Development, surface exposure, and embryo behavior affect oxygen levels in eggs of the red-eyed treefrog, Agalychnis callidryas

Oxygen stress can slow development, induce hatching, and kill eggs. Terrestrial anamniote embryos face a potential conflict between oxygen uptake and water loss. We measured oxygen levels within eggs to characterize the respiratory environment for embryos of the red-eyed treefrog, Agalychnis callidryas, a Neotropical frog with arboreal egg masses and plastic hatching timing. Perivitelline oxygen partial pressure (Po2) was extremely variable both within and among eggs. Po2 increased with air-exposed surface of the egg and declined over the developmental period before hatching competence. Through the plastic hatching period, however, average Po2 was stable despite continued rapid development. Development was synchronous across a wide range of perivitelline Po2 (0.5-16.5 kPa), and hatching-competent embryos tolerated Po2 as low as 0.5 kPa without hatching. The variation in Po2 measured over short periods of time within individual eggs was as great as that measured across development or surface exposure, including sharp transients associated with embryo movements. There was also a strong gradient of Po2 across the egg from superficial to deep positions. Ciliary circulation of fluid within the egg is clearly insufficient to keep it mixed. Embryos may maintain development under hypoxic conditions by strategic positioning of respiratory surfaces, particularly external gills, to exploit the patchy distribution of oxygen within their eggs.     

The effects of nest temperature,nest substrate,and clutch size on the oxygenation of embryos and larvae of the Australian moss frog,Bryobatrachus nimbus

The jelly around amphibian eggs presents a formidable barrier to oxygen diffusion. Therefore, egg capsules must be thin enough, and the dimensions of globular egg masses small enough, to avoid oxygen limitation leading to developmental retardation or death. The eggs of the Australian moss frog, Bryobatrachus nimbus, have the thickest jelly capsule known for any anuran amphibian. Laboratory measurements of respirometric variables predict that single prehatching embryos should be normoxic between 5 degrees and 20 degrees C, with Po(2 in) maintained above critical levels (10.2-17.0 kPa). However, numerical models of embryos amid larger egg masses (13-20 eggs) predict hypoxia at temperatures above 5 degrees C. Contrary to model predictions, however, B. nimbus embryos rarely experience hypoxia in natural nests, because embryos occur in one or two layers and the moss substrate permits aeration of the lower surface while photosynthesis probably supplies oxygen directly. After hatching, larvae move to oxygen-rich regions of the jelly mass and disperse more widely within the mass as temperatures increase. Although nest characteristics relieve diffusive constraints, small clutch sizes, low rates of embryonic and larval respiration, and the cool climate occupied by B. nimbus are the main characteristics that prevent hypoxia.     

THE EFFECT OF TEMPERATURE ON EGGS AND IMMATURE STAGES OF CULEX ANNULIROSTRIS SKUSE (DIPTERA: CULICIDAE)

Abstract
No immature stages of Culex annulirostris were found during field sampling in 1979–1980 when the average water temperature was < 17 °C; they reappeared when the average water temperature was 19 °C and reached the peak density (mean 107 immatures/cylinder) at 26.5 °C.
The effect of 6 temperatures (15–40°C) on egg hatching, development and survival of the immature stages of Cx annulirostris in the laboratory showed that at 15 and 40°C, eggs failed to hatch and larvae died in the first instars. The optimum temperatures for egg hatching and the survival of immature stages were 25 and 30°C. At these temperatures, 85 and 82% respectively of egg rafts hatched, the mean number of larvae per raft was 258 ± 9.8 and 260 ± 11.4 with immature survival of 83.5 and 79.0% respectively. Mean time to hatch at 20–35°C ranged from 1.2 d (35°C) to 2.9 d (20 °C). Developmental times from first instar to adult ranged from 7.1 d (35 °C) to 25.2 d (20 °C). The threshold for development of the immatures was 15.6 ± 2.5°C and the thermal constant was 142.9 ± 26.5 day—degrees (incubation temperatures 20–35°C). At less suitable temperatures of 20 and 35 °C, hatching (57.5 and 45%), number larvae per raft (mean 139.8 ± 9.8 and 102.6 ± 14.2) and survival were low.     

Biotic and abiotic factors affect the nest environment of embryonic leatherback turtles, Dermochelys coriacea

Clutches of leatherback turtles, Dermochelys coriacea, have lower hatching success than those of other sea turtles, but causes of high embryonic mortality are unknown. We measured characteristics of clutches along with spatial and temporal changes in PO(2) and temperature during incubation to determine the extent to which they affected the developmental environment of leatherback embryos. Minimum PO(2) in nests decreased as both the total number and mass of metabolizing embryos increased. Increases in both the number and mass of metabolizing embryos caused an increase in maximum nest temperature. However, neither PO(2) nor temperature was correlated with hatching success. Our measurements of relatively high nest PO(2) (lowest 17.1 kPa or 16.9% O(2)) indicate that hypoxia apparently does not cause the low hatching success of leatherback clutches. Oxygen partial pressure increased and temperature decreased from the center toward the periphery of leatherback nests. We inferred from these measurements that positions of eggs within nests vary in quality and potentially affect overall developmental success of entire clutches. The large metabolic mass of leatherback clutches and limits to gas flux imposed by the sand create a situation in which leatherback embryos collectively affect their own environment.     

Effects of temperature on energy cost and timing of embryonic and larval development of the terrestrially breeding moss frog, Bryobatrachus nimbus

The Australian moss frog, Bryobatrachus nimbus, oviposits four to 16 large eggs in terrestrial nests constructed in moss or lichen in subalpine regions of southern Tasmania. Nidicolous larvae overwinter beneath snow, reaching metamorphosis without feeding after 395 d, the longest development time known for an endotrophic anuran. However, a few clutches develop more quickly and metamorphose before winter. This study examines the effect of temperature on development time and energy expenditure by measuring temperatures and developmental stages in field nests as well as rates of oxygen consumption (Vo2), developmental stage, body mass, and energy content in the laboratory at three relevant temperatures (5 degrees, 10 degrees, 15 degrees C). Eggs and larvae reared at 5 degrees C differentiated very slowly, and their development time far exceeded those in natural nests, but development times at 10 degrees and 15 degrees C averaged 277 and 149 d, respectively, and were shorter than field incubation times. Generally, respiration rates of aquatic hatchlings were low in comparison with other species but increased with larval age and jumped about 25% higher near metamorphosis when larvae were able to air breathe. The mean energy density was 26.0 J mg(-1) for the dry ova and 20.6 J mg(-1) for a dry gut-free froglet, and total production efficiency was 61.5%. We developed a model based on the relationships between incubation temperature and V&d2;o2 to estimate the respiratory cost of development to metamorphosis, the first such study for an amphibian. The cost was 177 J at 15 degrees C, 199 J at 10 degrees C, and at least 249 J at 5 degrees C, and we predicted that continual development at 5 degrees C would lead to premature yolk depletion because it equalled the 249 J contained in fresh ova. Continuously logged field-nest temperatures and interpolation of laboratory data provided estimates of development rates, Vo2, and respiratory energy costs in field nests. Development to metamorphosis required between 185 and 234 J when larvae overwintered, but completion of metamorphosis before winter saved 123 J. However, the advantage of emergence in warmer months, when conditions are suitable for feeding and growth, may offset the greater energy cost of overwintering.     

Effects of hypoxia on embryonic development in two Ambystoma and two Rana species

Oxygen available to amphibian embryos fluctuates widely and is often very low. We investigated the effects of oxygen partial pressure (1. 3-16.9 kPa) on embryonic development and hatching of two salamander (Ambystoma) and two frog (Rana) species. In Ambystoma, chronic hypoxia resulted in slowed development, delayed hatching, and embryos that were less developed at the time of hatching. Although hypoxia was not lethal to embryos, temporary developmental abnormalities were observed in Ambystoma at oxygen partial pressures of 3.8 kPa and below. Posthatching survival decreased below 3.3 kPa. In Rana, hypoxia did not affect developmental rate, presumably because hatching occurs at a very early stage of development relative to Ambystoma. However, Rana embryos hatched sooner in hypoxia than in normoxia, resulting in less developed embryos at the time of hatching. The results suggest that embryonic hypoxia may negatively affect survival and fitness in these species.     

Developmental arrest during embryonic development of the common chameleon (Chamaeleo chamaeleon) in Spain

Embryonic development of the common chameleon, Chamaeleo chamaeleon, was monitored from oviposition to hatching at a field site in southwestern Spain and in the laboratory under five experimental temperature regimes. Embryos were diapausing gastrulae at the time of oviposition; developmental arrest in the field continued as cold torpor during winter. Postarrest development in the field commenced in April, and hatching occurred in August, for a total incubation period of 10.5 mo. In the laboratory, one group of eggs was incubated at a constant warm (26 degrees C) temperature. The remaining treatments simulated field conditions and consisted of initial periods of warm temperature of 0, 27, 46, and 71 d, a subsequent 4-mo period of cold winter (16 degrees C) temperature, and a final period of warm (26 degrees C) temperature. Embryos in the constant warm temperature treatment were in diapause an average of 3 mo, with clutch means ranging from 2 to 4 mo. Hatching among clutches occurred over 2 mo. In contrast, for field and experimental eggs that experienced cold winter conditions, hatching within treatments occurred over 2-14 d; "winter" conditions synchronized development. The length of time between the end of cold conditions and hatching did not differ among treatments; development thus resumed as soon as temperature was suitable regardless of the initial period of warm temperature. Diapause in nature thus insures that embryos remain gastrulae after oviposition despite nest temperatures that may be warm enough to support development.     

Globins and hypoxia adaptation in the goldfish, Carassius auratus

Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures. We investigated the contribution of respiratory proteins to hypoxia tolerance in C. auratus. We determined the complete coding sequence of hemoglobin alpha and beta and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin. Like the common carp (Cyprinus carpio), C. auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage. Myoglobin is also expressed in nonmuscle tissues. By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48 h at Po(2) ~ 6.7 kPa, 8 h at Po(2) ~ 1.7 kPa, 16 h at Po(2) ~ 6.7 kPa) at 20 degrees C. We observed small variations in the mRNA levels of hemoglobin, neuroglobin and cytoglobin, as well as putative hypoxia-responsive genes like lactate dehydrogenase or superoxide dismutase. Hypoxia significantly enhanced only the expression of myoglobin. However, we observed about fivefold higher neuroglobin protein levels in goldfish brain compared with zebrafish, although there was no significant difference in intrinsic myoglobin levels. These observations suggest that both myoglobin and neuroglobin may contribute to the tolerance of goldfish to low oxygen levels, but may reflect divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and hypoxia response (myoglobin).     

Metabolic heating and the prediction of sex ratios for green turtles (Chelonia mydas)

We compared incubation temperatures in nests (n=32) of the green turtle (Chelonia mydas) on Ascension Island in relation to sand temperatures of control sites at nest depth. Intrabeach thermal variation was low, whereas interbeach thermal variation was high in both control and nest sites. A marked rise in temperature was recorded in nests from 30% to 40% of the way through the incubation period and attributed to metabolic heating. Over the entire incubation period, metabolic heating accounted for a mean rise in temperature of between 0.07 degrees and 2.86 degrees C within nests. During the middle third of incubation, when sex is thought to be determined, this rise in temperature ranged between 0.07 degrees and 2.61 degrees C. Metabolic heating was related to both the number of eggs laid and the total number of hatchlings/embryos produced in a clutch. For 32 clutches in which temperature was recorded, we estimate that metabolic heating accounted for a rise of up to 30% in the proportion of females produced within different clutches. Previous studies have dismissed any effect of metabolic heating on the sex ratio of marine turtle hatchlings. Our results imply that metabolic heating needs to be considered when estimating green turtle hatchling sex ratios.     

Effects of hatching age on development and hatchling morphology in the red-eyed tree frog, Agalychnis callidryas

The red-eyed treefrog, Agalychnis callidryas , lays eggs on leaves overhanging ponds. Tadpoles hatch and enter the water at different ages, and late-hatched tadpoles survive aquatic predators better than do early-hatched tadpoles. Here I assess developmental consequences of hatching age through: (1) a morphological study of embryos and tadpoles through the plastic hatching period; (2) a behavioural assay for an effect of hatching age on feeding; and (3) a field experiment testing the effect of hatching age on growth to metamorphosis. Substantial development of feeding, digestive, respiratory and locomotor structures occurs in embryos over the plastic hatching period. Hatchling morphology thus varies with age, with consequences for behaviour and predation risk. Hatched tadpoles develop faster than embryos, and early-hatched tadpoles feed before late-hatched tadpoles. After all tadpoles have hatched, the effect of hatching age on size decreases. I found no evidence for an effect of hatching age on size at metamorphosis and only weak evidence for an effect on larval period. Hatching age affects the sequence of developmental change: early-hatched tadpoles lose external gills while otherwise more developed embryos maintain them. Plasticity in external gill resorption may be adaptive given differences in the respiratory environments of embryos and tadpoles. Early-hatched tadpoles also diverge from embryos in shape, growing relatively smaller tails. The study of functional morphology and developmental plasticity will contribute to understanding hatching as an ontogenetic niche shift.     

Impact of nest relocation on the reproductive success of Loggerhead Turtles,Caretta caretta,in the Göksu Delta,Turkey (Reptilia: Cheloniidae)

When the nests of marine turtles are at a risk of inundation, relocation of the nests are often used in the conservation measures. Here, I determined the effect of nest relocation on Loggerhead Turtle (Caretta caretta) egg hatching success during the 2013 and 2014 nesting seasons in the Göksu Delta, Mersin, Turkey. I compared natural and relocated clutches, including those relocated before and after inundation, and evaluated 102 (94.6%) and 63 (81.1%) of survived nests in 2013 and 2014 respectively. Relocated nests experienced a 30% decrease in hatching success and a more prolonged incubation period compared to nests left in situ. Egg failure in nests relocated before and after inundation was similar in early-stage embryos, whereas it was three-fold higher in mid-stage embryos and two-fold lower in late-stage embryos. Thus, there was no significant difference in overall hatching success between the two relocation types. Moreover, there was no effect of delayed relocation of nests after inundation on hatching success. Possible impacts specific to the nesting site should be considered and explored before using nest relocation as a conservation tool. The relocation approach is recommended for nests at a high risk of inundation when the loss of nests is inevitable.     

Phenotypic responses of hatchlings to constant versus fluctuating incubation temperatures in the multi-banded krait, Bungarus multicintus (Elapidae)

Most studies on egg incubation in reptiles have relied on constant temperature incubation in the laboratory rather than on simulations of thermal regimes in natural nests. The thermal effects on embryos in constant-temperature studies often do not realistically reflect what occurs in nature. Recent studies have increasingly recognized the importance of simulating natural nest temperatures rather than applying constant-temperature regimes. We incubated Bungarus multicintus eggs under three constant and one fluctuating-temperature regimes to evaluate the effects of constant versus fluctuating incubation temperatures on hatching success and hatchling phenotypes. Hatching success did not differ among the four treatments, and incubation temperature did not affect the sexual phenotype of hatchlings. Incubation length decreased as incubation temperature increased, but eggs incubated at fluctuating temperatures did not differ from eggs incubated at constant temperatures with approximately the same mean in incubation length. Of the hatchling phenotypes examined, residual yolk, fat bodies and locomotor performance were more likely affected by incubation temperature. The maximal locomotor speed was fastest in the fluctuating-temperature and 30 degrees C treatments and slowest in the 24 degrees C treatment, with the 27 degrees C treatment in between. The maximal locomotor length was longest in the fluctuating-temperature treatment and shortest in the 24 degrees C and 27 degrees C treatments, with the 30 degrees C treatment in between. Our results show that fluctuating incubation temperatures do not influence hatching success and hatchling size and morphology any differently than constant temperatures with approximately the same mean, but have a positive effect on locomotor performance of hatchlings.     

The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures

The hatching performance of embryos of the common carp (Cyprinus carpio L.) was examined after 1, 7, 14, 21, or 28 days of storage at -8, -6, -4, -2, 0, 2, or 4 degrees C with different concentrations of methanol (0.5-7.0 M in 0.5 M steps) or varying concentrations of methanol in 0.1 M sucrose or trehalose. Preserved embryos failed to hatch after storage at -8 and -6 degrees C, regardless of the duration of storage or the concentrations tested. Likewise, there was no hatching out above 5.0 M concentration of methanol, even with the addition of sucrose or trehalose. After storage at 2 or 4 degrees C, the hatching rate was higher with mixtures of methanol (1.5 M) and trehalose (0.1 M) than with methanol plus sucrose or methanol alone. At 4 degrees C, the solution containing 1.5 M methanol supplemented with trehalose gave the highest hatching response of embryos stored for 14 days. Comparison of hatching after 24h of storage at the effective temperatures (-4, -2, 0, 2, and 4 degrees C) revealed that low concentrations of methanol were effective at high temperatures and high concentrations at sub-zero temperatures. The combination of 0.1 M trehalose with 1.5 M methanol gave the highest percentage hatching out both at 4 and 2 degrees C. At 0 degrees C, the highest percentage hatching occurred with 0.1 M trehalose plus 2.5 M methanol and at -2 and 4 degrees C, the best results were with 0.1 M trehalose plus 3.0 M methanol.     

Oxygen uptake and local Po2 profiles in submerged larvae of phaeoxantha klugii (Coleoptera: Cicindelidae), as well as their metabolic rate in air

We studied whether oxygen uptake from the surrounding water might enhance survival in submerged third instar larvae of Phaeoxantha klugii, a tiger beetle from the central Amazonian floodplains. Local oxygen partial pressures (Po(2)) were measured with microcoaxial needle electrodes close to larvae submerged in initially air-saturated still water. The Po(2) profiles showed that the larvae exploit oxygen from the aquatic medium. Metabolism in the air of more or less resting larvae was determined by measuring the rate of CO(2) production (sV dot co2) with an infrared gas analyzer at 29 degrees C. The sV dot co2 was around 1.8 mu L g(-1) min(-1), equivalent to an oxygen consumption rate (sV dot o2) of 1.8-2.6 mu L g(-1) min(-1). Oxygen consumption (V dot o2) of individually submerged larvae measured in closed respiration chambers at 19-10.3 kPa Po(2) (initially air saturated, 29 degrees C) ranged between 0.05 and 0.2 mu L min(-1) and was not correlated with body mass. The sV dot o2 ranged between 0.1 and 0.4 mu L min(-1), that is, 4%-22% of the metabolic rate measured in air. Mean V dot o2 decreased with declining Po(2); however, some individuals showed contrary patterns. V dot o2 was additionally measured in dormant larvae, in larvae submerged for 1-2 d in open water or for 30-49 d within sediment, as well as in larvae exposed to anoxia before the measurements. The range of V dot o2 was similar in all groups, indicating that the larvae exploit oxygen from the water whenever available. Similar V dot o2 across the whole range of body mass investigated (0.31-0.76 g) suggests that oxygen uptake occurs by spiracular uptake. Assuming that larvae survive for some time at rates comparable to depressed metabolic rates reported for other insect species, it can be concluded that oxygen uptake from water can sustain aerobic metabolism even under quite severe hypoxia. It might therefore play an important role for survival during inundation periods.     

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.     

Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.     

Cryopreservation of Mexican fruit flies by vitrification: stage selection and avoidance of thermal stress

This report presents details of a vitrification methodology for the cryopreservation of embryos of the Mexican fruit fly, Anastrepha ludens. The overall summary of the data indicates that selecting the correct developmental stage for cryopreservation is the most important criterion. The key aspect in selection of the correct stage is to balance depletion of the gut yolk content against development of the embryonic cuticle. Embryogenesis was divided into four stages between 90 and 120 h after incubation at 21.7 degrees C. The classification was based on the intestinal yolk content and the initial development of mandibular-maxillary complex. Stages having low mid-gut yolk content and the appearance of mouth hooks were found to be the most suitable for cryopreservation. Embryos developing at 30 degrees C had premature cuticle formation relative to gut development and significantly lower hatching after cryopreservation. Vitrification of embryos by direct quenching in liquid nitrogen was less effective than quenching after annealing the samples in liquid nitrogen vapor. Quenched samples of vitrification solutions containing 1,2-ethanediol as the major component exhibited fractures. Fracturing occurred less frequently when the solutions were annealed and when containing polyethylene glycol. Hatching of vitrified embryos stored in liquid nitrogen for over 12 months was not statistically different from those held for only 15 min. Our protocol yielded normalized hatching rates that ranged as high as 61%. Selecting the exact stage for cryopreservation from a population of embryos obtained by collection from ovipositing females during a span of just 30 min resulted in nearly 80% of the embryos hatching into larvae.     

Physiological development of the embryonic and larval crayfish heart

The cardiovascular system is the first system to become functional in a developing animal and must perform key physiological functions even as it develops and grows. The ontogeny of cardiac physiology was studied throughout embryonic and larval developmental stages in the red swamp crayfish Procambarus clarkii using videomicroscopic dimensional analysis. The heart begins to contract by day 13 of development (at 25 degrees C, 20 kPa O(2)). Cardiac output is primarily regulated by changes in heart rate because stroke volume remains relatively constant throughout embryogenesis. Prior to eclosion, heart rate and cardiac output decreased significantly. Previous data suggest that the decrease in cardiac parameters prior to hatching may be due to an oxygen limitation to the embryo. Throughout development, metabolizing mass and embryonic oxygen consumption increased, while egg surface area remained constant. The surface area of the egg membrane is a constraint on gas exchange; this limitation, in combination with the increasing oxygen demand of the embryo, results in an inadequate diffusive supply of oxygen to developing tissues. To determine if the decrease in cardiac function was the result of an internal hypoxia experienced during late embryonic development, early and late-stage embryos were exposed to hyperoxic water (PO(2) = 40 kPa O(2)). Heart rate in late-stage embryos exposed to hyperoxic water increased significantly over control values, which suggests that the suppression in cardiac function observed in late-stage embryos is due to a limited oxygen supply.     

Effect of gossypol and its metabolite on in vitro early development of mouse embryos

The purpose of this study was to examine the effect of gossypol and its metabolite on early in vitro mouse embryo development. One hundred and thirty-eight excellent quality mouse blastocysts were randomly assigned to five different treatments. Culture media were supplemented with 10% (V/N) normal steer serum. The embryos were cultured at 37 degrees C with an atmosphere of 5% O(2), 5% CO(2) and 90% N(2), and embryo development was examined and recorded at 12-h intervals for 72 h. The percentage of embryos that developed to expanded blastocyst (92%), hatching blastocyst (84%), and hatched blastocyst (76%) stages in control Ham's F-10 media was not different from that of embryos cultured in media containing 0.1 and 5 mug of gossypol; however, none of the embryos treated with 265 ng of gossypol metabolite (GM) developed beyond the blastocyst stage. A substantial decrease in the percentage of embryos reaching hatching blastocyst (29%) and hatched blastocyst (29%) stages was observed in the embryos cultured with 5.3 ng of GM. At both light and electron microscopic levels, the embryos appeared to be affected even by a lower concentration of GM in vitro. Our results suggest that GM has a much greater potency than the parent gossypol in inhibiting the early development of mouse embryos in vitro.     

Survival and development of horseshoe crab (Limulus polyphemus) embryos and larvae in hypersaline conditions

The horseshoe crab Limulus polyphemus spawns in the mid- to upper intertidal zone where females deposit eggs in nests below the sediment surface. Although adult crabs generally inhabit subtidal regions of estuaries with salinities from 5 to 34 ppt, developing embryos and larvae within nests are often exposed to more extreme conditions of salinity and temperature during summer spawning periods. To test whether these conditions have a negative impact on early development and survival, we determined development time, survival, and molt cycle duration for L. polyphemus embryos and larvae raised at 20 combinations of salinity (range: 30-60 ppt) and temperature (range: 25-40 degrees C). Additionally, the effect of hyperosmotic and hypoosmotic shock on the osmolarity of the perivitelline fluid of embryos was determined at salinities between 5 and 90 ppt. The embryos completed their development and molted at salinities below 60 ppt, yet failed to develop at temperatures of 35 degrees C or higher. Larval survival was high at salinities of 10-70 ppt but declined significantly at more extreme salinities (i.e., 5, 80, and 90 ppt). Perivitelline fluid remained nearly isoosmotic over the range of salinities tested. Results indicate that temperature and salinity influence the rate of crab development, but only the extremes of these conditions have an effect on survival.