Addition of penicillamine, hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or in combination to bovine in vitro fertilization medium increases the subsequent embryo cleavage rate.

The cleavage rate of in vitro-matured bovine oocytes was compared after fertilization in 1) TALP medium alone (control); 2) in TALP+BOEC; 3) in TALP+PHE; or 4) in TALP+BOEC and PHE. The overall cleavage rate at 45 h post insemination was greater for embryos in Treatments 2 (52%), 3 (55%) and 4 (66%) than for Treatment 1 (32%). The oocyte cleavage rates for Treatments 2 and 3 were similar, but were lower than that of Treatment 4. Addition of PHE or BOEC, alone or in combination, to the fertilization medium resulted in more embryos at the 3- or 4-cell stage than the 2-cell stage by 45 h post insemination. After 5 d of co-culture with BOEC in M-199 medium, 21, 28, 25 and 35% of the cleaved embryos in Treatments 1, 2, 3 and 4, respectively, developed to the morula or blastocyst stage. The rate of development to morulae and blastocysts was similar among Treatments 1, 2 and 3, and between Treatments 2 and 4. Across treatments, a correlation of 0.98 was noted between the portion of embryos that had reached the 3- or 4-cell stage by 45 h post insemination and the percentage of embryos in each treatment that continued to develop to the morula or blastocyst stage in vitro.

Histological development and histochemical localization of enzymes in rumen and reticulum in bovine fetuses.

Thirty Holstein fetuses from 100 to 251 d after conception were utilized to study prenatal anatomical development of the epithelium of the rumen and reticulum. Four calves at birth were included in the study for comparison. Tissue sections were frozen and stained to locate specific enzymes. At 100 d, the epithelial layer of the rumen was differentiated into a thin basal zone and a thickened superficial zone of undifferentiated cells. The basement membrane was straight, and in both zones cells were perpendicular to it. At 120 to 141 d, low, primary undulations were detected in the basal zone, basement membrane and underlying lamina propria. At 150 to 166 d, secondary undulations and incipient papillae began to resemble the papillae of mature mucosa. In rumen papillae of 192-d to 215-d fetuses, shallow furrows began to separate papillae apexes from the mass of epithelium. In fetuses 244 to 251 d, the papillae began to be a separate entity. At birth, the basal position of the papillae still remained fused. An incipient separation between the papillae was seen. Several dehydrogenase enzymes, including those associated with the Krebs cycle and reductase associated with energy transformation, were observed in both ruminal and reticular tissue. Alkaline phosphatase activity was localized in the stratum corneum and in blood vessels. Development of the honeycomb configuration of reticular epithelium was evident in the 100-d fetus and progressed rapidly with age.

Effects of oocyte maturation length, sperm capacitation time, and heparin on bovine embryo development.

This study examined the effects of extending oocyte maturation 4 h beyond current methods and capacitating sperm with or without heparin 4 h before oocyte introduction to determine whether embryo development would increase after in vitro fertilization. Oocytes were aspirated from ovaries that were collected at slaughter. Cumulus-enclosed oocytes were matured in M-199 supplemented with serum from cows in standing estrus (20%), antibiotic-antimycotic solution (1%), HEPES (10 mM), and equine LH (30 micrograms/ml) in a humidified 5% CO2 atmosphere. Oocytes were matured for either 24 or 28 h and subsequently fertilized with sperm that had been capacitated 0 or 4 h (before oocyte contact) with or without heparin (0.2 microgram/ml). Data were analyzed as a 2 x 2 x 2 factorial design. Percentages of cleavage and development were transformed by the arcsin square root method before analysis of variance. An interaction of maturation length and sperm capacitation resulted because cleavage rate decreased with precapacitated sperm, but only within the 24-h maturation period. Heparin increased cleavage rate at 48 h after fertilization but did not affect further development. More oocytes developed to morulae when they matured for 24 h than when they matured for 28 h. In conclusion, a 24-h maturation length without precapacitated sperm was optimal for the subsequent development of cumulus-oocyte complexes.

Effects of media, serum, oviductal cells, and hormones during maturation on bovine embryo development in vitro.

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-17 beta (0, 1, and 2 micrograms/ml) and equine LH (0, 10, 20, and 30 micrograms/ml) on cumulus cell expansion and development after fertilization were determined. Cumulus cell expansion and blastocyst development decreased with estradiol-17 beta in the maturation medium, but LH in the medium enhanced expansion of cumulus cells and blastocyst development.