R.A. Godke

Production of goats by somatic cell nuclear transfer

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.

High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos taurus cows

Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P < or = 0.06) during the cool season than the hot season (45.0, 21.2, 6.0 for 2-cell, 8-cell and morula stages, respectively). In Experiment 2, Bos taurus cows (Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P < or = 0.003) percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stages. No difference (P > or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts, regardless of season.

Comparison of a solid-phase, no-extraction radioimmunoassay for progesterone with an extraction assay for monitoring luteal function in the mare, bitch, and cow

Abstract Equine, canine, and bovine plasma samples were assayed for progesterone (P 4 ) using extracted plasma in a liquid-phase assay and unextracted plasma in a solid-phase 125 I direct assay developed for human serum. The direct assay was also used to monitor P 4 levels in defatted milk. Results indicated that the direct-assay method was as reliable as the extraction assay for monitoring changes in plasma P 4 during the estrous cycle and early pregnancy. The regression equation specifying the relationship for the two methods was exponential. In this study, correlation coefficients from data subsets ranged from 0.93 to 0.99. The direct assay for P 4 did result in higher values than the extraction assay in plasma from diestrous animals. The use of diluted standards with stripped plasma from the species being assayed gave values that correspond more closely to the extraction assay. The comparisons in this study indicate that the direct radioimmunoassay (RIA) method offered a convenient, reliable method for monitoring luteal function in the species that were evaluated.

Comparing early embryo mortality in dairy cows during hot and cool seasons of the year

The objectives of this study were to identify the level and stage of embryonic mortality that occur in dairy cows during hot and cool seasons of the year. Experimental dairy cows, of varying ages, were artificially inseminated with frozen-thawed semen from proven Holstein sires. Females on each dairy unit were then randomly allocated to one of three experimental groups after partitioning by day of artificial insemination, days post partum, parity, and current milk production level. In Group I and Group II, nonsurgical embryo collection was performed on each cow using Dulbeccos phosphate-buffered saline as the flushing medium. Embryos from cows in Group I were collected on Days 6 or 7 post insemination during the hot (n=93) and cool (n=64) seasons. Embryos from cows in Group II were collected on Days 13 or 14 post insemination during the hot (n=97) and cool (n=63) seasons. In Group III, contemporary control cows were also inseminated during the hot (n=106) and cool (n=106) seasons, and fetal heart beat was evaluated via ultrasound between Days 25 and 35 following insemination. Embryo viability decreased (P<0.05) from 59% at Day 7 to 27% at Day 14 in the hot season, but was not decreased during the cool season (52 vs. 60%). Pregnancy rate at Days 25 to 35 was 21% in the hot season, which was less (P<0.05) than the 36% in the cool season. The percentage of unfertilized ova collected in both the hot and cool seasons suggests that fertilization failure was not affected by season of breeding. In summary, embryonic loss after Day 7 of pregnancy appears to be a problem in this hot, dry climate.

Assisted reproductive technology in nondomestic ungulates: A model approach to preserving and managing genetic diversity

Abstract The application of assisted reproductive technology (ART) to conservation biology has tremendous potential in the management and preservation of germ plasm from nondomestic ungulate species. Traditional approaches of superovulation and nonsurgical embryo recovery have been hampered in these species by inconsistent responses to commercially available gonadotropin preparations and by substantial interspecific variations in the details of reproductive regulatory processes. Although our general knowledge of reproductive physiology is improving, it appears that the production of embryos by IVF will more efficiently advance wildlife conservation efforts. This technology is already proving to be a powerful tool for rescuing gametes (sperm and oocytes) directly from the gonads of wildlife after death or gonadectomy. Other advanced procedures, like sperm microinjection and assisted hatching, may prove necessary for optimizing in vitro embryo production and in vivo developmental competence. Finally, there is a need to direct research attention to pathogen interactions with the zona pellucida (i.e. zona adherence and washing practices) to enhance the approved importation of valuable genetic material from free-ranging animals.

Transvaginal ultrasound-guided oocyte retrieval following FSH stimulation of domestic goats☆

The objectives of this study were to evaluate different ovarian stimulation protocols on donor goats and to develop a safe, repeatable method for harvesting oocytes from FSH-treated does (Experiment I). Based on the preliminary findings of the first experiment, 32 crossbred does were used in a second experiment (Experiment II), 16 that had not been previously aspirated and 16 that had undergone one previous aspiration, were used to fine tune the procedure. Females were randomly subjected to 1 of the 2 ovarian stimulation protocols: Treatment (A) does were implanted with a norgestomet ear implant. Starting 10 d post-implantation, does were administered FSH daily for 4 d. Does in Treatment (B) were treated similarly to those in (A) but were implanted for only 3 d before starting the FSH injections and implants were not removed prior to aspiration. Using a 2 x 2 factorial arrangement, fresh does (n=16), not previously aspirated, were then further randomly assigned to either a laparoscopic aspiration procedure (LAP) or a transvaginal ultrasound-guided aspiration procedure (TUGA). The LAP procedure was performed using a fiber optics. For the TUGA, the doe was placed in dorsal recumbency, and a 5 MHz human transvaginal transducer, attached to the ultrasound unit, was positioned vaginally for oocyte aspiration. In summary, there was no significant difference among treatment groups for parameters evaluated, with the exception of methods for oocyte collection. The number of follicles detected and oocytes harvested using TUGA (9.5 and 4.3, respectively) was less than for females obtained by LAP (17.4 and 14.4, respectfully). The percentage of oocytes recovered from does subjected to the TUGA (68%), however, was similar to those subjected to the LAP (69%). Unlike donor does subjected to a repeated LAP, there was no evidence of adhesions in donor does from the repeated TUGA group. The TUGA approach to oocyte collection should not be overlooked in an effort to decrease the chances of adhesions in valuable donor goats.

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.

Transferable embryo recovery rates following different insemination schedules in superovulated beef cattle

The objective of this study was to evaluate the transferable embryo recovery rates from superovulated donor cattle after different artificial insemination (AI) schedules. Sixty mixed-breed crossbred females were administered follicle stimulating hormone (FSH) and prostaglandin F(2)alpha (PGF(2)alpha) to induce a superovulatory response. At standing estrus, donor females were randomly allotted to one of five treatment groups for AI. Donors were inseminated with two units of high-quality or low-quality frozen semen at 12, 24, 36, or 48 h after the onset of estrus in treatment Groups I, II, III, and IV, respectively, or inseminated with two units at 12, 24, 36, and 48 h (eight units/donor) in control Group V. Donor females inseminated once at either 12 or 24 h after the onset of estrus did not differ from donors inseminated in Group V in overall fertilization and transferable embryo recovery rates. The highest fertilization rate (89.5%) and transferable embryo recovery rate (74.9%) per donor resulted when AI was performed with high-quality semen at 24 h after the onset of estrus. These findings indicate that repeated insemination of superovulated beef cattle is not necessary to attain optimal fertilization rates and production of transferable quality embryos in beef cattle.

Comparison of once daily and twice daily FSH injections for superovulating beef cattle.

Twelve cycling Angus-based crossbred cows were used in a crossover experimental design to evaluate two different injection schedules using Follicle Stimulating Hormone (FSH) for superovulating donor cattle. Females randomly assigned to Treatment (A) were given twice daily FSH injections of 5 mg each (12 hours apart) for five consecutive days starting on day 10 of the estrous cycle while those in Treatment (B) received the same daily dose level of FSH, except it was given in a 3.2% protein gelatin carrier vehicle and administered on a once daily injection schedule. Animals in both Treatments (A) and (B) were each given a 30 mg dose of commercially available prostaglandin-F(2alpha) agent 48 hours after the first FSH injection. Cows in estrus were initially handmated to a fertile bull then artificially inseminated 12 hours later with two units of frozen semen. All 12 animals (100%) given twice daily FSH injections and 11 of the females (91.6%) administered once daily FSH injections exhibited standing estrus within 5 days following injection of the luteolytic agent. On day 7 or 8 after the onset of standing estrus a laparotomy was performed to observe ovarian structures. When the superovulation response was evaluated, the mean number of corpora lutea per ovary ranged from 2.9 in the twice daily injection group to 4.1 in the once daily injected group. Unexpectedly, the once daily treated group had significantly more corpora lutea per animal (8.1 vs. 6.4) than those in the twice daily treated group. In addition, mean ovarian size score per animal increased significantly when pre-treatment scores were compared to those recorded following FSH treatment (laparotomy) in both Treatment (A) and (B), however, the post-treatment ovarian size scores were not different between these groups. When evaluating post-treatment follicular development, the once daily injection group had significantly more smaller follicles (<10 mm) and a greater number of ovulatory size follicles (>10 mm) than the twice daily injection group. Furthermore, viable appearing embryos were recovered from both treatment groups and no adverse reactions were observed with the gelatin carrier vehicle in Treatment (B). Since the once daily FSH injection schedule resulted in a superovulatory response equal to or greater than the twice daily FSH injection schedule, this approach to superovulation should not be overlooked by those involved in bovine embryo transplantation.