We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the fibrillar and intermediate configurations decreased significantly, and the proportions of degenerating and homogeneously fluorescent configurations increased. When cultured, 20% of oocytes classified as having fibrillar chromatin resumed meiosis, whereas 82% of intermediate and 81% to 86% of condensed chromatin oocytes did so. Meiotic resumption was higher in oocytes recovered immediately after slaughter, but these oocytes took longer to mature. Duration of maturation significantly affected blastocyst development rates in Cp oocytes recovered after a delay (13% and 38% for oocytes matured 24 and 36 h, respectively). Oocytes recovered after a delay had higher blastocyst development rates than did those collected immediately after slaughter. We conclude that the fibrillar and intermediate chromatin configurations may degenerate during ovary storage, resulting in decreased maturation rates, especially of Cp oocytes. Time of oocyte recovery and duration of maturation significantly affect the rate of blastocyst development. Oocytes with Cp and Ex cumuli have similar developmental competence to the blastocyst stage.

Genetic variants of Na(+)-taurocholate co-transporting polypeptide (NTCP; SLC10A1) and ileal apical sodium-dependent bile acid transporter (ASBT; SLC10A2), which greatly contribute to bile acid homeostasis, were extensively explored in the Korean population and functional variants of NTCP were compared among Asian populations. From direct DNA sequencing, six SNPs were identified in the SLC10A1 gene and 14 SNPs in the SLC10A2 gene. Three of seven coding variants were non-synonymous SNPs: two variants from SLC10A1 (A64T, S267F) and one from SLC10A2 (A171S). No linkage was analysed in the SLC10A1 gene because of low frequencies of genetic variants, and the SLC10A2 gene was composed of two separated linkage disequilibrium blocks contrary to the white population. The stably transfected NTCP-A64T variant showed significantly decreased uptakes of taurocholate and rosuvastatin compared with wild-type NTCP. The decreased taurocholate uptake and increased rosuvastatin uptake were shown in the NTCP-S267F variant. The allele frequencies of these functional variants were 1.0% and 3.1%, respectively, in a Korean population. However, NTCP-A64T was not found in Chinese and Vietnamese subjects. The frequency distribution of NTCP-S267F in Koreans was significantly lower than those in Chinese and Vietnamese populations. Our data suggest that NTCP-A64T and -S267F variants cause substrate-dependent functional change in vitro, and show ethnic difference in their allelic frequencies among Asian populations although the clinical relevance of these variants is remained to be evaluated.

Cyclodextrin, which stimulates cholesterol efflux from cells, was examined for its ability to induce capacitation of mouse spermatozoa. A chemically defined, protein-free medium was used for in vitro fertilization of cumulus-free mouse eggs. Fertilization did not occur in modified Krebs-Ringer bicarbonate medium (TYH) supplemented with 1 mg/ml polyvinylalcohol instead of BSA. However, fertilization was observed when spermatozoa were preincubated with methyl-beta-cyclodextrin (MBCD); fertilization rates increased dose-dependently from 0.25 to 0.75 mM MBCD. The fertilization rate decreased when 0.75 mM MBCD was added to both preincubation and fertilization media versus only the preincubation medium (21% vs. 53%); in sharp contrast, fertilization increased when 4 mg/ml BSA was present in both of the media versus the preincubation medium only (66% vs. 25%). At 0.75 mM, 2-hydroxy-beta-cyclodextrin had a lower ability to capacitate spermatozoa in vitro than MBCD (14% vs. 41%). Eggs fertilized by spermatozoa treated with MBCD (0.75 mM) developed to blastocysts (45%, 36 of 80) when cultured in KSOM. When 160 fertilized eggs were transferred to ICR recipients, 62 live offspring were born. After incubation of mouse spermatozoa for 90 min in 0.75 mM MBCD in TYH medium, the cholesterol content of the spermatozoa was significantly (p < 0.01) lower than that of the control (2.27 +/- 0.09 vs. 4.13 +/- 0.09 nmol unesterified cholesterol/10(7) sperm; mean +/- SEM, n = 5). The proportion of capacitated (B pattern) spermatozoa determined by chlortetracycline fluorescence was higher with MBCD treatment for 90 min than for the control (45% vs. 15%; p < 0.01). The proportion of acrosome-reacted (AR pattern) spermatozoa was not different between MBCD treatment and the control. Therefore, MBCD increased capacitation rather than the acrosome reaction of spermatozoa.

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We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect blastocyst development. Overall, transfer of eight blastocysts produced using roscovitine-treated donor cells and our standard activation protocol yielded three pregnancies, of which two (25% of transferred embryos) resulted in delivery of viable foals. Flow cytometric evaluation showed that roscovitine treatment significantly increased the proportion of cells classified as small, in comparison to growth to confluence or serum deprivation, but did not significantly affect the proportion of cells in G0/G1 (2N DNA content). Transfer of one blastocyst produced using roscovitine-treated donor cells, with addition of roscovitine injection at activation, yielded one pregnancy which was lost before 114 days' gestation. Transfer to recipients of two blastocysts produced using confluent donor cells with addition of cycloheximide at activation gave no resulting pregnancies. We conclude that roscovitine treatment of donor cells yields equivalent blastocyst production after nuclear transfer to that for confluent donor cells, and that direct injection of roscovitine-treated donor cells, followed by activation using sperm extract, is compatible with efficient production of viable cloned foals.

We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2.8%; P!0.05) and tended to be higher than that for injection of sperm extract alone (3.4%; PZ0.07). In experiment 2, there were no significant differences in blastocyst development among treatments with ionomycin once or twice, sperm extract then ionomycin, or ionomycin then sperm extract (range 4.6-7.3%). Overall, transfer of 26 blastocysts resulted in 16 pregnancies (62%) and 9 live foals (35% of transferred embryos). Treatment with sperm extract followed by ionomycin produced a live foal rate per embryo transferred of 5/10 (50%). One foal died of pneumonia 48 h post partum and one foal died at 1 week of age after complications during induction of anesthesia; the remaining seven foals are currently 10-14 months of age.

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