Estrous Cow Serum Research Articles

The influence of serum and gonadotropins on in vitro maturation and fertilization of bovine oocytes

The influence of estrous cow serum (ECS) or fetal calf serum (FCS) and their interaction with gonadotropins on in vitro bovine maturation and fertilization was evaluated. The addition of ECS or FCS to the medium significantly increased the percentage of oocyte maturation over that of Ham's F-10 medium alone (P<0.05). The addition of follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) or of FSH plus hCG to the medium provided no advantage in maturation over serum alone. However, FSH plus HCG added to the maturation medium significantly improved the frequency of pronucleus formation in both groups compared with a medium to which no gonadotropins were added (P<0.05). Both ECS and FCS (as medium supplements) promoted oocyte maturation. Although the addition of a combination of FSH plus hCG to the medium did not increase the rate of oocyte maturation, it did contribute to the high incidence of subsequent pronucleus formation.

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39°C under 5% CO 2 in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% ( 69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% ( 65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.

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-β (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.

Vitrification of ovine embryos

Our objective was to optimize in vitro maturation conditions of bovine oocytes as assessed by embryo development. In Experiment 1, cumulus-oocyte complexes were matured in either M-199 or RPMI-1640. Each medium was supplemented with an antibiotic-antimycotic solution (1%) and estrous cow serum (20%). Cumulus cell expansion after 24 h was greatest for cumulus-oocyte complexes matured in RPMI-1640. Morulae development on d 7 was greater (21.1%) for oocytes matured in M-199 than for oocytes that matured in RPMI-1640 (9.6%). In Experiment 2, cumulus-oocyte complexes were matured in M-199 supplemented with antibiotic-antimycotic solution (1%). Main effects were serum type (20%; estrous cow serum vs. superstimulated estrous cow serum) and coculture (with or without bovine oviductal epithelial cells). The percentage of oocytes developing into blastocysts (d 9) was higher for oocytes matured in estrous cow serum regardless of coculture. In Experiment 3, effects of estradiol-17β (0, 1, and 2 μg/ml) and equine LH (0, 10, 20, and 30 μg/ml) on cumulus cell expansion and development after fertilization were determined. Cumulus cell expansion and blastocyst development decreased with estradiol-17β in the maturation medium, but LH in the medium enhanced expansion of cumulus cells and blastocyst development.

Developmental stages of [formula omitted][formula omitted] embryos based on day of nonsurgical collection

Our objective was to optimize in vitro maturation conditions of bovine oocytes as assessed by embryo development. In Experiment 1, cumulus-oocyte complexes were matured in either M-199 or RPMI-1640. Each medium was supplemented with an antibiotic-antimycotic solution (1%) and estrous cow serum (20%). Cumulus cell expansion after 24 h was greatest for cumulus-oocyte complexes matured in RPMI-1640. Morulae development on d 7 was greater (21.1%) for oocytes matured in M-199 than for oocytes that matured in RPMI-1640 (9.6%). In Experiment 2, cumulus-oocyte complexes were matured in M-199 supplemented with antibiotic-antimycotic solution (1%). Main effects were serum type (20%; estrous cow serum vs. superstimulated estrous cow serum) and coculture (with or without bovine oviductal epithelial cells). The percentage of oocytes developing into blastocysts (d 9) was higher for oocytes matured in estrous cow serum regardless of coculture. In Experiment 3, effects of estradiol-17β (0, 1, and 2 μg/ml) and equine LH (0, 10, 20, and 30 μg/ml) on cumulus cell expansion and development after fertilization were determined. Cumulus cell expansion and blastocyst development decreased with estradiol-17β in the maturation medium, but LH in the medium enhanced expansion of cumulus cells and blastocyst development.

The effects of estrous cow serum on the in vitro maturation and fertilization of the bovine follicular oocyte

The effect of serum obtained from a cow at the time of standing estrus (serum A), at ovulation (serum B), and at 24 h after ovulation (serum C) on the in vitro maturation and fertilization of bovine oocytes was examined. Of 144 (Group A), 159 (Group B), and 158 (Group C) oocytes, 77 (53.4%), 82 (51.6%) and 82 (51.9%) oocytes were characterized by expansion of cumulus cells, respectively. There was no significant difference in the effect of the three types of cow serum on the cumulus expansion (P < 0.05). Of 461 oocytes, 316 oocytes were cultured with sperm for fertilization, and 145 oocytes were cultured without sperm for evidence of parthenogenetic development. Of 56 (Group A), 56 (Group B), and 62 (Group C) oocytes with expanded cumulus cells, 19 (33.9%), 7 (12.5%), and 11 (17.7%) oocytes were cleaved, respectively, after exposed to the sperm for 24 h. There was a significant difference in the effect of the three types of cow serum on the fertilization rate (P < 0.05). A total of 145 oocytes was cultured in the absence of sperm and no evidence of parthenogenetic division was observed. The effect of the three types of serum obtained from the cow on the maturation of oocytes was not significant, but a significant difference did exist in the fertilization rate of oocytes. Cow serum obtained at the time of standing estrus had a beneficial effect on the fertilization rate of oocytes in vitro.

Development to the late morula or blastocyst stage following in vitro maturation and fertilization of bovine oocytes

The developmental capacity of bovine oocytes matured and fertilized in vitro was investigated under five different culture systems (IVF-medium, BPM-G and TCM199 with bovine epithelial cells for in vitro culture; and rabbit and sheep oviducts for in vivo culture). Immature bovine oocytes were cultured for 24 h in TCM199 supplemented with 20% estrous cow serum and fresh granulosa cells. Fertilization in vitro was performed using pooled frozen-thawed semen from two bulls after swim-up and heparin treatments. Eighteen to 20 h after in vitro insemination, oocytes were transferred to rabbit or sheep oviducts, or were cultured in the three different media for 6 days. Recovery rates from the rabbit and sheep oviducts were 68.1% and 35.3%, respectively. In the oocytes recovered, the frequency of development to the late morula and blastocyst stages was not significantly different in rabbit ( 58 258 : 22.5%) and sheep ( 8 49 : 16.3%) oviducts. The frequency of in vitro development was higher in TCM199 with added bovine oviduct cells ( 44 361 : 12.2%) than in either IVF-medium ( 5 319 : 1.6%) or BPM-G ( 1 253 : 0.4%). Although the developmental capacity of oocytes in vitro was significantly ( P < 0.001) lower than in the rabbit (but not in the sheep) oviduct, the loss of oocytes in vivo counteracted this advantage of the oviduct system. The present results indicate that co-culture of embryos with bovine oviduct epithelial cells is an effective means of supporting the further development of bovine oocytes matured and fertilized in vitro.

Effects of Sera and Steroid Hormones on Development of Bovine Oocytes Matured and Fertilized in Vitro and Co-Cultured with Bovine Oviduct Epithelial Cells

The present experiment was designed to identify possible effects of sera and steroid hormones added to a co-culture with bovine oviduct epithelial cells on embryonic development in vitro. Bovine oocytes were matured in vitro for 24 h and then fertilized in vitro using swim-up and heparin-treated, frozen-thawed spermatozoa. At 18 and 20 h after insemination, oocytes were cultured for 3 or 7 d in a co-culture system with bovine oviduct epithelial cells containing either fetal calf serum (FCS) or estrous cow serum (ECS) and one of six hormonal additions (none, 1 or 10 micrograms/ml estradiol [E]; 1 microgram/ml progesterone [P]; 1 microgram/ml E + P; and 10 micrograms/ml E + P). A total of 2,666 oocytes were cultured for 3 d and examined for cleavage. Of those, 2,280 oocytes were cultured up to 7 d for development to the late morula or blastocyst stage. Greatest cleavage rates for 2- to 8-cell and 8-cell stages were observed in FCS (71 and 24%) and ECS (66 and 23%) without steroid addition. For development into blastocysts, no serum effect was observed. Greatest rates for development into blastocysts were observed in FCS (14%) and ECS (16%) without steroid addition. These results indicate that addition of E and P at the doses and combinations tested did not enhance developmental capacity of in vitro fertilized bovine oocytes. Compared with FCS, ECS tended to increase cleavage rates and development into blastocysts.

In vitro maturation, fertilization and development of bovine oocytes

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39°C under 5% CO2 in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69125), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (6569), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.

The effects of estrous cow serum on the maturation and fertilization of the bovine follicular oocyte in vitro

The effects of estrous cow serum on the maturation and fertilization of the bovine follicular oocyte in vitro