E.S. Critser

In vitro maturation and fertilization of bovine oocytes

Abstract Securing a plentiful and economical source of zygotes is central to capitalizing in the bovine on the many new genetic technologies currently available in both research and commercial settings. Using the fruits of the now mature A.I. industry and the many female germ cells wasted in numerous meat packing facilities, bovine zygotes can be produced entirely in vitro. Primary oocytes remoyed from antral follicles of abattoir-obtained ovaries can be induced to undergo in vitro the sequence of events found during in vivo periovulatory maturation of those oocytes in follicles selected to ovulate. Systems for in vitro maturation must ensure that the resulting oocyte has normally completed the first reduction division, is capable of undergoing normal fertilization and yields a zygote competent of developing to term after embryo transfer. Sperm from frozen-thawed semen can be capacitated in vitro with heparin to yield an in vitro fertilization system of high efficiency. By changing heparin dosage or sperm concentration, ejaculates can be adjusted to give comparable standards of fertilization. Ejaculates can also be characterized for precise timing of fertilization and developmental potential of zygotes produced. Our current knowledge concerning these aspects of gamete physiology as applied to the practical production of bovine zygotes in vitro will be discussed in this review.

Use of A Fluorescent Stain for Visualization of Nuclear Material in Living Oocytes and Early Embryos

Hoechst dyes 33342 and 33258 were used to visualize pronuclei and nuclei of early preimplantation embryos. Murine one-cell zygotes exposed to dye stained rapidly over a range of concentrations (0, 0.02, 0.04, 0.1 or 0.2 micrograms/50 microliter of media). Development to morula and blastocyst in vitro was reduced (39/70, 56%; p less than 0.05) compared to controls (44/57, 77%) but not completely blocked. Porcine and bovine zygotes and embryos could also be stained but required incubation times up to 4 hr. Porcine embryos exposed to Hoechst 33342 had limited (p less than 0.01) in vitro development (29/74, 39%) compared to unstained controls (49/64, 76%). Hoechst dyes stain embryos from different species but suitably adjusted incubation times are required. Limited preimplantation development in vitro may be expected following staining and exposure to ultraviolet light.

Developmental potential of bovine oocytes matured in vitro or in vivo

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.

Fertilization potential of follicular oocytes classified by stage of cycle and size of follicle

Bovine follicular oocytes, collected from two sizes of vesicular follicles and from donor animals from three stages of the estrous cycle, were matured and fertilized in vitro. Frequency of fertilization and ability to form male pronuclei after in vitro maturation were found to be independent of both estrual stage and follicular size.

Lower sodium lactate in Whitten's medium improves in vitro developmental capacity of one-cell mouse embryos

A modification of Whittens medium, involving a reduced content of Na-lactate syrup (0.2 ml/100 ml; 11.65 mM) and osmolarity (251 mOsm), was compared with normal Whittens medium (0.37 ml/100 ml; 21.6 mM) for ability to support mouse embryonic development in vitro from one-cell to the blastocyst stage. In a pilot study utilizing 10 ICR donor female mice, in vitro developmental capacity (IVDC; percentage of fertilized one-cell embryos developing to blastocysts in vitro per female donor) was significantly enhanced by the modified medium (68.0 versus 24.0%; P<0.001). In the main study, utilizing 134 ICR and 17 ICR x C57BL/6J F(1) donor females, the modified medium supported increased IVDC for both ICR (67.9 versus 51.1%; P<0.001) and F(1) females (98.5 versus 89.4%; P<0.05). A large degree of among donor-female variation in IVDC was observed for both media in the ICR stock (SD = 30.0). The beneficial role of the reduction of Na-lactate in Whittens medium may be related to an improved provision of energy requirements for first cleavage and/or a more suitable osmolarity for development.

The effect of inhibitors of prostaglandin synthesis on hematocrits of mice

Abstract Four experiments were performed to evaluate the effects of nonsteroidal anti-inflammatory drugs (NSAID) on hematocrit of mice. Indomethacin (experiment I) and meclofenamic acid (experiment II) reduced (P 2 partially reversed (P 2 α was without effect. The results of these studies indicate that inhibitors of prostaglandin synthesis decreased hematocrit by increasing plasma volume.