Spermatozoa from 21 mature minke whales (Balaenoptera acutorostrata) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris‐based diluent, and frozen at ‐ 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol‐17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%‐40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E2 levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E2 levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.

We established and maintained somatic cell nuclear transfer embryo-derived stem-like cells (SCNT-eSLCs) from the traditional Korean beef cattle species, HanWoo (Bos taurus coreanae). Each SCNT blastocyst was placed individually on a feeder layer with culture medium containing three inhibitors of differentiation (3i). Primary colonies formed after 2-3 days of culture and the intact colonies were passaged every 5-6 days. The cells in each colony showed embryonic stem cell-like morphologies with a distinct boundary and were positive to alkaline phosphatase staining. Immunofluorescence and reverse transcription-polymerase chain reaction analyses also confirmed that these colonies expressed pluripotent markers. The colonies were maintained over 50 passages for more than 270 days. The cells showed normal karyotypes consisting of 60 chromosomes at Passage 50. Embryoid bodies were formed by suspension culture to analyse in vitro differentiation capability. Marker genes representing the differentiation into three germ layers were expressed. Typical embryonal carcinoma was generated after injecting cells under the testis capsule of nude mice, suggesting that the cultured cells may also have the potential of in vivo differentiation. In conclusion, we generated eSLCs from SCNT bovine embryos, using a 3i system that sustained stemness, normal karyotype and pluripotency, which was confirmed by in vitro and in vivo differentiation.

Abstract. This study evaluated the pregnancy rates following either a controlled internal drug release (CIDR)-based timed artificial insemination (TAI) or an embryo transfer (TET) protocol compared with that following a single PGF2α injection and AI after estrus (AIE) in lactating repeat breeder dairy cows. Fifty-three lactating dairy cows diagnosed as repeat breeders were used in this study and were randomly assigned to the following three treatments. (1) Cows, at random stages of the estrous cycle, received a CIDR device and 2 mg estradiol benzoate (EB; Day 0), a 25 mg PGF2α injection at the time of CIDR removal on Day 7 and a 1 mg EB injection on Day 8. The cows then received TAI 30 h (Day 9) after the second EB injection using dairy semen (TAI group, n=13). (2) Cows, at random stages of the estrous cycle, received the same hormonal treatments as in the TAI group. The cows then received TET on Day 16 using frozen-thawed blastocysts or morula embryos collected from Korean native cattle donors (TET group, n=13). (3) Cows, at the luteal phase, received a 25 mg injection of PGF2α and AIE using dairy semen (control group, n=27). The ovaries of the cows in the TET group were examined by transrectal ultrasonography to determine ovulation of the preovulatory follicles, and blood samples were collected for serum progesterone (P4) analysis. The pregnancy rate was significantly higher in the TET group (53.8%) than in the control (18.5%) or TAI (7.7%) groups (P<0.05). The ultrasonographic observations demonstrated that all the cows in the TET group ovulated the preovulatory follicles and concomitantly formed new corpora lutea. Accordingly, the mean serum P4 concentration remained constant between Day 0 and Day 7 of the luteal phase, decreased dramatically on Day 8 (P<0.01) and subsequently increased by Day 16 (P<0.01). These data suggest that the CIDR-based TET protocol can be used to effectively increase the pregnancy rate in lactating repeat breeder dairy cows. Key words: Controlled internal drug release (CIDR), Lactating repeat breeder dairy cow, Pregnancy rate, Timed artificial insemination (TAI), Timed embryo transfer (TET) (J. Reprod. Dev. 53: [1313][1314][1315][1316][1317][1318] 2007) epeat breeding is a major cause of bovine infertility. Repeated inseminations, delayed conception and increased culling have a severe economic impact on dairy farms associated with repeat breeding [1]. The various problems that cause bovine infertility and delayed conception include reproductive tract abnormalities, estrous detection

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The uptake and synthesis of 19 amino acids by fresh or frozen-thawed bovine blastocysts produced by parthenogenesis (PT) or in vitro fertilization (IVF) were compared in the present study. Fresh blastocysts, 180 h after IVF or PT activation, and frozen-thawed blastocysts, 168 h old and cultured for 12 h post-thawing, were cultured in synthetic oviduct fluid medium (SOFM) containing polyvinyl alcohol (PVA) with both essential and non-essential amino acids (EAA and NEAA, respectively) (Medium 1: M1) or SOFM containing PVA with only EAA (Medium 2: M2). In Experiment 1, when fresh or frozen-thawed PT blastocysts were cultured in M1, the uptake of glutamate (in fresh only), aspartate and arginine, and the synthesis of glutamine and alanine were significantly enhanced. In the culture with M2, serine, asparagine, glutamate, glutamine, glycine, arginine and alanine were significantly taken up. It was found that the glutamine concentrations was significantly higher (P < 0.001) in the culture medium drops containing embryos than in the drops without embryos. In Experiment 2, when PT blastocysts were cultured in M1, the uptake of aspartate and synthesis of alanine were greater (P < 0.01) than those by IVF blastocysts. When M2 was used, a significant (P < 0.01) production of serine, asparagine, glutamate, glutamine and alanine, and the uptake of arginine by PT blastocysts were observed. In Experiment 3, when IVF blastocysts were cultured in M1, fresh blastocysts depleted more aspartate and glutamate, and produced more glutamine and alanine than frozen-thawed blastocysts. When cultured in M2, frozen-thawed blastocysts depleted more threonine (P < 0.01) than fresh blastocysts. These results indicate that the uptake and synthesis of amino acids were different in fresh or frozen-thawed bovine blastocysts derived from PT or IVF. These differences in amino acid metabolism may be related to the viability of the blastocysts.