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.     

The pattern of ovarian development in the prepubertal antarctic minke whale (Balaenoptera bonaerensis)

This study describes the morphological and morphometrical changes associated with prepubertal ovarian development in the Antarctic minke whale (Balaenoptera bonaerensis). Ovaries were harvested from 94 immature minke whales caught in the Antarctic Ocean during the summer feeding season (December-March). Notable differences in ovarian size and morphology were found among animals. Up to 10 folds difference in ovarian weight was found among prepubertal whales of similar body size. During the prepubertal period, ovaries grew slowly and approximately doubled their weight. The morphologies of right and left ovaries were almost identical while the growth of the ovary appears to occur preferentially on the right side. The most striking morphological feature was numerous small antral follicles less than 5 mm in diameter found in ovaries of younger immature whales. The occurrence of these ovaries was highest in whales less than 6 m long and gradually decreased as body length increased. In larger whales, the occurrence of ovaries with a smaller number of follicles up to 10 mm and thick tunica albuginea increased. Thus, the ovary of the Antarctic minke whale experiences bursts of small follicular development during the early prepubertal period before becoming a more developed ovary with fewer but larger follicles, and thick tunica albuginea.     

Attempt at in vitro maturation of minke whale (Balaenoptera Bonaerensis) oocytes using a portable CO2 incubator

The present study was conducted to investigate whether a portable CO2 incubator was effective for in vitro maturation (IVM) of bovine, porcine and minke whale oocytes, and the effect of maturation media supplemented with different hormones; porcine follicle stimulating hormone (pFSH), estradiol-17beta (E2), or pregnant mare's serum gonadotropin (PMSG): human chorionic gonadotropin (hCG) for minke whale immature oocytes was also examined. In vitro maturation rates of bovine and porcine oocytes cultured in the portable CO2 incubator were not significantly different from the standard CO2 incubator. In minke whale IVM culture using the portable incubator, the maximum expansion of cumulus mass was observed by pFSH/E2 and PMSG/hCG at the end of IVM culture. Moreover, the IVM culture period was shortened to 28-30 h from 96-120 h previously reported. The proportion of matured oocytes cultured in the medium supplemented with pFSH/E2 (26.7%) was significantly higher (P<0.05) than that with PMSG/hCG (6.9%). The present study indicates that a portable CO2 incubator is a useful device for minke whale IVM culture on a research base ship, and the addition of pFSH/E2 into an IVM medium enhanced cumulus expansion and the proportion of minke whale matured oocytes.     

毛喉素对猪卵母细胞降脂及冷冻保护效果研究

猪卵母细胞脂质含量高被认为是其冷冻效率低下的重要因素之一。本文通过在猪卵母细胞体外成熟过程中添加化学降脂剂毛喉素(forskolin),检测冷冻前后成熟卵母细胞的脂滴含量、脂滴超微结构、线粒体膜电位、活性氧水平、早期凋亡指标、冻后存活率及发育潜能变化等,研究其对猪卵母细胞降脂和冷冻保护的效果。结果显示,成熟过程中毛喉素处理可部分提高卵母细胞成熟率,但差异不显著(P>0.05)。尼罗红荧光染色显示,毛喉素处理后卵母细胞内脂滴数量及面积在冷冻前后均极显著低于未处理组(P<0.01);超微结构观察发现,经毛喉素处理的卵母细胞中非均质脂滴数量与均质脂滴数量的比例扩大,且比均质脂滴面积更大;冷冻后,卵母细胞中以非均质脂滴为主,脂滴变小变少,分布不均匀,毛喉素处理后非均质与均质间的脂滴数量比例进一步增加。毛喉素处理极显著上调冻后卵母细胞线粒体膜电位(1.04 vs. 0.51,P<0.01),减轻氧化应激,降低早期凋亡率(68.30%vs. 86.03%,P<0.01),从而有效提高了冷冻后卵母细胞的存活率(71.17%vs. 51.47%,P<0.01)和孤雌激活卵...     

Vitrification of fully grown and growing porcine oocytes using germinal vesicle transfer

The survival rate of vitrified germinal vesicle (GV) stage porcine oocytes is very low, and it is not known if the vitrification damages the nucleus, cytoplasm or both. We have evaluated the eventual GV or cytoplasmic damage in fully grown (FG) and growing vitrified oocytes. Fifty-five percent of nonvitrified FG cumulus-denuded oocytes reached the metaphase II (MII) stage in culture. When growing oocytes from preantral (PA) and early antral (EA) follicles were matured in vitro, almost all oocytes were arrested at the GV stage (GV stage: PA 88.9 and EA 79.5%, respectively). When fresh GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes and matured in vitro, some of them reached the MII stage (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). The maturation rate of vitrified FG oocytes was only 6.1% but increased dramatically when vitrified GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes (MII stage: VitFG/FG 43.9%, VitPA/FG 7.1% and VitEA/FG 26.3%, respectively). These results were not significantly different from those for the nonvitrified groups (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). We activated the reconstructed oocytes that received fresh or vitrified GVs (FG/FG, EA/FG, VitFG/FG and VitEA/FG) and examined their embryonic development. Cleaved embryos (nonvitrified groups 13.0-61.8%, vitrified groups 33.3-40.0%) and blastocysts (nonvitrified groups 0.0-18.2%, vitrified groups 0.0-2.9%) were obtained after activation. These results demonstrate that vitrified porcine GVs maintain maturational and developmental competence and that vitrification predominantly damages the cytoplasm.     

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.     

Changes in the amount of proteins, glycogen and lipids in porcine oocytes during in vitro meiotic maturation

Changes in the cytoplasmic inclusions during meiotic maturation were histochemically examined in cultured porcine oocytes. The oocytes contained a small amount of protein and glycogen granules throughout the maturation culture, as well as Sudanophilic lipids composed of small, medium and large droplets. Soon after collection, the amount of Sudanophilic lipid droplets of small and medium size was small and there were 167 ± 11.2 large droplets. After being cultured for 22 h, the number of large lipid droplets decreased remarkably, while the number of small and medium ones increased. There were no differences in the number of Sudanophilic lipid droplets of different sizes between ovulated oocytes and the oocytes cultured for 44 h. The oocytes always contained a large amount of neutral fats and lipoids, but not cholesterols. In the oocytes cultured for 22 h with olomoucine, both the resumption of nuclear maturation and the decrease in the size of the Sudanophilic lipid droplets were inhibited. From the present findings, it appears that the change in the size of the Sudanophilic lipid droplets in the cytoplasm of porcine oocytes is closely related to nuclear maturation.     

Comparative Effects of Porcine Oocytes Vitrified at the GV and MⅡ Stages

This study was designed to compare the efficiency of porcine oocytes vitrified at the GV and MⅡ stages. The vitrified GV oocytes were matured in vitro and then evaluated their cortical granule distribution with immunofluorescence, CD9 protein level with Western blotting and zona pellucida dissolution time with pronse digestion method, as compared to vitrified MⅡ oocytes.The results showed that the survival percentages of oocytes vitrified at the GV stage were significantly lower than those vitrified at the MⅡ stage after 2 h of warming (P<0.05);However, there was no difference in maturation rate between vitrified and fresh oocytes (P>0.05). The oocytes vitrified at MⅡ stage resulted in significantly decreased normal cortical granule distribution and CD9 protein level (P<0.05), but no such results were found in vitrified GV oocytes after maturation (P>0.05). In addition, vitrification did not affect the dissolution time of zona pellucida in GV and MⅡ oocytes (P>0.05). The data demonstrated that despite of lower survival rate compared with MⅡ oocytes, vitrified porcine GV oocytes after maturation showed normal polar body extrusion, cortical granule distribution and CD9 protein level.     

Relationship between serum sex hormone concentrations and histology of seminiferous tubules of captured baleen whales in the Western North Pacific during the feeding season

The present study was conducted to obtain new information on relationships among serum testosterone (T), estradiol-17 beta (E(2)), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) concentrations and histology of seminiferous tubules in captured common minke and Bryde's whales during the feeding season. Blood samples and testes were collected from common minke (n=39 for blood samples, n=15 for testes) and Bryde's (n=14 for blood samples, n=7 for testes) whales captured from May 2001 to August 2001 in the Western North Pacific. Serum T concentrations, in 35.9% of the common minke and 57.1% of Bryde's whales, were below the detection limit (< 2.5 pg/ml). There were no significant differences in the serum concentrations of E(2), FSH, and LH among immature, mature common minke and Bryde's whales except that LH levels of immature Bryde's whales was higher than those of common minke whales. In most seminiferous tubules of mature whales, only a single-layer of spermatogonia was observed. However, spermatozoa were observed in seminiferous tubules in 2/13 of mature common minke and 4/4 of mature Bryde's whales with the low or undetectable T levels. These results indicate that the low serum T concentrations reflect the inactivity of spermatogenesis in both baleen whales, and that it is not possible to assess gonadal activity in either common minke or Bryde's whales using serum sex hormone concentrations during the feeding season.     

猪卵母细胞在GV期与MⅡ期的冷冻保存效果比较研究

为比较猪卵母细胞在GV期与MⅡ期的冷冻保存效果,试验在这两个成熟阶段对其进行玻璃化冷冻,GV期卵母细胞解冻后培养至成熟,MⅡ期卵母细胞解冻后恢复2 h,然后采用免疫荧光标记、Western blotting和链霉蛋白酶溶解方法分别检测它们的皮质颗粒分布、CD9蛋白表达水平和透明带消化时间上的差异。结果表明,GV期卵母细胞在解冻后2 h的存活率显著低于MⅡ期卵母细胞（P<0.05）,但极体排出率与对照卵母细胞无明显差异（P>0.05）;在冷冻MⅡ期卵母细胞中,皮质颗粒的皮质区分布比例和CD9的蛋白表达水平显著下降（P<0.05）,但冷冻GV期卵母细胞经体外成熟后则无明显变化（P>0.05）;冷冻GV期与MⅡ期卵母细胞均不会影响透明带的消化时间（P>0.05）。由此可见,猪卵母细胞在GV期的冷冻存活率虽然较MⅡ期低,但其体外成熟后极体排出率、皮质颗粒分布和CD9蛋白表达水平均未受到冷冻的影响。     

The effect of resveratrol on the developmental competence of porcine oocytes vitrified at germinal vesicle stage

We tested the effects of resveratrol both as a pre‐treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre‐treatment before vitrification of oocytes for 3 hr with 2 μM resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non‐vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non‐vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification‐related damages.     

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.     

Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach

In this study, the fluorescent lipid dye Nile Red, was used to demonstrate that the lipid content of immature bovine oocytes is correlated with the morphological appearance of the ooplasm. Oocytes with a uniform dark cytoplasm contained significantly more intracellular lipids in lipid droplets compared with oocytes with a granulated or pale cytoplasm (p < 0.05). Furthermore, this lipid-analysing technique was applied for the first time on single bovine in vitro embryos, showing a significant increase of the lipid content in lipid droplets after culture in the presence of serum (p < 0.05).     

KIT-KIT ligand in the growth of porcine oocytes in primordial follicles

Mammalian ovaries are endowed with a huge number of small oocytes in primordial follicles (primordial oocytes). The mechanism regulating initiation of oocyte growth and follicular development is not well understood. Several growth factors and cytokines are known to be involved in oocyte growth and follicular development. Herein, the involvement of KIT, a receptor tyrosine kinase, and its ligand, KIT ligand (KL), in the initiation of porcine oocyte growth was examined. At first, KIT expression was examined immunohistochemically in primordial oocytes from neonatal (10-20 days) and prepubertal (about 6 months) pigs. Similar expression of KIT was detected in all oocytes from both the neonatal and prepubertal pigs. Next, to examine the growth of primordial oocytes, ovarian tissues containing primordial oocytes were xenotransplanted into immunodeficient SCID mice. Primordial oocytes from the neonatal pigs grew with follicular development as described previously, whereas those from the prepubertal pigs did not initiate growth in the xenografts after 2 months. To stimulate the growth of primordial oocytes from the prepubertal pigs, they were cultured in a medium supplemented with KL (50 and 100 ng/ml) for 1 or 3 days before xenografting. After 2 months, however, the oocytes did not grow, and the primordial follicles did not develop, although a higher number of primordial oocytes survived in the KL-treated tissues. These results suggest that KIT-KL might not be associated with the growth initiation of porcine primordial oocytes, although they do enhance the survival of the oocytes.     

Description and Analysis of the use of Cold Harpoons in the Norwegian Minke Whale Hunt in the 1981,1982 and 1983 Hunting Seasons

Until 1984, cold harpoons, i.e. harpoons with no detonating device, were used to hunt minke whales in Norway. To investigate the effectiveness of such harpoons and compare them with alternatives, data on kills using cold harpoons were collected as part of a project dealing with alternative killing techniques for whales. Data on 353 whale kills were collected in 1981–83. The criteria used to determine the time of death were cessation of flipper movement, that the mandible relaxed, or that the whale hung immobile from the harpoon line. These criteria do not take into account any movements caused by spinal reflexes. About 17% of the animals died instantaneously (≤10 s). The median survival time was 570 s. Animals died most rapidly if hit in the brain, heart or major blood vessels. If only the lungs were injured, minke whales died less rapidly than terrestrial mammals. For whales that did not die immediately, shooting range, animal size and the angle of the shot all influenced the time to death. The efficiency of cold harpoons could be improved, but their use was no longer considered acceptable, and they were replaced by harpoons with penthrite grenades in 1984.     

High Velocity Projectiles for Killing Whales. Hunting Trials using 20 mm High Velocity Projectiles for Minke Whales in 1982

Norway was requested by the International Whaling Commission (IWC) to explore the use of high-velocity projectiles to replace cold harpoon as killing device for minke whales (Anon 1980). Tests of suitable high-velocity projectiles for minke whales were therefore initiated in 1982 as part of a wider project with the purpose of studying alternative killing methods to the traditional cold harpoon used in the Norwegian minke whale hunt until 1984 (Øen 1995). The results of the trials have previously been presented in unpublished reports to the IWC (Øen 1982, 1983, 1992).     

Cryopreservation of isolated mouse germinal vesicles

The storage of unfertilized oocytes, either immature, maturing or mature, is still unsatisfactory. Here we describe an approach in which germinal vesicles isolated as karyoplasts from immature oocytes are vitrified by open the pulled straws (OPS) method in evacuated porcine zonae pellucidae. After thawing, their survival was almost absolute. Moreover, when thawed GV-karyoplasts were fused to immature oocyte cytoplasts the maturation of reconstructed cells resulted in the production of secondary oocytes--metaphase II.     

Changes in Zona Pellucida Surface after in vivo and in vitro Maturation of Caprine Oocytes

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The zona pellucida (ZP) surface features of ovulated, inmature and in-vitro -matured goat oocytes were evaluated by scanning electron microscopy. The in vitro maturation (IVM) process of the ZP surface of oocytes from prepubertal and adult goats were also compared. Ovulated oocytes were collected from superovulated adult goats. Immature oocytes were recovered from slaughterhouse ovaries of prepubertal and adult goats. In-vitro -matured oocytes from adult and prepubertal goats were obtained after culture in TCM199 supplemented with 20% oestrous goat serum + 10 μg/ml FSH + 10 μg/ml LH + 1 μg/ml estradiol 17β for 27 h at 38.5°C in 5% CO₂ in air. All oocytes were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. Before IVM, the ZP surface of immature oocytes showed a rough surface with tight holes (Type I ZP). After the maturation process, the ZP surface acquired a lattice-like appearance with the outermost layer characterized by the presence of shallower large holes (Type II ZP) . A higher percentage of oocytes showing the mature type II ZP surface was observed in ovulated than in in-vitro -matured oocytes (82.6 versus 56.7%, respectively, p < 0.05). No significant differences were observed in ZP surface features when the IVM process of oocytes (immature and in-vitro -matured oocytes) from adult and prepubertal females was compared. These results show that the morphology of the ZP surface is related to the oocyte maturity in caprine. The IVM process gives rise to an adequate and similar development of the ZP surface in oocytes from adult and prepubertal goats.     

Lipolysis and ultrastructural changes of intracellular lipid vesicles after cooling of bovine and porcine GV-oocytes

The aim of our investigation was to compare the ultrastructure of lipid droplets, and the effect of cooling on intracellular lipid vesicles of bovine and porcine GV oocytes. The lipid droplets in bovine GV oocytes have a homogeneous structure. The utilization of lipids takes place directly from these vesicles without formation of interim lipid compounds. In contrast, there are two kinds of lipid droplets in porcine GV oocytes: 'dark', homogeneous vesicles next to 'grey' vesicles with electron-lucent streaks. Vesicles of each specific group are connected to each other. After a 12-h culture, the formation of the cisternal smooth endoplasmic reticulum layer was always associated with 'grey' lipid vesicles. This is evidence that during oogenesis lipolysis takes place only in 'grey' vesicles. It is supposed that cytoplasmic lipolysis has two stages: 'dark' vesicles change into a 'grey' form followed by a utilization of these 'grey' lipids. Furthermore, both types of lipid droplets in porcine oocytes changed morphologically during cooling: they changed into a spherical form with lucent streaks. Lipid droplets in bovine GV oocytes revealed no visible morphological changes after cooling.