Circadian locomotor activity in Japanese sea catfish <Emphasis Type="Italic">Plotosus lineatus</Emphasis>

To find out whether or not the locomotor activity of the Japanese sea catfish, Plotosus lineatus, shows a circadian rhythmicity, the locomotor activity of this catfish was recorded in a laboratory. All of the catfish recorded (n = 20) showed a nocturnal locomotor activity rhythm under a light–dark (LD) cycle (LD 12:12). The locomotor activity started after the light was turned off, and declined during the period of darkness. In five of the six catfish we examined, locomotor activity shifted gradually over the course of 3–4 days in order to synchronize to the LD cycle after it was advanced by 6 h. Locomotor activity persisted within a constant darkness (DD) in all eight of the catfish. The average free-running period of locomotor activity under DD lasted for 24.2 ± 0.4 h (mean ± SD). Thus, the present results demonstrate that the Japanese sea catfish has an endogenous circadian oscillator entrained by an LD cycle.     

Diel feeding rhythms of greenback flounder Rhombosolea tapirina (Günther 1862): the role of light–dark cycles and food deprivation

The role of light–dark (LD) cycles and food deprivation in the regulation of diel feeding rhythms in greenback flounder Rhombosolea tapirina (Günther 1862) was examined. A computerized monitoring system was developed to record uneaten pellets, and food intake was estimated as the difference between pellets presented and those uneaten. Three groups of five fish each (75–281 g) were exposed sequentially to the LD cycles: LD 12:12, LD 12:12 (scotophase advanced by 9 h), LD 6:18, DD, LD 6:6:6:6, LL and LD 12:12 (return to the initial cycle). Illumination was 120 lux at the water surface (complete darkness at scotophase), and water temperature was 15 ± 1 °C. Fish fed mainly during the light hours under LD 12:12 and resynchronized to the scotophase-advanced LD 12:12 regime after about 12 h. Feeding started at the onset of light and extended to the early scotophase under LD 6:18. A circadian feeding rhythm was detected in fish under DD and LD 6:6:6:6 regimes. Under the LL regime, two groups of fish displayed arrhythmic feeding patterns and did not resynchronize to LD 12:12 for at least 13 days. In contrast, the third group of fish exhibited a circadian feeding rhythm under the LL regime and immediately re-entrained to LD 12:12. Fish were deprived of food for a single period of 26–57 h once feeding patterns had been established under LD 12:12, LD 12 : 12 (scotophase advanced by 9 h) and DD regimes, and feed delivery was resumed during the scotophase or subjective night; the timing of feeding rhythms was shifted by the reintroduction of feed, but progressively resynchronized to the LD cycles. The results suggest that the greenback flounder is a diurnal feeder, that a LD 12:12 cycle is a potent environmental cue to entrain circadian feeding rhythms, and that a biological clock is involved in the timing of feeding.     

Feeding activity rhythm in Japanese sea catfish <Emphasis Type="Italic">Plotosus japonicus</Emphasis>

To examine the feeding activity characteristics of Japanese sea catfish Plotosus japonicus, access to feed box (feeding activity) and the number of food pellets consumed (food intake) were recorded under laboratory conditions. When fed ad libitum under a 12:12 h light–dark (LD) cycle, all fish exhibited feeding activity and consumed food during the dark period. Feeding activity increased in the presence of food, and this increased activity level continued for several days after food removal. During restricted food availability within the light period of the LD cycle, seven of nine fish exhibited food-anticipatory activity (FAA) both before and after the 12:00 food-restricted time. This FAA persisted under constant light conditions (in nine of nine fish). The average circadian free-running period was 24 h. These results suggest that P. japonicus exhibits a nocturnal feeding activity rhythm, which may be synchronized by a food-restricted time.     

Endocrine (plasma cortisol and glucose) and behavioral (locomotor and self-feeding activity) circadian rhythms in Senegalese sole (Solea senegalensis Kaup 1858) exposed to light/dark cycles or constant light

The existence of daily rhythms under light/dark (LD) cycles in plasma cortisol, blood glucose and locomotor and self-feeding activities, as well as their persistence (circadian nature) under constant light (LL), was investigated in Senegalese sole (Solea senegalensis). For the cortisol and glucose rhythms study, 48 soles were equally distributed in 8 tanks and exposed to a 12:12 LD cycle and natural water temperature (experiment 1). After an acclimation period, blood was sampled every 3 h until a 24-h cycle was completed. Blood glucose levels were measured immediately after sampling, while plasma cortisol was measured later by ELISA. In experiment 2, the fish were exposed to LL for 11 days, and after this period, the same sampling procedure was repeated. For the study of locomotor and self-feeding rhythms (experiment 3), two groups of sole were used: one exposed to LD and the other to LL. Each group was distributed within 3 tanks equipped with infrared photocells for the record of locomotor activity, and self-feeders for feeding behavior characterization. The results revealed a marked oscillation in cortisol concentrations during the daily cycle under LD, with a peak (35.65 ± 3.14 ng/ml) in the afternoon (15:00 h) and very low levels during the night (5.30 ± 1.09 ng/ml). This cortisol rhythm persisted under LL conditions, with lower values (mean cortisol concentration = 7.12 ± 1.11 ng/ml) and with the peak shifted by 3 h. Both rhythms were confirmed by COSINOR analysis (p < 0.05). The synchronizing role of temperature and feeding schedule, in addition to light, is also discussed. Diel rhythms of glucose were not evident in LD or LL. As to locomotor and self-feeding activity, a very marked rhythm was observed under LD, with higher activity observed during the night, with acrophases located at 2:14 and 3:37 h, respectively. The statistical significance of daily rhythms was confirmed by COSINOR analysis. Under LL, both feeding and locomotor rhythms persisted, with an endogenous period (τ) around 22.5 h. In short, our findings described for the first time the existence of circadian cortisol and behavioral circadian rhythms in flat fish. Such results revealed the importance of taking into account the time of day when assessing stress responses and evaluating physiological indicators of stress in fish.     

Production of melatonin by the gilthead sea bream pineal: anin vivo andin vitro study

This study deals with the variations of plasma melatonin content and ofin vitro pineal melatonin secretion, in one year-old male sea bream. Plasma melatonin levels varied from almost undetectable levels during the day to 150–200 pg ml^–1 during the night, in animals maintained under a light/dark (12L/12D) cycle. The duration of the nocturnal surge corresponded to the duration of the night. A similar pattern of melatonin secretion was obtainedin vitro, with pineals cultured under a 24h L/D cycle. When the photoperiod cycle was advanced by 8h, the first nocturnal peak in melatonin secretion was more acute compared to the profile described above, but the rhythmic variations were immediately synchronized to the new L/D conditions. Under constant darkness (D/D), circadian variations in melatonin release were observed for no more than two 24h cycles. No melatonin was detected in the culture media from retinas cultured under L/D or under D/D. These results strongly suggest that the pineal is the major source of plasma melatonin in the gilthead sea bream, and that the 24h photoperiod cycle is a powerful synchronizer of the rhythmic production of melatonin by this organ.     

Effects of light intensity on daily activity rhythm of juvenile sea cucumber,Apostichopus japonicus (Selenka)

Eight light‐intensity treatments (natural light, continuous darkness, and 15, 30, 60, 125, 250 and 500 lx under LD 12:12 cycle) were used to investigate the effects of light intensity on the daily activity of 30.27±3.08 g sea cucumber Apostichopus japonicus (Selenka). Cyclic nocturnal activity patterns of behaviour were observed at different light intensities in the range of 15–500 lx under LD 12:12 cycle. And an ongoing nocturnal cycle persisted in DD cycle for up to 8 days, but longer feeding time and less marked rhythm occurred at continuous darkness. Under poor light conditions (I<5.18 lx), the daily activity rhythm of A. japonicus was governed by an innate biological clock and the effect of light intensity was not significant among different treatments. And more individuals tended to retreat to shelters (from 56.04% to 91.83%) with the increase of light intensity within the weak light condition (from 5.18 to 278 lx). However, the daily behaviours of A. japonicus were influenced under strong light conditions (>278 lx). Less than 8.17% individuals kept actively feeding and the proportion was not decreased with the increase of light intensity.     

Photic and circadian regulation of self-feeding activity in ayu

ABSTRACT:   The self-feeding rhythms of ayu Plecoglossus altivelis altivelis were examined. Individual ayu (mean body weight 40 g) were held in 60-L glass tanks equipped with self-feeders. Six of 14 fish learned self-feeding during the experiment. Under two different light–dark (LD) conditions (16 h:8 h and 8 h:16 h LD), self-feeding was synchronized to the LD cycle, and feeding occurred almost exclusively during the light phase. During exposure to constant light (LL), circadian feeding rhythms were observed. These results indicate self-feeding rhythms in ayu are restricted to the light phase under LD conditions and are controlled by the circadian clock under LL conditions.     

Performance of the heat of the hagfish,Eptatretus cirrhatus

The maximum power output of isolated perfused ventricles of the hafish (Eptatretus cirrhatus) averaged 0.367±0.031 mW g^–1 (n=9), considerably high than estimates for the heart of the Atlantic hagfish (Myxine glutinosa). Maximal minute volumes averaged 21.55±1.28 ml min^–1kg^–1, with a mean stroke volume of 0.71±0.14 ml kg^–1 body weight, values which are similar to those reported for many teleost and elasmobranch hearts.Ventricular output showed the characteristic dependence upon atrial filling pressure up to an optimum filling pressure ofc. 4 mm Hg. At output pressures exceeding 14 mm Hg the stroke volume and power output fell sharply. At these afterloads, the ventral aorta remained distended following semilunar valve closure and so the volume of fluid ejected on ventricular systole was reduced. There was little change in the frequency of the heart as either input or output pressures were varied.     

Circadian melatonin release <Emphasis Type="Italic">in vitro</Emphasis> by European sea bass pineal

Melatonin production by the fish pineal is under the control of photoperiod. In addition, in some fish species pineal melatonin production is self-sustained by an intrapineal circadian pacemaker, not in salmonids, though. The aim of this research was to test melatonin release in vitro by the pineal of sea bass held under LD 12:12 and DD conditions to determine the endogenous nature of melatonin rhythm. Circadian rhythmicity ( = 28.3 h) was found since rhythmic melatonin release persisted on DD.     

Cathepsin L in eggs and larvae of perch Perca fluviatilis: variations with developmental stage and spawning period

This study describes changes in cathepsin L activity during early development in Eurasian perch (Perca fluviatilis) and determines to which extend a precocious proteolysis of the yolk reserves by activated cathepsin L could be involved in the seasonal decline of egg and larval quality. During embryogenesis and early larval stages, samples were collected for determination of fertilization and hatching rates, assays of cathepsin L activity, and evaluation of larval resistance to stress (osmotic shock and fasting). Cathepsin L activity increased significantly during the embryonic development, reaching a maximum of 177.1±12.3 nmol min^–1mg fresh weight^–1 on day 1 post-hatching and significantly decreasing on day 3. Activity increased significantly during the spawning season and individual variations between egg strands became very high during the second half of the spawning period. An inverse relationship was established between the cathepsin L activity in 7-day old eggs and the decrease of hatching rate. A negative exponential regression was calculated between the cathepsin L activity of eggs and the resistance of corresponding newly-hatched larvae to osmotic shock. Resistance to fasting was significantly reduced in larvae from eggs with cathepsin L activity higher or lower than 20 nmol min^–1 mg^–1 (lethal time₅₀=4.7±0.7 and 3.5±0.4 days, respectively). The involvement of cathepsin L in the degradation of yolk reserves and its potential consequences on the quality of perch eggs and larvae are discussed in relation to results reported in salmonid fishes.     

Meagre’s melatonin profiles under captivity: circadian rhythmicity and light sensitiveness

The present study reveals the first characterization of the plasma melatonin rhythms of the meagre (Argyrosomus regius) under aquaculture conditions. Melatonin levels were monitored during a 24 h cycle under a photoperiod of 16 L:8D and under constant darkness (DD), respectively to characterize the daily rhythm of this indoleamine and to test its endogenous origin. Besides, to identify which light intensities are perceived as night or day by this species, the degree of inhibition of nocturnal melatonin production caused by increasing intensities of light was tested (3.3, 5.3, 10.5, and 120 μW/cm²), applying 1 h light pulses at Mid-Dark. The result for melatonin daily rhythm in plasma showed a typical profile: concentration remained low during all daytime points, increasing greatly during dark points, with maximum values at 16:00 and 22:00 h, zeitgeber time. Under DD conditions, the plasma melatonin profile persisted, with a similar acrophase but with a lower amplitude between subjective day and night periods, indicating this rhythm as being endogenously driven. Moreover, meagre seemed to be very sensitive to dim levels of illumination during the night, since an intensity of just 3.3 μW/cm² inhibited melatonin production. However, only the pulse of 5.3 μW/cm² caused a melatonin drop till daytime concentrations. Thus, the threshold of light detection by the pineal organ was suggested as being located between 3.3 and 5.3 μW/cm². Such results are an added value for this species biology knowledge, and in consequence to its adaptation to aquaculture conditions, allowing the improvement of culture husbandry protocols.     

Diel Rhythms of Serum Metabolites and Thyroid Hormones in Red Porgy Held in Different Photoperiod Regimes

Diel rhythms in serum glucose, lactate, cholesterol, triglycerides and thyroid hormones were studied in red porgy, Pagrus pagrus, held under different photoperiod regimes (constant darkness – DD, 8L:16D,12L:12D), at a constant temperature (17.1–18.7 °C) and fed with commercial pellets, by means of a self-feeder. A clear diurnal rhythm in feeding activity, regardless of the photoperiod regime was demonstrated. All serum components showed significant diel rhythms, although they were not necessarily consistent or circadian in periodicity. As well as this, differences in the average values among the varying treatments were observed. Fish held under the 12L:12D protocol displayed significantly higher average T₄, T₃ and lactate levels during the day rather than at night. Maximum glucose values occurred 8–12 h after dawn and maximum lactate concentrations at 06:00 or 14:00 h. Diel variations in cholesterol were evident only in the DD group with peak values inversely correlated with the rhythm of food intake. Triglycerides displayed a similar pattern of changes. Significant diel fluctuations in T₄ serum levels were observed only in fish exposed to the 12L:12D protocol, with peak values at dawn. A clear diurnal peak (10:00 h) in T₃ concentrations was observed in fish subjected to the 12L:12D regime, while fish exposed to the 8L:16D protocol showed two peaks: one in the photophase (10:00 h) and another in the scotophase (02:00 h). The light–dark alternation and the general activity rhythm of fish seem to be the main synchronizers of the diel rhythms observed in this study.     

Growth of Arctic charr (Salvelinus alpinus L.) under conditions of constant light and temperature

Arctic charr, Salvelinus alpinus, display distinct cycles of food intake, growth and conversion when reared in the laboratory under constant conditions (continuous light; temperature 10°C). Growth rates peaked in December–January and June–July and it is suggested that charr may have an endogenous growth rhythm with a 6-month cycle.     

Regulation of cardiac function in the horn shark by changes in pericardial fluid volume mediated through the pericardioperitoneal canal

The California horn shark (Heterodontus francisci), instrumented with a pericardial catheter and a ventral-aortic flow probe, was studied to determine the effect of complete pericardial chamber evacuation on the time course for restitution of pericardial fluid-volume and pressure, and the effects of both fluid removal and its restitution on cardiac output. Prior to evacuation, pericardial pressure was –0.02±0.02 kPa, and cardiac output was 18±2 ml min^–1 kg^–1. Evacuation reduced pericardial pressure to –0.73±0.14 kPa, and increased cardiac output to 23±4 ml min^–1 kg^–1. The time course for restoration of post-evacuation pressure is described by a non-linear asymptotic function. A large percentage of the pericardial pressure and volume recovery occurred within the first hour, while, complete restoration of pre-withdrawal conditions required about 11 h. Pericardial pressure-volume relationships, determined by incremental infusion of small volumes of elasmobranch saline into the pericardium, confirm previous findings that the operating pericardial pressure in the horn shark is at or near ambient pressure and that both pericardial fluid volume and cardiac stroke volume influence horn shark pericardial pressure.     

Diel changes in plasma arginine vasotocin, isotocin, and melatonin in rainbow trout (Oncorhynchus mykiss)

The diel changes in plasma AVT, IT and Mel in rainbow trout (Oncorhynchus mykiss) were studied to assess potential relationships. Blood was sampled at 05:00, 11:00, 16:00, 22:30 and 05:00 in freshwater-adapted fish and at 22:30 in brackish water-adapted fish maintained under natural photoperiod. A few of the FW-acclimated fish were assigned to one of two experimental groups and adapted to DD or LL lighting regimes. Blood samples were taken at 11:00 and 22:30. Hormones were extracted from plasma by solid phase extraction and determined by high-performance liquid chromatography. Marked diel variations in AVT and Mel were detected in fish maintained under natural photoperiod. Plasma AVT (fmol ml^–1) increased during the light to reach the maximal level at the end of that phase (261.7±23.1). Thereafter, AVT concentration decreased and became minimal at 05:00 (68.9±11.5) 3 h before the sunrise. Plasma Mel (pmol ml^–1) increased between 16:00 and 22:30 when a peak value was reached (1204.0±55.5). Thereafter, Mel levels decreased and were minimal after the onset of the light phase (242.8±37.0). IT levels displayed no significant diel changes. Linear regression analysis indicated the negative correlation between plasma Mel and AVT for five collecting times of the daily 24 h cycle in freshwater fish and at 22:30 in brackish water fish. A similar correlation occurred at 11:00 in the DD group and at 22:30 in the LL group. To elucidate the character of the Mel-AVT relationships further studies are required.     

Food-entrainable circadian oscillator in goldfish: multiple daily feeding times and food-anticipatory activity

The existence of a food-entrainable circadian oscillator (FEO) was examined in goldfish Carassius auratus. Single goldfish were exposed to light–dark (LD) 12:12 (lights on 06:00–18:00 h) and fed at 12:00 h (phase I). The photoperiod regime was shifted to constant light (LL) (phase II). The fish were then fed at 06:00 or at 18:00 h (phase III), and all fish were fasted (phase IV). In both groups, three out of eight fish exhibited food-anticipatory activity (FAA) in phases II and III, the free-running period (τ) of locomotor activity was approximately 24.0 h, and circadian rhythms of locomotor activity started to free-run from the previous FAA in phase IV, indicating the existence of an FEO. Next, single goldfish were exposed to LD 12:12 and were fed twice daily at 12:00 and 00:00 h (phase I). The photoperiod regime was shifted to LL (phase II), and the fish were fasted (phase III). In phase II, FAA was observed in three out of six fish for both feeding times. In phase III, a circadian rhythm of locomotor activity was observed in five fish, and circadian rhythms started to free-run from the previous feeding time (either 12:00 or 00:00 h) in three fish. These results suggest that individual goldfish can remember two daily feeding times.     

Photoperiodic mechanisms and rhythms of reproduction in the female rainbow trout

The present work investigates the importance of circadian and circannual rhythms in the photoperiodic control of reproduction in the rainbow trout. Maintenance of groups of 20–30 female trout under continuous light (LL), constant long (18L:6D) or short (6L:18D) days and conditions of constant temperature (8.5–9.0°C) and feeding rates (0.5% body weight, day^–1), starting in February, produced markedly different spawning periodicities during the first and subsequent years of treatment. At the end of the first year, spawning was advanced by up to 2 months in the fish under LL and 18L:6D and delayed by up to 5 months in those under 6L:18D when compared with the December spawning of control fish under ambient light. Continued exposure of the fish to the same photoperiod regimes produced cycles of spawning and peak levels of testosterone, oestradiol-17 and calcium (as an index of vitellogenin) at intervals of 150–170 days under LL and 18L:6D and 320–420 days under 6L:18D. The functional importance of these spawning cycles and their relationship to circannual rhythms and the control of reproduction is discussed.Exposure of fish to skeleton (6L:4D:2L:12D, 6L:6D:2L:10D and 6L:8D:2L:8D) and resonance (6L:42D, 6L:48D and 6L:54D) procedures produced ranges of spawning times up to two months in advance of control fish. Results with the resonance regimes, where fish received only a half the light-dark cycles and a quarter of the total daylight hours of those on ambient light cycles show that trout do not measure daylength and time by counting daily cycles or by hour-glass mechanisms. Collectively, these data support the proposition that rhythmic processes of photosensitivity are involved in the modulation of reproductive development in the rainbow trout and that circadian and circannual rhythms cooperate in the timing and entrainment of this cycle.     

The effects of temperature in the life cycle of two consecutive generations of the cuttlefish Sepia officinalis (Linnaeus, 1758), cultured in the Algarve (South Portugal)

We are presently culturing the 4^th generation of thecuttlefish, Sepia officinalis in our laboratory. A firstgeneration (F1) was grown from eggs collected from the wild (Ria Formosa–South Portugal) during the summer, at mean temperatures of 27°C ± 3°. In the present study, a second generation(F2), originated from eggs laid in the laboratory by females from F1 wascultured between the start of autumn and the end of spring, at meantemperaturesof 15 °C ± 4 °C. The life cycle ofcuttlefish from F2 was compared to F1. Populations of 30 cuttlefish were usedineach experiment. Cuttlefish were grown from one day old until the cycle wascompleted (when the last female in each population had died). Cuttlefish fromF2cultured at much lower temperatures had a longer life cycle, of almost 9 months(260 days) compared to cuttlefish from F1, which completed their cycle in lessthan 6 months (165 days). Cuttlefish from F2 grew significantly larger (U =0.00; p < 0.01) with mean weights of 343.3 ± 80.5 g and248 ± 33.1 g for males and females, respectively, comparedtoF1 (199.6 ± 40 g and 143.3 ± 30.9 g formales and females, respectively). Females from F2 had higher fecundity (225eggsfemale^–1) compared to females from F1 (144 eggs perfemale^–1), produced bigger eggs (t = 45.60752; p < 0.0001),weighing 0.74 ± 0.18 g, compared to 0.46 ± 0.11 fromF1,and bigger hatchlings (t = 7,144783; p < 0.0001), weighing 0.10 ±0.02g, compared to 0.09 ± 0.02 g for the summerpopulation.     

Basal H2-receptor stimulated and pH-dependent acid secretion from an isolated stomach mucosa preparation of the cod,Gadus morhua,studied using a modified pH-static titration method

Gastric acid secretion from isolated cod stomach mucosa was measured using a pH-static titration method. A basal acid secretion rate (BASR) of 6.0±0.6 nEqH⁺min^–1cm^–1 was measured when using 0.9% NaCl as luminal solution. There was a dose-dependent increase in response to histamine between 0.12 and 0.20 M (EC₅₀=0.15 M), above which gastric acid secretion plateaued at 13.5±1.8 nEqH⁺min^–1cm^–1. Ranitidine, a H₂-receptor antagonist, completely blocked the stimulatory effect of histamine and reduced the BASR. The H₁-receptor antagonist, clemastine, did not inhibit the response to histamine. Acid secretion rates decreased significantly when the pH of the luminal side of the mucosa was lowered from pH 5.75 to pH 4.50, indicating that a negative feedback mechanism was operating. Histological staining showed that oxynticopeptic cells were uniformly distributed throughout the cardiac stomach.It is concluded that the acid secretion in the isolated stomach mucosa of cod can be measuredin vitro with a pH-static titration method. The method was used to demonstrate that the BASR is downregulated by a decrease in pH. Furthermore, we conclude that the histamine receptor in the cod stomach mucosa resembles the mammalian H₂-receptor and that histamine is secreted under basal conditions.     

The effects of experimental anaemia on CO2 excretionin vitro in rainbow trout,Oncorhynchus mykiss

The effects of severe experimental anaemia on red blood cell HCO₃ ^– dehydrationin vitro were examined in rainbow trout,Oncorhynchus mykiss. After 5 days of anaemia (haematocrit=4.9±1.1%) induced by intraperitoneal injection of phenylhydrazine hydrochloride, fish displayed elevated arterial CO₂ tensions (anaemic PaCO₂=3.19±0.42 torrvs. control PaCO₂=1.35±0.17 torr) and a significant acidosis (anaemic pHa=7.73±0.04vs. control pHa=7.99±0.04). However, after 15–20 days of anaemia (hct=6.6±0.8%) induced by blood withdrawal, the arterial CO₂ tension was significantly lower than the control value, suggesting that physiological adjustments occurred within this time period to compensate for the lowered haematocrit. Compensation probably did not involve alterations in ventilation, which was unaffected by 5 days of anaemia (anaemic ;w=786±187 ml min^–1 kg^–1 vs. control ;w=945±175 min^–1 kg^–1), based on indirect Fick principle measurements.Potential adaptations to longer term anaemia at the level of the red blood cells were investigated using a radioisotopic HCO₃ ^– dehydration assay. Owing to the difference in haematocrits, the HCO₃ ^– dehydration rate for blood from anaemic fish was significantly lower than that for control fish following equilibration at the same CO₂ tension. This difference was eliminated when HCO₃ ^– dehydration rates were measured on blood samples adjusted to the same haematocrit, a result which implies that the intrinsic rate of CO₂ excretion at the level of the red blood cell was not up-regulated during anaemia. The difference was also eliminated by equilibrating the blood samples with CO₂ tensions appropriate for the group from which the sample was obtained,i.e., PCO₂=1.4 torr for control samples and PCO₂=3.2 torr for anaemic samples; each at the appropriate haematocrit. It is concluded that the elevated PaCO₂ helps to reset CO₂ excretion to the control level, but that some additional physiological adjustment occurs to lower the PaCO₂ after 15–20 days of anaemia.