Equilibrium vitrification of mouse embryos at various developmental stages

Previously, we developed a new method by which 2‐cell mouse embryos can be vitrified in liquid nitrogen in a near‐equilibrium state, and then kept at ?80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight‐cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol‐based solutions, named EFSc because of their composition of ethylene glycol (30–40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM‐70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at ?80°C. When 8‐cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at ?80°C, the survival rate was high even after 4 days in storage and remained high after re‐cooling in liquid nitrogen. On the other hand, the survival of vitrified‐expanded blastocysts kept at ?80°C was low. Therefore, 8‐cell embryos and morulae can be vitrified in a near‐equilibrium state using the same method as for 2‐cell embryos. A high proportion of C57BL/6J embryos at the 2‐cell, 8‐cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re‐cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near‐equilibrium vitrification method, which is effective for 2‐cell mouse embryos, is also effective for embryos at the 8‐cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice. Mol. Reprod. Dev. 79: 785–794, 2012. © 2012 Wiley Periodicals, Inc.     

Interspecific interactions in the marine rotifer microcosm

Copepods and protozoans often co-exist in marinerotifer mass cultures. Interspecific interactionbetween the rotifer Brachionus rotundiformisTschugunoff and eight other zooplankton species,namely Brachionus plicatilis O. F. Müller (rotifer),Diaphanosoma celebensis Stingelin (cladoceran),Tigriopus japonicus Mori, Acartia sp. (copepod),Euplotes sp., Vorticella sp., an unidentifiedprotozoan species (P1 strain) (protozoan) and Artemiasp. (anostracan) at two developmental stages (nauplii –0.95 mm, 0 days old; adults – 3.3 mm, 19 days old) wereinvestigated in the laboratory.There was no contaminating species that contributedto an increase in rotifer population growth during theexperiments. Four types of interspecific interactionswere seen between B. rotundiformis and otherco-existing zooplankton species. These include effectson population growth: (1) both species declined, (2) onespecies is promoted while the other is not influenced,(3) one species is declined while the other is notinfluenced and (4) one species is promoted while theother declined. The first type was exhibited by B. rotundiformis vs B. plicatilis, B. rotundiformis vsD. celebensis and B. rotundiformis vs Artemia sp. The second type was exhibited by B. rotundiformis vsVorticella sp. and the third type by B. rotundiformisvs Euplotes sp. and B. rotundiformis vs T. japonicus. The fourth type was exhibited by B. rotundiformis vs Acartia sp. and B. rotundiformis vs P1 strain.     

The concept of biological control methods in aquaculture

Microbial techniques of biocontrol using the interaction ofmicroorganisms to repress the growth of deleterious bacteria andviruses were developed. The bacterial strain used in this work alsoimproved the growth of fishes and crustaceans. Using the conceptandprocedures of the biocontrol method described here, the aquacultureproduction became stable and evenincreased.     

The fecundity patterns of S and L type rotifers of Brachionus plicatilis

Fecundity patterns of S and L type rotifers Brachionus plicatilis, which were previously found to have distinct growth ability, were analyzed using data of age-specific fecundity. The data were obtained by individual cultures of S and L type strains at 17, 24 and 34 °C. The pattern was analyzed by using normal probability functions. When the age was transformed into logarithmic value, the S and L types had an identical pattern of fecundity at every temperature. This fact indicates that the difference of the growth response to the temperature between S and L type strains stemmed only from the differences in net reproduction rate, not from the pattern.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Structural differences between the resting eggs of Brachionus plicatilis and Brachionus rotundiformis (Rotifers, Brachionidae): an electron microscopic study

Surface topography and layering of resting eggs of Brachionus plicatilis and B. rotundiformis are described, based on scanning and transmission electron microscope studies. In B. plicatilis, the resting eggs are spherical with smooth wavy ridges on the surface, whereas these ridges are small and condensed in B. rotundiformis. The distribution of pores on the egg surface clearly distinguishes the two species.Ultrastructure of the egg membranes of both species varies greatly with regard to size, shape and sculpturing of each membrane. The alveolar and dense sublayers, which constitute the outer egg membrane (S1) are very thick (10–12 µm) in B. rotundiformis compared to B. plicatilis (4–5 µm). Thus, each species has a characteristic surface and membrane architecture. The functional roles of these membranes, during the resting phase of rotifers, are discussed.     

Semi-mass culture of the dinoflagellate Gymnodinium splendens as a live food source for the initial feeding of marine finfish larvae

A technique was developed for the semi-mass culture ofthe unarmored dinoflagellate, Gymnodiniumsplendens under laboratory conditions. A maximumcell density of 4600 to 6800 cells ml^–1 was observedwithin 8 to 11 days of culture. An initial feedingtest for 8 days with three important marine finfishlarvae showed that red spotted grouper, Epinephelus akaara preferred G. splendensfed 200 cells ml^–1 with 44% survival. The Japanesestripe knife jaw, Oplegnathus fasciatus,attained 22% survival fed a combination of G. splendensand rotifers (200 cells ml^–1 and 5ind. ml^–1, respectively). Red sea bream, Pagrusmajor larvae did not respond well to the initialfeeding of G. splendens alone. Red seabream were observed to be solely dependent on rotifers(5 ind. ml^–1) as initial food. Gymnodiniumsplendens may be used as a live food in the initialfeeding of red spotted grouper larvae (E. akaara) toreduce mortality and to further enhancegrowth during the critical first few days ofrearing.     

Application of unicellular algae Chlorella vulgaris for the mass-culture of marine rotifer Brachionus

Condensed suspension of Chlorellavulgaris was used for the food of the rotifer Brachionus plicatilis and B. rotundiformis inplace of Nannochloropsis oculata. Thisreport describes the characteristics of C. vulgaris as arotifer food in comparison with N. oculata and thepresent status of this field.The cell components of C. vulgarissuch as protein content, amino acids, minerals andvitamins are generally similar to those of N. oculata. However, the taxonomic status of thesealgal species are different. Based on thesimilarity of cell components, the dietary value ofC. vulgaris is equal in value to that of N. oculata for rotifer growth. Dietary value ofC. vulgaris can be improved by addition ofvitamin B₁₂. This improved C. vulgaris is currently widely used as an indispensable food organism for rotifer culture. Recent investigationshave shown that the use of the condensed suspensionof C. vulgaris makes it possible tosignificantly increase the rotifer density atharvest. Application of condensed C. vulgaris has made rotifer culture quite easy because theculture of N. oculata is no longer required,and intensive rotifer production in aquaculture cannow be realized.     

Application of a growth-promoting bacteria for stable mass culture of three marine microalgae

The practical mass culture of marine microalgae, facesoccasionally unexpected problems or collapse. The effect of amarine bacterium, Flavobacterium sp., which was found topromote growth of a marine diatom Chaetoceros gracilisin the axenic culture condition, was examined on the masscultures of three marine microalgae.Three marine microalgae (C. gracilis, Isochrysisgalbana, and Pavlova lutheri) were mass cultured in 3 lflatbottom flasks (2.5 l capacity of culture medium), in anindoor culture room at a commercial pearl oyster hatchery. Themicroalgal cells and the bacterium were inoculated at the sametime, in the culture media. The specific growth rate andmaximal cell density were determined in treated cultures (withadded bacterial strain) and in controls (without addedbacterial strain). The specific growth rate of C.gracilis in treated cultures was significantly higher thanthat of control cultures, and the stationary growth phase inthe treated cultures lasted longer till the end of the cultureperiod. However, the bacterium had no apparent effect on theexponential growth phase of two phytoflagellates, I.galbana and P. lutheri, but kept longer the high celldensity in the stationary growth phases. The added bacterialstrain (Flavobacterium sp.) was the dominant species(more than 45%) among the bacterial flora during the cultureperiod.     

Developmental potential of bovine nuclear transfer embryos and postnatal survival rate of cloned calves produced by two different timings of fusion and activation

We compared developmental potential of somatic cell nuclear transfer (NT) embryos and postnatal survivability of cloned calves produced by two different fusion and activation protocols. As donor cells for NT, bovine cumulus cell-derived cultured cells of passage 5 were used following culture in serum-starved medium for 5-7 days. Enucleated oocytes were fused with donor cells at 21 or 24 hr post maturation. NT embryos fused at 21 hr were activated chemically 3 hr after fusion (DA group) and embryos fused at 24 hr were activated chemically immediately after fusion (FA group). Chemical activation was accomplished by calcium ionophore for 5 min and cytochalasin D + cycloheximide for 1 hr then cycloheximide alone for 4 hr. After in vitro culture in IVD101 medium for 7 days, embryo transfer was performed. Fusion rates were 86 and 84% in the DA and FA groups, respectively. Developmental rate to the blastocyst stage of NT embryos in the DA group was higher than in the FA group (42% vs. 28%). Pregnancy rate did not differ significantly between the DA and FA groups (11/13 and 5/7 at day 35), and 13 cloned calves (including 1 set of twins from a single embryo transfer) were born. High rates of postnatal mortality were observed in both groups. These results suggest that the DA method improves in vitro developmental potential of NT embryos, but the timing of fusion and chemical activation does not affect the pregnancy rate and the survivability of cloned calves.