Sensitivity to chilling of medaka (Oryzias latipes) embryos at various developmental stages

As an essential step toward cryopreservation of fish embryos, we examined the chilling sensitivity of medaka (Oryzias latipes) embryos at various developmental stages. Embryos at the 2-4 cell, 8-16 cell, morula, blastula, and early gastrula stages were suspended in Hanks solution. They were chilled to various temperatures (usually 0 degrees C), kept for various periods (usually 20 min), then cultured for up to 14 d to determine survival (assessed by the ability to hatch). Embryos at the 2-4 cell stage were the most sensitive to chilling to 0 degrees C, but sensitivity decreased as development proceeded. The survival rate of 2-4 cell embryos was affected after 2 min of chilling at 0 degrees C; although the rate decreased gradually as the duration of chilling increased, 38% of them still survived after 40 min of chilling. Embryos at the 2-4 cell stage were sensitive to chilling at 0 or -5 degrees C, but much less sensitive at 5 or 10 degrees C. The survival rate of 2-4 cell embryos subjected to repeated rapid cooling and warming was similar to that of those kept chilled. When early gastrula embryos were preserved at 0 or 5 degrees C, the hatching rate did not decrease after 12 and 24h of chilling, respectively, but then decreased gradually as storage was prolonged; however, 3-10% of the embryos hatched even after storage for 10 d. In conclusion, although later-stage medaka embryos would be suitable for cryopreservation (from the perspective of chilling sensitivity), chilling injury may not be serious in earlier stage embryos.     

Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos

Stage-dependent chilling sensitivity has been reported for many species of fish embryos. Most of these studies reveal that developmental stages beyond 50% epiboly are less sensitive to chilling, but the chilling sensitivity accelerates rapidly at subzero temperatures. In this study, the effects of methanol and developmental arrest on chilling injury were studied using zebrafish (Danio rerio) embryos at 64-cell, 50% epiboly, 6-somite, prim-6 and long-bud stages. Embryos were exposed to methanol or anoxic conditions before they were cooled to 0 or -5 degrees C with slow (1 degrees C/min), medium (30 degrees C/min) or fast ( approximately 300 degrees C/min) cooling rates and were held at these temperatures for different time periods. Embryo survival was evaluated in terms of the percentage of treated embryos with normal developmental appearance after 3-day culture. Experiments on the effect of methanol on chilling sensitivity of the embryos showed that the addition of methanol to embryo medium increased embryo survival significantly at all developmental stages and under all cooling conditions. Higher concentration of methanol treatment generally improved embryo survival when embryos were cooled at a fast cooling rate of 300 degrees C/min. Experiments on the effect of developmental arrest on chilling sensitivity of embryos showed that embryos at 50% epiboly and prim-6 stages underwent developmental arrest almost immediately after 15 min oxygen deprivation. After 4h in anoxia, the survival rates of the embryos were not significantly different from their respective aerobic controls. Anoxia and developmental arrest had no effect on the chilling sensitivity of zebrafish embryos.     

Incorporation of antifreeze proteins into zebrafish embryos by a non-invasive method

The cryopreservation of fish embryos is a challenge because of their structure, with multiple compartments and permeability barriers, and their high chilling sensitivity. Vitrification at advanced developmental stages is considered to be the more promising option. Nevertheless, all reported attempts have failed. Previous studies demonstrated a better ability for freezing in species that naturally express antifreeze proteins (AFPs). These proteins have been delivered into other fish embryos using time-consuming techniques like microinjection. In the present study, the introduction of FITC labelled AFPs was assayed in zebrafish embryos at early developmental stages (from 2-cell to high blastula stage), before the formation of the yolk syncytial layer, by an easy and non-invasive method and evaluated by fluorescence and confocal microscopy. Incubation with AFPs at 128-cell or high blastula stage provides incorporation of the protein in 50–90% of embryos without affecting hatching. Incubation in media containing protein is a simple, harmless and effective method which makes it possible to treat several embryos at the same time. AFPs remain located in derivatives from marginal blastomeres: the yolk syncytial layer, the most cryosensitive and impermeable barrier, and different digestive organs. Our findings demonstrate that delivery of AFP type I and AFP type III into zebrafish embryos by incubation in media containing protein is a simple and harmless method that may improve cryoprotection of the cellular compartment.     

Effect of cooling rate and partial removal of yolk on the chilling injury in zebrafish (Danio rerio) embryos

High chilling sensitivity is one of the main obstacles to successful cryopreservation of zebrafish embryos. So far the nature of the chilling injury in fish embryos has not been clear. The aim of this study is to investigate the effect of cooling rate and partial removal of yolk on chilling injury in zebrafish embryos. Zebrafish embryos at 64-cell, 50%-epiboly, 6-somite and prim-6 stages were cooled to either 0 degrees C or -5 degrees C at three different cooling rates: slow (0.3 degrees C/min or 1 degree C/min), moderate (30 degrees C/min), and rapid (approximately 300 degrees C/min). After chilling, embryos were warmed in a 26 degrees C water bath, followed by 3-day culturing in EM at 26 +/- 1 degrees C for survival assessment. When embryos were cooled to 0 degrees C for up to 30 min, 64-cell embryos had higher survival after rapid cooling than when they were cooled at a slower rate. When 64-cell embryos were held at -5 degrees C for 1 min, their survival decreased greatly after both slow and rapid cooling. The effect of cooling rate on the survival of 50%-epiboly and 6-somite embryos was not significant after 1 h exposure at 0 degrees C and 1 min exposure at -5 degrees C. However, rapid cooling resulted in significantly lower embryo survival than a cooling rate of 30 degrees C/min or 1 degree C/min after 1 h exposure to 0 degrees C for prim-6 stage or 1 h exposure to -5 degrees C for all stages. Chilling injury in 64-cell embryos appears to be a consequence of exposure time at low temperatures rather than a consequence of rapid cooling. Results also indicate that chilling injury in later stage embryos (50%-epiboly, 6-somite and prim-6) is a consequence of the combination of rapid cooling and exposure time at low temperatures. Dechorionated prim-6 embryos were punctured and about half of yolk was removed. After 24 h culture at 26 +/- 1 degrees C after removal of yolk, the yolk-reduced embryos showed higher embryo survival than did control embryos after rapid cooling to -5 degrees C for 10 to 60 min. Results suggest that cold shock injury after rapid cooling can be mitigated after partial removal of yolk at the prim-6 stage. These findings help us to understand the nature of chilling sensitivity of fish embryos and to develop protocols for their cryopreservation.     

DORMANCY IN SEEDS OF FRASERA CAROLINIENSIS (GENTIAN ACEAE)

In freshly matured seeds of the long-lived monocarpic perennial, Frasera caroliniensis Walt., the embryos are underdeveloped and physiologically dormant. Dormancy was broken by a long period of stratification (chilling) at 5 C. Seventy six percent of the seeds germinated at 20 C (day)/10 C (night) after 98 days of chilling at 5 C, while seeds kept at 5 C germinated to 87% after 205 days. A warm, moist pretreatment was not required for subsequent breaking of dormancy at 5 C. Embryos in fresh seeds averaged 1.3 mm long, but after 12 weeks of chilling they averaged 4.1 mm. Thus, the embryos require a period of chilling to become fully developed, after which seeds can germinate at the afterripening temperatures (5 C) or at some higher temperature. Seeds of F. caroliniensis fit Nikolaeva's (1977) morpho-physiological complex dormancy type.     

Preliminary experiments on the cryopreservation of penaeid shrimp (Penaeus japonicus) embryos, nauplii and zoea

To improve availability of penaeid seedstock during periods of high demand, experiments were conducted to determine the feasibility of stockpiling embryos by freezing them. Embryos were screened for developmental stage; cryoprotectants, chilling effects, and freezing regimens were likewise evaluated. Juvenile forms (embryos, nauplii and zoea) of Penaeus japonicus were exposed to various cryoprotectants, including dimethyl sulfoxide, glycerol, methanol, ethylene glycerol and polyethylene glycol 300 under ambient temperature (25 degrees C). Following this bioassay, maximum safe concentrations of each cryoprotectant were tested on the juveniles under chilling to 0 degree C and with 42 freezing regimens. Methanol was found to be relatively nontoxic. Early developmental stages were the most sensitive to chilling. Initial attempts to freeze P. japonicus juveniles were reported. The survival rate of nauplii and zoea treated with 10% methanol in natural sea water (35 ppt salinity) and frozen to -15 degrees C was 85%, and some nauplii and zoea survived freezing to -25 and -196 degrees C. However, no treatment yielded normal nauplii or zoea after freezing.     

Structural and ultrastructural characteristics of the yolk syncytial layer in Prochilodus lineatus (Valenciennes, 1836) (Teleostei; Prochilodontidae)

The yolk syncytial layer (YSL) has been regarded as one of the main obstacles for a successful cryopreservation of fish embryos. The purpose of this study was to identify and characterize the YSL in Prochilodus lineatus, a fish species found in southeastern Brazil and considered a very important fishery resource. Embryos were obtained through artificial breeding by hormonal induction. After fertilization, the eggs were incubated in vertical incubators with a controlled temperature (28 degrees C). Embryos were collected in several periods of development up to hatching and then fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Morphological analyses were carried out under either light, transmission or scanning electron microscopy. The formation of the YSL in P. lineatus embryos starts at the end of the cleavage stage (morula), mainly at the margin of the blastoderm, and develops along the embryo finally covering the entire yolk mass (late gastrula) and producing a distinct intermediate zone between the yolk and the endodermal cells. The YSL was characterized by the presence of microvilli on the contact region with the yolk endoderm. A cytoplasmic mass, full of mitochondria, vacuoles, ribosomes, endomembrane nets and euchromatic nuclei, indicated a high metabolic activity. This layer is shown as an interface between the yolk and the embryo cells that, besides sustaining and separating the yolk, acts as a structure that makes it available for the embryo. The structural analyses identified no possible barriers to cryoprotectant penetration.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

Characteristics and kinetics of subzero chilling injury in Drosophila embryos.

Drosophila embryos manifest unusually high sensitivity to chilling in that they are killed with increased rapidity by exposure to temperatures between 0 and -25 degrees C in the absence of ice formation. Thus, 50% of 15-h eggs succumb in 35, 4, and 1 h at 0, -9, and -15 degrees C, respectively. The sensitivity becomes substantially greater in embryos at stages of development earlier than 12 h, especially at 3 and 6 h. The killing kinetics at given subzero temperatures between 0 and -25 degrees C are characterized by a shoulder followed by a more-or-less linear decrease in survival with time. The lower the temperature, the shorter the shoulder and the faster the postshoulder decline. The rate of both components follows Arrhenius kinetics, i.e., plots of log rate vs 1/absolute temperature are linear, the slopes being proportional to the activation energy. In both cases the activation energy is high and negative; namely, -46.5 kcal/mol for the shoulder length and -24.7 kcal/mol for the postshoulder inactivation. Negative activation energies are unusual, and according to absolute reaction rate theory, they exist only when the entropy of activation is negative, which suggests that the activated state is more ordered. By combining the duration of the shoulder as a function of time and temperature with the rate of postshoulder inactivation, one can compute survival as a function of temperature for embryos cooled at various rates. For those cooled at less than or equal to 1 degree C/min, the computed curve of survival vs temperature agrees closely with observed survivals. But for embryos cooled at approximately 10 degrees C/min, the drop in survival occurs some 7 to 10 degrees above that computed. Embryos exposed to 0 degree C for greater than 5 min undergo conditioning that renders them more resistant to subsequent exposure to lower temperatures, and those cooled at 10 degrees C/min presumably lack sufficient time at 0 degree C to undergo such conditioning; hence the discrepancy between observed and computed survivals. As a test of the possibility that chilling injury is a consequence of the loss of synchrony of coupled reactions involved in embryological development, embryos were rendered anoxic prior to chilling, a treatment that has been shown by Foe and Alberts to reversibly halt development of early stages. Although anoxia somewhat reduced chilling injury in 6-h eggs, it had no effect on 15-h eggs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Alteration in ultrastructural morphology of bovine embryos following subzonal microinjection of bovine viral diarrhea virus (BVDV)

The aim of the present study was to evaluate the development and ultrastructure of preimplantation bovine embryos that were exposed to bovine viral diarrhea virus (BVDV) in vitro.The embryos were recovered from superovulated and fertilized Holstein-Friesian donor cows on day 6 of the estrous cycle. Compact morulae were microinjected with 20 pl of BVDV suspension (10(5.16) TCID(50)/ml viral stock diluted 1:4) under the zona pellucida (ZP), then washed in SOF medium and cultured for 24-48 h. Embryos were evaluated for developmental stages and then processed immunocytochemically for the presence of viral particles, using fluorescent anti-BVDV-FITC conjugate. Ultrastructure of cellular organelles was analysed by transmission electron microscopy (TEM).After microinjection of BVDV under the ZP, significantly more (p<0.001) embryos (83.33%) were arrested at the morula stage compared with the intact control (30.33%). Immunocytochemical analysis localized the BVDV-FITC signal inside the microinjected embryos. TEM revealed: (i) the presence of virus-like particles in the dilated endoplasmic reticulum and in cytoplasmic vacuoles of the trophoblast and embryoblast cells; (ii) the loss of microarchitecture: and (iii) abnormal disintegrated nuclei, which lacked reticular structure and the heterochromatin area. In all, the embryo nuclear structure was altered and the microarchitecture of the nucleolus had disappeared when compared with the nuclei from control embryos. Dilatation of the intercellular space and the loss of the intercellular gap junctions were often observed in bovine BVDV-exposed embryos.These findings provide evidence for the adverse effect of BVDV virus on the development of bovine embryos, which is related to irreversible changes in the ultrastructure of cell organelles.     

Cryopreservation of flounder (Paralichthys olivaceus) embryos by vitrification

Conventional cryopreservation of complex teleost embryos has been unsuccessful, possibly because their large size (1-7 mm diameter), multi-compartmental structure and low water permeability lead to intracellular ice formation and chilling injury. To overcome these obstacles, we have developed a vitrification procedure for cryopreservation of flounder (Paralichthys olivaceus) embryos. In initial toxicity tests, propylene glycol (PG) and methanol (MeOH) were less toxic to embryos than dimethylformamide (DMF) or dimethyl sulfoxide (Me2SO), whereas ethylene glycol (EG) and glycerol (Gly) were toxic to all tested embryos. Embryos between four-somite and tail bud stages were more tolerant to vitrifying solutions than embryos in other developmental stages. Four vitrifying solutions (FVS1-FVS4) were prepared by combining a basic saline solution (BS2) and cryoprotectants PG and MeOH in different proportions (FVS1: 67, 20 and 13%; FVS2: 60, 24 and 16%; FVS3: 55, 27 and 18%; FVS4: 50, 30 and 20% of BS2, PG and MeOH, respectively). Their impact on flounder embryos was then compared. FVS1 produced the highest survival rate; whereas deformation rate was highest for FVS4. Five-step equilibration of embryos in FVS2 resulted in higher survival rates than equilibration in 4, 3, 2 or 1 steps. Flounder embryos varying from the 14-somite to the pre-hatching stage were cryopreserved in the four vitrifying solutions in liquid nitrogen for 1-7 h. From eight experiments, 20 viable thawed embryos were recovered from 292 cryopreserved embryos. Fourteen larvae with normal morphology hatched successfully from the 20 surviving frozen-thawed embryos from five experiments. Embryos at the tail bud stage exhibited greater tolerance to vitrification than embryos at other stages. These results establish that cryopreservation of flounder embryos by vitrification is possible. The technology has many potential applications in teleost germplasm resource conservation.     

Cryomicroscopic observations of cattle embryos during freezing and thawing

A cryomicroscope was used to observe changes in the appearance of day 6 1 2 to 7 1 2 cattle embryos during cooling and warming in 1.4M glycerol/PBS. Embryos were cooled at various rates between 0.2 and 25 degrees C/min to temperatures between -25 and -60 degrees C and then cooled rapidly ( approximately 250 degrees C/min) to temperatures below -140 degrees C. The volume of the embryos calculated from the cross-sectional area during slow cooling decreased at -25 degrees C to about 50% of the isotonic volume. Fracture planes could be observed in the extracellular ice matrix surrounding the embryos after rapid cooling to approximately -140 degrees C. The fracture planes often touched the zona pellucida and sometimes caused cracks in the zona. Cracks in the zona pellucida were observed more often after rapid cooling from temperatures between -20 to -35 degrees C (9 13 ) than from temperatures between -36 to -60 degrees C (2 7 ). When embryos were warmed rapidly ( approximately 250 degrees C/min) from temperatures below -140 degrees C, no change was observed in the appearance of either the embryo or its surroundings except the melting of the extracellular ice. However, when embryos were warmed slowly (2 or 5 degrees C/min), a series of events was observed; first, at approximately -70 degrees C the cytoplasm and the extracellular space gradually darkened and reached maximum darkness at approximately -55 degrees C. Then, on continued slow warming, the dark material gradually disappeared and finally the large extracellular ice crystals melted.     

Freezing and cryoprotective dehydration in an Antarctic nematode (Panagrolaimus davidi) visualised using a freeze substitution technique

The pattern of ice formation during the freezing of Panagrolaimus davidi, an Antarctic nematode that can survive intracellular ice formation, was visualised using a freeze substitution technique and transmission electron microscopy. Nematodes plunged directly into liquid nitrogen had small ice crystals throughout their tissues, including nuclei and organelles, but did not survive. Those frozen at high subzero temperatures showed three patterns of ice formation: no ice, extracellular ice, and intracellular ice. Nematodes subjected to a slow-freezing regime (at -1 degrees C) had mainly extracellular ice (70.4%), with the bulk of the ice in the pseudocoel. Some (24.8%) had no ice within their bodies, due to cryoprotective dehydration. Nematodes subjected to a fast-freezing regime (at -4 degrees C) had intracellular (54%) and extracellular (42%) ice. Intracellular ice was confined to the cytoplasm of cells, with organelles in the spaces in between ice crystals. The survival of nematodes subjected to the fast-freezing regime (53%) was less than those subjected to the slow-freezing regime (92%).     

Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour.

Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC) > or =10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 degrees C and 15 degrees C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degrees C, but remained high at 25 degrees C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze-fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 degrees C (4 h) plus 1 d of further incubation at 25 degrees C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 degrees C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 degrees C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10-15 degrees C), are caused by gel phase formation.     

Studies on chilling sensitivity of zebrafish (Danio rerio) oocytes

Human activity in the last few decades has had a devastating effect on the diversity of fresh water and marine fish. Further decline of fish population may have serious economic and ecological consequences. One of the most promising techniques to preserve fish population is to cryopreserve their germ cells. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryo cryopreservation and fish oocyte cryopreservation has never been studied systematically. The aim of this study is to investigate the chilling sensitivity of fish oocytes. Experiments were conducted with zebrafish stage III (vitellogenic) and stage V (mature) oocytes, which were chilled at 10, 5, 0, -5 or -10 degrees C for 15 or 60 min using a low temperature bath. Control oocytes were kept at room temperature at 22 degrees C. Oocyte viability was assessed using three different methods: trypan blue staining (TB), thiazolyl blue tetrazolium bromide (MTT) staining and observation of germinal vesicle breakdown (GVBD). The results showed that zebrafish oocyte are very sensitive to chilling and their survival decreased with decreasing temperature and increasing exposure time periods. Normalised survivals assessed with TB staining after exposure to 0, -5 or -10 degrees C for 15 or 60 min were 90.1+/-6.0, 77.8+/-7.6, and 71.2+/-9.3%, and 60.2+/-3.8, 49.6+/-6.7, and 30.4+/-3.0%, respectively. The study found that the sensitivity of viability assessment methods increase in the order of MTT < TB < GVBD. It was found that stage III oocytes were more susceptible to chilling than stage V oocytes, and that individual female had a significant influence (p < 0.0001) on oocyte chilling sensitivity. Zebrafish oocyte chilling sensitivity may also be one of the limiting factors for development of protocol of their cryopreservation.     

Ultrasound enhanced methanol penetration of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos measured by permittivity changes using impedance spectroscopy

Studies on permittivity changes in fish embryos measured by impedance spectroscopy after ultrasound treatment during exposure to cryoprotectant is reported here for the first time. The permittivity changes of zebrafish embryos in cryoprotectant solutions before and after ultrasound treatment were measured using impedance spectroscopy. Zebrafish (Danio rerio) embryos at 50% epiboly stage were exposed to 2 M methanol for 25 min before ultrasound treatment for 5 min at 22 degrees C. Embryos were treated with ultrasound in different frequencies (24 and 48 kHz) and voltages (50, 100, 150 and 175 V) combinations. The results showed a clear increasing trend of permittivity from voltage 50 to 175 V over lower impedance frequency range of 10-10(3) Hz indicating increased methanol penetration into the embryos after ultrasound treatment. The embryo survival was not compromised after ultrasound treatment under conditions used in the present study. The use of impedance spectroscopy technique provides a useful none-invasive tool for detecting changes of cryoprotectant penetration in fish embryos after ultrasound treatment. The technique is especially useful for the selection of the suitable cryoprotectants in embryo cryopreservation and may also allow quantitative measurements in embryo membrane permeability studies.     

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 – 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.     

Optimization of protocols for microinjection-based delivery of cryoprotective agents into Japanese whiting Sillago japonica embryos

Microinjection has proven useful for introduction of low-permeability cryoprotective agents (CPAs) into fish eggs or embryos for cryopreservation. In this work, we examined the suitable conditions for single or combined microinjection into the perivitelline space (PS) and the yolk mass (YM) of embryos of the Japanese whiting, an alternative marine fish model for embryo cryopreservation studies. The parameters examined were injection volume, CPA type and concentration, vehicle (diluent), and suitable developmental stage. Somites and tail elongation embryos tolerated single or combined injection with 2.1 and 15.6 nl in the PS and YM, respectively, whereas earlier embryonic stages tolerated only up to 8.2 nl in the YM. The injected solutions diffused rapidly throughout the PS and YM and remained contained within each compartment unless in the case of structural damage caused by injection of larger volumes. Yamamoto solution was marginally better as a vehicle for microinjection of CPAs than fish Ringer and phosphate buffer saline whereas ¼ artificial sea water was clearly unsuitable. Ethylene glycol was well tolerated by embryos in all developmental stages whereas 1, 2-propylene glycol was suitable only for early embryonic stages. Overall, microinjection was efficient in delivering high loads of CPAs inside whiting embryos more swiftly than previously obtained for this species by immersion-based impregnation protocols. Embryos microinjected with CPAs showed a decrease in embryo nucleation temperature and an increase in chilling tolerance. CPA-microinjected embryos will provide valuable materials to optimize the remaining parameters that are critical for successful cryopreservation such as cooling and warming strategies.