The objectives of this study were to isolate bacteria from in vitro fertilized (IVF) bovine embryo product system with or without antibiotics (penicillin + streptomycin) and to determine whether bacterial contamination affected the development of IVF embryos. 10¹ to 10³ colony forming units (CFU)/ml of bacteria were isolated from transport medium and wash medium with antibiotics of slaughterhouse derived ovaries, but no bacteria were recovered from subsequent wash medium of oocytes and in vitro culture medium. In contrast, 10¹ to 10⁶ CFU/ml of bacteria were isolated from all media without antibiotics used in IVF embryo product system. The bacteria isolated from transport medium were mainly gram negative rods and resistant to antibiotics of penicillin, macrolide, tetracyclin and cephem line. The rates of cleavage and development to blastocysts of IVF embryos produced in IVF embryo product system with antibiotics were significantly higher than without antibiotics. It was concluded that effective antibiotics should be added to IVF bovine embryo product system for removing or killing bacteria adhered to ovaries, oocytes or embryos.

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A spermiogram obtained from a boar was found to show separation of the head and tail in 100% of the spermatozoa. The ejaculated spermatozoa and the testes were investigated by light and electron microscopy. The mean proportions of heads and tails in ejaculated spermatozoa were 17.6 ± 3.0% and 82.4 ± 3.0%, and the ratio of heads to tails was 1: 4-5. The mean proportion of mottle spermatozoa was 23.8 ± 4.8%. Most of the tails had a cytoplasmic-like swelling in the middle piece. Ultrastructurally, the ejaculated spermatozoa lacked a basal plate in both the head and the tail, but no other ultrastructural abnormality was observed. The cytoplasm in the middle piece of the tail contained vesicular or tubular structures. The right testis in this boar was located in the scrotum, but the left testis was intraabdominal. Electron micrographs of the scrotal testis showed that the abnormalities were first recognizable in spermatids at the cap phase. Pairs of centrioles were not observed on the nuclear membrane at the caudal pole of the nucleus. Since the centrioles failed to approach the nucleus, it appeared that mechanical connection between the proximal centriole and the nucleus was not established. In addition, the basal plate was not formed on the nuclear membrane. The spermatozoa in the lumen of the seminiferous tubules showed separation of the head from the tail. From these results, it is concluded that the heads were already detached form the tails of the spermatozoa in the testis.

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We examined the efficacy of methyl cellosolve (MC) as a cryoprotectant for cryopreservation of bovine embryos derived from in vivo and in vitro. In the presence of 1.3M-MC, in vitro fertilized (IVF) embryos were cooled from 0 C to -6 C at -1 C/min and seeded, held for 10 min, and then cooled either at -0.3 C/min or at -0.5 C/min to -30 C. They were plunged and stored in liquid nitrogen. While no significant difference in the survival rates after 48 h of post-thaw culture was observed beween the 2 cooling rates, the development rates to hatched blastocysts were significantly (P<0.01) higher in cooling at a rate of -0.3 C/min than at a rate of -0.5 C/min. When IVF embryos were cryopreserved in the presence of various concentration of MC or ethylene glycol (EG), the survival and the development rates to hatched blastocysts were higher in 1.3M -MC and 1.8M-EG than in other concentrations of the each cryoprotectant. Sixty one of in vivo embryos collected from 10 superovulated Japanese Black cows and 48 of blastocysts produced in vitro were cryopreserved in the presence of 1.3M-MC or 1.8M -EG, and transferred immediately after thawing into synchronized recipients without removing the cryoprotectant. The pregnancy rates originated from in vivo and in vitro embryos were 48.3% (14/29), 47.6% (10/21) using 1.3M-MC and 50.0% (16/32), 63.0% (17/27) using 1.8M-EG, respectively. These results indicated that MC has the efficacy as a cryoprotectant for direct transfer of bovine embryos.

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The relationship between thawing rate and the viability of frozen bovine blastocysts derived from in vitro fertilization and culture was examined. Embryos were frozen in straws with 1.4M glycerol+0.2M sucrose(GS), 1.6M 1,2-propanediol(PG) or 1.8M ethylene glycol(EG). They were thawed at various rates by placing the straws in 10, 20, 30, 40 or 50 C water bath. As soon as the crystallized medium in the straw melted, the contents were drained into a petri dish. The embryos were transferred into culture medium and co-cultured with cumulus cells for up to 72 h. When embryos were frozen in GS, there were no significant differences in the proportion of survived embryos which recovered the blastocoel among the various thawing rates, but hatching rate of the embryos increased with increasing the thawing rate. When embryos were frozen in PG, the survival (94%) and hatching rates (77%) of embryos thawed in 20 C water bath (876 C/min) were significantly higher (P<0.05) than those observed in other thawing rates. When embryos were frozen in EG, there were no significant differences in the rates of survival (82∼94%) and development (73∼91%) with all the thawing rates.

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[in Japanese]

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The objective of this study was to investigate the formation of bovine embryonic stem (ES) like colonies according to different stage (morula or blastocyst) and derivation (in vivo or in vitro fertilization) of bovine embryos. The zona pellucidae of morula stage embryos were removed manually by stainless steel blade (BIO-CUT, FEATHER), and blastomeres were disaggregated by trypsin. Thereafter, blastomeres were seeded onto mitomycin C-treated mouse embryonic fibroblasts feeder layers cultivated in DMEM containing 20%FBS, glucose, sodium bicarbonate, non-essential amino acid and β-mercaptoethanol. On the other hand, inner cell masses (ICM) were isolated from blastocyst stage embryos by BIO-CUT and seeded onto the same medium as described above. At 7 day after seeding, the formation rate of ES like colonies were determined. When morula stage embryos were used, ES like colony was not observed neither in vivo nor in vitro. In contrast,when blastocyst stage embryos were used, the formation rate of ES like colonies in vivo and in vitro were 16.0% and 17.1% respectively. There was no significant difference between these 2. In conclusion, our experiment demonstrated that blastocyst stage embryos were suitable for obtaining ES like colonies on our culture method.

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Natural mating in the dog is done by a special mechanism the ejaculated semen being transported directly from the vagina to the uterus depend on swelling and retention of the penis in the vagina, such mechanism defend backflow of semen. This trial was undertaken on transport the liquid (IODAMIDE MEGLUMIN) into the uterus of the estrous bitches. A balloon-like artificial penis made with latex rubber (Figs. 2, 3)was inserted into the vagina. Then adequate volume of air was infused into the artificial penis with a syringe through the tube for expansion. This prevents prolapse of the rubber penis by expansion of the rub- bercoital lock. Then about 10 ml of the liquid was infused through the other tube. And 5 minutes later, most of the liquid had been observed in the uterine horns by X-ray photograph (Fig.4) Hereafter, this method will be compared with the intravaginal insemination method elevating the hindquarters of the estrous bitch.

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Using the artificial penis, 6 bitches (the total number) were inseminated with fresh semen. As a result, they were all pregnant. These results indicate that an adequate amount of sperms had been transported into the uterus.This suggests that this technique can be utilized in the case of frozen semen.

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A convenient and simple defatting method using acetonitorile and n-hexan for estradiol-17β radioimmunoassay (RIA) is established. Using the present defatting method, plasma or serum estradiol-17β concentrations of various animals having low blood levels of estradiol-17β can be measured conveniently without the purification of estradiol-17β using LH20 column chromatography. Data of plasma from cows used the present defatting method were coincided well with data by defatting method using methanol and n-hexan. The specific steps in the procedure of this defatting methods were as follows. 1) Ether extraction: Standard solutions or plasma (serum) samples (1 ml) were transferred to glass tubes (13 × 100 mm) for ether extraction. Three ml diethyl ether was added to each tube. The tubes were agitated to extract estradiol-17β and stilled for 5 minutes. The tubes were subsequently dipped in dry ice-ethanol bath to freeze the water layer and ether layer was transferred to a new glass tube (12 × 75 mm) by decapitation. 2) Defatting: To remove substances that interfere with the estradiol assay, after drying the ether, a solution of a mixture of 1 ml n-hexane and 0.5 ml acetonitorile (V/V) was added, and the tubes were mixed. This procedure was repeated twice. After aspiration of the hexane phase, the methanol phase was dried with nitrogen gas and redissolved in 0.1 ml PBS (50 mmol/L, pH 7.5) containing 1% (W/V) BSA (BSA-PBS), and the solution was used for the estradiol RIA.

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[in Japanese]

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[in Japanese]

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[in Japanese]

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