Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes

The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.     

Effects of delayed settlement on post-settlement growth and survival of scleractinian coral larvae

Demographic connectivity requires both the dispersal of individuals between sub-populations, and their subsequent contribution to population dynamics. For planktonic, non-feeding marine larvae, the capacity to delay settlement enables greater dispersal distances, but the energetic cost of delayed settlement has been shown to adversely impact post-settlement fitness in several taxa. Here, we assess whether delayed settlement influences mortality rates or growth rates for the first 6 weeks following settlement of the scleractinian coral, Acropora tenuis. Coral larvae that were settled at 2, 4, and 6 weeks after spawning, and then deployed in the field, showed negligible effects of delayed settlement on post-settlement survival and time to initial budding for colony formation. Between-cohort differences in budding rate appeared to be explained by temporal variation in the post-settlement acquisition of zooxanthellae. The potential for coral larvae to remain in the pelagic zone for increased periods of time with little to no effect on post-settlement survival and growth suggests that the capacity for delayed settlement is likely to have meaningful demographic consequences for broadcast-spawning reef-building corals, and that the predicted trade-off between delayed settlement and post-settlement fitness is less applicable to reef-building scleractinian corals than other taxa with non-feeding larvae.     

Effects of cryopreservation on the ultrastructural anatomy of 3-segment Nereis virens larvae (Polychaeta)

Abstract. A transmission electron microscope study of fresh and cryopreserved Nereis virens larvae at the three chaetiger stage is described with special emphasis on examining the structure of the photoreceptors and surface ciliation of the head, the midgut epithelium, and muscle cells. Complex ectodermal structures such as the developing rhabdomeric adult eyes were unaffected by the cryopreservation procedure. Some loss of surface cilia on the prostomium was observed but is not life-threatening though it may restrict ciliary swimming in the recovered larvae. Loss of pigment from the prostomium is caused by osmotic stress. Structural damage was observed in the digestive tissues of the larvae cryopreserved before or after the optimum stage of development. This damage is potentially more serious and may account for the relatively short time period during development where cryopreservation can be successfully applied.     

Long-wavelength photosensitivity in coral planula larvae

Light influences the swimming behavior and settlement of the planktonic planula larvae of coral, but little is known regarding the photosensory biology of coral at this or any life-history stage. Here we used changes in the electrical activity of coral planula tissue upon light flashes to investigate the photosensitivity of the larvae. Recordings were made from five species: two whose larvae are brooded and contain algal symbionts (Porites astreoides and Agaricia agaricites), and three whose larvae are spawned and lack algal symbionts (Acropora cervicornis, Acropora palmata,and Montastrea faveolata). Photosensitivity originated from the coral larva rather than from, or in addition to, its algal symbionts as species with and without symbionts displayed similar tissue-level electrical responses to light. All species exhibited as much (or more) sensitivity to red stimuli as to blue/green stimuli, which is consistent with a role for long-wavelength visible light in the preference for substrata observed during settlement and in facilitating vertical positioning of larvae in the water column.     

Studies on the cryopreservation of Dictyocaulus viviparus (Nematoda) third-stage larvae

Various cooling (0.1-5,100 degrees C min-1) and warming (20-6,800 degrees C min-1) rates, stepped cooling schedules and four cryoprotective additives (dimethyl sulphoxide, methanol, ethanediol and glycerol) were investigated in cryopreservation studies with Dictyocaulus viviparus third-stage larvae. Exsheathment with sodium hypochlorite was essential to achieve significant survival. With uninterrupted cooling, highest survival (30% normally motile) was achieved with rates of 10-70 degrees C min-1. Survival was higher (50-75%) using 1 degree C min-1 to -10 degrees C followed by plunging into liquid nitrogen. The optimum warming rate was 6,800 degrees C min-1. The use of cryoprotectants led to marginally lower survival while varying the suspending media had no significant effect on survival. X-irradiated, exsheathed third-stage larvae cryopreserved by the optimum protocol yielded 38.3 +/- 4.2% survival. Two calves each infected with 45,000 (15,000 viable) exsheathed, unirradiated, cryopreserved third-stage larvae harboured 494 worms (1.1% infectivity) and 355 worms (0.8%) at necropsy. Numbers of first-stage larvae in the faeces reached 420/g and 105/g respectively 27 days after infection.     

Preliminary studies of sperm cryopreservation in the mushroom coral, Fungia scutaria

Coral species throughout the world are facing severe environmental pressures. Because of this, we began cryobiological studies on the sperm of the mushroom coral, Fungia scutaria. We determined that F. scutaria sperm had a mean length of 56 microm and head diameter of 2.5 microm, and a mean spontaneous ice nucleation temperature of -37.2 +/- 1.7 degrees C. When the sperm were exposed to the cryoprotectant glycerol for 5 or 20 min (at 10% v/v), no fertilized larvae were produced. However, when sperm were exposed for 20 min to propylene glycol (10% v/v), fertilizations were produced at the same rate as untreated control eggs and sperm (P > 0.05), but slightly less for dimethyl sulfoxide (10% v/v) (P < 0.05). Regardless, dimethyl sulfoxide caused less osmotic damage to the sperm membrane than did propylene glycol. Therefore, we used the dimethyl sulfoxide (10% v/v) to develop cryopreservation protocols that yielded good post-thaw morphology and motility (>95%) for coral sperm.     

Photo-movement of coral larvae influences vertical positioning in the ocean

Coral Reefs - Behaviour can have profound consequences for the dispersal potential of an organism. In the marine environment, larvae rely heavily on oceanic currents to migrate from one area to...     

Surface ultrastructure of the advanced third-stage larvae of Gnathostoma nipponicum

The surface ultrastructure of advanced third-stage larvae (AL3) of Gnathostoma nipponicum was studied using scanning electron microscopy. The larvae were recovered from the grass snake Rhabdophis tigrina in the Republic of Korea. Parasites had a globular head bulb with a pair of lips at the anterior end and 2 labial papillae and an amphid on each lip. The head bulb was characteristically armed with 3 transverse rows of hooklets, averaging 36, 38, and 43 in number, increasing posteriorly. A total of 213-232 minute unidentate cuticular spines were present along the entire length of the larvae, forming the transverse striations. Two pairs of cervical papillae were located between the 8th and 12th transverse striations, and a pair of body papillae was seen laterally on the posterior third of the body. A pair of caudal phasmids was recognized near the posterior extremity. The surface ultrastructure of AL3 of G. nipponicum is unique compared with that of other species.     

Auditory sensitivity in settlement-stage larvae of coral reef fishes

The larval phase of most species of coral reef fishes is spent away from the reef in the pelagic environment. At the time of settlement, these larvae need to locate a reef, and recent research indicates that sound emanating from reefs may act as a cue to guide them. Here, the auditory abilities of settlement-stage larvae of four species of coral reef fishes (families Pomacentridae, Lutjanidae and Serranidae) and similar-sized individuals of two pelagic species (Carangidae) were tested using an electrophysiological technique, auditory brainstem response (ABR). Five of the six species heard frequencies in the 100–2,000 Hz range, whilst one carangid species did not detect frequencies higher than 800 Hz. The audiograms of the six species were of similar shape, with best hearing at lower frequencies between 100 and 300 Hz. Strong within-species differences were found in hearing sensitivity both among the coral reef species and among the pelagic species. Larvae of the coral reef species had significantly more sensitive hearing than the larvae of the pelagic species. The results suggest that settlement-stage larval reef fishes may be able to detect reef sounds at distances of a few 100 m. If true hearing thresholds are lower than ABR estimates, as indicated in some comparisons of ABR and behavioural methods, the detection distances would be much larger.     

Water contamination reduces the tolerance of coral larvae to thermal stress

Coral reefs are highly susceptible to climate change, with elevated sea surface temperatures (SST) posing one of the main threats to coral survival. Successful recruitment of new colonies is important for the recovery of degraded reefs following mortality events. Coral larvae require relatively uncontaminated substratum on which to metamorphose into sessile polyps, and the increasing pollution of coastal waters therefore constitutes an additional threat to reef resilience. Here we develop and analyse a model of larval metamorphosis success for two common coral species to quantify the interactive effects of water pollution (copper contamination) and SST. We identify thresholds of temperature and pollution that prevent larval metamorphosis, and evaluate synergistic interactions between these stressors. Our analyses show that halving the concentration of Cu can protect corals from the negative effects of a 2-3°C increase in SST. These results demonstrate that effective mitigation of local impacts can reduce negative effects of global stressors.     

Effects of cryopreservation on the epigenetic profile of cells

Effective cryopreservation protocols are essential for long-term storage of cells and their subsequent clinical application. Freezing protocols are generally considered as safe; however, putative effects on epigenetic marks have not yet been studied in detail. While post-thaw cell survival rates have been used to evaluate the success of cryopreservation protocols, increasing evidence suggests that freezing may be associated with deviations from the physiological epigenetic marks with putative long-term effects on the cells and/or their derivatives. A better understanding of the underlying mechanisms would be beneficial for improving safety and effectiveness of freezing protocols. The purpose of this review is to provide current information regarding epigenetic alterations (DNA methylation and histone modification patterns) associated with cryopreservation.     

Chloroplast ultrastructure of Hypericum perforatum plants regenerated in vitro after cryopreservation

The ultrastructure of leaf mesophyll cells of in vitro cultured Hypericum perforatum L. plants regenerated after cryopreservation was studied. Electron microscopy analysis revealed that the chloroplasts in plants pretreated with abscisic acid and regenerated after cryopreservation were round, with increased amount of starch, rather small volume of the thylakoid system, and destroyed envelope. Plants pretreated with 0.3 M mannitol and cooled at rates of 0.1 or 0.3 °C min^?1 possessed chloroplasts with high starch content that resulted in a reduction of a membrane system. However, the pretreatment with 0.3 M mannitol and cooling at a rate of 0.2 °C min^?1 was the best as chloroplast ultrastructure resembled the controls regenerated without cryopreservation.     

Effects of pressure on swimming behavior in planula larvae of the coral Porites astreoides (Cnidaria, Scleractinia)

Mechanisms governing the behavior of coral planulae are not well understood, particularly those manifesting themselves between the time when the larvae are released and when they settle. Larvae from the hermatypic coral Porites astreoides Lamarck were exposed to different levels of hydrostatic pressure—103.4, 206.9, 310.3, 413.8, and 517.1 kPa (including ambient pressure). Data were collected at stops of the above pressures for 15 min each, respectively. This was done in both an increasing sequence and a decreasing one. When exposed to increases in pressure from 103.4 kPa, larvae swam upward (negative barotaxis) in a spiraling motion. Upon exposure to decreasing pressure from 517.1 kPa, larvae moved downward (positive barotaxis), but the magnitude of the vertical movement was much less than in the case of increasing pressure. This suggests that these larvae are more sensitive to increased pressure than decreasing pressure. High variance was also observed in the responses of these larvae at both the intra- and inter-colony levels. Thus, this behavioral trait is variable within the population. The trait may be genetically based, and thus may be susceptible to alteration by natural selection, although this remains to be demonstrated. This study is the first to document these behavioral mechanisms in coral larvae.     

Effects of cryopreservation procedures on sperm membranes

Empirical approaches to semen cryopreservation have resulted in the production of young in a broad range of species. However, acceptable levels of fertility in most domestic animal species has not been achieved. In this review, an attempt has been made to describe the complexity of the sperm plasma membrane and the many steps in a cryopreservation procedure where membrane perturbations can occur. Improvement in sperm cryopreservation procedures will require a careful consideration of the complexity of the sperm plasma membrane, the interaction of its components and the influence of cooling, freezing and thawing on these interactions.