Structure and functions of the placenta in common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales.

The structure and functions of placentas were examined in 3 species of rorqual whales, common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales, with the aim of confirming the structural characteristics of the chorion, including the presence of the areolar part, and clarifying steroidogenic activities and fetomaternal interactions in the placentas of these whales. Placentas were collected from the second phase of the Japanese Whale Research Program under Special Permit in the North Pacific (JARPN II). Histological and ultrastructural examinations revealed that these whale placentas were epitheliochorial placentas with the interdigitation of chorionic villi lined by monolayer uninucleate cells (trophoblast cells) and endometrial crypts as well as folded placentation by fold-like chorionic villi. Moreover, well-developed pouch-like areolae were observed in the placentas, and active absorption was suggested in the chorionic epithelial cells of the areolar part (areolar trophoblast cells). Berlin blue staining showed the presence of ferric ions (Fe(3+)) in the uterine glandular epithelial cells and within the stroma of chorionic villi in the areolar part. An immunohistochemical examination revealed tartrate-resistant acid phosphatase (TRAP; known as uteroferrin in uteri) in the cytoplasm of glandular cells and areolar trophoblast cells. This result suggested that, in cetaceans, uteroferrin is used to supply iron to the fetus. Furthermore, immunoreactivity for P450scc and P450arom was detected in trophoblast cells, but not in areolar trophoblast cells, suggesting that trophoblast cells synthesize estrogen in whale placentas. Therefore, we herein immunohistochemically revealed the localization of aromatase and uteroferrin in cetacean placentas during pregnancy for the first time.

Areolae of the placenta in the Antarctic minke whale (Balaenoptera bonaerensis).

In this study, we examined the existence and structure of areolae and the steroidogenesis of areolar trophoblast cells in the Antarctic minke whale placenta morphologically and immunohistochemically. Placentas were collected from the 15th, 16th and 18th Japanese Whale Research Program under Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. The opening and cavity of fetal areolae formed by taller columnar trophoblast cells (areolar trophoblast cells) with long microvilli and a bright cytoplasm, as compared with the trophoblast cells of the chorionic villi interdigitating with the endometrial crypts, were recognized in observations of serial sections. The opening of the areolar cavity was hidden by chorionic villi with areolar trophoblast cells. Furthermore, a closed pouch-like structure lined by tall columnar cells similar to areolar trophoblast cells within the stroma of chorionic villi was noticed and continued to the areolar cavity, with the opening seen on serial sections. In a surface investigation of the chorion and endometrium by SEM, maternal (endometrial) areolae irregularly surrounded by endometrial folds were obvious. Moreover, we distinguished areolar trophoblast cells with long microvilli attached with many blebs from trophoblast cells. In our immunohistochemical observations, a steroidogenic enzyme, cytochrome P450 side chain cleavage enzyme (P450scc), was detected with strong immunoreactivity in trophoblast cells. However, areolar trophoblast cells showed weak or no immunoreactivity for P450scc.

Variation of cholesterol contents in porcine cumulus-oocyte complexes is a key factor in regulation of fertilizing capacity.

The aims were to explore substances affecting maturation of porcine oocytes and effects of cholesterol efflux by methyl-ß-cyclodextrin (MBCD). Cumulus-oocyte complexes (COCs) were collected from ovaries with or without corpora lutea (CL). Ovarian cholesterol content was determined and histologic sections were prepared for immunostaining of lanosterol 14α-demethylase, a catalytic enzyme during cholesterogenesis. In addition, COCs collected from ovaries without a CL (prepubertal gilts) were subjected to in vitro maturation with MBCD for 22 hours, followed by maturation without MBCD for 22 hours. Fertilizability and developmental competence of matured oocytes were monitored. The cholesterol content in COCs from the ovaries with CL (2.73 µg/µg protein) was higher (P < 0.05) than that from the ovaries without CL (1.88 µg/µg protein). Immunoreactive lanosterol 14α-demethylase was localized mainly in cells within a CL and in proximity to the CL. In COCs from ovaries without a CL, the cholesterol content just before in vitro maturation was 1.29 µg/µg protein, but it was decreased (P < 0.05; 0.51 µg/µg protein) by culturing in MBCD-containing medium for 22 hours, and subsequently increased (1.55 µg/µg protein) by culturing in MBCD-free medium for 22 hours. When oocytes were matured with MBCD for 22 hours and then matured without MBCD for the next 22 hours, the fertilization rate improved (P < 0.05) to 76.9%, and the blastocyst rate (9.5%) decreased (P < 0.05; fertilization and blastocyst rates were 69.6% and 26.3%, respectively, in the control group). We concluded that ovarian cholesterogenesis depended on sexual maturity of the donor and that variation in cholesterol content in COCs during in vitro maturation of porcine oocytes affected their ability to be fertilized.

Structure and steroidogenesis of the placenta in the Antarctic minke whale (Balaenoptera bonaerensis).

There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.

A novel method for detection of chromosomal integrity in cryopreserved livestock spermatozoa using artificially fused mouse oocytes.

PURPOSE: The aim of the present study was to investigate the effect of mouse oocyte volume on the efficiency of chromosomal analysis in livestock spermatozoa. METHODS: Oocytes were injected with bull, ram, boar and dog sperm heads, and then fused with enucleated mouse oocytes. RESULTS: The increment of oocyte volume increased the rates of morphologically normal oocytes after sperm injection, which induced much higher rates of overall chromosome detection in bull, ram and dog spermatozoa. The recipient oocyte volume did not affect the chromosomal integrity. Furthermore, in bull, the chromosomal integrity detected by fused mouse oocytes was similar to that derived from a homologous system. On the other hand, chromosomal plates of boar spermatozoa could not be detected despite the use of fused oocytes. CONCLUSION: These data indicate that fused mouse oocytes improved the efficiency of chromosome detection in bull, ram and dog spermatozoa.

Factors affecting the fertility of ewes after intrauterine insemination with frozen-thawed semen during the non-breeding season.

In this study, two successive field trials were conducted during the non-breeding season to investigate various factors affecting on fertility of Suffolk ewes after intrauterine insemination with frozen-thawed semen. In the first year (Experiment 1), three sperm numbers per insemination dose (0.25, 0.5 and 1 million sperm) and five sheep farms were used, and in the second year (Experiment 2), parity, age, body weight, body condition score (BCS) and postpartum days were investigated to compare pregnancy and lambing rates. High pregnancy and lambing rates (70.6 and 70.6%, respectively) were obtained with 0.25 million sperm per dose. There were no significant differences in the pregnancy and lambing rates among the five farms, but there was a tendency for one farm to have higher pregnancy (75.8%, P=0.065) and lambing (72.7%, P=0.077) rates than those (46.7-53.3% and 45.2-53.3% for the pregnancy and lambing rates, respectively) of the other farms. In Experiment 2, ewe age significantly affected both the pregnancy and lambing rates. Nulliparous ewes had a higher lambing rate (72.0%) than that (44.2%) of multiparous ewes, but a significant difference was not revealed. Regardless of body weight, BCS tended to be an important factor influencing on fertility of ewes. Body weight and the postpartum days did not affect the fertility of ewes. It was concluded from these results that the fertility of Suffolk ewes after intrauterine insemination with frozen semen was significantly influenced by sperm number per dose and ewe age. Nulliparous ewes at less than three years of age and with a BCS of more than 3.0 are expected to have higher fertility than other ewes.

Reproductive ability after uterine artery embolization in a sheep model: observation over 2 seasons.

OBJECTIVE: To investigate the mid-term effect of uterine artery embolization (UAE) on fertility after bilateral UAE with either tris-acryl gelatin microspheres (TAGM) or gelatin particles (GP). MATERIALS AND METHODS: Fertility was compared in 6 ewes that underwent UAE with TAGM, 6 ewes that underwent UAE with GP, and 6 control ewes. All ewes were artificially inseminated or naturally bred for 2 consecutive breeding seasons after UAE. Pregnancies in each breeding season were investigated. RESULTS: Overall, 36 lambs, all normal in appearance, were delivered after 2 breeding seasons. All 18 ewes delivered lambs in at least the first or second breeding season, with 13 having lambs in both the first and second breeding seasons. In the first breeding season after UAE, all 12 ewes in the UAE group delivered lambs, while 5 (83.3%) of the 6 ewes in the control group did. In the second breeding season, 9 (90%) of the 10 ewes that were alive in the UAE group delivered lambs, while 5 (83.3%) of 6 ewes in the control group did. There were no significant differences in the rate of ewes delivering in the first and second breeding season between control and UAE groups (P = .3333; first season, P > .9999; second season, Fisher exact probability test). CONCLUSION: The mid-term influence of UAE on reproductive ability in sheep was minimal.

Effects of seasonal changes on in vitro developmental competence of porcine oocytes.

The aim of this study was to investigate whether seasonal changes affected in vitro developmental competence of porcine oocytes. The relationship between atmospheric temperature and embryonic development of in vitro matured porcine oocytes following intracytoplasmic sperm injection was examined throughout the year. The blastocyst rate (31.1%) in winter (mean atmospheric temperature during December to February: -3.8 C) was significantly higher (P<0.05) than those of other seasons in 2008/2009 (19.7-23.5%; 6.3-17.5 C). The monthly mean blastocyst rates were negatively correlated with the temperatures (r=-0.5944, P<0.05). The results of the present study suggest that porcine embryos could be produced throughout the year, but the in vitro production efficiency was significantly affected by season, i.e., atmospheric temperatures. Furthermore, the results showed that winter is a favorable season for blastocyst production in the region of Obihiro, Hokkaido, Japan.

Fertilizability, developmental competence, and chromosomal integrity of oocytes microinjected with pre-treated spermatozoa in mice.

The aim of the present study was to investigate the safety of sperm pre-treatment during the ICSI procedure using a mouse model. Mouse spermatozoa were treated with methyl-beta-cyclodextrin, lysolecithin, Triton X-100, and dithiothreitol (DTT), and injected into mouse oocytes. The injected oocytes were monitored for chromosomal integrity and pre- and post-implantation development. The chromosomal integrity of the injected oocytes was impaired by in vitro incubation and chemical antagonism. Particularly in the 60-min DTT group, severe chromosome damage increased. Despite the chromosomal damage, the resultant embryos frequently developed to the blastocyst stage. However, the embryos in the 60-min DTT group had significantly higher chromosomal damage and decreased developmental competence to live fetuses. These results indicate that excessive sperm pre-treatment such as DTT for 60 min generates severe chromosome damage in injected oocytes, and that the damage decreases developmental competence to live fetuses but not to blastocysts.

Fertility after uterine artery embolization: investigation using a sheep model.

OBJECTIVE: To investigate the influence of uterine artery embolization (UAE) on fertility after bilateral UAE with either tris-acryl gelatin microspheres (TAGM) or gelatin particles (GP). MATERIALS AND METHODS: Six ewes that underwent UAE with TAGM, 6 ewes that underwent UAE with GP, and 6 control ewes were compared. After hormonal synchronization of the menstrual cycle, artificial insemination (AI) was performed. When pregnancy did not result, ewes were naturally inseminated. RESULTS: After AI, progesterone concentrations in blood increased and were maintained at >1.0 ng/mL in 9 ewes (3 per group). Three ewes became pregnant after AI. The abortion rate was higher in the UAE group. The remaining 15 sheep were naturally inseminated, with 14 delivering 15 lambs. Mean period of term gestation in UAE group ewes was 155.7 versus 158.6 days in control group ewes. Lambs' body weight, body length, and withers height after birth did not differ between those from UAE group and control group. Lambs from ewes embolized with GP tended to be smaller and had lower body weight than those from other groups. CONCLUSION: Uterine artery embolization influenced reproductive ability in sheep and UAE with GP could lead to intrauterine growth retardation.

Production of Sei whale (Balaenoptera borealis) cloned embryos by inter- and intra-species somatic cell nuclear transfer.

The objectives of this study were to choose an effective embryo reconstruction method and an effective post-activation agent for in vitro production of sei whale (Balaenoptera borealis) interspecies somatic cell nuclear transfer (iSCNT) embryos. Moreover, trichostatin A (TSA) treatment of whale iSCNT embryos was performed to improve the in vitro embryo development. In Experiment 1, the fusion rate was significantly higher (88.1%) in embryos reconstructed using the intracytoplasmic cell injection method (ICI) than that (48.7%) in the subzonal cell insertion (SUZI) counterpart. The rates of pseudopronucleus (PPN) formation (77.4 vs. 77.2%) and cleavage (24.5 vs. 37.0%) did not vary between ICI and SUZI. However, the PPN formation and cleavage rates were significantly (P<0.05) lower in the iSCNT embryos than in the parthenogenetic control (95.7% and 64.4%, respectively). Although 21.5% of the bovine parthenogenetic embryos developed to the blastocyst stage, no iSCNT embryo developed beyond the 6-cell stage. In Experiment 2, the cleavage rate did not vary between the TSA (50 nM)-treated and non-treated whale iSCNT embryos (30.5 vs. 32.3%, respectively). Moreover, it did not vary between the TSA-treated iSCNT and SCNT embryos (30.5 vs. 32.0%, respectively). Only one TSA non-treated iSCNT embryo developed to a compacted morula with 20 nuclei. One TSA-treated whale SCNT embryo developed to the 8-cell stage, and out of five whale iSCNT embryos, a 6-cell stage embryo was positive for whale DNA. In conclusion, bovine oocytes have the ability to support development of sei whale nuclei up to the 6-cell stage.

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes.

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60 approximately 81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Effects of the presence and the numbers of corpora lutea in non-delivered and delivered pigs on in vitro oocyte maturation and embryonic development.

The aim of this study was to investigate the effects of the presence and the numbers of corpora lutea (CL) in porcine ovaries on in vitro oocyte maturation and embryonic development following intracytoplasmic sperm injection (ICSI). At oocyte collection, the ovaries of non-delivered and delivered pigs were classified into four groups by CL presence. The effect of the number of CL was also investigated following re-division of the non-delivered groups into four groups. In addition, the progesterone (P(4)) concentrations in follicular fluid (FF) of all the groups were measured to confirm the relationship between the presence and numbers of CL. Throughout the present study, the oocytes recovered from the CL-holding ovaries showed high (P<0.05) oocyte maturation rates, blastocyst rates and P(4) concentrations in FF. Furthermore, in the non-delivered groups, the blastocyst rates and P(4) concentrations in FF seemed to coincide with the CL numbers in each ovary. From these findings, we concluded that the presence and number of CL in the ovary can be used as an indicator for estimation of the developmental competence of porcine oocytes. Additionally, the present study suggests that P(4) in FF influences in vitro oocyte maturation and embryonic development in porcine in vitro production.

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes.

PURPOSE: The aim of the present study was to investigate the fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa. METHODS: After canine spermatozoa were injected into mouse oocytes, the rates of oocyte activation, male pronuclear formation and chromosomal aberrations were investigated. RESULTS: The rates of oocyte activation were comparable (90.6-100%), no matter the sperm type injected. The percentage of male pronuclear formation was higher (P < 0.001) in the freeze-dried spermatozoa (92.3%) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. However, the chromosomal damage in the oocytes injected with freeze-dried spermatozoa was higher (72.9%: P < 0.001) than with fresh (26.9%) and frozen-thawed (21.4%) spermatozoa. CONCLUSIONS: These data indicate using mouse oocytes that freeze-dried canine spermatozoa may potentially fertilize canine oocytes although chromosomal damage is frequently generated.

Effects of semen extenders and storage temperatures on characteristics of frozen-thawed Bryde's (Balaenoptera edeni) whale spermatozoa.

The present study investigated effects of three semen extenders and storage temperatures on post-thaw characteristics of Bryde's whale spermatozoa. Spermatozoa were collected from the vasa deferens of three mature Bryde's whales captured during the Japanese whale research in the north-west Pacific (May to August 2007) after death. The three semen extenders used for freezing were 1) a commercialized synthetic extender (AndroMed: AM), 2) Tris-based + 10% bovine serum albumin (BSA) and 3) Tris-based + egg yolk (EY). The sperm samples from the three whales were frozen with the three extenders, and the post-thaw spermatozoa were stored at three different temperatures (35 C; 20-25 C, room temperature; and 5 C) for 0, 6, 12, 24, 48 and 96 h. At each time-point, total and progressive motility (PM), viability (live or dead), the hypo-osmotic test, defective acrosomes and malformation were examined. Immediately after thawing, AM resulted in similar recovery rates (60.4 and 83.3%) in 2 of the 3 whales examined and had comparable post-thaw recovery rates to those obtained using the EY and BSA extenders. Immediately after thawing, the proportion of PM in EY (17.6%) was higher (P<0.05) than that in BSA (15.0%). In the hypo-osmotic test, the proportions of AM (26.0%) and BSA (25.2%) were higher (P<0.05) than that of EY (17.3 %). The three extenders had similar viabilities (36.7, 37.9 and 32.1%, respectively), but the viability of BSA was higher (P<0.05) than that of EY. The present study showed that a synthetic semen extender, AndroMed, could be used for cryopreservation of whale spermatozoa in addition to Tris-based extenders containing bovine serum albumin or egg yolk. Storage of the post-thaw Bryde's whale spermatozoa was better at 5 C than at room temperature or 35 C. The frozen-thawed Bryde's whale spermatozoa maintained their motility and viability for at least two days at room temperature and for four days at 5 C.

Fertility after different artificial insemination methods using a synthetic semen extender in sheep.

The present study aimed to investigate the fertility of ewes artificially inseminated with three different methods using a synthetic semen extender, AndroMed. The three methods of artificial insemination (AI) were cervical AI with fresh-diluted or frozen-diluted semen at observed estrus, and an intrauterine AI with frozen-thawed semen. A total of 80 ewes were treated with a controlled internal drug release (CIDR) containing 0.3 g progesterone per device for 12 days. In Experiment 1 (26 Suffolk ewes), superovulation was induced with 20 mg follicle-stimulating hormone and 250 IU equine chorionic gonadotropin (eCG) two days and one day before CIDR removal, respectively, during the non-breeding season. In Experiment 2 (54 Suffolk and Suffolk crossbred ewes), an intramuscular injection of 500 IU eCG was administered one day before CIDR removal to synchronize estrus and ovulation during the breeding season. In Experiment 1, fresh-diluted or frozen-thawed semen was deposited into the cervical orifice after estrus detection, and an intrauterine AI with frozen-thawed semen was performed by laparoscopy at a fixed-time basis without estrus detection. Embryos were recovered by uterine flushing 6 days after AI, and the rates of recovered, fertilized (cleaved) ova and embryos at the morula or blastocyst stage were compared among the three AI methods. In Experiment 2, the pregnancy rates after the three AI methods were compared. In Experiment 1, the rates of recovered ova were not significantly different among the three AI methods (52.5-56.7%). The rate of fertilized ova (81.0%) by laparoscopic AI with frozen-thawed semen was significantly higher compared with cervical AI of fresh-diluted (25.5%) or frozen-thawed (3.5%) semen, but the rate of embryos at the morula or blastocyst stage (17.6%) was significantly lower than that of the cervical AI with fresh-diluted semen (69.2%). The rates of ewes yielding fertilized ova were not significantly different among the three groups (44.4, 11.1 and 62.5% for cervical AI with fresh-diluted and frozen-thawed semen and intrauterine AI with frozen-thawed semen). In Experiment 2, the pregnancy rate of ewes intrauterinally inseminated with frozen-thawed semen (72.2%) was significantly higher than those of ewes inseminated cervically with fresh-diluted (5.5%) or frozen-thawed (0.0%) semen. The present results showed that acceptable fertilization and pregnancy rates could be obtained by an intrauterine AI with frozen-thawed semen using a synthetic semen extender (AndroMed), but not sufficient by the cervical AI with either fresh or frozen semen.

Fertility after artificial insemination using a soybean-based semen extender in sheep.

The present study aimed to compare the fertility of ewes intrauterinally inseminated with frozen-thawed semen using a soybean-based semen extender (AndroMed) with those of ewes intrauterinally inseminated with frozen-thawed semen using a Tris-based extender containing either egg yolk or BSA. Suffolk ewes (n=104) were treated with an intravaginal sponge containing 40 mg fluoroprogesterone acetate (FGA) for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin to induce estrus and ovulation during the non-breeding season (July, 2007). Intrauterine insemination was carried out 40-46 h after removal of the FGA sponge (n=90), regardless of the incidence of estrus. The pregnancy rates were not significantly different among the semen extenders containing egg yolk (64.5%) or BSA (58.6%) and AndroMed extender (56.7%). The lambing rates (64.5, 55.2 and 56.7% for the semen extenders containing egg yolk, BSA and AndroMed, respectively) and prolificacy (1.59 to 1.75) were also not significantly different. The present results indicate that an egg yolk-containing semen extender can be replaced with the non-animal derived extender AndroMed, which could be used for intrauterine insemination using frozen-thawed ram semen without reducing fertility.

Chemical properties of epidermal lipids, especially sphingolipids, of the Antarctic minke whale.

It is well known that sphingolipids specifically exist in the terrestrial mammal epidermis and correlate with skin barrier functions. However, the lipid properties of the marine mammal epidermis have not been examined in detail. We thus investigated the chemical composition of lipid components, especially sphingolipids, in the black epidermis (outer skin) of Antarctic minke whales (six mature and six immature specimens). Complex lipid fractions mainly contained cerebroside (CE), cholesteryl sulfate and sphingomyelin (SM), as well as two glycerophospholipids. Moreover, in the superficial layer of the black epidermis, CE was richly abundant but phospholipids were scarce. As component fatty acids, the non-hydroxy monounsaturated very long chain fatty acids (VLFA) within 34 carbons were generally present in CE and SM in the black epidermis. CE also consisted of alpha-hydroxy fatty acids with monounsaturation within C34 (17%) and a slight proportion of omega-hydroxy ones (32:1 and 34:1), the latter being probably derived from acyl-CE. Component sphingoid bases of both sphingolipids were predominantly 4-sphingenine (64%), followed by a C16 analogue (21%). When comparing these by different maturities, mature whales showed sphingolipid profiles with higher levels of unsaturated fatty acids and with shorter sphingoid base chains than those of immature ones. Component analysis revealed that CE sugars were 67% glucose and 33% galactose, and alpha-hydroxy fatty acids only bound to galactose.

Follicle size-dependent changes in follicular fluid components and oocyte diameter in Antarctic minke whales (Balaenoptera bonaerensis).

The concentrations of various components of follicular fluid were compared among three groups of follicles (small, <5 mm; medium: 5-10 mm; large, >10 mm) with a control that consisted of the components of umbilical serum using seven pregnant Antarctic minke whales. Follicular oocytes recovered from the follicles were also used for measurement of oocyte diameter after removing the cumulus cells. The mean diameter of the ooplasm in the oocytes from the large follicles (143.2 microm) was significantly greater than those from the small (127.1 microm) and medium (131.7 microm) follicles, although there were no significant differences in diameter of the whole oocyte and thickness of the zona pellucida among the three follicular sizes. The osmolarity of the follicular fluid from the small follicles (363.3 mOsmol) was significantly lower than that of the medium follicles (388.9 mOsmol) and tended to be lower than that of large (381.9 mOsmol) follicles, respectively, both of which were similar to that of the umbilical serum (379.5 mOsmol). There was no significant difference in the concentrations of all components of the follicular fluid between the medium and large follicles. As compared with the values of the umbilical serum, the total-protein, glucose, albumin and chlorine concentrations of the follicular fluid from the medium and large follicles were significantly higher, and the total cholesterol and calcium concentrations were significantly lower. The concentrations of lactic acid (85.3-136.0 mg/dl) of the follicular fluid from the three groups of follicles were significantly lower than that of the umbilical serum (360.0 mg/dl). Among the follicles, the follicular fluid from the small follicles (136.0 mg/dl) contained a significantly higher concentration of lactic acid than that from the large follicles (85.3 mg/dl). The progesterone concentrations were not significantly different among the fluid from the three group of follicles and the umbilical serum: however, the estradiol 17-beta concentrations of the follicular fluid increased with the size of the follicle (14.3 and 34.6 ng/ml for small and large follicles, respectively). These results offer new information concerning whale reproductive physiology, especially for improvement of in vitro oocyte maturation and related technologies in whales.

Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey.

The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.