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)     

Effects of fetuin on zona pellucida hardening,fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment I, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P<0.05) were formed when FCS was used compared to PVA and 0.25mg/ml of fetuin. In Experiment II, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P<0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P<0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P<0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes.     

Evaluation of culture systems containing bovine oviduct epithelial cells or granulosa cells to mature and maintain the developmental competence of bovine oocytes in vitro

The effects of estrous cow serum (ECS), bovine oviduct epithelial cells (BOEC), and bovine granulosa cells (GC) on in vitro maturation (IVM) of immature oocyte-cumulus complexes (OCCs) were evaluated. Selected OCCs were cultured for 24 to 26 h in microdroplets of culture medium (CM; TCM 199 + 25 mM HEPES + 100 mug gentamicin sulfate/ml) or in CM medium supplemented or conditioned with 20% ECS, BOEC +/- 20% ECS or GC + 20% ECS. Supplemented media were incubated for 2 h before addition of OCCs, whereas media were conditioned by incubation with 20% ECS or BOEC +/- 20% ECS for 6 d, or with 20% ECS +/- GC for 24 or 48 h before addition of OCCs. The developmental competence of oocytes after TVM was assessed by insemination with glass wool separated, frozen-thawed bovine spermatozoa in microdroplets of modified medium (TALP) containing heparin (5 mug/ml) and BOEC for 18 h. The presumptive zygotes were cultured in microdroplets of CM medium + 20% ECS + BOEC for 7 to 9 d to assess embryo development to morula and blastocyst stages. The percentages of OCCs undergoing IVM (85 to 94%) and in vitro fertilization (IVF) (66 to 80%) were high, irrespective of the IVM conditions. Only after the IVM of OCCs in CM medium alone was the percentage of oocytes undergoing IVF significantly lower (66%; P<0.05). The proportion of IVF oocytes developing to blastocysts with a normal complement of cells (126 to 138) increased significantly (P<0.05) when the OCCs were matured in supplemented or conditioned CM medium containing ECS and/or somatic cells (18 to 28%) compared with those in CM medium alone (9%). When the CM medium was supplemented or conditioned with GC + 20% ECS, the proportion of fertilized oocytes developing to blastocysts increased significantly (28%; P<0.05). These results indicate that the potential of immature OCCs to be fertilized and to complete embryonic development to the blastocyst stage in vitro is enhanced by maturation in CM medium containing 20% ECS and/or BOEC or GC.     

Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers

In the present study, comparison of 2 different culture media (Ham's F-12 and M-199) for supporting in vitro maturation of goat oocytes, and their subsequent embryonic development was evaluated in the presence or absence of sera (estrous goat serum, EGS and fetal calf serum, FCS) and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol, 1 ug/ml). Neither medium (Ham's F-12 or M-199) when supplemented with EGS and hormones showed any notable changes in the maturation rate nor in cleavage and blastocyst development. The mean cell number for blastocysts was also significantly low (P < 0.05). However, Ham's F-12 medium supplemented with FCS and hormones showed a considerable increase in the maturation rate, but subsequent embryonic development was not appreciably increased. However, maturation, cleavage and blastocyst development rates of oocytes matured in M-199 medium in combination with 10% FCS and hormones were significantly higher (P < 0.05). Mean cell number per blastocyst was also significantly increased in this latter treatment compared with that of the other groups (P < 0.05). The results thus indicated that both the culture medium and serum have a marked effect on maturation and subsequent embryonic development. Further, the results also showed that the combination of M-199 with FSH, LH and E2 supplemented with 10% FCS was the most efficacious medium for in vitro maturation and subsequent embryonic development of the media, sera and hormone combinations studied.     

Effects of epidermal growth factor on maturation, fertilization and development of bovine follicular oocytes

When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 0 to 50 ng/ml EGF, the rate of metaphase II oocytes of 30 ng/ml EGF (97%) was significantly (P < 0.05) higher than that of the control (77%), 10 (85%), and 50 ng/ml EGF (82%). After in vitro fertilization, the rate of monospermic oocytes of 30 ng/ml (75%) and 50 ng/ml EGF (77%) was significantly (P < 0.05) higher than that of the control (56 %). When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 30 ng/ml EGF and/or 10% FCS and fertilized with frozen-thawed spermatozoa, the rate of monospermic oocytes was significantly (P < 0.05) higher in EGF + FCS (82%) than in EGF (61%) and FCS (67%). The rate of oocytes with 2 pronuclei was significantly (P < 0.05) higher in EGF + FCS (54%) than in EGF (27%). When in vitro-fertilized bovine embryos were cultured for 8 d with granulosa cells in TCM 199 containing 0, 10, 30 and 50 ng/ml EGF, the rate of embryos developing to the blastocyst stage was not significantly different among the control (22%), 10 ng/ml (20%), 30 ng/ml (18%), and 50 ng/ml (20%) EGF groups. These results indicate that EGF has a beneficial effect on in vitro maturation and fertilization of oocytes and that EGF plus FCS also have a beneficial effect on normal fertilization of oocytes. However, EGF had no beneficial effect on in vitro development of embryos when they were co-cultured with granulosa cells in medium with FCS.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media

This study examined the role of protein supplementation at the various steps of the in vitro production of bovine embryos derived from two different morphological categories of COC. The basic medium was TCM 199 and was supplemented with hormones during maturation in vitro and either estrous cow serum (ECS), bovine serum albumin (BSA) at various concentrations or polyvinyl-alcohol (PVA). Fertilization in vitro was carried out using frozen-thawed semen or one bull in Fert-talp containing heparin, hypotaurin and epinephrine and either 6 mg/ml BSA or 1 mg/ml PVA. In vitro culture up to the blastocyst stage was performed in TCM 199 supplemented with either ECS, BSA or PVA. The first experiment investigated the influence of different medium-supplements (ECS, BSA or PVA) on nuclear maturation and revealed no significant differences among treatment groups nor between categories of COC (63.9% to 74.9% and 48.9% to 77.0%, respectively). The time course of in vitro fertilization was elucidated in Experiment 2 in medium supplemented with either protein or PVA during maturation and fertilization. Penetration was not affected (70.9% to 79.3% penetration 12 h after onset of oocyte-sperm-co-incubation), but formation of pronuclei was decreased (P < 0.05) 12 and 19 h after onset of oocyte-sperm-co-incubation and was retarded in medium supplemented with PVA (12 h: 63.8 vs 21.4 %; 19 h: 57.5 vs 20.8 %, respectively) while cleavage was not affected. In Experiment 3, six treatment groups were formed in which the two different morphological categories of cumulus-oocyte-complexes (COC) were incubated in basic medium supplemented with 1) ECS during maturation and embryo culture and BSA during fertilization; 2) PVA during maturation and embryo culture, fertilization medium with PVA; 3) PVA during maturation and embryo culture, fertilization medium with BSA; 4) BSA (1 mg/ml) during maturation, fertilization and embryo culture; 5) BSA (6 mg/ml) during maturation, fertilization and embryo culture; and 6) BSA (10 mg/ml) during maturation, fertilization and embryo culture. The rates of cleavage and the development to morulae or blastocysts did not differ (P > 0.05) among treatment groups and between both categories of COC and were showing a high degree of variability (cleavage 54.0% to 65.1% and 41.3% to 55.7%, respectively; morulae 25.3% to 53.0% and 26.0% to 51.2%, respectively; blastocysts 5.4% to 24.7% and 0.6% to 20.3%, respectively). Parthenogenetic activation only rarely occurred in medium containing PVA throughout all steps of in vitro production of bovine embryos (Experiment 4) and led to early cleavage stages (8%), but no development to morula- or blastocyst-stages was observed. It is concluded that 1) formation of pronuclei was retarded in medium lacking protein-supplementation, indicating that BSA is required for regular fertilization in vitro and 2) under our experimental conditions, protein-supplementation is not necessary for maturation and development up to the blastocyst stage in vitro.     

Effects of platelet-derived growth factor (PDGF) on the in vitro production of bovine embryos in protein-free media

The purpose of our experiments was to explore the effects of platelet-derived growth factor (PDGF)-supplementation at the various steps of in vitro production of bovine embryos using protein-free media. Cumulus-oocyte-complexes (COC) were collected by slicing abattoir ovaries and then dividing the COC into 2 morphological categories. After maturation for 24 h in TCM-199 supplemented with hormones and either 20% estrous cow serum (ECS) or 1 mg/ml polyvinyl-alcohol (PVA), oocytes were co-incubated for 19 h with frozen/thawed spermatozoa from bull of proven fertility. The semen was diluted in Fert-Talp supplemented with heparin, hypotaurine and epinephrine and either 6 mg/ml bovine serum albumin (BSA) or 1 mg/ml PVA. Presumptive zygotes were transferred into embryo culture medium containing either 20% ECS or 1 mg/ml PVA for a total of 10 d. The PDGF was added at concentrations of 1, 10 or 100 ng/ml to the maturation medium (Experiment 1), fertilization medium (Experiment 2) or culture medium from Day 1 on (Experiment 3), respectively, or at 1 ng/ml PDGF to both the fertilization and culture medium from Day 3 on (Experiment 4), with each medium supplemented with PVA. Oocytes/embryos incubated in the absence of PDGF in media supplemented with either ECS or PVA served as controls. An average of 20 COC was incubated in 1 droplet under silicone oil, and each experiment contained 4 to 6 replicates. No significant differences were found among the various concentrations of PDGF, nor did PDGF-supplementation during maturation (Experiment 1) or embryo culture on Day 1 (Experiment 3) significantly affect development of oocytes/embryos (34.7 +/- 3.5 to 40.4 +/- 2.5% morulae, 11.9 +/- 2.4 to 18.8 +/- 2.5% blastocysts; and 23.2 +/- 2.3 to 27.5 +/- 3.4% morulae, 11.5 +/- 2.6 to 12.7 +/- 2.3% blastocysts, respectively; x +/- SEM). In the presence of 10 ng/ml PDGF in the fertilization medium development to morulae and blastocysts was similar to that of the ECS-group, and was higher (P < 0.05) than that of the PVA-control (ECS: 32.1 +/- 4.6 and 13.8 +/- 2.7%; PVA: 17.5 +/- 0.8 and 6.1 +/- 1.3%; PDGF: 30.6 +/- 3.0 and 14.0 +/- 2.2%, respectively). Development to morulae/blastocysts was increased, and was at the same level as in the ECS-group when the fertilization and/or embryo culture medium on Day 3 contained PDGF compared with the PVA-control group (morulae: ECS 25.3 +/- 4.4%, PVA 13.9 +/- 2.2% [P < 0.05], PDGF 16.7 +/- 3.2 to 19.1 +/- 1.1%; blastocysts: ECS 5.3 +/- 2.1%, PVA 5.0 +/- 1.7%, PDGF 7.1 +/- 1.6 to 9.1 +/- 1.7%, respectively). These results indicate that under our laboratory conditions PDGF can elevate low rates of development and the addition of PDGF to the fertilization medium enhances bovine preimplantation embryonic development. Thus, PDGF can be potentially an important factor in a completely defined medium to substitute the effects of serum.     

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-beta (E(2), 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro.

Efforts to improve bovine embryonic development in vitro involved study of effects of thyroid stimulating hormone (TSH) alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF), and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control), TSH (0.1, 0.5, or 1.0 micrograms/ml) or PRL (10, 100, or 1000 micrograms/ml), luteinizing hormone (LH) (0, 10, or 100 micrograms/ml) + TSH (0.1 or 0.5 micrograms/ml), and serum (20%, v/v) + 0.5 micrograms TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P less than 0.05). All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 micrograms TSH/ml (53.4%) than for other TSH treatments (P less than 0.05). The combination of TSH (0.1 and 0.5 micrograms/ml) with 10 micrograms LH/ml for IVM enabled higher proportions (P less than 0.05) of ova to fertilize (67.4 and 69.2%) than did medium alone (28.3%), LH (10 micrograms/ml) alone (54.1%) or serum + 0.5 micrograms TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 micrograms LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.     

Effects of maturation and co-culture treatments on the developmental capacity of early bovine embryos.

A total of 901 cumulus-oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n = 451), were cultured in TCM-199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n = 450) were cultured in TCM-199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P less than 0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co-cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co-cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co-cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co-culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P less than 0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16-cell stage was also greater (P less than 0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P less than 0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co-culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of sera, bovine serum albumin and follicular cells on in vitro maturation and fertilization of porcine oocytes

The effects of fetal calf serum (FCS), estrus gilt serum (EGS) BSA, dispersed granulosa cells, hemi-sections of follicular wall, and replacement of medium after 24 hours on in vitro maturation and fertilization of porcine oocytes were studied. The results indicate that the use of BSA for 24 or 48 hours inhibited the expansion of cumulus cells and the maturation of oocytes. An incubation of 24 hours culture in FCS followed by a second 24 hours in BSA containing medium did not decrease the rate of maturation but significantly decreased the polyspermy and mean number of spermatozoa penetrated/oocyte. Renewing the medium with or without removal of cumulus cells during the second incubation increased the maturation rate. Removal of cumulus cells decreased the penetrability, the polyspermy rates of the oocyte and the mean number of spermatozoa/oocyte penetrated. The EGS-supplemented medium, dispersed granulosa cells or hemi-sections of follicular wall did not affect the maturation or fertilization rates. In conclusion, BSA, a protein supplement in maturation medium, inhibited cumulus cell expansion and maturation of porcine oocytes. After resumption of meiosis triggered by FCS, BSA did not influence maturation. The FCS-BSA treatment reduced the incidence of polyspermy and the mean number of spermatozoa penetrated/oocyte without decreasing the rate of maturation and fertilization.     

Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro

The role of follicle cells in the acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eight-cell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of culture medium and protein supplementation on in vitro oocyte maturation and fertilization in the domestic cat

Domestic cat oocytes were cultured either in Waymouth MB 753/1 Medium (WAY) or in Eagle's Minimum Essential Medium (MEM) containing FSH, LH and estradiol-17beta and supplememted with one of the following: 5% fetal calf serum (FCS); 4 mg/ml bovine serum albumin (BSA); or 3 mg/ml polyvinylalcohol (PVA, a non-protein control). The oocytes were evaluated for: nuclear maturation after 48 hours of culture (in vitro maturation, IVM); fertilization and cleavage 24 to 30 hours postinsemination (in vitro fertilization, IVF); and early embryo development 48 hours postinsemination. Maturation rates were similar (P>0.05) for WAY + BSA (29.4%), MEM + BSA (46.7%) and MEM + PVA (43.3%), but were different (P<0.05) from the other treatments (range, WAY + FCS, 9.6% to WAY + PVA, 14.9%). Fertilization and cleavage rates were also similar (P>0.05) for WAY + BSA (51.4%, 30.5%), MEM + BSA (45.8%, 40.1%) and MEM + PVA (56.1%, 37.4%) and were greater (P<0.05) than all other treatments. These IVM/IVF oocytes were capable of culturing beyond 2-cells, with the highest proportion of 4- and 8- cell embryos forming in WAY and MEM media in the presence of BSA or in MEM medium containing PVA. In the domestic cat IVM/IVF system: both the type of culture medium and protein supplement influence the proportion of oocytes reaching Metaphase II; the type of protein supplement has a more significant (P<0.05) impact than medium on fertilization, cleavage and early embryo development; and nuclear maturation and fertilization in vitro can proceed in this species in the absence of supplementary protein.     

In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis).

Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.     

The influence of different types of media supplement on the meiotic maturation of bovine oocytes in vitro

This work was designed to evaluate the influence of different types of media supplements in the maturation of preovulatory oocytes in cattle. We studied 4 different supplements: serum from estrous cattle (ECS), serum from fetal calves (FCS), amniotic fluid from cattle (bAF) and follicular fluid from cattle (bFF). Preovulatory bovine oocytes recovered from ovaries of mature cows after slaughter were cultured for 24 h in TCM-199 medium containing 20% of one of these protein supplements. The percentages of oocyte maturation were significantly higher (P < 0.001) in ECS (78%), FCS (79%) and bAF (77%) than in bFF (52%). These data indicate that supplementation with ECS, FCS or bFF is adequate for maturation of bovine oocytes without addition of other compounds.