Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Nitrogen distribution and excretion during embryonic post-yolk sac development inZoarces viviparus

In the viviparous teleostZoarces viviparus (L.) embryonic post-yolk sac development in the ovary is characterized by significant increases in dry weight and nitrogen per embryo, thus indicating an extensive matrotrophic relationship. In the ovarian fluid surrounding the embryos during their intraovarian development, sources of nitrogen were shown to derive from amino acids, urea, ammonia and various cellular components. The level of urea in the ovarian fluid increased significantly from 3.68±0.25 mol urea-N·ml^-1 during early post-yolk sac embryonic development to 6.14±0.44 in late development. The corresponding level of ammonia-N in the ovarian fluid increased from 0.25 to 0.45 mol·ml^-1. An estimation of embryonic nitrogen loss was made by measuring urea and ammonia-N excretion in vitro by post-yolk sac embryos or larvae (i.e. seawater-acclimated embryos). Urea-N constituted an average of 65% of the total nitrogen excreted by the embryos into the ambient medium during a 5-h time-course compared to only 35% in the larvae. Urea-N was excreted at maximum rates during the first hour of the experiment, 0.54±0.09 mol N·g^-1·h^-1 by embryos and 0.35±0.02 by larvae, and then declined to lower levels in both embryos and larvae. A decline after 1 h was also observed for excretion rates of ammonia. In conclusion, the capacity for urea excretion by post-yolk sac embryos ofZ. viviparus may be of adaptive significance for their prolonged stay in ovario. The capacity for excretion of urea seems to decrease after acclimation to sea water.Abbreviations aa amino acid(s) - bm body mass - dw dry weight - NPS ninhydrin-positive substances - sw sea water - ww wet weight     

Effects of footwear on three-dimensional tibiotalar and subtalar joint motion during running

Running is a popular form of recreation, but injuries are common and may be associated with abnormal joint motion. The objective of this study was to determine the effect of three footwear conditions – barefoot (BF), an ultraflexible training shoe (FREE), and a motion control shoe (MC) – on 3D foot and ankle motion. Dynamic, biplane radiographic images were acquired from 12 runners during overground running. 3D rotations of the tibiotalar and subtalar joints were quantified in terms of plantarflexion/dorsiflexion (PF/DF), inversion/eversion (IN/EV) and internal/external rotation (IR/ER). Across the early stance phase (defined as footstrike to heel-off), BF running demonstrated greater tibiotalar joint range of motion for PF/DF (28.2±8.3°) and IR/ER (7.0±1.4°) than the shod conditions (FREE: PF/DF=15.1±5.9°, IR/ER=4.8±2.1°; MC: PF/DF=15.0±6.2°, IR/ER=4.3±0.7°). Also at the tibiotalar joint, BF running resulted in a position significantly more plantarflexed (BF: 2.0±12.5°, FREE: 15.7±12.2°, MC: 16.5±9.3°) and internally rotated (BF: 12.9±4.5°, FREE: 10.7±4.3°, MC: 10.6±3.9°) at footstrike compared to both shod conditions. No differences were detected between the shod conditions at any point in the early stance phase at the tibiotalar joint. The MC condition demonstrated significant differences compared to FREE at several points throughout the early stance phase at the subtalar joint, with the greatest differences seen at 30% in PF/DF (MC −1.4±8.8°: FREE: −0.5±9.0°), IN/EV (MC −8.1±5.7°: FREE −6.3±5.5°) and IR/ER (MC −9.5±5.3°: FREE: −8.7±5.2°). These findings indicate that footwear has subtle effects on joint motion mainly between BF and shod conditions at the tibiotalar joint and between shod conditions at the subtalar joint.     

Enhancement of supercooling capacity and survival by cold acclimation, rapid cold and heat hardening in Spodoptera exigua

Insects can increase their resistance to cold stress by prior exposure to non-lethal cold temperatures. Here, we investigated the supercooling capacity and survival of eggs, 3rd and 5th instar larvae, and pupae of Spodoptera exigua (Lepidoptera: Noctuidae) during CA, and responses to various pre-treatment protocols, including constant temperatures, thermoperiods, and RCH, RHH, RCH + RHH and RHH + RCH combined with thermoperiods. Only acclimated eggs demonstrated a significant decrease in SCP, from −20.7 ± 0.3 to −22.9 ± 0.3 °C, among all experimental groups compared to non-acclimated stages. Survival increased by 17.5% for eggs, 40.0% and 13.3% for 3rd and 5th instar larvae, and by 20.0% for pupae after CA. Compared to controls, survival of eggs under the conditions of thermoperiod (5:15 °C), thermoperiod (5:15 °C) + RHH, and thermoperiod (5:15, 10:20, and 15:25 °C) + RCH significantly increased. In addition, survival of 3rd and 5th instar larvae and pupae increased under the conditions of thermoperiod (5:15 °C) and thermoperiod (5:15 °C) + RCH, possibly due to the induction of heat shock proteins or cryoprotectants. However, the pre-treatments of thermoperiod + RCH + RHH and thermoperiod + RHH + RCH did not significantly enhance survival of any developmental stage. These adaptive responses may allow S. exigua to enhance supercooling capacity and survival in response to seasonal or unexpected diurnal decreases in environmental temperatures.     

Effect of temperature, salinity and delayed attachment on development of the solitary ascidian Styela plicata (Lesueur)

The solitary ascidian Styela plicata (Lesueur) is a common member of epibenthic marine communities in Hong Kong, where seawater experiences extensive seasonal changes in temperature (18-30 °C) and salinity (22-34‰). In this investigation, the relative sensitivity of different developmental stages (i.e., duration of embryonic development, larval metamorphosis and post-larval growth) to various temperature (18, 22, 26 and 30 °C) and salinity (22‰, 26‰, 30‰ and 34‰) combinations is reported. Fertilized eggs did not develop at lower salinities (22‰ and 26‰). At higher salinities (30‰ and 34‰), the duration of embryonic development increased with decreasing temperature (18 °C: 11.5±0.3 h; 30 °C: 8.5±0.3 h). More than 50% of larvae spontaneously attached and metamorphosed at all the levels of temperature and salinity tested. At higher temperatures (22, 26 and 30 °C) and salinities (30‰ and 34‰), functional siphon developed in about 72 h after hatching, whereas at low temperature (18 °C), siphon developed only in <30% of individuals in about 90 h. However, none of the metamorphosed larvae developed subsequently at low salinity (22‰). When forced to swim (or delayed attachment), larvae lost about 0.27 mJ after 48 h (about 22% of the stored energy). Such a drop in energy reserves, however, was not strong enough to cause a significant impact on post-larval growth. This study suggests that temperature and salinity reductions due to seasonal monsoon may have significant effect on the embryo and post-larval growth of S. plicata in Hong Kong.     

Preliminary studies on cryopreservation of snakehead (Channa striata) embryos

This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1 M dimethyl sulfoxide (Me₂SO), 1 M ethylene glycol (EG), 1 M methanol (MeOH) and 0.1 M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and −2 °C for 5 min, 1 h and 3 h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and −2 °C at 1 h and 3 h exposure in each treatment. Heartbeat stage was more tolerant against chilling at −2 °C for 3 h exposure in Me₂SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.     

An investigation of oxidative stress and antioxidant biomarkers during Greenshell mussel (Perna canaliculus) oocyte cryopreservation

Oxidative damage to proteins and lipids, the enzymatic and nonenzymatic antioxidants' response, and the fertilization and development capability of Perna canaliculus oocytes were investigated at critical treatment steps in a previously published controlled-rate cryopreservation protocol. The cryoprotectant (CPA) from this protocol comprises 10% ethylene glycol (v:v) and 0.2 M trehalose (wt/vol) final concentration. Critical treatment steps included (1) seawater control, (2) CPA addition, (3) CPA addition followed by cooling to −6 °C, (4) CPA addition and cooling to −10 °C, and (5) CPA addition and cooling to −35 °C and immersion in liquid nitrogen (LN). The percentage of fertilized oocytes was 53.8 ± 13.3% in the seawater control but was reduced to 26.0 ± 15.6% after −35 °C + LN treatment, whereas development to D-larvae was 21.0 ± 6.4% in the seawater control reduced to 4.8 ± 2.9% after cooling to −6 °C, and was zero at all the subsequent cooling steps. All oxidative damage biomarkers, protein carbonyls (PCs) and lipid hydroperoxides (LPs), and antioxidants, superoxide dismutase (SOD), catalase, glutathione peroxidase, percent reduced glutathione (%GSH), and total glutathione (defined as glutathione; reduced [GSH] plus glutathione disulphide; derived from two molecules of GSH [GSSG]) were measured over all treatments on unfertilized oocytes over a post-treatment recovery period of 0 to 240 minutes in seawater. An ANOVA showed that both treatment and post-treatment periods had significant effects on the concentrations of all biomarkers (P < 0.05). Protein carbonyls and LPs increased very little after CPA addition and cooling treatments, when compared with the seawater control, but large increases up to sixfold occurred between 0 and 240 minutes for the −35 °C + LN treatment. Concentrations of SOD, catalase, total glutathione, and %GSH at 0 minutes decreased by −31.2%, −26.9%, −21.9%, and −25.0%, respectively, between the seawater control and the −35 °C + LN treatment. In contrast, glutathione peroxidase concentrations at 0 minutes increased by 34.3% between the seawater control and the −35 °C + LN treatment. Although most biochemical biomarkers showed an increasing trend over time (0–240 minutes), total glutathione decreased in concentration over time in all treatments, with the greatest decrease after the −35 °C + LN treatment (−41.2%). Significant correlations between biomarkers and D-larvae yield were negative for LPs and positive for SOD, total glutathione, and %GSH (P < 0.05). This is the first report of an investigation using these oxidative stress biomarkers and antioxidant responses on mussel oocytes, and the results have proved useful in characterizing cellular injury during the cryopreservation process.     

Cryoprotective effects of antifreeze proteins delivered into zebrafish embryos

Fish embryo cryopreservation, which is useful in aquaculture or biodiversity conservation, is still far from being achieved. Structural barriers reduce the entrance of cryoprotectants into embryo compartments. Previous studies demonstrated a better ability for freezing in Arctic species which naturally express antifreeze proteins (AFPs). In this study, AFPs were delivered in early zebrafish embryos by incubation in media containing protein. Their cryoprotective effects were then analyzed. Chilling sensitivity was evaluated at 4 °C and −10 °C. Survival rates significantly increased in embryos incorporating AFPI and kept at −10 °C. To analyze their effects on cryopreservation, 5-somite embryos were vitrified. Incorporation of AFPI reduced the percentage of embryos that collapsed at thawing (14.2% of AFPI-treated embryos and 48.9% of controls). Cellular damage caused by vitrification was assessed after thawing by cell dissociation and further analysis of cell survival in culture (SYBR-14/IP labeling). The percentage of viable cells at thawing ranged from 25 to 50%, considered incompatible with embryo development. Cells recovered from frozen-control embryos did not survive in culture. However, the incorporation of AFPs allowed survival similar to that of cells recovered from non-frozen embryos. Blastomere cryopreservation trials incorporating AFPI in the extender also demonstrated a significant increase in viability after freezing. Our findings demonstrated that delivery of AFPs into zebrafish embryos by incubation in media containing protein at early stages is a simple and harmless method that increases cryoprotection of the cellular compartment. This beneficial effect is also noticed in blastomeres, encouraging their use in further protocols for embryo cryopreservation.     

Hypoxia and temperature: Does hypoxia affect caiman embryo differentiation rate or rate of growth only?

In crocodilians, the rate of embryonic development and consequently many posthatch attributes are affected by temperature. Since temperature exhibits strong influences on fitness (embryo survivorship and phenotype) by shaping development, we manipulated oxygen concentration in order to uncouple the effects of developmental rate from the direct effects of temperature. Here we consider whether oxygen constrains either differentiation rate (progression from one stage to the next) or embryonic growth (size). Thus, we incubated Caiman latirostris eggs at various oxygen concentrations, and at two temperatures (31 °C, 100% female-producing temperature, and 33 °C, 100% male-producing temperature). We monitored the developmental stages of these embryos within the thermosensitive period (stages 20–24), and assessed several physiological and morphological hatchling traits. While embryonic size was strongly influenced by oxygen, differentiation rate did not seem to be affected. Very low oxygen concentrations and high temperatures inhibited embryo survival. In addition, oxygen availability affected incubation period and hatchling size, whereas temperature did not cause a significant variation in hatchling size. By investing energy in differentiation hypoxic embryos decreased their size.     

Development of cell-to-cell relationships in the thyroid gland of the chick embryo

Summary Thyroid glands of 7 to 21 day-old chick embryos were examined by electron microscopy using freeze-fracture and thin-section preparations. The primitive follicle lumen first appears between two adjacent epithelial cells in 8 day-old embryos, and is formed in the region of a focal tight junction (macula occludens). The focal tight junction develops into the zonula occludens when the primitive follicle lumen first forms.The zonula occludens is, at first, composed of 4.6 ± 2.45 strands, but with increasing embryonic age the number of strands increases to 5.9 ± 1.41 in 13 day-old, and 8.0 ± 1.75 in 19 day-old embryos.Thyroglobulin stored within the embryonic gland lumina is isolated from the mesenchymal tissue even at the first appearance of these follicle cavities.Well developed gap junctions already occur in the thyroid gland of the 7 day-old embryo, so that an intimate relationship and communication between these cells already exists at the time of their functional differentiation.This study was supported by a grant from the Japan Ministry of Education     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Effects of vascular endothelial growth factor on porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer

This study examined the effects of vascular endothelial growth factor (VEGF) on porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) at different developmental stages. Four sets of experiments were performed. In the first, supplementation of the in vitro culture medium with 5 ng/mL VEGF was suitable for porcine IVF embryo development, and the blastocyst formation rate was significantly higher than the control and other groups (57.73 ± 6.78% (5 ng/mL VEGF) vs. 43.21 ± 10.22% (control), 42.16 ± 10.24% (50 ng/mL VEGF) and 41.91 ± 11.74% (500 ng/mL VEGF); P < 0.05). The total cell number after supplementation with 5 ng/mL VEGF was significantly higher than the control and other groups (151.85 ± 39.77 (5 ng/mL VEGF) vs. 100.00 ± 34.43 (control), 91.2 ± 31.51 (50 ng/mL VEGF), and 112.53 ± 47.66 (500 ng/mL VEGF); P < 0.05). In the second experiment, when VEGF was added at different developmental stages of IVF derived embryos (early stage, days 1-3, late stage, days 4-7), the blastocyst formation rate and total cell number were significantly higher at the late stage (47.71 ± 9.13% and 131.5 ± 20.70, respectively) than in the control (34.32 ± 7.44% and 85.50 ± 20.41, respectively) and at the early stage (33.60 ± 5.78% and 86.75 ± 25.10, respectively; P < 0.05). There was no significant difference in the blastocyst development rate or total cell number between the whole culture period (days 1-7) and the late stage culture period after supplementation with 5 ng/mL VEGF (P > 0.05). In the third experiment, the cleavage rate was significantly higher when SCNT embryos were cultured with VEGF during the whole culture period than in the late stage (63.56 ± 15.52% vs. 39.72 ± 4.94%; P < 0.05), but there was no significant difference between the control and the early stage culture period (P > 0.05). The blastocyst formation rate was significantly higher at the late stage culture period with VEGF than at the early stage culture period (34.40 ± 15.06% vs. (16.07 ± 5.01%; P < 0.05). There was no significant difference in the total cell number between the groups (P > 0.05). In experiment 4, using real-time PCR, VEGF mRNA expression was detected in all the developmental stages of IVF and SCNT embryos, but the expression level varied according to the developmental stage. VEGF receptor, KDR mRNA was detected in all stages IVF and SCNT embryos. However, flt-1 mRNA was not expressed in all embryonic stages of IVF and SCNT embryos. These data suggest that VEGF supplementation at the late embryonic developmental stage might improve the developmental potential of both IVF and SCNT preimplantation porcine embryos through its receptors.     

Chromosome abnormalities in early chicken (Gallus domesticus) embryos

Chromosome studies were undertaken to determine if early embryonic mortality in chicken (Gallus domesticus) embryos is associated with chromosome aberrations. A rapid cytological technique was developed for screening large numbers of embryos for euploidy and aneuploidy. — Of 115 embryos examined, 6 or 5.2% had aberrant chromosome complements. All of these chromosome aberrations occurred in embryos that were phenotypically abnormal. Of 45 macroscopically abnormal embryos, 13.3 % were chromosomally aberrant. These included two cases of haploidy (A-Z), one case of trisomy-1, a case of trisomy-2 and two cases of triploidy (3A-ZZW and 3A-ZWW). — Possible modes of origin for euploid and aneuploid embryos are discussed and consideration given to the significance of these aberrations in relation to embryo viability, constancy of chromosome numbers and nucleolar organization.     

Closed pulled straw vitrification of in vitro-produced and in vivo-produced bovine embryos

The objective of this study was to evaluate the efficiency of the closed pulled straw (CPS) method for cryopreserving in vitro-produced and in vivo-produced bovine (Bos taurus) embryos. Based on the open pulled straw (OPS) protocol, the top end of a CPS was closed by tweezers (heated in a flame) to prevent the cryoprotectant medium containing embryos from contacting the liquid nitrogen. Bovine in vitro or in vivo morulae and early blastocyst embryos were frozen by slow cryopreservation, OPS vitrification, or CPS vitrification. Morphology of postthawed embryos was evaluated, and normal embryos were used for successive culture for 72 h. There were no significant differences between OPS and CPS freezing groups in postthawed in vitro-produced embryos with respect to rates of morphologically normal embryos (mean ± SD, 87.9 ± 5.2% vs. 85.4 ± 4.9%), survival at 24 h (58.0 ± 6.8% vs. 56.3 ± 4.4%), and survival at 72 h (35.2 ± 6.0% vs. 34.9 ± 6.7%). However, both OPS and CPS vitrification resulted in higher postthaw rates of morphologically normal embryo and survival at 24 and 72 h than those of the slow-freezing method (P < 0.05). Similar results were obtained for in vivo-derived embryos. We concluded that CPS vitrification was a feasible method to cryopreserve both in vitro-derived and in vivo-derived bovine embryos. This method not only eliminated the risk of embryo contamination by preventing contact with liquid nitrogen but also retained the advantages of the OPS vitrification method.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 ± 0.33 × 10⁻¹⁵ mol s⁻¹, 1.38 ± 0.58 × 10⁻¹⁵ mol s⁻¹, and 3.44 ± 2.07 × 10⁻¹⁵ mol s⁻¹, respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).     

Chill sensitivity and cryopreservation of eggs of the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae)

There is an increasing need for methods of cryopreservation of arthropods. In particular, Lepidoptera are extremely important in entomological applications for the protection of agricultural crops and forest ecosystems and also in many aspects of biodiversity conservation. Yet, few studies have dealt with cryopreservation techniques in species of this insect order.The aim of this study was to examine the chill sensitivity of eggs of the greater wax moth Galleria mellonella (L.) and the possibility to cryopreserve the eggs by vitrification methods.One day-old eggs were dechorionated with water solutions of 1.25% sodium hypochlorite and 0.04% Tween 80, treated with cryoprotective agents in two steps, subjected to rapid cooling by immersion in LN and stored in a mechanical freezer for 48 h at −140 °C. They exhibited survival rates of 1.6 ± 0.5% after being cooled in LN and 0.6 ± 0.2% after being stored in the mechanical freezer. 92.9% of the larvae that hatched from cryopreserved eggs completed development regularly, producing adults that bred and laid fertile eggs.The hatching rate of eggs in the F1 and F2 generations was higher than 90%. Adult emergences of the progeny of eggs stored at ultra-low temperatures allowed us to establish a laboratory colony.     

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16–22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16–22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16–22 somite stage embryos survived when cooled at −25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at −25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at −140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (−196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.     

Pattern duplications in larvae of the polyembryonic wasp Copidosoma floridanum

 Copidosoma floridanum is a polyembryonic wasp that undergoes total cleavage of the egg followed by proliferation of blastomeres to produce up to 2,000 embryos from a single egg. This unusual mode of development raises several questions about how axial polarity is established in individual embryonic primordia. By examining embryonic development of larvae with duplicated structures (conjoined larvae), we determined that conjoined larvae form by mislocalization of two embryonic primordia to a common chamber of the extraembryonic membrane that surrounds individual embryos. Analysis of an anterior marker, Distalless, in mislocalized early embryos indicated that anterior structures form independently of one another. This suggests each embryonic primordium has some intrinsic polarity. However, during germband extension embryos usually fuse in register with each other, resulting in conjoined larvae with heads facing each other. Analysis of the posterior segmental marker, Engrailed, in conjoined embryos suggested that fusion in register initiates during germband extension. Thus, even though embryonic primordia initially have a random axial orientation, conjoined larvae usually possess a common orientation due to reorientation during germband extension. These observations suggest that differential cellular affinities during segmentation play an important role in embryo fusion. Received: 13 June 1996 / Accepted: 15 August 1996     

Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis

Summary Eggs ofAlligator mississippiensis were incubated at 30 °C and 33 °C throughout incubation up to hatching. Every four days several eggs were opened and the albumen, yolk and extra-embryonic fluids removed and weighed. The embryo was removed and fixed prior to being staged, weighted and measured for various morphometric criteria. Development at 33 °C was accelerated compared with 30 °C in terms of yolk and albumen utilization and embryo growth. Significant losses in yolk mass did not occur until stage 22 at 33 °C but occurred at stage 18 at 30 °C. Different patterns in growth were observed in embryos at the two temperatures at similar morphological stages: between stages 18 and 22 embryos at 33 °C were smaller (in mass and length) compared with embryos at 30 °C despite being morphologically similar. The differences in growth and physiology between embryos at 30 °C (females) and 33 °C (males) were dependent on incubation temperature but not sex. Incubation at 33 °C accelerated both growth and development inAlligator; initially morphogenesis was accelerated by the higher temperature but later, growth rate was accelerated.