Rat, goat and cattle embryos were bisected by a specially devised metalmicroblade, and subsequent development of the bisected embryos was examined after 0.5-3 h in culture.
1). High proportions of the embryos from rat, goat and cattle were successfully bisected, but bisection of the goat early blastocysts was comparatively difficult due to that the embryo at this stage still has coarse zona pellucida.
2). In the goat, it was clarified that the late blastocyst having soft zona pellucida is easily bisected without serious damage of inner cell mass.
3). The rate of the paired embryos developed subsequently in culture was lower in the rate (29/62, 47%) than in goat (6/6, 100%) or cattle (10/13, 77%).
These results indicated that bisection of zona intact embryo by metal blade used at the present study is a simple and conventional method, in which both procedures of holding embryo and softening treatment of zona pellucida are not necessary. This method is successfully applied for bisection of goat and cattle late blatocysts, at this stage the zone pellucida has been soft.

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Histochemical examinations of proteins, neutral and acid polysaccharides, and lipids were made using the oocytes in primordial, secondary and antral follicles and the embryos at the stages of 1-8 cell, morula and blastrocyst of Japanese field voles, Microtus montebelli.
1). The cytoplasm of both oocytes and embryos always contained acrolein-Schiff reactive proteins, but not neutral polysaccharides that were periodic acid-Schiff reactive and salivary test-resistant. The cytoplasm of oocytes in primordial and secondary follicles possessed no alcian blue positive acid polysaccharides, while that in antral follicles and the cytoplasm of embryos contained some of them. The zonae pellucidae of both the oocytes and embryos always contained proteins and neutral polysaccharides. The zonae pellucidae of oocytes had no acid polysaccharides, while those of embryos possessed some of them.
2). Glycogen granules and lipid droplets were stored in the eggs as the cytoplasmic inclusions. The oocytes and embryos always contained a small amount of periodic acid-Schiff reactive glycogen granules. The oocytes in primordial and secondary follicles contained no sudanophilic lipid droplets, while the oocytes in antral follicles and the embryos at every developmental stage possessed a small amount of lipid droplets.

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The effect of two different freezing velocities (1 °C/min and 6 °C/min) on fertilizing ability of spermatozoa from the roosters selected for high fertility with frozen-thawed spermatozoa and the randomly selected control males was determined. Significantly (P<0.05) higher fertility was obtained when semen of the selected male was frozen at a rate of 1°C/min to -70°C. In contrast, semen frozen at 6°C/min until -70°C showed no significant difference in fertility between two male lines. The results of the present study indicate the possibility of male selection for higher freezability of spermatozoa. Better viability and fertilizing capacity of frozen-thawed fowl spermatozoa were suggested to be obtained with a freezing rate of 1°C/min.

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The present study was undertaken to examine whether the 'in vitro 2-cell block' in the mouse was controlled by the pronuclei or by the cytoplasm by using pronuclear transplantation technique. In the first experiment, enucleation and karyoplast fusion with inactivated Sendai virus were performed between 'blocking' (CD-1) and 'non-blocking' (F1: C57BL/6 × CBA) pronuclear eggs. In the second experiment, the effect of fused cytoplasm from Fl embryos at various stages on the subsequent development of CD-1 zygotes was examined. In the third experiment, the effect of fused cytoplasm from CD-1 zygotes on the development of Fl zygotes was examined. The results obtained are as follows.
1). Significantly high proportion of enucleated Fl zygotes with CD-1 pronuclei de-veloped beyond the 2-cell stage (72.4%) as compared to the enucleated CD-1 eggs received F1 pronuclei (40.4%) or control CD-1 zygotes (42.3%).
2). The proportion of CD-1 zygotes developed beyond 2-cell stages was significantly increased (from 42.9 to 72.3%) by the fusion with cytoplasma from F 1 2-cell embryos.
3). The fusion of cytoplasm from CD-1 zygotes did not significantly suppress the development of F 1 zygotes.
4). These results demonstrate that the preimplantation development of mouse zygotes in vitro is controlled by cytoplasm, but not by the pronuclei, and suggest that the cytoplasmic factor emerges at the 2-cell stage to maintain the subsequent development.

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The present study was undertaken to find out the suitable methods for blastomerefusion of mouse 2-cell embryos using electric stimulus. Two-cell mouse em-bryos were treated with electric stimuli under different conditions, and those that showed blastomere fusion were cultured for 3 days to observe their subsequent development. The results obtained are as follows.
1). The proportion of embryos fused was not different after the stimuli of 100 to 200 μsec at 1.0 to 2.0 kV/cm field strength (67 to 100%), except 100 μsec at 1.0 kV/cm (39%). However, theproportion of fused embryos developed to blastocysts after the treatment of 1.0 kV/cm field strength (84 to 100%) was significantly higher than that obtained after the stimuli of 1.5 and 2.0 kV/cm (0 to 45%). The maximum rates of fusion anddevelopment of fused embryos were obtained by 150 to 200 μsec at 1.0 kV/cm.
2). The differences in the media (electrolyte vs non-electrolyte solution), the tempera-tures of the solution (15°C and 27°C) and strains of the mouse used atthe present experiment (F1, C57BL/6 vs BALB/c) did not significantly affect the fusionfrequency and the developmental ability of fused embryos.
3). In vitro development of the fused embryos was one step behind the control embryos upto 4-cell stage. The compaction of the fused embryos was observed at 4-cell stage, and blastoceal formation occurred around at the same time as observed in the control embryos.

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In order to analyze mechanisms through which genes integrated into whole mice influence the expression of mouse genes, we introduced the E1A gene of human adenovirus type 12 into the pronuclei of fertilized mouse eggs. Three (12%) of 26 andthree (6%) of 48 mice that developed from eggs injected with Charon10-gE1A-2 DNA and pSV2-gpt-gEl A DNA, respectively, were found to carry the injected sequences. Multiple copies of the injected DNAs were found integrated into the genomesof these mice. They were arranged mainly in tandem, a head-to-tail form. These mice carrying Charon10-gE1A-2 transmitted the integrated sequences to their progeny. However, the patterns of the transmission were different from mouse to mouse. On the other hand, the mice carrying pSV2-gpt-gE1 A have not so far been confirmed to the capability of transmitting the sequence to their progeny. Comparative analysis of proteins extracted from 6 tissues by two-dimensional gel electrophoresis showed that several proteins were different in their quantities when a mouse carrying Charon10-gE1A-2 was compared to its normal littermate. The MW/pI of these spots were, 27kd/6.4 of lung, and 26kd/5.1, 28kd/5.1, 49kd/5.1, 66kd/5.9, 47kd/6.6, and 24kd/7.1 of pancreatic proteins

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Ovariectomized goats, pretreated with subcutaneous implants of proges-terone andestradiol, were subjected to estradiol infusion for 60 hr with an infusion rate gradually increasing from 0.5 to 5.0 μg/hr, thereby mimicking the increase of estradiol secretion during the follicular phase. Plasma LH profiles thus induced were indistinguishable from those seen in normal estrous cycles; preovulatory-like LH surge occurred about 40 hr after the initiation of estradiol infusion concomitant with the onset of estrus

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Three females born with acardius amorphus in cattle were cytogenetically examined. Two females showed the sex chromosomal chimera (XX/XY), but the other female did not show any aberrations. The present study indicated that freemartinism can also occur in females born with acardius amorphus, in addition to the females of heterosexual twins and multiple births or single-born animals. The acardius amorphus and its twin were found to be dizygotic twins of different sex with XX/XY chimerisms.

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The present experiment was conducted to determine the temperature and time of sperm preparation described by Cheng (1985) on in vitro fertilization of sheep. Method A was followed by that of Cheng (1985) except 1 h storage at a concentration of 1×10⁸ spermatozoa /ml in modified 199-sheep serum medium (pH 7.4) at 39°C under 5% CO₂ in air. In methods B and C, the sperm suspension at a concentration of 2×10⁸ cells/ml in 2 ml of the medium (pH 7.8) was kept for 40 min either at 39°C under 5% CO₂ in air or room temperature (20°C) under an aerobic condition. Follicular oocytes obtained from ovaries stimulated with FSH or from ovaries of animals killed at an abattoir, were matured in vitro.
In vitro maturation rate was 92.7% (153/165). Fertilization rates were 85.2%, 74.5% and 80.4% for methods A, B and C, respectively. There was no significant differences in the fertilization rates among and between the methods. The present results indicated that temperature (39°C or 20°C) for the high pH treatment was not an important factor, and that the high pH treatment for 40 min might be not necessary if the final sperm suspension (1 × 10⁶ cells /ml) in the medium (pH 7.4) was stored at 39°C for 1 h before in vitro insemination.

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The efficiency of two types of filters for bovine embryo collection was ex-amined. A total of 60 degenerated bovine embryos were used for each filter and 59 embryos (93%) were recovered for each of the filter. With Immuno Systems, Inc. filter, no embryo was recovered from the washings. However, 4 embryos (6.7%) were recovered from the washings when Continental Plastic Corp. filter was used. Hence, it is important to wash the filters after filtration.

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