The motor activity of the chick embryo and amnion during embryogenesis

Studies have been made on the motor activity of amnion and chick embryo from the 5th to the 14th day of development. Between the 5th and the 8th day of embryogenesis, when embryonic movements are rather poor, amnion contractions are mainly observed, their frequency being maximum to the 7th day. On further development (8-14 days), with the increase in the mass of the limbs which account for embryonic movements (body extremities), the increase in the intensity of their motor activity is paralleled by the decrease in the frequency of amnion contractions. Therefore, during the intensive growth and development of mainly frontal part of the embryo, the deficiency of motor activity of rather undeveloped body and extremities is presumably compensated by temporal motor activity of the amnion. Between the 8th and the 10th day, synchronous movements of embryo and amnion are observed. Possible mechanisms of synchronization are discussed.     

Role of motility in embryonic development I: Embryo movements and amnion contractions in the chick and the influence of illumination

This study provides a quantitative analysis of the active movements of the chick embryo and of the contractions of the amnion over the entire developmental period of 21 days. Four types of embryo movements are distinguished. The motor activity of the embryo shows two characteristic peaks, with maximum contraction frequencies on the 12th and on the 16th day. In contrast, the amnion activity is higher at earlier stages and decreases as the body activity increases. The amnion activity is largely independent of the body activity. Illumination has a strong influence on embryo movements. It is shown that increases of light intensity affect the patterns of activity of both the embryo and the amnion. While the effect of light on the embryo can be interpreted as being transmitted via the optic system, the mechanism of the amniotic response is unclear. The results suggest that the amnion itself may be sensitive to light. J. Exp. Zool. (Mol. Dev. Evol.) 291:186-194, 2001.     

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.     

The motor activity of the chick embryo amnion in the late stages of development. The role of serotonin and noradrenaline]

Experiments with 13- to 19-day-old chicken embryos maintained at 37 degrees C were conducted using direct registration of embryonic movements and amnion pulsation. It is shown that in the non-innervated and lacking blood vessels non-striated-muscled amnion, motoric activity could be observed nearly up to the end of the embryonic development, not only during 5 to 14 days old interval as it was supposed earlier. In accordance with our previous results indicating important role of biogenic amines (serotonin and noradrenaline) in the regulation of motoric activity of the chicken embryo amnion at earlier and middle ages, this study provides some evidences of the humoral regulatory mechanism even at later embryonic stages. After being injected into amniotic fluid of older embryos, the serotonin stimulates and noradrenaline inhibits amnion motoric activity (the both are taken at final concentration of 10(-7) to 10(-6) M). Serotonin's receptors blocator, the cyprogeptadin, suppress while beta-adrenoreceptors' blocator, the propranolol, activates intact amnion motorics ( both are taken at final concentration of 10(-7) to 10(-5) M).     

Ultrasonography for detection of early pregnancy following embryo transfer in unknown breed of Bos indicus cows

Real time, B mode ultrasound was used from Days 18 to 64 to detect and monitor the conceptus in 30 unknown breeds of Bos indicus recipients following embryo transfer. The embryonic vesicle was first visible within the uterus between Days 18 and 20 (mean +/- SD 18.5 +/- 0.7d) d). The embryo proper was detected at Day 19.5 +/- 0.7. The heartbeats were detected at Day 22.6 +/- 0.9. The average day of first detection of the allantois, the C - shaped embryo and the amnion was 23.1 +/- 0.8, 23.8 +/- 1.4 and 25.1 +/- 1.4 d, respectively. The fore limb, the limb buds, the spinal cord and the optic area were observed on Days 32.7 +/- 1.3, 32.9 +/- 1.3, 33.0 +/- 1.5 and 33.6 +/- 1.4, respectively. Fetal movements could be detected at Day 50.7 +/- 1.0. Ribs and vertebrae were detected on Day 60.9 +/- 1.7. The mean length of the embryo proper was 4.5 +/- 0.8 mm. on Day 19. At Day 60 it was 52.5 +/- 7.0 mm. The growth of the embryo proper increased steadily until Day 39, growing rapidly thereafter. Embryonic death was detected in one recipient on Day 32. The echongenic area started increasing and recipient appeared in estrus on Day 40. Sixteen recipients exhibited estrus on Days 22 to 24. An increase in intrauterine fluid was observed on Days 18 to 24.     

Embryonic behaviour in the lizard, Lacerta vivipara

The behaviour of embryos of the lizard Lacerta vivipara has been studied in cultured eggs, removed from the mother. Spontaneous movements begin at a time when the embryo is still unresponsive to touch, and about two days before the appearance of reflex responses. The earliest movements consist of lateral flexion which gradually gives place to dorsiventral flexion. Embryonic movements are not necessarily related to contractions of the amnion. The activity of the embryo, as measured by the number of times movements were initiated and the amount of time spent in activity during a half hour period, rises to a plateau and then decreases sharply as the time of hatching approaches. Activity of the tail is prominent, particularly at times when other movements occur in rapid succession. Serpentine movements were not observed in embryos younger than stage 39; some prematurely born animals showed such activity in response to contact with the substratum. During embryonic life any part of the animal may move individually or in combination with any other part, such combined movements being unco-ordinated. In these respects the spontaneous motility of the lizard embryo closely resembles that of the chick. Statistical analysis of the embryonic movements provides evidence of rhythmic activity within the developing central nervous system.     

Cryomicroscopic observations of cattle embryos during freezing and thawing

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

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

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

Pancreatic enzyme spectrum in chronic stress]

In acute experiments on white male rats the enzyme spectrum of pancreas homogenate was studied in the process of 30-day adaptation after 3-hour exposure to the muscular loading (compulsory swimming in the water at a temperature of 32 +/- 1 degree C), the heat (overheating at 40-41 degrees C) and the cold (cooling at -3-4 degrees C). It was shown that the initial periods of adaptation to the factors mentioned (2d-12th day) were characterized by a considerable decrease in the activity of all the enzymes under study. With subsequent adaptation of the animal body to these stresses the enzyme activity level recovered to the initial value (18-24th day) and remained more or less stable up to the end of the experiment (30th day). It is suggested that the alteration of the pancreatic enzyme spectrum proceeds with the participation of the hypothalamic-hypophyseal-adrenal systems as a general adaptation syndrome.     

The cholinergic reaction of the amnion in the chick embryo

Studies have been made of the spontaneous contractions of the amnion and acetylcholine sensitivity of amniotic membrane in 8--14-day chick embryos. In 12--14-day embryos, the spontaneous rhythmic contractions were rather rare as compared to those in 8--9-day ones, their frequency being also lower. On the basis of kinetic analysis, it was concluded that both the dissociation constant (K) and the value of Pmax do not exhibit significant changes for tonotropic reaction from the 8th to the 14th day and for chronotropic reaction--from the 8th to the 10th day of incubation. After the 10th day of incubation, dose-effect chronotropic reaction not expressed. The spontaneous activity of the amnion and acetylcholine sensitivity of the amniotic membrane depend on the temperature being maximal at 38 degrees C. Possible regulatory mechanisms of contractile activity in chick amnion are discussed.     

Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN(2)) from -10, -20, or -33 degrees C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN(2) from -33 degrees C, very rapid thawing (10 s in 30 degrees C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30 degrees C for 10 s). By contrast, oocytes plunged into LN(2) from -10 or -20 degrees C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (-20 to -33 degrees C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to -33 degrees C could be plunged to -196 degrees C with little or no further damage.     

Catecholamines and DOPA in the amniotic fluid and amnion of the chick embryo

Identification and quantitative fluorimetric assay have been made on the content of DOPA, dopamine, noradrenaline and adrenaline in the amniotic fluid and amnion of the developing chick embryos. Significant increase in the content of DOPA, noradrenaline and adrenaline in the amniotic fluid was observed between the 6th and the 13th days of incubation; dopamine content sharply decreases at the 13th day. The content on amines in the amnion tissue remained essentially constant throughout the investigated period. The role of catecholamine in amniotic fluid in regulation of contractile activity of amniotic membrane in the developing chick embryo is discussed.     

Size-dependent pigmentation-pattern formation in embryos of Alligator mississippiensis: time of initiation of pattern generation mechanism

The pigmentation pattern of Alligator mississippiensis was examined. The number of white stripes on the dorsal side of embryos (stages 21-28) and hatchlings from eggs incubated at 30 degrees C (100% females) and 33 degrees C (100% males) was recorded. Total length, nape-rump length and tail length were recorded for each embryo and hatchling. The number of white stripes was affected by incubation temperature but not sex; hatchlings incubated at 33 degrees C had two more white stripes than those at 30 degrees C, despite being the same length. Five female hatchlings produced at 33 degrees C by manipulation of the temperature, had the same number of stripes as males that developed under the same incubation temperatures. The appearance of the pigmentation was accelerated in embryos incubated at 33 degrees C, occurring eight days earlier than at 30 degrees C. At the time just before the first signs of pigment deposition, embryos from 33 degrees C were longer than those at 30 degrees C. If the stripe formation is size dependent this explains why hatchlings at 33 degrees C have more stripes than hatchlings from 30 degrees C. The mechanism that produces the stripe patterns is unknown. We describe key elements a pattern formation mechanism must possess to produce such stripes and suggest a possible mechanism, based on cell movement driven by chemotaxis. We apply the mathematical model to dorsal patterning on A. mississippiensis. We show how length at pattern formation is the prime factor in determining stripe number and how the pattern can be formed in the observed anterior-posterior sequence. We present numerical simulations and show that the qualitative behaviour is consistent with the experimental results.     

Study of cardiac, respiratory, and motor activities in rat fetuses

Development of the cardiac, respiratory, and motor activities in rat fetuses with preserved placental circulation was studied at the 16th, 18th, and 20th gestation days. The presence of three main movement types has been found: complexes of generalized activity, local movements, and jerks. In development of respiratory function, there is observed a gradual transition from individual inspirations to series of breathing movements and then to formation of periodic breathing episodes. At the studied period, the heart rate has been found to increase. The existence of the slow-wave modulations of the heart rate with a period of 2040 s has been revealed. Analysis of interrelations between the respiratory and motor systems has shown that in the 16-day fetuses, each breathing movement is accompanied by extensor jerk. By the 20th days of embryonic development (E20), uncoupling of the respiratory and motor activities occurs. Comparison of the activity observed in the cardiac and somatomotor systems has shown that at E16, the cardiac rhythm fluctuations do not depend on the motor excitation jerks. In the 18-day fetuses, brief slowing down (decelerations) of the cardiac rhythm appeared during the motor activity jerks, whereas at E20, on the contrary, an increase of frequency (accelerations) of the cardiac rhythm occurred.     

The role of the temporal neocortex in the origin of convulsive activity]

Experiments on cats with cooling capsules implanted over different areas of the neocortex have shown that cooling of different intensity applied to the temporal neocortex may result in both stimulation and switching off effects. Cold stimulation (temperature dropping to 27-33 degrees C) manifested in generalized epileptiform brain electrical activity and paroxysmal states. The functional switching off the temporal area observed during its deeper cooling (20-21 degrees C) discontinues the paroxysmal state already developed and prevents the appearance of seizures, regrardless of the localization of the epileptogenic focus. The paroxysmal state weakens and ceases after repeated cooling of the temporal neocortex. The temporal neocortex, involved in the integrated activating brain system, plays a decisive role in the emergence of paroxysmal states.     

The effects of artificial analogue peptide TSKY isolated from the brain of hibernating ground squirrels in rats and mice

Elevation of the i.c.v. injection dose of TSKY from 4 to 8 microg increased the movement activity of rats; in EEG theta- and beta-rhythms were enhanced and alpha-rhythm was suppressed. On the contrary, after treatment of 15 microg the rats fell into sleepy-like state; theta- and beta2-rhythms suppression, delta-, alpha- and beta1-rhythms were increased. Exposure under hypoxia-hypercapnia conditions reduced body temperature of mice to 18-19 degrees C, and maintain this state about 3-4 h after transferring into conventional gas medium. Preliminary cooling mice were administrated with TSKY that at dose 100 microg intraperitonally induced a prolonged hypothermia up to 12 h. Analogous injection without cooling raised mice temperature by 1.2 degrees C during about 2 h.     

Effect of temperature on cold-hardiness and tissue ice formation in the adult chrysomelid beetle Melasoma collaris L

The freeze-tolerant chrysomelid beetle Melasoma collaris overwinters in plant litter on windswept ridges or covered with snow for 8-9 months in the Norwegian alpine region. Lower lethal temperature, supercooling and melting point depression were correlated to accumulation of glycerol. The lower limit of freeze tolerance was associated with the freezing of 73-75% body water. About 23-15.5% of the body water was osmotically inactive, and the highest percentage was revealed in individuals depleted of glycerol at 21 degrees C. A shift in cooling rate from 1 degrees Cmin(-1) to 1 degrees C every 13.5min lowered nucleating temperature markedly. The alteration in nucleating activity probably arises from the structure of the haemolymph nucleating agent that functions to slow embryo growth at the slow cooling rate. An enhanced supercooling is particularly beneficial in autumn before M. collaris has accumulated glycerol, since supercooled individuals accumulate glycerol in higher concentrations than frozen ones. Freezing at higher temperatures is probably a better survival strategy during brief intervals with pronounced decrease in air temperature.     

Effects of extender composition, cooling rate and freezing on the fertilisation viability of spermatozoa of the Pacific oyster (Crassostrea gigas)

The aim of this research was to optimise protocols for freezing spermatozoa of the Pacific oyster. All the phases of the cryopreservation procedure (choice of cryoprotectant, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa to restore on thawing the morphological and physiological characteristics of fresh semen. The choice of type and concentration of cryoprotectant in which semen is incubated before freezing is fundamental for a successful cryopreservation: the cryoprotectants (dimethylsulfoxide--Me(2)SO, ethylene glycol--EG, propylene glycol-PG, and glycerol in concentrations between 5 and 15%) were tested for their toxicity on the semen exposed up to 30 min at +26 degrees C (room temperature) by evaluating its ability to fertilise and the embryo development to the regular D larval stage. The best cryoprotectants, Me(2)SO, EG, and PG 5, 10, and 15% respectively, were used for the pre-cooling (adaptation/cooling) tests. Two different adaptation/cooling procedures were tested: (A) from +26 degrees C to 0-2 degrees C (2.6 degrees C/min) and (B) at +26 degrees C for 15 min. Lastly, using the cryoprotectants and the adaptation procedure (B) that had given the best results in the preceding stages of the experiment, four cooling rates were tested: 6, 11, 16, and 21 degrees C/min. It was seen that the semen that was incubated with EG 10%, adapted at +26 degrees C for 15 min, and then cooled at a rate of 6 degrees C/min showed a percentage of regular D larvae on thawing comparable to that of fresh semen (p > 0.05).     

Metabolic responses of chicken embryos to graded, prolonged alterations in ambient temperature

1. Chicken embryos aged 12, 16, 18 and 20 (externally pipped) days of incubation were exposed to graded reductions (2 degrees C) in ambient temperature from 38 to 28 degrees C, exposure to each temperature lasting up to 9 hr. 2. Oxygen uptake was measured first at 38 degrees C and then in the quasi-equilibrium state at lowered temperatures. The temperature coefficient (Q10) was calculated for each egg. 3. For mild cooling (32 degrees C), the Q10 in 18-day-old embryos was about 1.5, while 12- and 16-day-old embryos had a Q10 value of about 2, indicating that a feeble homeothermic metabolic response to cooling appears in late prenatal embryos. It became more marked in externally pipped embryos and further augmented in hatchlings.     

The effects of lowered temperature on spontaneous eye movements in a teleost fish

A new opto-electronic method has been used to measure spontaneous eye movements in a lightly restrained unanaesthetized marine teleost fish (Parore). The normal scanning pattern of eye movement is similar to that previously described in goldfish. The effects of cooling on eye movements were investigated by 2 degrees C step changes down from ambient temperature (13-14 degrees C). Lowered temperature altered the scanning pattern, decreased saccade velocity, increased mean saccade amplitude and impaired the ability of the fish to hold the eye stationary between saccades. All eye movements stopped at temperatures around 6 degrees C, but could be restored by subsequent warming.