Development of heart rate responses to acoustic stimuli in Muscovy duck embryos

Heart rate (HR) of Muscovy duck embryos (Cairina moschata f. domestica) was continuously recorded from the 21st day of incubation (E21) until hatching (E35). During that period, embryos were exposed to different acoustic stimuli (species-specific maternal and duckling calls, music, rectangular and sine waves, white noise). Sudden HR changes occurred at the onset of acoustic stimulation (on-response), as well as spontaneously. From E27 onwards, the response rate was significantly higher than the rate of spontaneous HR changes. The on-response rate increased further until E30. Most responses were elicited by maternal calls and music, but rarely by duckling calls. On-responses could be classified into: HR increase (36.4%), HR decrease (37.9%) and an increase in instantaneous HR variability (23.2%). The increase in HR variability occurred only in response to sounds, but not spontaneously. HR increases were mainly observed when the baseline HR was lower than the long-term HR trend. On-response duration was no longer than 3 min in 90% of all observations. The hourly mean HR and standard deviation did not change, even during phonoperiods composed of several sound patterns and lasting several hours. We conclude that Muscovy duck embryos are able to perceive exogenous acoustic stimuli, and that the acousto-sensory-->cardiac axis is functional from E27.     

Cardiac rhythms in developing chicks

Instantaneous heart rate (IHR) of chicks was determined by electrocardiogram measured non-invasively from the day of hatch to day 6 for continuity of investigation of HR fluctuations from embryos and for ascertainment of HR diurnal rhythms. In Experiment I, IHR was determined for 1-h periods twice a day, in daytime and at night, to investigate development of heart rate fluctuations (variability and irregularities). Chick IHR was substantially more arrhythmic than embryonic HR and spontaneous acceleration dominated HR fluctuations. Chick HR fluctuations were categorized into three types; [1] Type I as a widespread baseline HR (20-50 bpm) due to respiratory arrhythmia, with a mean oscillatory frequency of 0.74 Hz (range 0.4-1.2 Hz); [2] Type II as low frequency oscillations of baseline HR, at a mean of 0.07 Hz (range 0.04-0.10 Hz), and [3] Type III as non-cyclic irregularities, dominated by frequent transient accelerations. In Experiment II, continuous measurements of HR were made under conditions of a natural photoperiod, thermoneutrality and with feed available throughout the first week after hatching and circadian rhythms of HR were ascertained. HR was very variable in the daytime (250-500 bpm), due in part to feeding and activity, and decreased to a diurnal low (200-350 bpm) at night when mean HR was relatively stable. HR fluctuations persisted throughout the diurnal cycle.     

Heart rate responses to altered ambient oxygen in early (days 3–9) chick embryos in the intact egg

Normal heart rate (HR), and the HR responses to hypoxia and hyperoxia during early heart development in chick embyros have not been studied in detail, particularly in undisturbed embryos within the intact egg. HR was measured in day 3–9 chick embryos at 38 °C using relatively noninvasive impedance cardiography. Embryos were exposed to air (control) and to hypoxic (10% O₂) or hyperoxic (100% O₂) gas for a 2-h or 4-h period, during which HR was continually monitored. Control (normoxic) HR increased from about 150 beats per min (bpm) on day 3 to about 240 bpm on days 7–9. HR in very early embryos showed a variety of moderate responses to hypoxia (all survived), but as development progressed beyond day 6, hypoxic exposure induced a profound bradycardia that frequently terminated in death before the end of the measurement period. In contrast to the marked developmental changes in hypoxic sensitivity, HR showed little response to hyperoxia throughout development, suggesting no “hypoxic drive” to HR. We speculate that hypoxia has little effect early in development because of the embryo's small absolute O₂ demand, but as the embryo grows, hypoxia represents a progressively more severe perturbation. Although general trends were identified, there was considerable variation in both HR and HR responses to ambient O₂ changes between individuals of the same developmental stage. Accepted: 16 December 1998     

Long-term measurement of heart rate in chicken eggs

Taking advantage of acoustocardiogram (ACG), we measured the heart rate (HR) of chick embryos continuously from day 12 until hatching and then investigated the development of HR irregularities (HRI), HR variability (HRV), and the existence of a circadian rhythm in mean HR (MHR). HRI comprised transient bradycardia and tachycardia, which first developed on day 14 and 16 in most embryos, respectively. Transient bradycardia increased in frequency and magnitude with embryonic development and occurred over periods of up to 30 min in some embryos. MHR was maximal on around days 14-15 and thereafter decreased to about 250-260 bpm on days 16-18. Baseline HRV, which is an oscillation of the MHR baseline, occurred as HR decreased from days 15-16 and became predominant on days 17-18. The magnitude of the baseline oscillations reached up to 50 bpm in some embryos and the period ranged between about 40-90 min (ultradian rhythm). A circadian rhythm of MHR was not found in late chick embryos. On days 18-19, embryonic activities were augmented and then breathing movements began to occur, disturbing ACG signals and thus making it difficult to measure the HR. Instead, the development of breathing activities was recorded. Breathing frequency was irregular at first and then increased to a maximum of about 1.5 Hz prior to hatching.     

Membrene turnover in imbibed and dormant embryos of the wild oat (Avena fatua L.)

A comparative study of protein synthesis has been carried out with embryos excised from dormant (D) and non-dormant (ND) caryopses of the wild oat. Although D embryos imbibed in water or ND embryos imbibed in abscisic acid do not germinate, they incorporate [¹⁴C]leucine into TCA-insoluble material for the first 48 h as readily as embryos that do germinate (ND embryos imbibed in water, or D embryos imbibed in gibberellic acid). Pulsechase experiments with [¹⁴]leucine show that in both D and ND embryos the proteins associated with the membranes undergo turnover. The rates of decay of incorporated radioactivity are similar in both dormant and germinating embryos up to 98 h following embryo excision. Fractionation of the membrane proteins in SDS-polyacrylamide gels indicates that the different polypeptides have different rates of turnover. It is concluded that membrane proteins in imbibed D embryos are in a state of constant turnover, and that this is a part of the replacement processes necessary to maintain the integrity of hydrated cells. The continuation of such synthetic events could account for long term survival of dormant Avena fatua in the imbibed state.Abbreviations CCRSE cytochrome relative stain equivalents - D dormant - ND nondormant - ABA abscisic acid - GA gibberellic acid GA₃     

Ontogeny of salinity tolerance and hyper-osmoregulation by embryos of the intertidal crabs Hemigrapsus edwardsii and Hemigrapsus crenulatus (Decapoda, Grapsidae): survival of acute hyposaline exposure

The adults of Hemigrapsus edwardsii and Hemigrapsus crenulatus are euryhaline crabs and strong hyper-osmoregulators. Their embryos are carried externally attached to the abdominal pleopods of female crabs, where they are exposed to temporal and spatial changes in salinity associated with their intertidal and estuarine habitats. Although embryos lack the branchial and excretory organs responsible for adult osmoregulation, post-gastrula embryos were highly tolerant of exposure to hypo-osmotic sea water. Detached eggs (embryos+envelopes), of both species, at all developmental stages between gastrulation and hatching, exhibited 80-100% survival for periods up to 96 h in sea water (osmolality, 1050 mmol kg(-1)) and in dilutions to 50%, 10%, and 1%. Cleavage stages were less tolerant of dilution; H. edwardsii, <50% survived 24 h in 10% sea water; H. crenulatus <50% survived 6 h in 10% sea water. Post-gastrulation stages strongly hyper-osmoregulated but cleavage stages were hyper-osmoconformers (maintaining internal osmolality approximately 150 mmol kg(-1) above external). Osmoregulatory capacity was reduced just prior hatching, particularly in H. crenulatus, although salinity tolerance remained high. Gastrulation therefore marks a critical stage in the ontogeny of osmoregulation and salinity tolerance. Total Na+/K(+)-ATPase activity increased greatly during embryogenesis of H. crenulatus (undetectable in blastulae; gastrulae 0.31+/-0.05 pmol P(i) embryo(-1) min(-1); pre-hatching 16.4+/-1.0 pmol P(i) embryo(-1) min(-1)). Na+/K(+)-ATPase activity increased in embryos exposed to dilute sea water for 24 h implicating regulation of this transporter in a short-term acclimation response.     

Effects of Music Composed by Mozart and Ligeti on Blood Pressure and Heart Rate Circadian Rhythms in Normotensive and Hypertensive Rats

There is continuing discussion on the effect of music (“Mozart effect”) on numerous functions in man and experimental animals. Radiotelemetry now allows one to monitor cardiovascular functions in freely‐moving unrestrained experimental animals. Radiotelemetry was used to monitor systolic and diastolic blood pressure (SBP, DBP), heart rate (HR), and motor activity (MA) in male normotensive WKY and hypertensive SHR animals. Rats were synchronized to a 12 h light (L): 12 h dark (D) regimen in an isolated, ventilated, light‐controlled, sound‐isolated animal container. Music (Mozart, Symphony # 40; Ligeti, String Quartet # 2) were played for 2 h at 75 dB in the animal cabin starting at the onset of L or D in a cross‐over design. Data were collected every 5 min for 24 h under control conditions and during and after music. In addition, plasma concentrations of norepinephrine (NE) were determined in unrestrained animals at 3 h intervals over 24 h. In both WKY and SHR, highly significant circadian rhythms were obtained in SBP, DBP, HR, and MA under control conditions; HR was lower and BP higher in SHR than in WKY. NE was circadian rhythmic in both strains with higher values in D; the increase in NE with immobilization was much more pronounced in SHR than in WKY. The music of Mozart had no effect on either parameter in WKY, neither in L nor in D. In contrast, in SHR, the music of Mozart presented in L significantly decreased HR and left BP unaffected, leading to a small decrease in cardiac output. The music of Ligeti significantly increased BP both in L and in D and reflexively reduced HR in L, the effects being long‐lasting over 24 h. Interestingly, white noise at 75 dB had no effect at all on either function in both strains. The effects of both Mozart and Ligeti cannot be attributed to a stress reaction, as stress due to cage switch increased HR and BP both in WKY and SHR. The study clearly demonstrates that music of different character (tempo, rhythm, pitch, tonality) can modify cardiovascular functions in freely‐moving rats, with SHR being more sensitive than normotensive animals. The relative contribution of the characteristics of the two pieces of music, however, needs further evaluation.     

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.     

An in vitro method for recording the electrical activity of the isolated heart of the adult Drosophila melanogaster

A recording chamber for monitoring the electrophysiological properties of the isolated heart of adult Drosophila melanogaster has been developed. Spontaneously generated field potentials of constant amplitude can be recorded for 6-8 h (n = 14); in very few cases, records were maintained stable for over 10 h (n = 4), and in some cases below 6 h (n = 5). The chamber consists of the tip of a micropipette, which allows for monitoring the field potential generated by the spontaneously contracting heart. The method can produce accurate information about the heart rate and the amplitude of the cardiac action potential. The preparation can be used for pharmacological studies on the heart of D. melanogaster since it responds, with an increase in the heart rate, to unusually low concentrations of octopamine, 1 nM, a compound with cardioaccelerating properties for insect heart. The recording system can be easily modified for experiments on the heart of other insects. Finally, the isolated heart of D. melanogaster provides a simple method for identifying mutations that affect heart physiology.     

The Na+-Ca2+ Exchanger Is Essential for Embryonic Heart Development in Mice

The cardiac Na⁺–Ca²⁺ exchanger 1 (NCX1) is thought to be the major calcium extrusion mechanism and to play an important role in the regulation of intracellular calcium in the heart. The Na⁺–Ca²⁺ exchanger is particularly abundant in the heart, although it is found in a variety of other tissues. To investigate the role of NCX1, we have generated NCX1-deficient mice. Mice heterozygous for the NCX1 mutation showed no discernable phenotype, grew normally, and were fertile; however, no viable homozygote was observed among 175 offspring obtained from intercrosses of heterozygotes. All the homozygous mutant mice died in utero before E10.5. Morphological analysis indicated that homozygotes of NCX1 mutation at E9.5 died with an underdeveloped heart with a dilated pericardium. Microscopic analysis of these embryos showed myocardial cell loss due to apoptosis. The apoptosis was first observed in E8.5 mutant heart. Areas outside the heart appeared normal in the mutant embryos at E8.5. In contrast, at E9.0, various regions of mutant embryos showed extensive cell loss. These results suggest that mutant embryos die owing to cardiac abnormalities caused by apoptotic cell loss, indicating that NCX1 is essential for normal development of the heart.     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

Protection of Embryos from Freshening and Drying in Layings of the Gastropod Littorina sitkana

Protection from wetting (freshening) and drying of layers of the intertidal gastropod Littorina sitkana Philippi, 1846, specified by direct development was studied. It has been shown that the jellylike material of the layings did not protect the developing embryos from changes in the environmental salinity, but rather protected them from drying periods up to 12 h.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

Alterations in Ca2+-channels during the development of diabetic cardiomyopathy

In order to examine the status of Ca²⁺ channels in heart sarcolemma during the development of diabetes, rats were injected intravenously with 65 mg/kg streptozotocin and hearts were removed 1, 3 and 8 weeks later. Crude membranes from the ventricular muscle were prepared and the specific binding of ³H-nitrendipine was studied by employing different concentrations of this Ca ²⁺-antagonist. A significant decrease in both dissociation constant and maximal number of 3H-nitrendipine binding was observed in 3 and 8 weeks diabetic preparations. No such alterations were evident in diabetic brain membranes. Treatment of diabetic animals with insulin prevented the occurrence of these changes in the myocardium. The altered ³H-nitrendipine binding characteristics in diabetic heart membranes may not be due to the high levels of circulating catecholamines in this experimental model because no such changes were seen upon injecting a high dose (40 mg/kg) of isoproterenol in rats for 24 hr. The reduced number of ³H-nitrendipine binding sites may decrease Ca²⁺-influx through voltage sensitive Ca²⁺ channels and partly explain the depressed cardiac contractile force development in chronic diabetes whereas the increased affinity of Ca²⁺ channels may partly explain the increased sensitivity of diabetic heart to Ca ²⁺.     

Ontogeny of heart rate responses to exogenous melatonin in Muscovy duck and chicken embryos

One of the major physiological effects of melatonin is coupling the internal clock with different organ functions. Despite the long list of functional responses to melatonin discovered in the past, it has been unclear when responsiveness to melatonin develops during ontogeny. The aim of the present study was therefore to investigate in Muscovy duck and chicken embryos, when they start to exhibit heart rate (HR) changes to exogenous melatonin. HR was recorded continuously in Muscovy duck embryos from day 24 of incubation (D24) and in chicken embryos from D17 until hatching. Every day four doses of 10 microg melatonin were injected into each egg at 30 min intervals. In Muscovy duck embryos HR responses to melatonin were first observed on D25; from D27 all embryos responded. In all cases HR decreased immediately after the injection. HR deviation from baseline values and duration of decreased HR period increased during the experimental period. Chicken embryos showed similar responses as Muscovy ducks from D17 onwards; from D18 the response rate was 100%. On D19 dose-response data revealed a partial responsiveness to exogenous melatonin at doses of 0.1 to 1 ng and full responsiveness from 10 ng to 10 microg. The time of the first occurrence of HR responses to melatonin coincides with published results on the start of periodic pineal melatonin secretion. These data suggest that the output signal of the avian internal clock, periodic plasma melatonin fluctuations, could result in periodic cardiac function already prenatally in these two avian species.     

Increased yolk testosterone facilitates prenatal perceptual learning in Northern bobwhite quail (Colinus virginianus)

Prenatal learning plays an important role in the ontogeny of behavior and birds provide a useful model to explore whether and how prenatal exposure to hormones of maternal origin can influence prenatal learning and the development of behavior. In this study we assessed if prenatal exposure to yolk testosterone can influence auditory learning in embryos of Northern bobwhite quail (Colinus virginianus). We experimentally enhanced testosterone concentrations in bobwhite quail eggs prior to incubation. The embryos from these T-treated eggs as well as control embryos that had received the vehicle-only or were non-treated were exposed to an individual bobwhite hen's maternal call for 120 min over the course of the day prior to hatching. All chicks were tested at 24 h following hatching for their auditory preference between the familiar bobwhite maternal call versus an unfamiliar bobwhite maternal call. T-treated chicks spent significantly more time in proximity to the familiar call compared to the unfamiliar call and also showed shorter latencies to approach the familiar call than control birds. Increased emotional reactivity, i.e. propensity to express fear responses, was also found in T-treated chicks. Baseline heart rates recorded in a second group of T-treated embryos and control embryos did not differ, which suggests no effect of yolk testosterone on baseline arousal level. To our knowledge this is the first demonstration of the influence of prenatal exposure to testosterone on auditory learning.     

Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor

Compared to non-embryogenic callus, proembryonic mass, globular, and heart-shaped embryos of Eleutherococcus senticosus had higher levels of endogenous reduced glutathione (GSH). GSH content declined during the course of the embryo development (torpedo and cotyledon). Similarly, glutathione reductase that is involved in the recycling of GSH providing a constant intracellular level of GSH was also higher in globular and heart-shaped embryos. The transient increase in GSH contents also correlated with the changes in measured γ-glutamylcysteine synthetase activity over the same period. The endogenous levels of oxidized glutathione showed similar trend during development of the somatic embryos, whereas it declined in maturing somatic embryos. A pronounced increase in glutathione-S-transferase, glutathione peroxidase, catalase, and guaiacol peroxidase activity was observed during somatic embryo maturation. Ascorbate-glutathione cycle enzymes (ascorbate peroxidase; dehydroascorbate reductase and monodehydroascorbate reductase) activities also induced indicated that antioxidant enzymes played an important role during embryo development. These results suggested that the coordinated up-regulations of the antioxidant enzymes and glutathione redox system provide protection during somatic embryo development in E. senticosus. Antioxidant responses through alterations of the glutathione redox systems, have been described in the present studies have a significant role in somatic embryo development.     

Embryonic communication in the nest: metabolic responses of reptilian embryos to developmental rates of siblings

Incubation temperature affects developmental rates and defines many phenotypes and fitness characteristics of reptilian embryos. In turtles, eggs are deposited in layers within the nest, such that thermal gradients create independent developmental conditions for each egg. Despite differences in developmental rate, several studies have revealed unexpected synchronicity in hatching, however, the mechanisms through which synchrony are achieved may be different between species. Here, we examine the phenomenon of synchronous hatching in turtles by assessing proximate mechanisms in an Australian freshwater turtle (Emydura macquarii). We tested whether embryos hatch prematurely or developmentally compensate in response to more advanced embryos in a clutch. We established developmental asynchrony within a clutch of turtle eggs and assessed both metabolic and heart rates throughout incubation in constant and fluctuating temperatures. Turtles appeared to hatch at similar developmental stages, with less-developed embryos in experimental groups responding to the presence of more developed eggs in a clutch by increasing both metabolic and heart rates. Early hatching did not appear to reduce neuromuscular ability at hatching. These results support developmental adjustment mechanisms of the 'catch-up hypothesis' for synchronous hatching in E. macquarii and implies some level of embryo-embryo communication. The group environment of a nest strongly supports the development of adaptive communication mechanisms between siblings and the evolution of environmentally cued hatching.