The zona pellucida (ZP) of mammalian ova plays an important role during maturation, fertilization, and early embryonic development. The hardness of ZP of mammalian ova has been mainly evaluated by a biochemical method, such as a difference in ova dissolution speed with an enzyme or an acid reagent treatment. However, the physical hardness of ZP in bovine embryos is largely unknown. Recently, we developed a system measuring the physical hardness of ZP of mammalian ova with a micro bio-sensor (MBS). The objective of this study was to examine the physical ZP hardness of in vivo- and in vitro-derived bovine embryos using an MBS. In the measurement system, the thin glass needle was connected at the tip of a piezo-electric ceramic tactile sensor, and the sample was pushed into a sensor at fixed speed under the computer-controlled micromanipulation system. The change in frequency at the time of displacement of the sample into the sensor was recorded using the computer program. Measurement of gelatin samples of known concentration (4 to 8%) was used to determine the basic characteristic of a sensor and to make a standard curve. In vivo-generated embryos were recovered from 6 superovulated Japanese Black cows with multiple injection of FSH. On Day 7 after insemination, morulae (M), early blastocysts (EB), and blastocysts (BL) were recovered by nonsurgical flushing of uterine horns. In vitro-generated embryos were produced as described earlier (Yoshida et al. 1998 J. Vet. Med. Sci. 60, 549–554). The M, EB, and BL at Days 5 to 7 of post-insemination in vitro were used for measurement of ZP hardness. When a sensor made contact with a harder gelatin sample, the change in frequency was large; the change in frequency has the characteristic of being small for a softer gelatin sample. By comparison with a standard curve, ZP hardness converted into gelatin concentration for each stage of bovine embryos generated in vivo was 3.95% (M: n = 9), 4.14% (EB: n = 32), and 3.92% (BL: n = 14), respectively. On the other hand, ZP hardness of bovine embryos generated in vitro was 3.42% (M: n = 56), 3.33% (EB: n = 36), and 3.25% (BL: n = 23), respectively. There was a significant difference (P < 0.01) in the hardness of ZP between in vivo- and in vitro-generated bovine embryos.
Effect of zona pellucida removal on early development of in vitro produced bovine embryos