S.A. Voelkel

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbeccos phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Hams F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (</=12 hours) and long term (up to 72 hours) culture of halved bovine embryos.

Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves

Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.

An attempt to isolate Brucellaabortus from uterine flushings of brucellosis-reactor donor cattle

Abstract Two experiments were conducted to test for the recovery of brucella organisms from uterine flushings and harvested embryos of sero-positive embryo donor females. In Experiment I, 16 sero-positive cows were superovulated with FSH treatments and artificially inseminated at 12, 24 and 36 hours following the onset of estrus with brucella-free semen. At 48 hours after the onset of estrus, one half the potential donor females were administered an intrauterine inoculation of 3.3 to 4.6 × 10 4 Brucella abortus (strain 2308) organisms while the remainder received a control inoculation. In Experiment II, the same 16 cows were similarly administered superovulatory treatments and inseminated following estrus. The uterine inoculation was increased to 1.5 to 2.5 × 10 8 organisms administered 48 hours following estrus. Samples of recovered flushing medium and homogenized embryo residues were placed into a validated in vitro culture system to detect the presence of brucella bacteria. Uterine flushings and embryos recovered from 31 females exhibiting estrus following FSH treatments were free from either field strain or the inoculated B. abortus (strain 2308) contamination. The flushings obtained from a single female, which did not respond with estrus following FSH treatment but was inoculated at appointment, did contain B. abortus which was identified as the inoculated strain 2308 and not field strain organisms. These results indicate that brucella contamination of flushing media and harvested embryos will not likely be incurred when collecting embryos from sero-positive donor females. These findings offer further encouragement for the use of embryo transplantation as a method to produce brucella-free offspring from infected cows.

The effect of the beef recipient female on embryo transplant offspring

Abstract Embryo transfer techniques were employed to investigate the importance of prenatal and postnatal maternal breed effects on calves of high growth potential (Chianina) and low growth potential (Angus). Six crossbred cow breed groups of known producing ability were used as recipients. These crossbred recipient females had been produced by artificia ly inseminating Angus and Hereford cows with semen from Chianina, Maine Anjou and Simmental bulls. Prior production records on the recipients cipients were used to calculate Most Probable Producing Ability (MPPA) estimates for each cow for birth weight and weaning weight. Donor breed differences were evaluated (P