The purpose of present study was to examine the ability of potential de-velopment of splitted mouse embryos at 2-(1/2) and 4-cell (2/4) stage.
Two and 4-cell embryos were collected from the superovulated F₁ (C57BL/6J × C3H/HeN) mice 48 hr after hCG injection. After removal of zona pellucida (Hank's solution + 0.5% pronase), the embryos were separated into two halves by pipetting in Ca²⁺ or Ca²⁺. Mg²⁺-free BMOC-3 medium containing 0.02% EDTA. Splitted embryos were cultured in BMOC-3 medium in Multiplate Terasaki for 48, 72 or 96 hr. Then the embryos developed into blastocysts were transferred to recipients on Day 2 or Day 3 of pseudopregnancy. These recipients were killed at Day 17 of gestation and the existence live fetuses, dead fetuses and swellings were examined. Furthermore, a mono-zygotic pair of 1/2 embryos developed into blastocysts after 48 hr culture were transferred to recipients on Day 2 of pseudopregnancy or pregnancy.
The results were as follows:
1) Success rate of splitting 2-and 4-cell embryos by pipetting were 93.5-100% and 88.2%, respectively.
2) Most of 1/2 and 2/4 embryos were developed into blastocysts after 72 hr culture (93.9-100%).
3) The propotion of live fetuses after transfer of 1/2 embryos were 14/56 (25.0%), 4/52 (7.7), 12/60 (20.0) and 3/74 (4.1), and those of 2/4 embryos were 14/49 (28.6), 12/57 (21.2), 11/57 (19.3) and 3/43 (7.0) in the case of 48 hr→Day 2, 72 hr→Day 2, 72 hr→Day 3 and 96 hr→Day 3 combinations, respectively.
4) One or six sets of monozygotic twins were obtained after transfer of a pair of splitted embryos into pseudopregnant or pregnant recipients.
These results indicate that the zona pellucida is not necessary for the development of embryos in vitro and it is possible to produce monozygotic twins from 2-cell stage embryos.

View full abstract

Effects of destruction of follicles (by cauterization or puncture) or ovaries (by resection) on maintenance of pregnancy were examined in pregnant rabbits which received unilateral ovariectomy (ULO) on day 23 of pregnancy.
Normal delivery was produced after ULO in 2 of 3 pregnant does. Pregnant does which received ULO aborted completely after destruction of all visible follicles (≥ 1.0 mm in diameter) in residual ovaries by cauterization or puncture. Fewer than 2 corpora lutea or 10 large follicles (≥ 1.5 mm in diameter) in residual ovaries of pregnant does which received ULO could not maintain pregnancy. Resection of more than one-third of the residual ovary also resulted in abortion.
Plasma progesterone levels in pregnant does which received ULO decreased after follicular or ovarian destruction. The levels after treatment in does with normal delivery were significantly higher than those in does which aborted. Minimal concen-tration of progesterone for maintenance of pregnancy seemed to be about 5 ng/ml in peripheral plasma on day 23 of pregnancy.

View full abstract

Progesterone enzyme immunoassay kit using progesterone-3-CMO-β-galacto-sidase (P-β-Gal) as a tracer and polystyrene bead coated with rabbit γ-globulin raised against 11α-OH-progesterone-hemisuccina te-BSA as a solid phase (Teikoku Hormone Mfg. Co. Ltd., Tokyo) was applied for direct measurement of progesterone in defatted milk from cows. Time required for whole procedure was 3 hours in maximum. The least detectable amount of progesterone in this kit was 11 pg./tube (0.225 ng/ml). Intra-assay coefficients of variation (CVs) were 5.9-9.2% and inter-assay CVs were 7.3-13.1%. Recovery rates of progesterone added to defatted milk were 80.0-109.4% (mean = 92.6%). There was highly significant correlation (Y=0.959X+0.054, r=0.939, P<0.01, n=40) between progesterone levels measured by P-EIA kit and by already established double antibody EIA. A progesterone level in defatted milk 1.0 ng/ml or higher indicates the presence of the functional corpus luteum. Accuracy of pregnancy diagnosis based on progesterone levels, 1.0 ng/ml or higher for pregnant cows and lower than 1.0 ng/ml for not pregnant cases, in defatted milk measured by P-EIA kit 21-24 days after AI was 86.2% (81/94) for positive cases and 100% (39/39) for negative cases. These results indicate that the P-EIA kit is the reliable and practical tool for measuring progesterone levels in defatted milk and for the assessment of presence of the corpus luteum.

View full abstract

In our previous study we have reported a new method for inducing ice crystal formation in extracellular solution using silver iodide. Rabbit morulae survived freezing with the silver iodide seeding method. The present study examined the viability of frozen bovine embryos using the silver iodide seeding procedure after transfer to appropriate heifers.
Seven-days excellent and good embryos recovered from superovulated heifers by FSH were used in the present study. Plastic straws were used for frozen storage of the embryos. Each straw was filled by successive aspiration of the following fractions: 70 mm high of 10% v/v glycerol containing one embryo, 3 mm of air and 10 mm of 1% suspension of silver iodide in distilled water (Fig. 1). The straws were cooled to -7°C at 1°C/min, and held at -7°C for 10 min without initiating seeding. They were then cooled to -35°C at 0.3°C/min and plunged into liquid nitrogen. After rapid thawing, the embryos recovered were immersed in 0.75 M or 1.0 M sucrose solution for 10 min and washed by modified phosphate buffered saline solution. The seven embryos were transferred to the total 7 recipients synchronized. This resulted in two pregnant cows which delivered male normal calf, respectively.

View full abstract

In vitro and in vivo survival of the mouse embryos from one-cell to blasto-cyst stage frozen by vitrification method (Rail and Fahy, 1985) was investigated in the present study. The embryos were plunged into LN₂, directly after exposure to the vitrification solution (VS1). The results were obtained as follows.
1) The viavility of embryos after freezing and thawing was high in 8-cell embryos (86%) and morulae (77%), and the lowest in one-cell eggs (0%) frozen by pro-cedure A.
2) The live young were obtained after transfer of frozen-thawed embryos at all stages to recipients.
3) Although the viavility of blastocysts diluted gradually by VS1 solution was low (29%-44%), it was enhanced remarkably (75%) when they were diluted with 2.0 M glycerol-0.5 M sucrose and then 0.5 M sucrose-PBI solution at room temper-ature.

View full abstract

The present study was undertaken to produce chimaeras by using nuclear transplantation technique in the mouse. The nuclear transplantation was performed between BALB/c×BALB/c embryo (GPI-1, a/a and albino) and F1 (C57BL/6J×CBA) ×CBA embryo (GPI-1, b/b and non-albino) at 2-cell stage.
The results obtained are as follows.
1. The proportion of Fl embryos which received BALB nucleus and developed to blastocyst stage was significantly higher than that of BALB embryos receiving Fl nucleus (62% vs 42%).
2. Chimaeras were obtained after transfer of Fl embryos receiving BALB nucleus to the uterus of recipient CD-1 females (38%). On the other hand, BALB embryos receiving F1 nucleus did not produce chimaeric mice.
3. The results of the analysis of chimaeric state showed that BALB/F1 ratio of coat color patterns almost coincided with GPI-1 a/b ratio in the red blood cells but not in the germ cell line.

View full abstract

The ductus epididymidis of adult dogs (1.5-2 years) could be histologically divided into four segments.
1) Segment I occupied the greater part of the caput epididymidis with the ductuli efferentes, corresponding to "initial segment" (Glover & Nicander, 1971). This segment showed a higher epithelium with longer stereocilia. Principal cells had well-developed Golgi apparatus and abundant rough endoplasmic reticulum.
2) Segment II was situated in the dorsal part between the caput and corpus epididymidis, corresponding to the proximal one-third of "middle segment" (Glover & Nicander, 1971) and "subsegment I" (Sinowatz et al., 1979a). Spermatozoa were rather few. Principal cells had numerous vacuoles containing various electron-dense flocculent material.
3) Segment III occupied the greater part of the corpus, corresponding to the distal two-thirds of "middle segment" (Glover & Nicander, 1971) and "subsegments II to IV" (Sinowatz et al., 1979a). This segment showed a narrow lumen containing numerous spermatozoa. Principal cells had many lysosomes, some fragments of spermatozoa and huge vacuoles.
4) Segment IV was located in the cauda, corresponding to "terminal segment" (Glover & Nicander, 1971). This segment showed a wider lumen and a lower epithelium with shorter stereocilia. Spermatozoa were densely crowded. Principal cells had many lysosomes.

View full abstract

In vitro fertilization was performed by using gametes of diabetic obese mouse, C57BL/KsJ-dbm that is incapable of reproducing due to homozygous autosomal recessive diabetis(db). Newly ovulated eggs from db/db mice treated with 5 i.u. PMSG and 5 i.u. hCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis. At 6 hr after insemination, the oocytes were examined for the extrusion of 2nd polar body and the male and female pronucleus. The percentages of fertilized oocytes were 24%, 41%, 24% and 99% when oocytes were inseminated with db/db, adrenalectomized db/db(Adrex), db/+ and (C57BL/6J × C3H/HeN)F1 spermatozoa, re-spectively. The adrenalectomy could not make db/db males regain fertility but signifi-cantly increase in vitro fertilization rate. Number of abnormal spermatozoa in Adrex was significantly fewer than that of intact db/db. Only 28% of intact db/db and 39% of Adrex db/db cultured in the Whitten's medium containing lOOμM EDTA developed into the blastocyst stage 120 hr after insemination. As a result of the transfer of 2-cell embryos which were fertilized with Adrex spermatozoa, eleven newborn (F:M=3:8) were obtained. These results indicated that in vitro fertilization technique can be ap-plied for amelioration of defected reproductive ability in diabetic obese (db/db) mouse.

View full abstract

Intact preantral follicles at Stage 1-5 were isolated from the rat ovary by enzymatic dissociation, and collected separately at every stage of development, with filtration by stainless wire meshes consisting of various pore size. The follicle with 2 layers of granulosa cells (Stage 2) developed rapidly following increase of granulosa cells in vitro.

View full abstract

Three Japanese Black heifers were starved for 7 days and examined for changes in body weight and in NEFA and progesterone levels of blood, and fertility. The following results were obtained.
1. Body weight decreased in three heifers during starvation.
2. The NEFA level of blood increased markedly in three heifers during starvation.
3. After starvation of 7 days, the estrus returned on 22 days, 28 days and 62 days, respectively. As the last one was the youngest (11 months old at the start of experiment) and had the lowest body weight (198 kg), the starvation would have brought about the most strong effect on estrous cycle.
Although their different periods for estrous return, all three heifers conceived at the second insemination.

View full abstract