In vitro fertilization of follicular oocytes harvested from ovaries and matured in vitro was attempted for 55 minke whales (Balaenoptera acutorostrata) captured for Japanese research purposes in the Antarctic Ocean during the period from November 1995 to March 1996. In Experiment 1, effects of culture duration (96 h or 120 h) on maturation of follicular oocytes and addition of caffeine (5 mM) and/or heparin (100 pg/ml) on sperm penetration and pro‐nuclear formation were investigated. Spermatozoa recovered from the vasa deferentia of four mature males were diluted (5‐fold) and frozen at ‐ 80°C. The post‐thawed and pooled spermatozoa were used for in vitro insemination. A higher (P < 0.05) proportion of the oocytes cultured for 120 h (34.2% of 260) progressed beyond the second metaphase stage than of the oocytes cultured for 96 h (26.0% of 262). For the matured oocytes, higher rates of penetration (P < 0.05) and pronuclear formation (P < 0.01) were obtained in the oocytes cultured for 120 h (55.1% and 40.4%) than in those cultured for 96 h (32.4 % and 20.6%). Addition of caffeine and heparin did not show a significant effect. In Experiment 2, follicular oocytes matured for 120 h and then inseminated were cultured to examine the subsequent development in two culture systems (with and without co‐cultured cumulus cells). Of 448 inseminated oocytes, cleaved embryos (2–16 cells) were observed with (5.8%) and without (4.9%) co‐cultured systems. No cleavage was observed in 54 ova without insemination. These results indicate that in vitro fertilization of minke whale in vitro matured follicular oocytes with cryopreserved spermatozoa is possible, yielding cleaved embryos.

Factors affecting in vitro maturation (IVM) of minke whale (Balaenopetra acutorostrata) follicular oocytes were investigated. In experiment 1, recovery rates for oocytes from follicles of different sizes (small, 1-5 mm; medium, 6-10 mm; large, > or = 11 mm) were similar in both immature (54.7%) and mature (53.5%) females, and the follicular sizes did not affect recovery rate. Approximately half the oocytes recovered from small follicles in immature (55.5%) and mature (52.1%) whales were surrounded by at least a few layers of cumulus cells. Before culture, 71.7% and 61.2% of oocytes from immature and mature whales, respectively, were at the germinal vesicle stage. For IVM, effects of serum type, hormones, and additional cumulus cells (experiment 2) and effects of culture durations (24-120 h, experiment 3) were investigated. The three factors investigated in experiment 2 did not affect maturation rates. TCM199 supplemented with fetal whale serum, hormones, and additional cumulus cells showed the highest rate (21.6%) of matured oocytes and resulted in a significant difference from the rate in medium with only fetal calf serum added (6.6%). The first oocyte with an extruded polar body was observed after 84 h of culture. The maximum rate (27.3%) of matured oocytes was obtained by 96 h of culture, but there was no significant difference in the proportions of matured oocytes between 90 and 120 h in culture. These results indicate that in vitro nuclear maturation of immature follicular oocytes in minke whales can be induced.

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Abstract. The morphology, motility and viability of frozen-thawed southern minke whale (Balaenoptera acutorostrata) spermatozoa was evaluated. The whales used in this study were captured during the period from December 1994 to February 1995 for the Japanese Whale Research Programme under Special Permit in the Antarctic. Each sperm sample recovered from the vasa deferentia of eleven whales was diluted to a 1:9 ratio with Tris (300 mM)-citrate (94.75 mM)-glucose (27.75 mM)-egg yolk (15%; v/v)-glycerol (5%; v/v) diluents. The diluted samples were cooled gradually at 5 C for 2 h, frozen at 80 C and kept in liquid nitrogen. After thawing, sperm motility was evaluated subjectively. Sperm viability was assessed by eosin-nigrosin staining. Sperm abnormality and head morphology were assessed by carbol-fuchsin staining using phase-contrast microscopy. Sperm dimensions were measured using a calibrated projection microscope. Sperm motility and viability respectively varied from 1.0 to 20% (7 out of 11 samples), and 1.0 to 11.6%. Sperm concentration ranged from 13 to 585.3 × 10 6 spermatozoa/ml. The mean proportion of morphologically abnormal spermatozoa was 84.0 ± 6.7%. Seven morphotypes of sperm heads were observed, and the most common types were conical and elliptic in shape. The mean total length of spermatozoa was 56.7 ± 0.5 µm, with their sperm heads measuring 5.2 ± 0.1 µm for length and 3.0 ± 0.0 µm for width; the mean length of sperm tail was 51.7 ± 0.5 µm.

Minke whale (Balaenoptera acutorostrata) follicular oocytes were cryopreserved by a slow-step freezing procedure using ethylene glycol. The morphologically viable proportion of postthawed minke whale follicular oocytes was 39.7%. The maturity of the animals (immature and mature whales) or the presence or absence of cumulus cells (CC) did not affect the proportion of morphologically viable oocytes. Postthawed oocytes were examined for nuclear status after in vitro maturation. The presence of CC (29.1%) significantly enhanced (P < 0.05) the proportion of oocytes at metaphase I/anaphase I/telophase I stages compared to results with the absence of CC (13.5%). A total of 4 of 194 postthawed oocytes matured to the second metaphase stage after culture for 5.5 days with or without CC. The cryopreserved immature oocytes obtained from immature and mature whales were processed to examine the ultrastructure by transmission electron microscopy. Varying ultrastructural damage to the cytoplasm was observed as a result of the cryopreservation procedures. These results show that 20-30% of cryopreserved minke whale follicular oocytes can resume meiosis in vitro, but damage induced by the freezing and thawing procedures was observed.