Using hormone-treated pregnant cows as a potential source of oocytes for in vitro fertilization.

In vivo collection of oocytes during pregnancy may be alternative method of obtaining gametes for in vitro fertilization (IVF) from genetically superior gestating cattle. The objectives of this experiment were to induce follicular growth in mature beef cows during each trimester of pregnancy, and then to collect oocytes and verify oocyte competency by IVF and subsequent embryo culture in vitro. Cyclic beef cows in Treatment A and pregnant cows in Treatment B were administered a total dose of 40 mg of FSH in descending dose levels (6, 5, 4, 3 and 2 mg) twice daily for 5 consecutive days. Cows in Treatment A were administered 25 mg of PGF(2)alpha and in Treatment B an equal volume of 0.9% saline at the seventh FSH injection. Pregnant cows in Treatment C were administered neither FSH nor PGF(2)alpha and served as a control group. Following a gonadotropin treatment, the ovaries of each female were evaluated for follicular development by ultrasonography. Oocytes were collected by follicle aspiration from cows in the first trimester. Following IVF procedures, the embryos were co-cultured on caprine oviductal cells, or in the chicken embryo co-culture system, or were placed in goat oviducts in vivo. The mean number of follicles per ovary 12 hours after FSH treatment was not different for cows in Treatments A and B, (8.1 vs 7.7) and both numbers were greater (P<0.05) than the 1.1 follicles per ovary for the control cows in Treatment C. Oocytes collected in vivo and exposed to IVF, resulted in 20% cleaving, and of these embryos 50% developed to the morula stage in culture. In summary, stimulating supplemental follicular development with FSH treatment during pregnancy and collecting the oocytes for IVF may be an alternative method for obtaining supplemental gametes from valuable donor cattle.

Effect of heat-stress on bovine embryo development in vitro.

Chronic elevation of uterine temperature has long been known to increase embryo mortality in dairy cattle. Short-term elevation in temperature of mouse embryos to 43 degrees C (acute) has been shown to induce intracellular production of heat-shock proteins. In this study, in vitro development of bovine embryos was assessed during short-term (60 h) coculture with oviduct epithelial cells at 38.6 degrees C (T1), 40 degrees C (T2), 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 5% CO2 (T3), or 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 100% CO2 (T4). During incubation, embryos cocultured at 40 degrees C had a greater (P < .05) mean embryo development score at 36 h than embryos cocultured at 38.6 degrees C. At 60 h of incubation, embryo development scores were greater (P < .05) for embryos cultured at 38.6 degrees C than for those cocultured at 40 degrees C. The number of embryos hatched at 60 h was similar after coculture at 38.6 degrees C (T1) or a prior pulse treatment with 5% CO2 and 43 degrees C (T3), but the embryo development score at 60 h was greater (P < .05) for the pulse-treated embryos. Embryos in T4 had greater (P < .05) embryo development scores than did T1 embryos from 36 through 60 h. Pulse treatment (T4) resulted in a greater (P < .05) number of hatched embryos at 60 h than T1, T2, and T3. These results indicate a detrimental effect of a chronic elevation in temperature that was evident shortly after embryo hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells.

Early stage caprine embryos were incubated with goat oviduct and uterine cells to evaluate whether these cells could be used as a somatic cell culture system to enhance development through the developmental block at the 8- to 16-cell stage during in-vitro culture. Following gonadotrophin treatment and natural mating, 2- to 4-cell embryos were surgically recovered from donor females for in-vitro culture studies. In Experiment 1, embryos were equally and randomly allotted to culture treatments of either culture medium plus caprine oviduct cells or culture medium alone. In both treatment groups, embryos were incubated in Medium-199 with 10% fetal bovine serum, 0.25% lactalbumin and 1% antibiotic-antimycotic at 37 degrees C in a humidified atmosphere of 5% CO2 in air. In Experiment 2, similar embryos were cultured in the same medium with either caprine oviduct cells, caprine uterine cells or sequentially incubated with oviduct cells and then uterine cells during a corresponding incubation interval. The culture conditions in Experiment 2 were the same as in Experiment 1. Following 72 h in culture, (Experiment 1), significantly more embryos developed through the in-vitro developmental block into blastocysts and hatched blastocysts when cultured with oviduct cells compared with no embryos developing through the in-vitro block when incubated with medium alone. In Experiment 2, caprine embryos co-cultured with oviduct cells alone resulted in more embryos developing into blastocysts and hatched blastocysts compared with those co-cultured with uterine cells alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Culturing two- to eight-cell caprine embryos using domestic chicken eggs.

Early-stage caprine embryos were placed in the chick embryo amnion to determine if this culture method would support the development of embryos from a farm animal species. Following superovulation and natural mating, two- to eight-cell embryos were surgically collected from crossbred donor goats. Embryos were allotted to in vitro culture treatments across two different experiments (EXP). In EXP-I, embryos allotted to Treatment A (control) were cultured in Ham's F-10 with 10% fetal calf serum and 1% antibiotic-antimycotic (HF-10). Embryos in Treatment B were placed on a bovine fetal uterine fibroblast monolayer in HF-10, embryos allotted to Treatment C were agarose embedded and injected into the amniotic cavity of a day-4 chick embryo and those placed in Treatment D were co-cultured in HF-10 with day-15 caprine trophoblastic vesicles. In EXP II Treatments A, B, and C were the same; however Treatment D was omitted. EXP-I and EXP-II also differed in that chick embryo co-culture was for 72 hr in EXP-I but was extended to 96 hours in EXP-II. Additionally, the monolayer co-culture was limited to 96 hr in EXP-II; whereas, embryos in EXP-I remained on monolayer culture for 96 hr plus an additional 72 hr for subsequent embryo evaluation. Results indicate that the amniotic cavity of the developing chick embryo enhanced the development of two- to eight-cell caprine embryos through to hatching blastocysts when compared with that of the control medium alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Using the amniotic cavity of the developing chick embryo for the in vivo culture of early-stage mammalian embryos.

The fertile chicken egg may provide an effective, inexpensive method for promoting the development of early-stage embryos from other species. Presently, the loss of viability associated with the in vitro culture of mammalian embryos is hindering the use of in vitro fertilization with farm animals. Consequently, alternative in vitro laboratory methods are needed for the culture of mammalian embryos. A new method has been developed that involves the culture of mammalian embryos in the amniotic cavity of a developing chick embryo. Chick embryos were placed into shell-less incubation (37 C) at the 72-h developmental stage. After 24 h of shell-less incubation, agarose-embedded mammalian embryos were injected into the amniotic cavity of the chick embryo. The mammalian embryos were first placed into a drop of liquid agarose. One to four embryos were then aspirated into a beveled injection pipette and cooled, allowing the agarose to harden. Following penetration of the amnion with the beveled pipette, the agarose cylinder containing the embryos was expelled into the amniotic cavity. The shell-less culture system was then returned to incubation at 37 C for an additional 72 to 96 h. Following incubation, the amniotic cavity containing both chick and mammalian embryos was isolated and the agarose-embedded mammalian embryos were harvested. Significantly more embryos developed in the chick embryo amnion than in the control medium alone. Results obtained using this method on laboratory animals (mice) and on domestic mammals (goats and cattle) indicate that the chick-embryo amnion can support the development of early-stage, mammalian embryos to the blastocyst stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)