In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media.

Development of bovine embryos derived from in vitro-matured/in vitro-fertilized (IVM/IVF) oocytes was examined in two culture media: hamster embryo culture medium (HECM), a relatively simple, chemically defined, protein-free medium containing 20 amino acids; and tissue culture medium (TCM)-199, a more complex medium designed for culture of somatic cells. The first experiment studied (1) effects of glucose and/or phosphate (Pi) using HECM and (2) the development of bovine IVM/IVF embryos in four different conditions: HECM, TCM-199, TCM-199 + 10% unheated bovine calf serum (BCS), and oviduct cell-conditioned TCM-199 + 10% BCS. After IVF, 45% of the inseminated oocytes developed to the morula/blastocyst stages (M&B) when cultured in HECM; blastocyst development was depressed in the presence of glucose and Pi when compared to Pi alone. When the four culture conditions were compared, there was no significant difference in M&B development (42-51% of inseminated oocytes). However, blastocyst development in TCM-199 supplemented with 10% BCS (29.7%) or with BCS + oviduct cell-conditioned medium (21.6%) was significantly greater than in nonsupplemented HECM (9.7%) or TCM-199 (13.8%). In the second experiment, the effects of serum supplementation and/or oviduct cell conditioning on HECM and TCM-199 were examined in a 2 x 2 x 2 factorial experiment. Oviduct cell conditioning of either HECM or TCM-199 without serum supplementation did not enhance bovine embryo development. Serum supplementation exhibited a biphasic effect, with inhibition at the first cleavage and stimulation of morula compaction and blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

牛卵母细胞体外受精及体外发育的研究

本研究利用从屠宰场收集到的牛卵巢,获取卵母细胞,经体外成熟后进行体外受精,其受精率为73±2％。受精卵分别用发育培养基TCM—199+10％FCS(MA)、颗粒细胞单层(MB)和卵管上皮单层(MC)进行培养,其8—16细胞的发育率分别为52％、54％和61％;而囊胚的发育率分别为20％、28％和42％。相比较而言,MB和MC培养基内突破8—16细胞阻断发育到囊胚的发育率优于MA。     

Methods for collecting and maturing equine oocytes in vitro

The objective of our study was to develop an effective method for collecting and maturing equine oocytes. In Experiments 1 and 2, oocytes were collected from excised ovaries obtained via colpotomy. In Experiment 3, oocytes were collected from ovaries obtained after slaughter. Follicles were aspirated and flushed with various treatments to recover the oocytes, which were then cultured and stained to observe the stage of meiosis. In Experiment 1, the aspiration treatments consisted of 0.5 ml of modified Dulbecco's PBS with 0, 100 or 500 lU/ml hyaluronidase. There was no increase (P>0.05) in oocyte recovery with the addition of hyaluronidase. The oocytes were cultured in either TCM-199 or Ham's F-10 medium containing 0.5 ug/ml FSH, 1 ug/ml LH, 1 ug/ml estradiol 17β, 250 uM Na-pyruvate and 10% estrual mare serum for 0, 24, 36 or 48 h. Maturation rates were higher (P<0.05) at 36 h for oocytes cultured in TCM-199 (79%) than for those in Ham's F-10 (21%). There was no difference (P>0.05) in the percentage of maturation of oocytes between the 2 media at 48 h of culture. In Experiment 2, a single aspiration was performed with no flushing medium (dry aspiration) in 0.5 ml of PBS or in PBS with 1000 IU/ml hyaluronidase. The oocytes were then cultured in TCM-199 for 24, 30 or 36 h. There was an increase (P<0.05) in oocyte recovery when follicles were flushed with PBS, with or without hyaluronidase. There was also a difference (P<0.05) in the percentage of maturation of oocytes between 30 and 24 h (86 vs 48%), but no further increase was seen by 36 h (84%). In Experiment 3, follicles were aspirated with PBS 5 to 6, 6 to 7 or 7 to 8 h after slaughter. The oocytes were cultured for 30 h in TCM-199 either with or without 100 IU/ml eCG. There was no effect of eCG or time from slaughter on oocyte maturation or cumulus expansion (P>0.05).     

Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos

To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages.     

Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws

Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.     

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.     

Bacteria in semen used for IVF affect embryo viability but can be removed by stripping cumulus cells by vortexing

Bacterial contamination of in vitro vs in vivo produced embryos presents a particular danger because of the alteration of the zona pellucida and the use of various biological products during culture. Our objective was to investigate the effects of semen contaminated with bacteria on IVF of bovine oocytes and to determine if removal of cumulus cells by vortexing as opposed to pipetting would reduce contamination and improve subsequent embryonic development. Semen from 5 bulls of the Native Korean breed (Bulls A, B, C, D, E) was used for IVF of matured oocytes. Preliminary studies had shown that the semen from Bulls A, B, D and E but not Bull C was contaminated with various species of common bacteria. After IVF, the cumulus cells surrounding the oocytes were removed either by pipetting or vortexing. Viability and cleavage rates of the resulting zygotes was assessed after 44 h in culture. When cumulus cells were removed by pipetting, only zygotes derived from oocytes that were fertilized with uncontaminated semen from Bull C developed to morula and blastocyst stages; zygotes derived from oocytes that were fertilized with contaminated semen from Bulls A, B, D and E started to degenerate, and the culture media became noticeably turbid. When cumulus cells were removed by vortexing, zygotes derived from oocytes fertilized with either contaminated or uncontaminated semen showed good rates of development (16 to 32%) to morula or blastocyst stages. From these results it can be concluded that the bacteria introduced with the semen contaminated the in vitro system and severely reduced the viability of the embryos. In contrast, complete removal of the cumulus cells with vortexing, as opposed to pipetting, reduced the contamination of the culture medium, allowing embryonic development to take place.     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.     

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.     

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22-24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.     

A serum-free medium for use in a cumulus cell co-culture system for bovine embryos derived from in vitro maturation and in vitro fertilization

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the >or= 2-cell (51 to 60%), >or= 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.     

Development of IVM-IVF produced 8-cell bovine embryos in simple,serum-free media after conditioning or Co-culture with buffalo rat liver cells

Development of 8-cell bovine embryos derived from in vitro matured/in vitro fertilized (IVM/IVF) oocytes was evaluated in two simple, serum-free media (CZB and SOM) with buffalo rat liver cells co-culture (BRLC) or after conditioning compared to a commonly used, serum-supplemented complex medium TCM-199. In a 3 x 4 factorial design, 578 eight-cell embryos were randomly assigned to 12 treatment groups. The factors were: first, type of culture medium (M199/FBS, CZBg and SOM), and second, the use of BRLC (as co-culture or to condition media for 24 hr and 48 hr) and unconditioned media. Development to morula was not affected by the type of medium, but co-culture and 48 hr conditioning within media type resulted in better development when compared to the 24-hr conditioned or unconditioned groups. Blastocyst development in SOM (38.9%) was different (P < 0.05) than in CZBg (46.6%) and M199/FBS (48.7%) and was lowest in the unconditioned group (27.8%) followed by 24 hr conditioned (33.3%), 48 hr (56.3%), and co-culture (59.6%). No blastocyst expansion was observed with unconditioned media and 24 hr conditioned SOM. Significant differences (P < 0.05) were found among all treatment groups except the co-culture and 48-hr conditioned groups. Hatching occurred only with co-culture and 48-hr conditioned groups of M199/FBS and CZBg media. These data show that CZB with glucose conditioned by BRLC monolayers for 48 hr can support the development of IVM/IVF produced bovine embryos to blastocyst compared to culture in TCM-199 with serum. © 1994 Wiley-Liss, Inc.     

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.     

Peritoneal fluid from rabbits or goats as media for in vitro maturation, fertilization and initial culture of caprine oocytes

The efficacy of peritoneal fluid from rabbit and goat for in vitro maturation, fertilization and initial culture of embryos from caprine oocytes was evaluated. Peritoneal fluid was collected from adult female goats (n = 9) or rabbits (n = 9). Good quality oocytes were subjected to in vitro maturation and fertilization in three different media viz. Tissue Culture Medium (TCM-199), goat Peritoneal Fluid (gPF) and rabbit Peritoneal fluid (rPF). Maturation rates were 74.7+/-2.07% and 63.6+/-5.28% in TCM-199, gPF 65.8+/-2.54% and 55.6+/-3.79%, and rPF 57.7+/-1.78% and 44.6+/-3.01% when evaluated on the basis of cumulus cell expansion and the achievement of metaphase-II stage, respectively. However, no significant differences were observed in respect of maturation rate between the control and gPF and between gPF and rPF groups. Freshly ejaculated buck semen was treated with heparin (10 microg/ml) and after 45 min incubation with heparin, 8.0% sperm were live and acrosome reacted. The proportions of fertilized oocytes based on male and female pronuclei formation or on cleavage development were 50.5+/-5.03, 42.3+/-3.15 and 34.2+/-1.98%; 31.0+/-2.80, 27.9+/-2.12 and 21.8+/-1.69% for TCM, gPF and rPF, respectively. It was concluded that peritoneal fluids either from goats or rabbits could be used as an alternative medium to TCM-199. However, further research is required to confirm its efficacy for embryo development up to the blastocyst stage.     

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.     

Effect of holding technique and culture drop size in individual or group culture on blastocyst development after ICSI of equine oocytes with low meiotic competence

The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured, fertilized and cultured in groups at 5microl medium per embryo. In Experiment 3, oocytes were held in the EH treatment, then were matured and fertilized. In Study 3.1, injected oocytes were cultured individually in drop sizes as for Experiment 1; in Study 3.2, groups of 2-7 oocytes were cultured in fixed drop sizes of 5 or 50microl. Blastocyst development rates of individually-cultured embryos were not significantly different among drop sizes in either Experiment 1 or 3 (15-29%). In Experiment 2, blastocyst rates were not significantly different between holding treatments (17-23%). In Experiment 3, for group-cultured oocytes, blastocyst development was not significantly different between 5 and 50microl drops (39 and 27%, respectively). In conclusion, compact-cumulus oocytes may be effectively held in the EH treatment before maturation, and single culture of equine embryos yields acceptable blastocyst development. The greatest blastocyst rate (39%) was obtained with group culture in a 5microl droplet.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Effect of homologous preovulatory follicular fluid on in vitro maturation of equine cumulus-oocyte complexes

The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would improve cumulus expansion was supported. More (P < 0.05) compact COCs incubated in 50% or 100% follicular fluid developed a moderately to completely expanded cumulus after 24 and 36 h of incubation and more (P < 0.05) expanded COCs incubated in 100% follicular fluid developed a moderately to completely expanded cumulus after 36 h of incubation compared to control medium. The hypothesis that incubating COCs in medium containing follicular fluid would improve nuclear maturation was not supported. Although more (P < 0.05) compact COCs incubated in 50% follicular fluid reached polar body-stage compared to those in control medium, the nuclear maturation rate in the control medium was lower than it was when the same medium was used in a preliminary experiment (described in main text); therefore, the apparent superiority of 50% follicular fluid must be interpreted cautiously. Based on these results, future studies are warranted to further address the value of adding preovulatory follicular fluid to equine IVM culture systems.