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H. Miyamoto and T. Ishibashi

Summary. Eight-cell mouse embryos were frozen rapidly to −196°C in the presence of glycerol by a two-step procedure; the lower two-thirds of each tube containing embryos was placed directly from 0°C into crushed solid CO2 for some time before transfer to liquid nitrogen. The cooling rate between −10 and −60°C was ~30°C/min. Suitable conditions for the survival of embryos in this fashion were found to be: stepwise addition of glycerol and no seeding; 1 ·8 M-glycerol; 5–30 min equilibration time; 10–30 min holding time in crushed solid CO2; ~ 500°C/min thawing rate. A relatively high proportion (62%) of frozen embryos survived after rapid thawing, but none survived slow thawing. Eight-cell embryos frozen–thawed in this fashion and transferred to recipients developed into normal young.

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H. Miyamoto and T. Ishibashi

Summary. Mouse morulae were frozen rapidly to − 196°C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from − 7°C after seeding into liquid nitrogen vapour at −170 to − 180°C and then into liquid nitrogen 10–15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 m-glycerol, 2 m-propylene glycol, 2 m-ethylene glycol; 5–30 min equilibration time at 0°C; 3–60 min holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen–thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69–74%) were obtained after rapid freezing by liquid nitrogen vapour. Morulae frozen in this fashion, cultured and transferred to recipients developed into normal young.

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H. Miyamoto and T. Ishibashi

Survival of mammalian embryos after storage at very low temperatures was first achieved in the mouse with suitably slow rates of cooling and thawing and in the presence of the dimethyl sulphoxide (DMSO) or glycerol (Whittingham, Leibo & Mazur, 1972; Wilmut, 1972; Leibo, Mazur & Jackowski, 1974). Similar procedures have been applied for the embryos of cattle (Wilmut & Rowson, 1973), rabbit (Bank & Maurer, 1974; Maurer & Haseman, 1976), rat (Whittingham, 1975), and sheep (Willadsen, Polge, Rowson & Moor, 1976) in the presence of DMSO. A report (Whittingham, 1971a) of the survival of mouse embryos frozen and thawed in medium containing polyvinylpyrrolidone (PVP) has not been confirmed (Whittingham et al., 1972; Wilmut, 1972) and no survival has been achieved for rabbit embryos frozen in the presence of PVP or glycerol (Bank & Maurer, 1974). Greater survival was obtained for mouse embryos frozen in the presence of DMSO than in glycerol (Whittingham et al., 1972). It therefore seems that DMSO is more effective than glycerol for the protection of mammalian embryos from freezing damage. This communication reports the survival of mouse and rat embryos after freezing and thawing by using ethylene glycol as the cryoprotective agent.

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H. Miyamoto and T. Ishibashi

Summary. Eight-cell mouse and rat embryos were frozen to −79°C or −196°C in the presence of ethylene, diethylene, triethylene, propylene or polyethylene glycol. Ethylene glycol was the most effective cryoprotectant for mouse and rat embryos and considerable protection against damage during freezing and thawing was also afforded by propylene glycol. The degree of protection given by the other glycols was relatively low. Mouse embryos survived freezing after only 0·1 min exposure to ethylene glycol at 0°C but exposure for longer periods was necessary with the other glycols. Mouse embryos that survived freezing and thawing with a glycol as the protective agent were capable of developing to full-term fetuses.

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H. MIYAMOTO and T. ISHIBASHI

Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan

(Received 14th April 1975)

In attempts to define suitable conditions for the in-vitro fertilization of mammalian eggs, it has been shown that the osmolality of the medium affects the fertilization rate of mouse and hamster eggs (Miyamoto & Chang, 1973b), and the pH values of the medium affect the fertilization rate of hamster eggs (Bavister, 1969; Miyamoto et al., 1974), mouse eggs (Iwamatsu & Chang, 1971; Miyamoto et al., 1974) and rat eggs (Miyamoto et al., 1974). Iwamatsu & Chang (1971) showed that calcium ions affect the capacitation of spermatozoa and fertilization of mouse eggs in vitro in the presence of bovine follicular fluid. It has recently been shown that guinea-pig spermatozoa fail to fertilize eggs in calcium-free media (Yanagimachi & Usui, 1974). The achievement of fertilization in vitro by epididymal spermatozoa of mice (Toyoda et al., 1971) and

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H. MIYAMOTO and M. C. CHANG

Summary.

About 64% of 854 superovulated mouse eggs were fertilized in vitro by epididymal spermatozoa and were capable of cleaving in chemically defined media. Approximately 10% of such eggs fertilized in vitro or in vivo could develop to the blastocyst stage in culture. When two-cell eggs fertilized in vitro and in vivo were transferred to the oviducts of pseudopregnant mice, only 13% and 16%, respectively, developed into normal fetuses.

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H. MIYAMOTO and M. C. CHANG

Summary.

The removal of follicular cells decreased the proportion of mouse eggs penetrated by epididymal spermatozoa, but did not affect the proportion of hamster eggs penetrated, whether the spermatozoa were taken from the epididymis or from the uterus.

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H. MIYAMOTO and M. C. CHANG

Summary.

Hamster spermatozoa recovered from the epididymis or the uterus were deposited into the uterine horns at various times before ovulation. It was found that the fertilizing life of hamster spermatozoa in the female tract is about 13 hr.

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H. MIYAMOTO and M. C. CHANG

Summary.

The acrosome reaction of epididymal spermatozoa and fertilization in vitro of mouse eggs in chemically defined media without tissue fluid were investigated. About 8 to 10% of motile spermatozoa lost their acrosome but no eggs were penetrated when the spermatozoa and eggs were incubated in a basic medium (modified Krebs-Ringer bicarbonate solution containing glucose) for 5 to 7 hr. Addition of a single metabolic intermediate, such as sodium oxaloacetate or sodium pyruvate, to the basic medium increased the proportion of motile spermatozoa without an acrosome (19 to 34%) and the proportion of eggs penetrated (3·2 to 25·5%). Incubation of spermatozoa and eggs in the basic medium containing serum albumin of various species caused a further increase in the proportion of motile spermatozoa without an acrosome (50 to 65%) and in that of penetrated eggs (60·7 to 86%). The best medium for sperm capacitation and fertilization of mouse eggs in vitro, however, was the basic medium containing bovine serum albumin, sodium lactate and sodium pyruvate. The time required for sperm capacitation was 1 hr in this medium, and 2 hr in the medium containing only serum albumin. Certain components present in the oviducal fluid and in the cumulus egg clots, probably similar to serum albumin and sodium lactate or sodium pyruvate, appeared to be beneficial for the capacitation of spermatozoa and fertilization of eggs. It was concluded that serum albumin, sodium lactate and sodium pyruvate can be substituted for tissue fluid in the induction of capacitation of spermatozoa and fertilization of mouse eggs in vitro.

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H. MIYAMOTO and M. C. CHANG

Summary.

Sperm penetration was not observed when newly ovulated eggs, together with spermatozoa suspended in Tyrode's solution, were deposited into uteri of hamsters. When epididymal spermatozoa preincubated for 1 to 3 hr in Tyrode's solution containing an equal volume of heated serum were used, 4 to 11% of eggs were penetrated 5 to 7 hr later, but signs of egg degeneration were observed.