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The maturation of ovarian oocytes in vitro and fertilization after transfer to mated animals have been reported for rabbits (Chang, 1955), sheep (Crosby, Ryan & Gordon, 1971), pigs (Motlik & Fulka, 1974; Leman & Dziuk, 1971) and cattle (Sreenan, 1970; Hunter, Lawson & Rowson, 1972). In-vitro maturation of follicular oocytes followed by successful fertilization in vitro has been reported for the mouse (Cross & Brinster, 1970; Iwamatsu & Chang, 1972; Mukherjee, 1972), rat (Niwa & Chang, 1975), hamster (Barros & Munoz, 1974), guinea-pig (Yanagimachi, 1974), rabbit (Brackett, Mills & Jeites, 1972) and man (Edwards, Bavister & Steptoe, 1969). There does not, however, appear to be any report on the fertilization in vitro of follicular oocytes matured in vitro for large domestic animals such as cattle. The present experiment was therefore performed to examine the possibility of capacitation of bovine spermatozoa in reproductive tracts isolated from oestrous cows or in the uteri in situ of oestrous rabbits.
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Summary. Fertilization of rat eggs in vitro could not be achieved when epididymal spermatozoa were preincubated and eggs in clots incubated in a chemically defined medium without D-glucose. Very high proportions (84–100%) of eggs examined were undergoing fertilization when 2·78–8·34 mM-D-glucose were included in the medium. The substitution of D-fructose or D-galactose for D-glucose resulted in very poor penetration rates (0–4%), but D-mannose was effective for fertilization (59–99% penetration). Incubations for sperm capacitation and egg fertilization in different media containing the various hexoses showed that rat epididymal spermatozoa could be partly capacitated without hexose, that D-glucose, D-mannose or D-galactose but not D-fructose is effective for sperm capacitation and that only D-glucose and D-mannose supported penetration of eggs in vitro.
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Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l−1 (NaH2PO4), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l−1. This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l−1 and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001–0.01 μmol phosphate l−1, but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 μmol phosphate l−1. In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l−1 (67%) and 10.0 mmol glucose l−1 (60%) were not statistically different from the percentage at 5.0 mmol glucose l−1 (46%), but was significantly greater than the percentage at 2.5 mmol l−1 (33%). When osmolarity of the medium with 5.0 mmol glucose l−1 was varied by adjusting the amount of NaCl added, more (82–98%) of the one-cell embryos developed to the four-cell stage at 212–276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.
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Summary. Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen–thawed spermatozoa in Medium BO with caffeine (5 mm) and heparin (10 μg/ml). Very high penetration rates (95–100%) were obtained in all oocytes which had been cultured for 0–20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20–22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.
Keywords: in-vitro fertilization; immature oocytes; maturation; bovine; sperm nuclear decondensation
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It has been reported that rat eggs can be fertilized in vitro (Miyamoto & Chang, 1973; Toyoda & Chang, 1974). Further study has revealed that the eggs from immature rats are easier to fertilize in vitro with a lower concentration of spermatozoa (Niwa & Chang, 1973) and that the capacitation of rat spermatozoa can be better achieved in a lower concentration than in a higher concentration of spermatozoa (Niwa & Chang, 1974). The present experiment was designed to determine the optimal sperm concentration and the minimal number of spermatozoa for the fertilization of rat eggs in vitro.
The medium for fertilization was prepared according to the description of Toyoda & Chang (1974). The eggs recovered from naturally ovulating mature rats or from immature rats which had been induced to superovulate were inseminated with epididymal spermatozoa from mature rats, incubated and examined as described by Niwa & Chang (1973). In order
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Summary. Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4·5–5 h at 37°C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17–20 h after insemination 60·6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10·0 and 16·7% with ejaculated spermatozoa, and 3·3, 19·6 and 26·4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27·8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.
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Summary.
Ovulated eggs from mature rats and superovulated eggs from immature rats were incubated with various concentrations of epididymal spermatozoa from mature rats. Few eggs from mature rats and only 16 to 20% of the superovulated eggs from immature rats were fertilized when the sperm concentration was 1·8 to 3·7 × 106 spermatozoa/ml. At concentrations of 0·7 to 1·5 × 106 spermatozoa/ml, 2 to 8% of the eggs from mature rats and 96% of the eggs from immature rats were fertilized. It appears that the eggs from the immature rats are much easier to fertilize in vitro than those from the mature rats, but the optimal concentration of spermatozoa also plays an important rôle.
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Summary. Boar spermatozoa were preincubated for various times in the isolated uterus and oviduct from a maturing gilt and used to inseminate zona-free hamster eggs. The proportions of eggs penetrated and activated were increased, and the interval between insemination and sperm penetration was shortened when the spermatozoa were preincubated for 4–5·5 h instead of 2–3·5 h. Overall penetration rates were higher and sperm penetration occurred about 1 h earlier when the eggs were inseminated with spermatozoa preincubated in the uterus than in the oviduct. It is concluded that the change in ability of boar spermatozoa to penetrate zona-free hamster eggs is due to capacitation which requires 4–4·5 h and 5–5·5 h of preincubation in the isolated uterus and oviduct, respectively.
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Summary. The effects of cryoprotectants and freeze—thawing procedures on the survival of frozen rat morulae were examined. In the samples frozen and thawed in the presence of DMSO, ethylene glycol or glycerol, higher proportions of the embryos developed into blastocysts in culture when they were frozen slowly (50–71%) than when they were frozen rapidly (20–39%), but the thawing rates of the slowly frozen samples did not affect the viability of the embryos. When erythritol was used as a cryoprotectant, all of the embryos frozen—thawed slowly were killed but rapidly thawed embryos survived regardless of the freezing rate (36–52%). The morulae frozen slowly with ethylene glycol or glycerol and those frozen rapidly with DMSO or erythritol were transferred to recipients after thawing and full-term young were obtained with all 4 cryoprotectants.
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Summary. The effects of DMSO, ethylene glycol, glycerol, erythritol, dimethylformamide and sucrose on the survival of unfrozen and frozen mouse morulae were examined. All the agents had a deleterious effect on survival of the unfrozen morulae at 20°C. The harmful effect of erythritol was lower at 0°C and sucrose had a protective effect at that temperature. Dimethylformamide and DMSO were more harmful than ethylene glycol and glycerol at both temperatures. Higher proportions of the morulae frozen in DMSO developed into expanded blastocysts when slowly frozen samples were thawed slowly (82%) than when they were thawed rapidly (66%) and when rapidly frozen samples were thawed rapidly (85%) than when they were thawed slowly (4%). When the samples were frozen in the presence of ethylene glycol or glycerol, higher survival rates were obtained after slow (80–94%) than rapid (1–59%) freezing. When erythritol was used, only embryos thawed rapidly developed (31–50%) in subsequent cultures. Sucrose and dimethylformamide did not afford cryoprotection. Morulae frozen–thawed rapidly in the presence of ethylene glycol, glycerol or erythritol and transferred to recipients developed into normal young.
