Amphetamines reduce embryonic size and produce caudal hematomas during early chick morphogenesis

Experiments were designed to study some of the similarities and differences in the effects of amphetamines and trypan blue on early chick morphogenesis. Both dextroamphetamine sulfate (0.5 mg/egg) and methamphetamine hydrochloride (1.0 mg/egg) were capable of inducing, in 3-day chick embryos, caudal hematomas which were similar in appearance and location to those routinely observed following treatment with trypan blue. It was found, too, that both dextroamphetamine and methamphetamine treated embryos frequently exhibited a significant decrease in crown rump length and cross-sectional area of the notochord, neural tube, dorsal aortae and whole body section, when compared with unopened or saline injected controls. Trypan blue treated embryos had only a rare decrease or increase in the size of structures when compared to either control group. These findings suggest that the amphetamines have an ability to decrease or retard embryonic growth in the chick.     

A Rapid Stain Method for Detecting Certain Plant Virus Inclusions

The trypan blue method for detecting certain virus crystallin inclusions is greatly facilitated by a pre-stain treatment with a diethyl-ether-ethyl-alcohol solution made up in physiological saline (5-ml. of diethyl ether and 10 ml. of ethyl alcohol diluted to 100 ml. with physiological saline solution). Maceration of the study material in trypan blue solution also gives good results.     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.     

Hypoxic adaptations of hemoglobin in Tibetan chick embryo: high oxygen-affinity mutation and selective expression

Tibetan chicks (Gallus gallus) survived with high hatchability (35.0%) and Recessive White Feather broilers (RWF) from low elevations survived rarely and with a low hatchability (3.0%) after simulated incubation under hypoxia of 13% O2. The functional mutation of Met-32D(B13)-Leu of alpha(D) globin chain was related with hypoxia based on allele distribution, homology model building and oxygen affinity assay. Whole embryos on days 3-8 and whole blood on days 9-18 were collected to investigate the stage expression profiles of all seven globins and HIF-1alpha by real-time PCR. Under hypoxia (12.0% O2) on days 3-8, HbE was overexpressed, HbA was expressed earlier and HbP expression was restricted, which completely overturned the expression profile under normoxia. The amount of hemoglobin expression in Tibetan chicks was remarkably higher than that of RWF. HIF-1alpha expression peaked early in both breeds, with. In conclusion, the special hypoxic expression profile on days 3-8 certainly is a common molecular mechanism of hypoxia tolerance in surviving Tibetan chick and RWF embryos; the mutation Met-32D(B13)-Leu and increasing hemoglobins are important mechanisms of hypoxia adaptation in Tibetan chick embryos, and we suggest that HIF-1alpha could be responsible for the hypoxic expression profile.     

A study of the effects of trypan blue injected into amphibian zygotes

Summary Trypan blue was injected (0.001–0.002 mg/egg) into zygotes ofRana. The trypan blue-injected series showed much greater mortality than the controls. A probable suppression of gastrulation was seen. 29.7% of the dye-injected and 50.3% of the control embryos developed beyond stage 14. Abnormalities seemed to appear only after neurulation began. Microcephaly, trunk and tail abnormalities occurred though in a small number. Sections of a few embryos revealed disorganized brain and degeneration of the neural tissue. There were no cases of mesodermalization of the notochord.The results are discussed from the view point that trypan blue alters the physical state of proteins in the cytoplasm of the egg.     

Opposing effects of hypoxia on catecholaminergic locus coeruleus and hypocretin/orexin neurons in chick embryos

Terrestrial vertebrate embryos face a risk of low oxygen availability (hypoxia) that is especially great during their transition to air‐breathing. To better understand how fetal brains respond to hypoxia, we examined the effects of low oxygen availability on brain activity in late‐stage chick embryos (day 18 out of a 21‐day incubation period). Using cFos protein expression as a marker for neuronal activity, we focused on two specific, immunohistochemically identified cell groups known to play an important role in regulating adult brain states (sleep and waking): the noradrenergic neurons of the Locus Coeruleus (NA‐LC), and the Hypocretin/Orexin (H/O) neurons of the hypothalamus. cFos expression was also examined in the Pallium (the avian analog of the cerebral cortex). In adult mammalian brains, cFos expression changes in a coordinated way in these areas. In chick embryos, oxygen deprivation simultaneously activated NA‐LC while deactivating H/O‐producing neurons; it also increased cFos expression in the Pallium. Activity in one pallial primary sensory area was significantly related to NA‐LC activity. These data reveal that at least some of the same neural systems involved in brain‐state control in adults may play a central role in orchestrating prenatal hypoxic responses, and that these circuits may show different patterns of coordination than seen in adults. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1030–1037, 2014     

Influence of euthanasia method on blood and gill variables in normoxic and hypoxic Gulf killifish Fundulus grandis

In many experiments, euthanasia, or humane killing, of animals is necessary. Some methods of euthanasia cause death through cessation of respiratory or cardiovascular systems, causing oxygen levels of blood and tissues to drop. For experiments where the goal is to measure the effects of environmental low oxygen (hypoxia), the choice of euthanasia technique, therefore, may confound the results. This study examined the effects of four euthanasia methods commonly used in fish biology (overdose of MS‐222, overdose of clove oil, rapid cooling and blunt trauma to the head) on variables known to be altered during hypoxia (haematocrit, plasma cortisol, blood lactate and blood glucose) or reflecting gill damage (trypan blue exclusion) and energetic status (ATP, ADP and ATP:ADP) in Gulf killifish Fundulus grandis after 24 h exposure to well‐aerated conditions (normoxia, 7·93 mg O₂ l^?1, c. 150 mm Hg or c. 20 kPa) or reduced oxygen levels (0·86 mg O₂ l^?1, c. 17 mm Hg or c. 2·2 kPa). Regardless of oxygen treatment, fish euthanized by an overdose of MS‐222 had higher haematocrit and lower gill ATP:ADP than fish euthanized by other methods. The effects of 24 h hypoxic exposure on these and other variables, however, were equivalent among methods of euthanasia (i.e. there were no significant interactions between euthanasia method and oxygen treatment). The choice of an appropriate euthanasia method, therefore, will depend upon the magnitude of the treatment effects (e.g. hypoxia) relative to potential artefacts caused by euthanasia on the variables of interest.     

Modification of actions of heat shock on development and apoptosis of cultured preimplantation bovine embryos by oxygen concentration and dithiothreitol

Preimplantation embryos exposed to elevated temperatures have reduced developmental competence. The involvement of reactive oxygen species in these effects has been controversial. Here we tested hypotheses that (1) heat shock effects on development and apoptosis would be greater when embryos were cultured in a high oxygen environment (air; oxygen concentration = approximately 20.95%, v/v) than in a low oxygen environment (5% oxygen) and (2) that these effects would be reversed by addition of the antioxidant dithiothreitol (DTT). Heat shock of 41 degrees C for 9 hr reduced development of two-cell embryos and Day 5 embryos to the blastocyst stage embryos when in high oxygen. There was no effect of heat shock on development when embryos were in low oxygen. Furthermore, induction of TUNEL-positive cells in Day 5 embryos by heat shock only occurred when embryos were in high oxygen. Addition of DTT to two-cell embryos either did not reduce effects of a heat shock of 41 degrees C for 15 hr on development or caused slight protection only. In contrast, treatment of Day 5 embryos with DTT reduced effects of heat shock on development and apoptosis. In summary, oxygen tension was shown to be a major determinant of the effects of heat shock on development and apoptosis in preimplantation bovine embryos. Protective effects of the antioxidant DTT were stage specific and more pronounced at later stages of development.     

The developmental potential of frozen-thawed hamster preimplantation embryos following embryo transfer: Viability of slowly frozen embryos following slow and rapid thawing

Hamster preimplantation embryos were slowly frozen (0.33°C/min) and seeded above 10°C in TC-199 containing 1.5 M-DMSO. These embryos were thawed either slowly (1.5°C/min) or rapidly (90°C/min). The thawed embryos were examined by morphology, trypan blue exclusion and viability after embryo transfer. Slow thawing gave significantly higher viability compared to rapid thawing. The early preimplantation embryos demonstrate higher sensitivity to freezing. The three tests of viability (morphology, trypan blue exclusion and embryo transfer) were found to be positively correlated.     

The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus auratus

Experiments were designed to assess the use of the vital dyes trypan blue and fluorescein diacetate as indicators of the viability of hamster ova and embryos. Exclusion of trypan blue and fluorescence with fluorescein diacetate showed high correlations with uptake of [3H]uridine by ova and further development of embryos in vitro. Ova killed by freezing and thawing incorporated [3H]uridine at background levels. Trypan blue exclusion and fluorescein diacetate uptake were highly correlated with each other (r = 0.99). Trypan blue and fluorescein diacetate serve as excellent indices of viability in ova and early embryos of hamsters.     

The effect of isoproterenol on cardiovascular function in the stage 24 chick embryo

The developing cardiovascular system of the chick embryo is susceptible to teratogenic effects of catecholamines. Yet the mechanism for the teratogenetic action is unclear. Since catecholamines affect cardiovascular physiology, we studied the acute effect of the beta-agonist isoproterenol on mean atrial pressure, heart rate, mean dorsal aortic blood flow, mean arterial pressure and vascular resistance in stage 24 chick embryos. Dorsal aortic blood velocity was measured with a 20-MHz pulsed-Doppler velocity meter and intravascular pressure was measured with a servo-null pressure system. Isoproterenol in doses of 2 X 10(-4) micrograms (2.5 micrograms/kg), 8 X 10(-4) micrograms (10 micrograms/kg), and 1.2 X 10(-3) micrograms (15 micrograms/kg) was injected intravenously in 5-microliters aliquots of chick Ringer's solution. Additional groups of embryos were treated with the beta-antagonist propranolol, and isoproterenol plus propranolol. Control embryos received 5 microliters chick Ringer's solution to assess the hemodynamic effects of a volume injection. We found that isoproterenol caused no change in mean atrial pressure, heart rate, or mean arterial pressure. However, isoproterenol caused a dose-related decrease in dorsal aortic blood flow and a 2.5-fold increase in vascular resistance. The effects of isoproterenol were blocked by propranolol, which suggested that the increase in vascular resistance was mediated by beta-receptor stimulation.     

Chlorazol Fast Pink B And Trypan Blue Tests for Virus and Other Proteinaceous Inclusions

A 1% solution of chlorazol fast pink B in 0.9% NaCl can be used like trypan blue to detect virus inclusions and proteinaceous entities in peelings from leaves or thin sections taken from living plant tissue. Like trypan blue, a solution of the pink dye causes somatic nuclei to swell and thus facilitates observation of their structure. The two dyes combine into a beautiful differential bicolored stain. Mix 5 ml of 0.5% trypan blue stock solution with 35 ml of 1% chlorazol pink B in 0.9% NaCl. Stain fresh tissue 1-2 minutes. The combination stain is superior to either dye alone for differentiating virus entities.     

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO₂ levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO₂. Increased incubation temperature caused an increase in the developmental rate at high PO₂s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO₂. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO₂s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.     

伊红、台盼蓝检测河蟹精子存活率的比较

对台盼蓝和伊红染色法检测河蟹(Eriocheir sinensis)精子存活率的方法进行了评价研究。结果表明,两种染色法死、活精子分别呈现出明显不同的染色特征:活精子无色透明,顶体中央凸起呈圆锥状,光镜下辐射臂及细胞边界清晰;死亡精子顶体着色,且中央有一染色较深的圆斑,核杯染色不明显,细胞体积变大,边界模糊。通过不同染色时间和不同染料浓度的比较发现,两种染色法最适染液浓度分别是0·25%的伊红和0·5%的台盼蓝,染色时间均以15min为佳。在此基础上,将新鲜精子和60℃水浴处理致死精子以不同的体积比混合,配成含致死精子比例为10%～90%的9个梯度样品,用伊红和台盼蓝分别测定各样品精子死亡率,并进行相关性分析。结果发现,各样品实测精子死亡率均略高于样品的理论死亡率,同时两种染色法实测值与样品理论值呈显著正相关(P<0·05),两种染色法之间亦呈显著正相关(P<0·05)。上述结果表明,伊红和台盼蓝可用于河蟹精子的活体染色,且两种染色法在对河蟹精子染色中具有一定的稳定性和可比性。     

The Use of Vital Dyes to Assess Embryonic Viability in the Hamster, Mesocricetus Auratus

Experiments were designed to assess the use of the vital dyes trypan blue and fluorescein diacetate as indicators of the viability of hamster ova and embryos. Exclusion of trypan blue and fluorescence with fluorescein diacetate showed high correlations with uptake of [³H]uridine by ova and further development of embryos in vitro. Ova killed by freezing and thawing incorporated [³H]uridine at background levels. Trypan blue exclusion and fluorescein diacetate uptake were highly correlated with each other (r = 0.99). Trypan blue and fluorescein diacetate serve as excellent indices of viability in ova and early embryos of hamsters.     

Cardiac rhythms of late pre-pipped and pipped chick embryos exposed to altered oxygen environments

During the final stages of embryonic development in chickens, diffusive gas exchange through the chorioallantoic membrane (CAM) is progressively replaced by pulmonary respiration that begins with internal pipping (IP) of the CAM. Late chick embryos going through the transition from CAM respiration to pulmonary respiration were exposed to hyperoxic (100% O(2)) and hypoxic (10% O(2)/N(2)) environments for 2-h and the responses of baseline heart rate (HR), and HR fluctuation patterns were investigated. 16- and 18-day-old (referred to as 18-d) embryos and 20-d externally pipped (EP) embryos were examined as pre-pipped embryos and pipped embryos, respectively. 19-d embryos were divided into two groups: embryos that had not yet internally pipped (Pre-IP embryos) and embryos that had internally pipped (IP embryos). IP was identified by detecting the breathing signal with a condenser microphone attached hermetically on the eggshell (i.e. acoustorespirogram) on day 19 of incubation. In the hyperoxic environment, HR baseline of pre-pipped embryos remained unchanged and that of pipped embryos was depressed. In the hypoxic environment, HR baseline of 16-d pre-pipped embryos was depressed and that of pipped (IP and EP) embryos elevated. These different responses in pipped embryos might be partially attributed to increased cholinergic input from the vagus nerve in hyperoxia and increased adrenergic response in hypoxia. While hyperoxia did not induce marked modification of instantaneous heart rate (IHR) fluctuation patterns, hypoxia tended to augment transient decelerations of IHR in late pre-pipped embryos and markedly depressed HR fluctuations in pipped embryos.     

Factors affecting the survival of one- and two-cell rabbit embryos cryopreserved by vitrification

The effects of equilibration time, glycerol (GLY), and 1,2-propanediol (PROH) concentration, and of vitrification and sucrose solution on the viability of 1- and 2-cell rabbit embryos were investigated. After collection, the embryos were equilibrated for 5 or 10 minutes in phosphate buffered saline (PBS) containing 10% GLY-20% PROH and were exposed for 30 seconds at 4 degrees C or were exposed and vitrified in one of two vitrification solutions 35% GLY-35% PROH or 20% GLY-50% PROH. The in vitro survival rates of 1-cell embryos equilibrated for both 5 and 10 minutes were lower (34.0 and 48.0%, respectively) than those of 2-cell embryos (78.8 and 68.5%, respectively; P<0.01). No differences were noted in the viability of embryos exposed to the 2 vitrification solutions. Following vitrification in a mixture of 35% GLY-35% PROH, the survival rates of 1- and 2-cell embryos were 18.3 and 13.7% and 19.6 and 10.4% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1- and 2-cell embryos vitrified in a solution of 20% GLY-50% PROH were 25.7 and 35.4% and 26.2 and 21.3% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1-and 2-cell embryos stored in 1M sucrose solution were 63.8 and 84.0%, respectively. In conclusion, the viability of vitrified 1- and 2-cell rabbit embryos was reduced as a consequence of their equilibration before vitrification, the exposure to vitrification solution and the dilution in a sucrose solution rather than of the vitrification process itself.     

Effects of periodic hypoxia on mortality, feeding and predation in an estuarine epifaunal community

The York River Estuary, a tributary of the Chesapeake Bay, USA, experiences periodic low oxygen stress (hypoxia), yet epifaunal species form dense communities there. We studied hypoxia tolerance of common epifaunal species in the York River by exposing sessile and mobile epifauna to high and low oxygen concentrations in laboratory aquaria. Mortality in hypoxia varied among species, ranging from 0% to 100%, with trends of decreased tolerance by mobile species relative to sessile species. While most species tested experienced some mortality after being exposed to hypoxia (at 1 mg O(2)/l or 0.5 mg O(2)/l) for 5 days, many species had a median lethal time (LT(50)) in hypoxia greater than 1 week (3 of 6 species at 1 mg O(2)/l and 6 of 14 species at 0.5 mg O(2)/l), the maximum duration of typical hypoxic episodes in the York River, suggesting that hypoxia may cause little mortality for some species in this system. However, hypoxia had sub-lethal effects on behavior in all species tested. Epifaunal animals responded to hypoxia with behaviors that moved them higher in the water column or by entering resting states until hypoxia passed. Feeding and predation by a variety of taxa (the hydroid Obelia bicuspidata, the mud crab Neopanope sayi, juvenile blue crabs Callinectes sapidus, the flatworm Stylochus ellipticus, and the nudibranch Doridella leucolena) decreased during hypoxia, despite varying mortality responses to low oxygen stress, suggesting that short hypoxic episodes may create predation refuges for prey species. At least one highly tolerant species (O. bicuspidata) showed substantially decreased growth in hypoxia. Although relatively high tolerance of hypoxia by many estuarine epifaunal species limits serious disturbance during brief hypoxic episodes, hypoxia's greatest impact on York River epifaunal communities might be through its indirect effects on behavior and predation.     

Pathogenesis of persistent truncus arteriosus induced by nimustine hydrochloride in chick embryos

Nimustine hydrochloride (ACNU) is a nitrosourea derivative anticancer agent which has been shown to cause persistent truncus arteriosus in chick embryos. The objective of this study was to confirm the teratogenic effects of ACNU on the cardiovascular system of chick embryos and to determine whether ACNU induces persistent truncus arteriosus by interfering with neural crest cells. Various doses of ACNU ranging from 10 to 200 micrograms were injected under the chorioallantoic membrane of chick embryos on the third day of incubation. Saline solution was used as the control. After 10 to 11 days of incubation, 242 (46%) survivors of the 524 treated eggs were obtained. The survival rates of the embryos and the frequencies of cardiovascular anomalies were dose dependent. Of 146 embryos with cardiovascular anomalies, 104 (71%) had persistent truncus arteriosus. Ventricular septal defect and double-outlet right ventricle were seen in 37 (25%) and one (1%), respectively. Aortic arch anomalies were seen in 116 embryos (79%). Quail-chick chimeras (chick embryos with quail cardiac neural crest) were treated with 50 micrograms of ACNU and examined histologically 24 hours later. These chimeras showed dying neural crest cells in the pharyngeal arches. Dying cells were also noted in the neural tube, cranial ganglia, retina, and otocyst. These results suggest that persistent truncus arteriosus in chick embryos treated with ACNU is induced by neural crest cell death.