Effect of egg turning and incubation time on carbonic anhydrase gene expression in the blastoderm of the Japanese quail (Coturnix c. japonica).

1. The gene expression of carbonic anhydrase, a key enzyme for the production of sub-embryonic fluid (SEF), was assessed in turned and unturned eggs of the Japanese quail. The plasma membrane-associated isoforms CA IV, CA IX, CA XII, CA XIV, and the cytoplasmic isoform CA II, were investigated in the extra-embryonic tissue of the blastoderm and in embryonic blood. 2. Eggs were incubated at 37.6 degrees C, c.60% RH, and turned hourly (90 degrees ) or left unturned. From 48 to 96 h of incubation mRNA was extracted from blastoderm tissue, reverse-transcribed to cDNA and quantified by real-time qPCR using gene-specific primers. Blood collected at 96 h was processed identically. 3. Blastoderm CA IV gene expression increased with the period of incubation only in turned eggs, with maxima at 84 and 96 h of incubation. Only very low levels were found in blood. 4. Blastoderm CA II gene expression was greatest at 48 and 54 h of incubation, subsequently declining to much lower levels and unaffected by turning. Blood CA II gene expression was about 25-fold greater than in the blastoderm. 5. The expression of CA IX in the blastoderm was the highest of all isoforms, yet unaffected by turning. CA XII did not amplify and CA XIV was present at unquantifiable low levels. 6. It is concluded that only gene expression for CA IV is sensitive to egg turning, and that increased CA IV gene expression could account for the additional SEF mass found at 84 to 96 h of incubation in embryos of turned eggs.

Role of carbon dioxide and ion transport in the formation of sub-embryonic fluid by the blastoderm of the Japanese quail.

1. The explanted blastoderm of the Japanese quail was used to explore the role of ions and carbon dioxide in determining the rate of sub-embryonic fluid (SEF) production between 54 and 72 h of incubation. 2. Amiloride, an inhibitor of Na+/H+ exchange, at concentrations of 10(-3) to 10(-6) M substantially decreased the rate of SEF production when added to the albumen culture medium. N-ethylmaleimide, an inhibitor of V type H+ ATPase, also decreased this rate but only to a small extent at the highest dose applied, 10(-3) M. Both inhibitors had no effect on SEF production when added to the SEF. 3. The inhibitors of cellular bicarbonate and chloride exchange, 4-acetamido-4'-isothiocyano-2,2'-disulphonic acid (SITS) and 4,4' diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), had no effect upon SEF production. 4. Ouabain, an inhibitor of Na+/K+ ATPase, decreased SEF production substantially at all concentrations added to the SEF (10(-3) to 10(-6) M). Three sulphonamide inhibitors of carbonic anhydrase, acetazolamide, ethoxzolamide and benzolamide, decreased SEF production when added to the SEF at concentrations of 10(-3) to 10(-6) M. Benzolamide was by far the most potent. Neither ouabain nor the sulphonamides altered SEF production when added to the albumen culture medium. 5. Using a cobalt precipitation method, carbonic anhydrase activity was localised to the endodermal cells of the area vasculosa. The carbonic anhydrase activity was primarily associated with the lateral plasma membranes, which together with the potent inhibitory effect of benzolamide, suggests the carbonic anhydrase of these cells is the membrane-associated form, CA IV. 6. The changes in SEF composition produced by inhibitors were consistent with the production of SEF by local osmotic gradients. 7. It is concluded that a Na+/K+ ATPase is located on the basolateral membranes of the endodermal cells of the area vasculosa, and that a sodium ion/hydrogen ion exchanger is located on their apical surfaces. Protons for this exchanger would be provided by the hydration of CO2 catalysed by the membrane-associated carbonic anhydrase. Furthermore, it is proposed that the prime function of the endodermal cells of the area vasculosa is the production of SEF.

Effect of egg turning and fertility upon the sodium concentration of albumen of the Japanese quail.

1. The effects of egg turning and fertility upon sodium concentration of albumen of the Japanese quail is described for up to 72 h incubation. 2. For incubated eggs the sodium concentration of albumen adjacent to the yolk sac was lower than that from albumen next to the shell. Static incubation increased the magnitude of this difference, such that albumen adjacent to the yolk sac was substantially depleted of sodium. This was found at the yolk equator and the yolk vegetal pole of both fertilised and unfertilised eggs. 3. Unincubated eggs also had a lower sodium concentration of albumen adjacent to the yolk sac compared with albumen next to the shell. 4. After 48 h of incubation yolk sodium concentration was substantially lower than albumen sodium concentration in both fertilised and unfertilised eggs, whether eggs were turned or not. 5. It is concluded than in unturned eggs the depletion of sodium from albumen adjacent to the vitelline membrane is not produced by ion transport processes but results from a passive movement of sodium into the yolk. Egg turning reduces the magnitude of the depletion of sodium from the albumen adjacent to the yolk sac by stirring the albumen, so permitting the full expression of ion and water transport across the blastoderm into the yolk sac.

The role of ion transport in the formation of sub-embryonic fluid by the embryo of the Japanese quail.

1. Using a modification of the method of New (1955) explanted blastoderms of the Japanese quail were cultured for up to 72 h, and the role of ions in the formation of sub-embryonic fluid (SEF) investigated. 2. Culture media deficient in either sodium or chloride ions reduced the volume of SEF secreted by up to a quarter. Ionic composition of the fluid was little altered, and sodium was transported against a concentration gradient. 3. Amiloride, an inhibitor of Na+/H+ exchange, reduced SEF production by half, whereas ionic composition and osmolality of the fluid was unchanged. 4. Likewise, acetazolamide, an inhibitor of carbonic anhydrase, reduced SEF production by the blastoderm, and left the ionic composition and osmolality of the fluid unaltered. 5. Neither furosemide or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid at the concentrations used changed the volume of SEF formed or its ionic composition. 6. It is concluded that the secretion of SEF is dependent upon the active transport of sodium across the blastoderm; an amiloride-sensitive Na+/H+ exchanger and carbon dioxide hydration catalysed by carbonic anhydrase are also involved. 7. Furthermore, it is proposed that fluid transport across the blastoderm is the result of local osmotic gradients, not from a sub-embryonic fluid hyperosmotic to albumen, as has been suggested previously.

The effect of water loss upon the urate, urea and ammonia content of the egg of the Japanese quail Coturnix coturnix japonica.

1. The urate, urea and ammonia content of the whole egg of the Japanese quail was measured in late incubation in eggs subject to different rates of water loss. 2. High rates of water loss substantially increased egg urate content, but had little or no effect on urea or ammonia content. 3. Allopurinol, an inhibitor of urate synthesis, reduced egg urate content to low levels, but produced no effect on urea content, and a small reduction in ammonia content. 4. The urea concentration of the embryo was lower than in allantoic fluid. 5. It is concluded that urate production by the avian embryo is primarily concerned with the modification of allantoic fluid composition.

Seminal plasma composition in budgerigars (Melopsittacus undulatus).

Seminal plasma composition was studied in budgerigars. Semen was obtained from adult male budgerigars by applying gentle pressure to both sides of the cloaca. Pooled samples were centrifuged at 15,000 g for 2 min, and the seminal plasma separated for biochemical analysis. Osmolality, Na+, K+, Cl-, pH, glucose and fructose values were determined. The biochemical composition of budgerigar seminal plasma obtained in this study was: Osmolality 329.9 +/- 14.5 mOs/kg; Na+ 158.6 +/- 8.4 mEq/l; K+ 16.39 +/- 6.24 mEq/l; Cl- 109.2 +/- 7.4 mEq/l; pH 8.20 +/- 0.18 glucose 4.25 +/- 0.96 mmol/l; fructose 0.59 +/- 0.29 mmol/l. The results are discussed in relation to the values reported for the domestic fowl. This forms part of a reproductive biology study of non-domesticated avian species.

The effects of a lowered ambient temperature on oxygen consumption and lung ventilation in the perinatal quail (Coturnix c. japonica).

The ambient temperature of embryos of pipped eggs and hatchings was reduced from 38 to 28 degrees C for a period of 1 hr. The weight-specific rate of oxygen consumption (VO2) of embryos and hatchlings at 38 degrees C was less than the predicted value for adults of the same weight. The Q10 value for mean weight-specific VO2 was 2.08 for pipped eggs, 0.98 for 4-hr-old hatchlings and 0.67 for 1-day-old chicks. The Q10 values for VO2 of individual embryos fell in the range 1.69-2.42, but the Q10 values for ventilatory frequency fell between 1.22 and 4.65. There was no correlation between the Q10 values for VO2 and ventilatory frequency for the same embryos. When ambient temperature was returned from 28 to 38 degrees C VO2 and ventilatory frequency returned to pre-cooling values within a period of 1 hr. The results are discussed in relation to the factors which influence gaseous exchange during the perinatal period.