted to compare the influence of different culture systems and the oviduct donor's cycle phase on the developmental potential of co-cultured bovine embryos derived from IVM/IVF oocytes and to establish an efficient freezing method for oviduct epithelial cells. In the first experiment, the effects of media (Menezo B2, synthetic oviduct fluid SOF); sera (no serum, fetal calf serum FCS, human serum HS); and the presence or absence of monolayer of bovine oviduct epithelial cells (BOEC) on developmental capacity of bovine embryos were investigated. In the second experiment, the influence of oviduct donor's hormonal status (superovulated versus unstimulated) and the cryopreservation of oviductal tissue on the support of developmental competence of bovine IVM/IVF-derived zygotes were examined. Oviduct epithelial cells were cryopreserved according to the modified two-step method previously applied to rabbit embryos. For zygotes co-cultured with a monolayer of BOEC the following blastocyst development rates were obtained: 40.1% (63/157); 34.5% (60/174); 13.0% (7/54); and 19.2% (14/73), respectively, in B2 serum-free medium, B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS medium. In the absence of BOEC the rates were 12.3% (10/81); 41.4% (36/87); and 8.9% (6/67), respectively, in B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS. It was shown that the source of oviduct epithelial cells and previous freezing had no influence on the proportion of cleaved zygotes (approximately 70%) or on the percentage of blastocysts (approximately 20%).     

Simulation of intrafollicular conditions prevents GVBD in bovine oocytes: a better alternative to affect their developmental capacity after two-step culture

To increase the developmental competence of bovine oocytes isolated from small, medium, and large follicles (2-3, 3-4, and 4-6 mm in diameter, respectively), we tried to modify the conditions for their in vitro culture. The first step involved conditions maintaining at least for 48 hr a reversible inhibition of the germinal vesicle breakdown (GVBD) and the second step stimulated the resumption of meiosis and completion of nuclear and cytoplasmic maturation during the subsequent 20-22 hr of culture. The effectiveness of this model depended mainly on the medium composition (reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity by agar, the reduction of oxygen concentration (within 6%-8%), the reduction of the proportion between the number of cumulus-oocyte complexes (COCs), and the reduction of the amount of a medium (within 6-7 mul per COC) to amplify the GVBD-inhibitory effect of oocyte surrounding granulosa cells. The COCs were incubated in clumps of 6-7 COCs. The effectiveness and reversibility of GVBD inhibition depended also on the duration of COCs isolation. The full reversibility of the GV block was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 hr) maturation technique was evaluated by the percentage of total and hatched blastocysts at day 9. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from growing follicles, mainly from the smallest category (13.9% vs. 7.1% and 9.2% vs. 3.3% for total blastocysts and hatched, respectively). However, the two-step culture of oocytes from large regressing follicles substantially reduced the blastocyst yield (9.7% vs. 39.1% and 4.9% vs. 26.7% for total blastocysts and hatched, respectively). The transfer of ten blastocysts (developed after two-step culture) to ten recipients resulted in seven pregnancies.     

Noncytopathic bovine viral diarrhea virus in a system for in vitro production of bovine embryos

Techniques for in vitro production of bovine embryos have evolved to the extent that applications for the commercial production of calves have been proposed. However, little is known about the epidemiological implications of the procedures. One concern is the introduction of noncytopathic bovine viral diarrhea virus (BVDV). In this study, follicular oocytes (n=247) collected from 10 cows were matured and fertilized in vitro and presumptive zygotes were cultured for 7 d. Primary cultures of bovine oviductal epithelial cells for use during in vitro fertilization and culture were divided into 2 groups. Treated oviductal cells were infected with BVDV while control cells were not exposed to the virus. Two approximately equal groups of mature oocytes from each cow were inseminated, and the presumptive zygotes were cultured with infected or noninfected oviductal cells. After 7 d in culture, zona pellucida-intact morulae/blastocysts and degenerated ova were washed, sonicated and assayed for the presence of virus. The rates of cleavage and development were also compared by Chi-square analysis. After washing, virus was not isolated from morulae and blastocysts but was isolated from some groups of degenerated ova. Infections of oviductal cells were inapparent and did not significantly (P>0.05) affect rates of cleavage or development.     

Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.     

Pregnancies established from water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured, in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells

Buffalo ovaries were collected immediately after slaughter and were transported to laboratory in sterile saline at 37 degrees C. Follicular oocytes with the cumulus mass aspirated from 2 to 6 mm in diameter follicles were cultured in TCM-199 medium supplemented with 10% buffalo estrus serum (BES) in 5% CO(2) at 38.5 degrees C. After 20 to 24 h of incubation, the oocytes were inseminated with precapacitated frozen thawed spermatozoa for 6 h. The fertilization rate was 78.15% of the matured oocytes. Over an in vitro culture period of 3 to 9 d, 4.02% of the inseminated oocytes developed to the morula stage when cultured with cumulus cells alone and 17.83% when cumulus cells plus oviductal epithelial cells were used. The percentage of developed blastocysts was very low (0.57%) when the oocytes were co-cultured with cumulus cells from the original oocytes. However, 8% of the inseminated oocytes that were denuded 3 d after insemination developed to the blastocyst stage when they were co-cultured with cumulus and oviductal epithelial cells. Sixteen early/expanded blastocysts were transferred non-surgically to 16 recipients. Four of the 16 recipients became pregnant, of which 2 delivered normal buffalo male calves.     

Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.     

Oocyte secreted factors improve embryo developmental competence of COCs from small follicles in prepubertal goats

Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (≥3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement.     

Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos

The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification‐warming, exposure to P4 improved the proportion of embryos that re‐expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI‐TOF mass spectrometry, a total of 242 phospholipid masses of 400–1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.     

Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro

The objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro by using morphometric analysis. Blastocysts produced in vivo (multiple ovulations, MO) were obtained from superovulated Holstein cows. For blastocysts produced in vitro, cumulus-oocyte complexes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into one of three culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi, followed by TCM-199 + 10% ECS from 72 to 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF + 0.6% BSA. At 168 hpi, six or seven grade 1 blastocysts from each of the four treatments (MO, IVPS, IVPSR, and mSOF) were fixed and prepared for transmission electron microscopy. Random micrographs of each blastocyst were used to determine the volume density of cellular components. Overall, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular space decreased (P < 0.05) and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased (P < 0.05). Across treatments, the proportional volumes of nuclei and inclusion bodies were increased in inner cell mass cells compared with trophectoderm cells for mid- and expanded blastocysts. For blastocysts produced in vitro, the volume density of mitochondria was decreased (P < 0.05) as compared with that of blastocycts produced in vivo. The proportional volume of vacuoles was increased (P < 0.05) in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts from all three in vitro treatments, the volume density of lipid increased (P < 0.05) and the volume density of nuclei decreased (P < 0.05) compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations in volume densities of organelles associated with cellular metabolism as well as deviations associated with altered embryonic differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro embryo production.     

The effect of macromolecular supplementation on the surface tension of TCM-199 and the utilization of growth factors by bovine oocytes and embryos in culture

The objectives of this study were to determine the surface tension of bovine follicular fluid (BFF) and TCM-199, and the effects of the synthetic surfactant, Twin-80, or FCS, in TCM-199 on surface tension measurements and subsequent effects on bovine oocyte and embryo development. The surface tension of BFF was determined to be approximately 45.5 mN m(-1) at 25 degrees C and approximately 42.7 mN m(-1) at 39 degrees C, which was comparable to the surface tension of TCM-199 containing Twin-80 (45.7 and 43.2 mN m(-1), respectively). There was no difference in surface tension measurements of BFF from follicles 2-7 mm or 8-15 mm in diameter. Both Millipore water and phosphate buffered saline (PBS) had a surface tension measurement of 69.5 mN m(-1) at 39 degrees C. Although the presence of Twin-80 in TCM-199 resulted in a reduction in surface tension measurement as compared with unsupplemented TCM-199, there was no effect on the number of oocytes reaching metaphase II. However, the addition of epidermal growth factor (EGF) to TCM-199 containing Twin-80 did result in increased maturation rates of oocytes in vitro. There was no effect of insulin, transferrin, and selenium (ITS), or EGF on surface tension measurement of TCM-199, but significantly more zygotes cleaved and developed to morulae/blastocysts in TCM-199 containing both Twin-80 and growth factors. The addition of 20 microg EGF ml(-1) to TCM-199 containing Twin-80 was as efficacious in supporting bovine embryos in culture as was the addition of 5% or 10% fetal calf serum. This study demonstrates that surface-active components in culture media positively affect bovine oocyte maturation and embryo development in culture. Data also suggest that non-ionic surfactants, such as Twin-80 in TCM-199, may successfully replace the surface-active properties but not the embryotrophic properties of serum in embryo maturation/culture media. Although commercial TCM-199 (containing Twin-80) did not require the addition of other surface-active compounds to lower surface tension, it did benefit from the addition of growth promoting factors, which were also provided by serum.     

Effects of timing of oocyte cryopreservation on in vitro development of nuclear‐transferred bovine zygotes

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuclear transfer (NT) was examined. In vitro‐matured (IVM), metaphase‐II oocytes were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro‐produced day‐5 morulae by a DC electropulse, and then cultured up to 8 days (non‐frozen controls; group I). Oocytes were frozen‐thawed in 1.5‐M ethylene glycol and 0.1‐M sucrose before enucleation (group II), after enucleation (group III), after enucleation and aging culture (group IV), or after activation (group V). In group I, 91% of IVM oocytes could be used for NT and 89% of them fused successfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups IV and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher than those in groups II, III, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respectively) were higher than the rate in group II (75%). Rates of development into blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V). Developmental kinetics and cell numbers of the blastocysts were similar among the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos in cattle. Mol. Reprod. Dev. 54:81–85, 1999. © 1999 Wiley‐Liss, Inc.     

Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)