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A. Hanada and H. Nagase

Summary. Semen was diluted 1:9 with egg yolk–citrate medium containing 0·31– 3·1 m (final concentration) formamide, butyramide, acetamide, propionamide, dimethylformamide, lactamide, malomide, ethylene glycol, trimethylene glycol, dimethylsulphoxide (DMSO) or glycerol. After 30 min incubation at 20°C, sperm motility was superior in hypertonic solutions of acetamide, lactamide, dimethylsulphoxide, trimethylene glycol and ethylene glycol. Some of these compounds were added to semen diluted 1:2 in an isotonic egg-yolk–glucose– lactose–raffinose solution and frozen by the pellet method. Relatively good survival of motility was obtained in 1·0 m-DMSO, -lactamide or -acetamide. Dimethylformamide (0·5 m), ethylene glycol (0·5–1·5M), trimethylene glycol (1·5 m) and propionamide (0·75 m) also gave some protection. Insemination of does with semen frozen and thawed with 1·0 m-DMSO, -lactamide or -acetamide gave fertilization rates of 68–88%, and 84% (38/45) of does gave birth to an average of 5·3 young.

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A. Hanada and M. C. Chang


Hamster eggs with follicular cells were fertilized by epididymal spermatozoa in two chemically defined media. The proportion of penetrated eggs was significantly higher in a medium for rabbit (16%) than in a medium for rat eggs (6%), but much lower than in Tyrode's solution containing follicular fluid or blood serum as reported by others. The optimal sperm concentration for sperm penetration ranged from 0·5 to 5 × 106/ml but penetration of denuded eggs failed in these media. When exposed to hamster spermatozoa in the rabbit medium containing living or dead spermatozoa of guinea-pig, rat, mouse or hamster, high proportions of denuded eggs (24-96 %) and eggs with follicular cells (93%) were penetrated. By exposure of denuded hamster eggs to hamster spermatozoa in supernatant fluid of frozen-thawed guinea-pig spermatozoa, 97% of eggs were penetrated in 8 hr compared to 0% in the control group. Sperm capacitation was also efficiently induced by preincubation of hamster spermatozoa in the supernatant fluid. The fertilizing capacity of hamster spermatozoa was maintained for 12 hr during incubation with frozen-thawed guinea-pig spermatozoa when the concentration of hamster spermatozoa ranged between 10 and 20 × 106/ml. The beneficial factor of guinea-pig spermatozoa appeared to be from spermatozoa themselves, not from the vasal or epididymal fluids. The presence of follicular cells, blood serum, bovine serum albumin, or even polyvinylpyrrolidone in the media is essential for the capacitation and acrosome reaction of hamster spermatozoa. The components of guinea-pig spermatozoa appear to maintain the fertilizing capacity of hamster spermatozoa and stimulate the process of capacitation.

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A. Hanada and M. C. Chang

Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545, U.S.A.

In a study of the penetration of zona-free eggs by foreign spermatozoa in the mouse, rat and hamster, it was concluded that the incorporation of eggs with foreign spermatozoa may depend on sperm capacitation and the physiological affinity between the spermatozoa and the vitellus of different species, and that the zona pellucida appears to be the major block for interspecific fertilization (Hanada & Chang, 1972). Yanagimachi (1972) has reported that hamster zona-free eggs can be penetrated readily by capacitated but not by uncapacitated guinea-pig spermatozoa. Based upon our recent studies of sperm capacitation and fertilization of rat eggs in vitro (Toyoda & Chang, 1974; Niwa & Chang, 1974, 1975), this paper describes the penetration of hamster and rabbit zona-free eggs by mouse and rat spermatozoa as affected by the capacitation of spermatozoa.

The medium used for the manipulation of gametes was

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In spite of advancement in the knowledge of the biochemistry of semen (White & Macleod, 1963; Mann, 1965), the physiological significance of seminal plasma for the fertilizing capacity of spermatozoa, except as a vehicle for sperm transport, is still obscure. When superovulated rabbits were inseminated with a minimal effective number of rabbit spermatozoa suspended in fructose Ringer solution or in rabbit seminal plasma, there was no significant difference in the proportion of eggs fertilized. When these sperm suspensions were allowed to stand at room temperature for 1 hr before insemination, however, the proportion of eggs fertilized was significantly higher for the spermatozoa suspended in rabbit seminal plasma (Chang, 1949). The present experiment was designed to determine whether seminal plasma, due to its various contents, might be beneficial for sperm transport when insemination was carried out

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T. Nagai, T. Takahashi, H. Masuda, Y. Shioya, M. Kuwayama, M. Fukushima, S. Iwasaki, and A. Hanada

Summary. In Exp. 1 pig oocytes matured in vitro were used to evaluate the fertilizability in vitro of frozen epididymal (4 boars) and ejaculated (3 boars) spermatozoa that were preincubated in modified TCM-199 for 4 h at 37°C. The percentages of penetrated oocytes with the frozen epididymal spermatozoa were 0–40%. In contrast, none of the occytes were penetrated with the frozen ejaculated spermatozoa. In Exp. 2, oocytes matured in vivo were inseminated in vitro with the frozen epididymal spermatozoa that were known to penetrate oocytes matured in vitro. The penetration rate was 79% and the percentage of polyspermic oocytes was 57%. Culture for 30 h of oocytes matured in vivo and fertilized in vitro resulted in 51% (34/67) developing to the 2-cell stage. These embryos were transferred to 2 recipient gilts. One gilt became pregnant and a litter of 3 (1 live and 2 dead) was born. These results indicate that frozen epididymal spermatozoa can be used for in-vitro fertilization in the pig.

Keywords: in vitro; oocyte; fertilization; pig; embryo transfer