Functional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light.

Functional enucleation is removal or denaturation of an oocytes DNA without piercing the zona pellucida. Two experiments were conducted in this study to determine the effects of centrifugation, and ultraviolet (UV) light on metaphase II bovine oocytes. Experiment 1 evaluated the effects of centrifugation (12,000 x g for 4 min) on the cleavage rate of in vitro matured oocytes. Centrifugation decreased (P < 0.05) the cleavage rate of oocytes (79.5 vs 70.4%). In addition, it was noted that there were two types of ooplasm after centrifugation, stratified and granular. Developmental potential, as represented by cleavage percent, of the two types of ooplasm was not significantly different. Experiment 2 was conducted to determine the interactive effects of centrifugation (as above) and UV light (254 nm) on cleavage rate of oocytes exposed as metaphase II oocytes. The UV light decreased (P < 0.07) oocyte cleavage rates (35.4 vs 25.2%). Centrifuging metaphase II oocytes also decreased (P < 0.07) cleavage rates (34.1 vs 26.5%). In addition, we determined the fate of chromosomes of oocytes centrifuged and(or) exposed to UV light. Both centrifugation and UV light alone affected (P < 0.05) chromosome placement at 42 +/- 3 h after fertilization. Furthermore, centrifugation and UV light interactively increased (P < 0.05) the percentage of non-cleaved oocytes with their DNA located in the perivitelline space (17.4, 15.5, 13.1, and 49.2, respectively, for control, UV exposed, centrifuged, and UV *centrifuged). Collectively, these data indicate that bovine oocytes at the metaphase II stage can be functionally enucleated with centrifugation and exposure to UV light; however, developmental potential may be diminished by those techniques.

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.

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium.

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P=0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (Por=0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P=0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P<0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P=0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P>or=0.535).

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.