Raymond W. Wright

Bovine embryo morphology and evaluation.

The following paper briefly reviews the morphology of the bovine embryo and presents a retrospective analysis of bovine embryo transfer results accumulated from April to December of 1982 at a commercial embryo transfer center. Of particular interests were bovine embryo morphology, assessment of embryo quality, and recipient-donor, recipient-embryo synchrony requirements. Embryos were recovered from superovulated donors five to nine days after estrus (estrus = day O). All embryos were individually examined at 200X for cell stage of development and embryo quality. Embryos were nonsurgically transferred to recipients that were within two days of estrous cycle synchrony with the donor. Attempts were made to synchronize estimated developmental age of embryos to the day of the recipient cycle. A high degree of variability was observed in morphological development and embryo quality within and among donors. Embryo recovery in individual donors resulted in a wide range of embryonic cell stages, often differing in estimated developmental ages from 24 to 48 hours. A total of 783 embryos were transferred, resulting in 308 pregnancies. Stage of embryonic development (16-cell through hatched blastocyst) had little effect on pregnancy rates. Embryo quality was a more accurate predictor of success. Embryos of excellent, good, fair and poor categories resulted in 45%, 44%, 27% and 20% pregnancy rates, respectively. Recipient-donor estrous cycle synchrony of two days in either direction did not significantly alter pregnancy rates. However, 88% of 258 pregnancies (584 total transfers) occurred with a +/-1 day recipient-embryo synchrony compared to 74% based on +/-1 day recipient-donor cycle synchrony (P<0.001). Results suggest that transfer of bovine embryos based on synchrony between day of recipient cycle and state of embryonic development provides higher pregnancy rates than transfers based on recipient-donor cycle synchrony.

Morphological and physiological differences between in vivo- and in vitro-produced preimplantation embryos from livestock species

Abstract Almost fifty years have passed since 1949 when Dowling (14) used an egg-saline medium, developed by Hammond (26) for mouse embryos to culture 8-cell bovine embryos to the 16-cell stage. These investigators certainly must have queried the normality of the cultured embryos with their variations in cell shape and color observed. With the advances of in vitro systems and the improvement of instrumentation, investigators are faced with the same fundamental questions asked some fifty years ago. Namely, how viable are embryos that are manipulated in vitro, as compared to embryos in vivo? Compounding this difficult question is the lack of basic information on metabolic processes of in vivo embryos. Once the embryos are removed from the maternal environment for study, they are exposed to myriad in vitro conditions which can negatively effect metabolism or morphology. This manuscript is a review of the literature concerning studies that compared in vitro-manipulated to in vivo-produced preimplantation embryos from the farm species. The authors apologize in advance for studies which were inadvertently omitted and acknowledge the diligent work of investigators who have published in this most challenging and important area of embryo development.

Development of one-cell ovine embryos in two culture media under two gas atmospheres☆

The objective of this study was to develop a successful system for culturing one-cell ovine embryos through several cleavage divisions. One hundred and four one-cell embryos were collected from synchronized, FSH-treated ewes 48 hr after the onset of estrus and randomly placed in one of four culture treatments. The effect of glucose supplementation and reduced oxygen tension (20% vs. 5%) on embryo development was studied. Embryo development was quantitated by a cleavage index based on the number of completed cell divisions. The number of embryos completing at least two cell divisions when cultured in Brinsters Pyruvate Medium (BPM) was 7 26 and 9 26 , under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while 22 26 and 20 26 embryos divided when cultured in BPM supplemented with 0.1% glucose (BPM-G) under similar atmospheres. Mean cleavage indices for embryos cultured in BPM were 1.2 and 1.6 under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while embryos cultured in BPM-G had mean cleavage indices of 4.6 and 4.0, respectively. Results of this study indicate that one-cell ovine embryos can be successfully cultured through several cleavage divisions. Glucose supplementation was beneficial for one-cell ovine embryo development. Reducing the oxygen tension from 20 to 5% had no effect on embryo development, and there was no media x gaseous atmosphere interaction.

Determination of pentose phosphate and Embden-Meyerhof pathway activities in bovine embryos

Quantitative determination was made of the activity of pentose phosphate pathway (PPP) and Embden-Meyerhof pathway (EMP) in individual bovine embryos from the six-cell to the hatched blastocyst stage. Embryos were collected from superovulated cross-bred heifers and classified into good and poor categories. A single embryo in 1 microl of medium was mixed with 2 microl of medium containing 3 to 30 nCi radiolabeled glucose previously placed on a detached lid of the 1.5-ml polypropylene microcentrifugé vial. The lid was then fitted to its vial which had been loaded in advance with 1.5 ml of 0.1 N NaOH. Vials were then incubated at 37 degrees C for 3 h. At the end of the incubation period, a 1.5-ml NaOH trap was inverted and placed into a 20-ml scintillation vial containing 10 ml of aqueous counting solution and counted in a liquid scintillation spectrophotometer. The PPP activity in good-quality embryos was greatest at the six-cell stage and decreased with increasing embryo development. The EMP activity showed the reverse trend. Poor- quality embryos had a lower glucose metabolism and higher PPP activity. Similar measurements were made on embryos following 24 h of culture, and total glucose metabolism and percentage of PPP activity were increased. In conclusion, these data suggest that in good quality bovine embryos total glucose utilization is low until 16-cell stage, with PPP being the predominant pathway. Total glucose utilization increases significantly at the morula stage; EMP activity increases with increasing embryo development; and PPP activity increases significantly in poor quality embryos and in embryos 24 h in culture.

Effects of breed and sire on carcass characteristics and fatty acid profiles of crossbred wagyu and angus steers.

In a two-year experiment, 54 steers sired by seven Wagyu bulls [American Wagyu Association (AWA) sire numbers 331, 384, 388, 411, 429, 433 and 488] and 15 steers sired by two Angus bulls, all out of Angus-Hereford cows, were used to evaluate the effects of sire and breed on carcass characteristics and fatty acid composition. Steers were given ad-libitum access to a high-concentrate diet (15 % alfalfa cubes and 85 % barley supplement) for at least 170 days. Breed and individual sire effects were analysed. Wagyu-sired steers had higher marbling, maturity and quality scores, more estimated kidney, pelvic and heart fat, larger longissimus dorsi muscle areas, lower fat thicknesses and yield grades than Angus-sired steers (p < 0.05). Steers sired by 388, 411 and 433 had lower fat thicknesses than steers sired by Angus, 429 and 488 (p < 0.05). Steers sired by 384 and 388 had higher marbling scores per cm subcutaneous fat than steers sired by Angus, 429 and 488, and lower fat thickness per 100 kg of carcass weight than Angus-sired steers (p < 0.05). For both subcutaneous fat and longissimus dorsi muscle, Wagyu-sired steers had higher (p < 0.05) percentages of 14:0, 14:1, 16:0, 16:1, and lower percentages of 18:0 than Angus-sired steers. The genetic differences in carcass characteristics among Wagyu sires may enable us to select for improved marbling with less fat in the Wagyu breed. Some statistically significant (p < 0.05) but small differences existed in fatty acid profiles between breeds and among sires.

Effects of biological source on cooking and palatability attributes of beef produced for the Japanese market

Boneless beef loin samples from five biological sources (Japanese Wagyu, American Wagyu (3 4 - 7 8 Wagyu), Angus, Longhorn and US Choice) were evaluated for cooking and palatability attributes as shabu-shabu, steaks and roasts. Japanese Wagyu beef was superior in palatability compared to Angus, Longhorn and US Choice beef when prepared as shabu-shabu or as steaks. Very palatable beef was produced for the Japanese market when the Wagyu breed and a controlled, extended feeding period were utilized. The results were more equivocal when the beef was prepared as roasts, but it is unlikely that a substantial demand for roasts will develop in Japan due to high retail costs and traditions in cookery.

Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural changes seen during culture in media alone.

OBJECTIVE To compare sperm chromatin structural changes seen in media only culture or in coculture with bovine oviduct epithelial cells. DESIGN Three freshly ejaculated and three cryopreserved sperm samples in media culture or in oviduct epithelial cell coculture. Sperm in each treatment were evaluated by the sperm chromatin structure assay during a 72-hour time course. SETTING An academic research laboratory. PATIENT(S) Normospermic donors with children. INTERVENTION(S) Semen collection through masturbation after 48 hours of abstinence. MAIN OUTCOME MEASURE(S) The sperm chromatin structure assay using flow cytometry to detect the susceptibility of sperm in either treatment to denaturation of DNA in situ. RESULT(S) The sperm chromatin structure assay data differed for sperm type (fresh or cryopreserved), over time, and between treatments within 6 hours of culture. In oviduct epithelial cell coculture, fresh sperm chromatin structure assay values for fresh sperm were stable, whereas in control medium higher chromatin degeneration levels were seen by 10 hours. For cryopreserved sperm, chromatin degeneration had increased by 1 hour postthaw in both treatments, although levels were higher in the control treatment thereafter. CONCLUSION(S) Sperm chromatin structural changes occur over time in culture. Such changes were observed within 2 hours for cryopreserved sperm. Coculture of sperm with oviduct epithelial cells results in a stabilizing effect for sperm against chromatin changes.

Relationship between bull field fertility and in vitro embryo production using sperm preparation methods with and without somatic cell co-culture

Experiments were designed to compare rates of embryonic development following oocyte exposure to cryopreserved spermatozoa from bulls of varying proven fertility, utilizing 3 different sperm preparation methods prior to oocyte introduction. These included 1) sperm co-culture with bovine oviductal epithelial cells (BOEC); 2) sperm co-culture with buffalo rat liver cells (BRLC); or 3) control culture in a routine, cell-free culture system. Semen from 9 bulls was classified by lifetime 60- to 90-d nonreturn rates as having either (mean +/- SEM) high (n=3) 73.2 +/- 3a, medium (n=3) 70.3 +/- 2b or low (n=3) 65.8 +/- 3c field fertility ((ac)p< 0.01; (bc)p< 0.05). There was no difference in embryo cleavage rates for spermatozoa from the high (58 +/- 18%), medium (57 +/-23%) or low (57 +/- 18%) fertility groups. Development to morula or beyond of oocytes fertilized with high (53 +/- 30%) or low (58 +/- 27%) fertility semen tended (P<0.10) to be higher than of those fertilized with medium fertility (33 +/- 28%) semen. This lack of relationship between in vivo fertility and in vitro embryo outcome was consistent across all sperm preparation methods. Therefore, pooled data were used to evaluate the effect of sperm preparation on embryo outcome. There was no difference in embryo cleavage rates between BOEC monolayers (51 +/- 22%), BRLC monolayers (60 +/- 20%) and the cell-free controls (60 +/- 17%). Subsequent embryonic development to compact morula and beyond was higher (P<0.01) with the BRLC monolayer treatment (61 +/- 28%) than with the BOEC monolayers (42 +/- 33%) or control culture (39 +/- 24%). In conclusion, these studies suggest that there is no predictive relationship between bull field fertility (in the ranges evaluated here) and in vitro embryo cleavage or development rates. However, oocytes inseminated with sperm cells co-cultured on BRLC monolayers develop to the morula stage or beyond at a higher rate than oocytes inseminated with spermatozoa from the BOEC or cell-free system.

Potential Functions of Retinoic Acid Receptor A in Sertoli Cells and Germ Cells During Spermatogenesis

Abstract: Elucidation of the retinoid signaling circuitry in the testis is critical to understanding how male germ cells develop to spermatozoa. Retinoic acid receptor A protein (RARA) is an essential mediator of retinoid signaling in the testis, as shown by a sterility phenotype observed for retinoic acid receptor A gene (Rara) knockout male mice. The seminiferous tubules of Rara knockout mice showed varying degrees of germ‐cell degeneration. A dramatic increase in apoptosis of early meiotic prophase spermatocytes was observed in these tubules compared to the wild‐type tubules. Germ‐cell loss was dependent on the stages of the spermatogenic cycle: germ‐cell loss was negligible in stages I–V, but severe after stages VIII and IX of the spermatogenic cycle. Using spermatogonial transplantation, the individual function of RARA in Sertoli cells or germ cells was determined. The wild‐type donor germ cells, transplanted into Rara knockout testes, colonized and proliferated in the RARA‐deficient microenvironment. The donor‐derived cells were mostly early meiotic prophase spermatocytes, with few more advanced germ cells detected. Conversely, when Rara‐deficient germ cells were injected into the microenvironment that express RARA, establishment of donor‐derived germ‐cell colonies was rare, but remarkably, once colonized, Rara‐deficient germ cells progressed normally through spermatogenesis. These results together suggest that RARA may function in Sertoli cells to promote the survival and development of early meiotic prophase spermatocytes, whereas RARA in germ cells functions to increase the proliferation and differentiation of spermatogonia, prior to meiotic prophase.

Superovulation of cattle with equine pituitary extract and porcine ESH

Superovulation has been practiced in cattle for more than 50 years but the results have been highly variable. Scientists at six locations compared a horse pituitary extract (HAP) with a single batch of porcine FSH (pFSH) to determine the efficacy of these hormones to induce superovulation and to test for variability in the superovulatory response. Acetone-dried equine pituitaries were suspended in 40% ethanol containing 6% ammonium acetate, and the supernatant was mixed with 2.5 volumes of cold ethanol. The resulting precipitate was washed with cold ether and dried. Total doses of 18 mg of HAP and 36 mg of pFSH were injected intramuscularly (i.m.) over 4 days, two injections per day, and prostaglandin (PGF(2)alpha; 25 mg, i.m.) was administered on Day 3. Injections were begun on Days 6 to 13 of the estrous cycle. The overall ovulation rates (mean +/- SEM) for HAP and FSH were 8.8 +/- 0.7 and 15.1 +/- 1.0, respectively (n=231; P<0.01). Location interacted (P<0.01) with the type of gonadotropin for the ovulation rate. When expressed as a proportion of the number of corpora lutea, the total number of embryos recovered was greater (P=0.03) for pFSH than for HAP, but there was no difference in the number of Quality 1 and 2 embryos. The results show that HAP can induce a satisfactory superovulatory response, but there was no evidence of reduced variability of response to HAP compared with pFSH.