Slow intrinsic oscillations in thick neocortical slices of hypoxia tolerant deep diving seals.

Direct evidence that the mammalian neocortex is an important generator of intrinsic activity comes from isolated neocortical slices that spontaneously generate multiple rhythms including those in the beta, delta and gamma range. These oscillations are also seen in intact animals where they interact with other areas including the hippocampus, thalamus and basal ganglia. Here we show that thick isolated neocortical slices from hooded seals intrinsically generate persistent spontaneous activities, both repetitive non-rhythmic activity with activity states lasting for several minutes, and oscillating activity with rhythms that are much slower (<0.1 Hz) than the rhythms previously described in vitro. These intrinsic activities were very robust and persisted for up to 1 h even in severely hypoxic conditions. We hypothesize that the remarkable hypoxia tolerance of the hooded seal nervous system made it possible to maintain functional integrity in slices thick enough to preserve intact neuronal networks capable of generating these slow oscillations. The observed activities in seal neocortical slices support the notion that mammalian cortical networks intrinsically generate multiple states of activity that include oscillatory activity all the way down to <0.1 Hz. This intrinsic neocortical excitability is an important contributor not only to sleep but also to the default awake state of the neocortex.

Development of myoglobin concentration and acid buffering capacity in harp (Pagophilus groenlandicus) and hooded (Cystophora cristata) seals from birth to maturity.

Pinnipeds rely on muscle oxygen stores to help support aerobic diving, therefore muscle maturation may influence the behavioral ecology of young pinnipeds. To investigate the pattern of muscle development, myoglobin concentration ([Mb]) and acid buffering ability (beta) was measured in ten muscles from 23 harp and 40 hooded seals of various ages. Adult [Mb] ranged from 28-97 to 35-104 mg g tissue(-1) in harp and hooded seals, respectively, with values increasing from the cervical, non-swimming muscles to the main swimming muscles of the lumbar region. Neonatal and weaned pup muscles exhibited lower (approximately 30% adult values) and less variable [Mb] across the body than adults. In contrast, adult beta showed little regional variation (60-90 slykes), while high pup values (approximately 75% adult values) indicate significant in utero development. These findings suggest that intra-uterine conditions are sufficiently hypoxic to stimulate prenatal beta development, but that [Mb] development requires additional postnatal signal such as exercise, and/or growth factors. However, because of limited development in both beta and [Mb] during the nursing period, pups are weaned with muscles with lower aerobic and anaerobic capacities than those of adults.

When the brain goes diving: glial oxidative metabolism may confer hypoxia tolerance to the seal brain.

Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cystophora cristata remain much longer active in hypoxic conditions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cytochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O(2) affinity. Moreover, there is no significant difference in total neuroglobin protein levels in mouse, rat and seal brains. However, in terrestrial mammals neuroglobin resided exclusively in neurons, whereas in seals neuroglobin is mainly located in astrocytes. This unusual localization of neuroglobin is accompanied by a shift in the distribution of cytochrome c. In seals, this marker for oxidative metabolism is mainly localized in astrocytes, whereas in terrestrial mammals it is essentially found in neurons. Our results indicate that in seals aerobic ATP production depends significantly on astrocytes, while neurons rely less on aerobic energy metabolism. This adaptation may imbue seal neurons with an increased tolerance to hypoxia and potentially also to reactive oxygen species, and may explain in part the ability of deep diving mammals to sustain neuronal activity during prolonged dives.

Size and distribution of oxygen stores in harp and hooded seals from birth to maturity.

Pinnipeds rely primarily on oxygen stores in blood and muscles to support aerobic diving; therefore rapid development of body oxygen stores (TBO(2)) is crucial for pups to transition from nursing to independent foraging. Here, we investigate TBO(2) development in 45 harp (Pagophilus groenlandicus) and 46 hooded (Cystophora cristata) seals ranging in age from neonates to adult females. We found that hooded seal adults have the largest TBO(2) stores yet reported (89.5 ml kg(-1)), while harp seal adults have values more similar to other phocids (71.6 ml kg(-1)). In adults, large TBO(2) stores resulted from large blood volume (harp169, hood 194 ml kg(-1)) and high muscle Mb content (harp 86.0, hood 94.8 mg g(-1)). In contrast, pups of both species had significantly lower mass-specific TBO(2 )stores than adults, and stores declined rather than increased during the nursing period. This decline was due to a reduction in mass-specific blood volume and the absence of an increase in the low Mb levels (harp 21.0, hood 31.5 mg g(-1)). Comparisons with other phocid species suggests that the pattern of blood and muscle development in the pre- and post-natal periods varies with terrestrial period, and that muscle maturation rates may influence the length of the postweaning fast. However, final maturation of TBO(2) stores does not take place until after foraging begins.

Isolation and partial characterization of a parapoxvirus isolated from a skin lesion of a Weddell seal.

A solitary skin lesion was found on the neck of a Weddell seal (Leptonychotes weddellii), chemically immobilized in Queen Maud Land (70 degrees 09'S, 05 degrees 22'E) Antarctica 2001. The lesion was elevated and 3cm in diameter, consisting of partly fresh and partly necrotic tissue, and proliferative papilloma-like structures were seen. Electron microscopy on a biopsy from the lesion revealed typical parapoxvirus particles. Polymerase chain reaction (PCR; B2L gene) generated amplicons of approximately 594 base pairs, comparable to Orf-virus, the prototype parapoxvirus. A comparison of these B2L PCR amplicon DNA sequences with corresponding sequences from other parapoxviruses, showed that the Weddell seal virus resembled isolates from grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) more than parapoxvirus from red deer (Cervus elaphus), sheep, cattle and Japanese serows (Capricornis crispus). It is thus concluded that the Weddell seal parapoxvirus belong to the tentative seal parapoxvirus species. Since parapox and orthopoxviruses may cause similar clinical diseases, we suggest that the term sealpox should be restricted to the clinical disease, whereas seal parapoxvirus should be used when caused by a parapoxvirus, rather than the general term "sealpox virus". This is the first verified case of parapoxvirus infection in a Weddell seal, and also the first report of any such infections in the Antarctic.

Click sounds produced by cod (Gadus morhua).

Conspicuous sonic click sounds were recorded in the presence of cod (Gadus morhua), together with either harp seals (Pagophilus groenlandicus), hooded seals (Cystophora cristata) or a human diver in a pool. Similar sounds were never recorded in the presence of salmon (Salmo salar) together with either seal species, or from either seal or fish species when kept separately in the pool. It is concluded that cod was the source of these sounds and that the clicks were produced only when cod were approached by a swimming predatorlike body. The analyzed click sounds (n = 377) had the following characteristics (overall averages +/- S.D.): peak frequency = 5.95 +/- 2.22 kHz; peak-to-peak duration = 0.70 +/- 0.45 ms; sound pressure level (received level) = 153.2 +/- 7.0 dB re 1 microPa at 1 m. At present the mechanism and purpose of these clicks is not known. However, the circumstances under which they were recorded and some observations on the behavior of the seals both suggest that the clicks could have a predator startling function.

Seasonal changes in heart rate and food intake in reindeer (Rangifer tarandus tarandus).

This study tested the hypothesis that the annual cycle in heart rate (HR) in reindeer is, at least in part, a consequence of seasonal fluctuation in voluntary-food intake. Heart rate and daily dry matter voluntary-food intake (DDMVFI) were recorded in two captive female reindeer (Rangifer tarandus tarandus) from April 1995 to August 1996. Heart rate was measured continuously in each animal for 20-24 h for 7 days each month using Polar(R) Sport Testers (PST); DDMVFI was measured in each animal daily for 17 months. Modal daily heart rate (MDHR) and DDMVFI fluctuated seasonally in close synchrony, both reaching maxima in July and minima in January. The relationship between HR and DDMVFI was investigated experimentally by manipulating the level of feeding in a stepwise manner in May, when appetite was low and in August, when DDMVFI was close to maximum. Heart rate showed stepwise changes in close synchrony with the changes in levels of feeding. These results suggest that the seasonal increase in HR in summer is a consequence of increased food intake and, likewise, decreased HR in winter is a consequence of reduced food intake. The observed relationship between food intake and HR presumably reflects changes in cardiac output and/or the rate of flow of blood to the gastrointestinal tract which are influenced by meal size.

Panting in reindeer (Rangifer tarandus).

Two winter-insulated Norwegian reindeer (Rangifer tarandus tarandus) were exposed to air temperatures of 10, 20, 30, and 38 degrees C while standing at rest in a climatic chamber. The direction of airflow through nose and mouth, and the total and the nasal minute volumes, respectively, were determined during both closed- and open-mouth panting. The animals alternated between closed- and open-mouth panting, but the proportion of open-mouth panting increased with increasing heat load. The shifts from closed- to open-mouth panting were abrupt and always associated with a rise in respiratory frequency and respiratory minute volume. During open-mouth panting, the direction of airflow was bidirectional in both nose and mouth, but only 2.4 +/- (SD) 1.1% of the air was routed through the nose. Estimates suggest that the potential for selective brain cooling is markedly reduced during open-mouth panting in reindeer as a consequence of this airflow pattern.

Chitinolytic bacteria in the minke whale forestomach.

Minke whales consume large amounts of pelagic crustaceans. Digestion of the prey is initiated by indigenous bacteria in a rumen-like forestomach system. A major structural component of the crustacean exoskeleton is chitin, the beta-1,4-linked polymer of N-acetyl-D-glucosamine. The exoskeletons appear to dissolve completely in the non-glandular forestomach. Bacteria in the forestomach fluid of six krill-eating minke whales were enumerated and isolated using an anaerobic habitat-simulating culture medium. Median viable population densities ranged between 6.0 x 10(6) and 9.9 x 10(9) bacterial cells per mL forestomach fluid. Bacterial isolates (n = 44) cultured from the forestomach fluid of one minke whale mainly resembled strains of Eubacterium (25%), Streptococcus (18%), Clostridium (14%), and Bacteroides (11%). As much as 12% of the bacterial isolates were chitinolytic, while beta-N-acetylglucosaminidase activity was demonstrated in 54% of the isolates, and utilisation of N-acetyl-D-glucosamine was observed in 73%. The chitinolytic isolates resembled strains of Bacteroides, Bacteroidaceae, Clostridium, and Streptococcus. Scanning and transmission electron microscopy of partly digested krill from the minke whale forestomach revealed bacteria close to and inside the chitinous exoskeleton. The bacterial chitinase may act on the chitinous crustacean exoskeletons, thereby allowing other bacteria access to the nutritious soft inner tissues of the prey, and thus initiating its degradation and fermentation.

On the direction and velocity of blood flow in the extradural intravertebral vein of harp seals (Phoca groenlandica) during simulated diving.

Ronald et al. (1977) suggested that blood flow in the caudal/lumbar sections of the extradural intravertebral vein (EIV) of seals changes direction from running towards the head before diving, to the opposite during diving. The possible advantage would be that the oxygen-depleted venous effluent from the brain is routed via the EIV to the posterior parts of the hepatic sinuses and the inferior caval vein and, hence, is prevented from mixing with the more oxygen-rich venous blood in their anterior parts. We have re-examined this hypothesis by use of Doppler flowmetry. A catheter-tip flow probe was introduced into the EIV of two similar-sized juvenile harp seals, and flow direction and rate determined before, during and after simulated dives lasting for 5 min, at three positions (caudal, lumbar and thoracic) along the EIV. Regardless of probe position, blood was mainly flowing towards the head in 11 of 13 experiments prior to diving, in 8 of 13 experiments during diving and in 11 of 13 experiments during recovery after diving (and away from the head in the remaining experiments). Flow direction was most variable in the caudal position. Mean blood velocity in the EIV was substantially lower during diving (0.10 +/- 0.22 cm s-1 (n=5) in thoracic position) than in the pre-dive (3.98 +/- 3.32 cm s-1 [n=5]) and post-dive (5.75 +/- 4.07 cm s-1 [n=5]) situations. Thus, the direction and rate of flow in the EIV was variable, particularly during diving, as is to be expected in a system of anastomosing, valveless veins. We conclude that the hypothesis of Ronald et al. (1977) most likely is false.

Simulations of the effect of currently used grenade harpoons for the killing of whales using a pig-model.

Physical model experiments, as well as simulations of the effects of grenade harpooning on anaesthetized pigs fully immersed in water suggest that the shock effect of the blast from the currently used grenades is relatively minor. Also the animals are not stunned to death, but loose consciousness and subsequently die from hemorrhage. Survival time is therefore very short if the animals are hit in the thorax, and is likely to be further reduced if the charge which is currently used is increased, or, even better, if shrapnel (fragment scattering) grenades are used instead of blast grenades.

The effect of body fat on basal metabolic rate in adult harp seals (Phoca groenlandica).

Three adult harp seals (Phoca groenlandica) were fed different daily amounts of capelin (Mallotus villosus), and their body composition determined by use of the tritiated water method at different levels of fattening. Basal metabolic rate (BMR) was measured after 5 days of fasting by indirect calorimetry, and was on average 1.1 W.kg-1 when 45% of body mass (BM) was fat and 2.3 W.kg-1 when body fat was reduced to 13% of BM. This suggests that body fat contributes little to BMR in these animals. It follows, that predictions of BMR on the basis of BM is questionable in seals, in which body fat may change seasonally between 20 and 60% of BM.

Effects of adrenergic and cholinergic drugs on splenic arteries and veins from hooded seals (Cystophora cristata).

Isolated ring preparations of arteries and veins from hooded seal spleens were subjected in vitro to adrenaline (A), noradrenaline (NA), isoprenaline (Iso), and acetylcholine (ACh), alone or in combination with the blockers phentolamine (Phe), propranolol (Pro), and atropine (Atr). Both arteries and veins constricted in response to A (the estimated effective dose required for half-maximal response (ED50) was 3.3 and 0.2 microM, for arteries and veins, respectively) and NA (estimated ED50 was 1.5 and 0.6 microM, for arteries and veins, respectively), but these effects were abolished when the drugs were given in combination with the alpha-adrenoceptor blocker Phe. The responses of arteries and veins to ACh and the beta-adrenoceptor agonist Iso were minor and inconsistent, and were completely abolished when combined with their respective blockers (Atr and Pro, respectively). The ED50 for both A and NA are quite high in relation to normal plasma levels of A and NA in seals. This implies that these vessels (and, hence, the supply of blood to the spleen) primarily are subjected to neurogenic, rather than humoral physiological control.

Rapid brain cooling in diving ducks.

Hypothermia may limit asphyxic damages to the brain, and many small homeotherms have been shown to use anapyrexic strategies when exposed to asphyxic conditions. Larger homeotherms do not seem to use the same strategy, but could save oxygen and prevent hypoxic brain damage by employing selective brain cooling (SBC) in connection with asphyxia. To test the hypothesis that selective brain cooling may take place in connection with asphyxia, we have recorded brain [hypothalamic (THyp)] and body [colonic (TC)] temperatures and heart rates in four Pekin ducks during 5-min simulated (head submersion) diving in cold water (10 degrees C). Diving resulted in a drop in THyp (3.1 +/- 1.4 degrees C) that continued into the recovery period (P < 0.001). Restricting heat loss from the buccal cavity and eyes during diving compromised brain cooling in an additive manner. TC was not influenced by diving. Control cooling of the head with crushed ice during a 5-min period of undisturbed breathing had no effect on THyp. Warm water (35 degrees C) markedly reduced brain cooling, and dive capacity was reduced by approximately 14% (P < 0.05) compared with diving in water at 10 degrees C. The data suggest that SBC is used in ducks during diving, and we propose that this mechanism may enable the bird to save oxygen for prolonged aerobic submergence and to protect the brain from asphyxic damages.

Volume capacity and contraction control of the seal spleen.

Volume changes in the spleens of hooded seals (Cystophora cristata) and harp seals (Phoca groenlandica) were measured plethysmographically in vitro in response to epinephrine, norepinephrine, isoprenaline, phentolamine, and acetylcholine. Dilated spleens contracted forcefully within 1-3 min of alpha-adrenoceptor activation with 1.0-5.0 micrograms epinephrine/kg body mass, whereas stimulation of beta-adrenoceptors and cholinergic receptors had little effect. The mass of dilated hooded seal spleens corresponded to 2-4% (n = 7) of body mass, with volume (V; ml) relating to body mass (M; kg) as follows: V = 12.0M + 910 (r2 = 0.96, n = 4). Thus the spleen of a 250-kg hooded seal maximally expels 3.9 liters, or 13%, of its estimated total blood volume. Average hematocrit in splenic venous outflow from dilated spleens was 90 +/- 3% (n = 3) in hooded seals and 85% (n = 2) in harp seals. From these data we have estimated that the aerobic diving limit of a 250-kg hooded seal increases only 105 s, at the most, if complete emptying of the spleen occurs during diving, while the corresponding estimate for a 112-kg harp seal is 80 s.

Pineal and thyroid functions in newborn seals.

Daily variations of pineal and plasma melatonin and plasma thyroid hormones were measured in harp seals (Phoca groenlandica), grey seals (Halichoerus grypus), and hooded seals (Cystophora cristata), ranging in age from newborn to 14 days. In newborn harp seals the mean mass of the pineal gland was 273 mg (+/- 45 SEM, n = 11), containing 49 ng (median) melatonin. In newborn, 4- and 10-day-old grey seals, the pineal mass was similar, weighing on average 337 mg (+/- 74, n = 6) and containing 90 ng melatonin. Two newborn hooded seal pups had pineals weighing 520 and 1289 mg, with 254 and 7600 ng melatonin, respectively. There were no day-night differences in the pineal contents of melatonin or in the number of pineal beta-adrenergic receptors measured in newborn harp seals, and, in newborn, 4- and 10-day-old grey seals, there were no day-night or age differences in pineal melatonin content. Plasma melatonin levels were 10 times higher in newborn seals than in two 10-day-old grey seals and one 14-day-old harp seal pup. In all seal pups, the levels exhibited a 24-hr rhythmicity, with increasing night- and decreasing daytime concentrations. Plasma levels of thyroxine (T4) and triiodothyronine (T3) were generally higher in newborn seals than in 10- and 14-day-old seals or in adult females. There was no apparent 24-hr rhythmicity, but the thyroid hormone levels generally declined throughout each sampling sequence. High pineal and thyroid activities may play a thermoregulatory role in newborn seals, but the results do not indicate a stimulatory action of melatonin in the peripheral conversion of T4 to T3. It is speculated that the large and active pineal gland, particularly in newborn seals, may be related to aspects of their diving habit.

Daily energy expenditure in free living minke whales.

Six minke whales (Balaenoptera acutorostrata) were instrumented with VHF-radio transmitters and four with sonic speed-depth transmitters off the west coast of northern Norway and Svalbard and followed within view for up to 24 h. During such periods their respiratory rate was continuously recorded and their energy expenditure estimated according to Folkow & Blix (1992) at different swimming speeds and types of activity. We found that cost of swimming is remarkably low in these large animals and that their estimated daily energy expenditure on average only amounts to 80 kJ kg-1 day-1.

Digestibility of krill (Euphausia superba and Thysanoessa sp.) in minke whales (Balaenoptera acutorostrata) and crabeater seals (Lobodon carcinophagus).

Apparent digestible efficiency (% DE) was studied by use of dietary Mn as an inert marker, in minke whales (Balaenoptera acutorostrata) and crabeater seals (Lobodon carcinophagus) which had been eating krill. Median % DE in minke whales (n5) eating krill of the genus Thysanoessa sp. (energy density (ED) 23.8 kJ/g) was 93 (range 87-93). Median % DE in crabeater seals (n6) eating krill of the species Euphausia superba (ED 20.8 kJ/g) was 84 (range 79-85), which is significantly lower than the % DE of krill in minke whales (P = 0.008). Since the chemical composition in E. superba and in Thysanoessa sp. is similar, it is suggested that the complex multi-stomached system of minke whales, which contains both chitinase (EC 3.2.1.14)-producing as well as several other types of bacteria, is superior to the single-stomached system of crabeater seals with regard to krill digestion. It is worth noting, however, that the % DE of krill in the crabeater seal is still very high.

In vitro digestibility of different prey species of minke whales (Balaenoptera acutorostrata).

Information on diet composition, daily energy expenditure, energy storage and the utilization of energy in the prey are important factors when evaluating the food consumption of minke whales (Balaenoptera acutorostrata) during their summer stay in northern waters. The purpose of the present study was in this context to obtain information on the digestible energy (DE) of different prey selected by minke whales. An in vitro three-stage digestion technique, simulating the different compartments of the digestive system, has been developed. The initial step simulated the anaerobic microbial fermentation of substrate in the forestomach. The next stage included the addition of pepsin (EC 3.4.23.1)-HCl, simulating ventricle enzymic decomposition, and finally, in the third step, fresh extract from duodenal contents was used to simulate enzymic intestinal degradation of the remaining components of the food. The inoculum was normally obtained from animals which had recently eaten the prey to be tested. In such tests we obtained a dry matter disappearance (DMD) and a DE for herring (Clupea harengus) of 80.4 (SD 5.0)% (n 18) and 92.1 (SD 3.7)% (n 16) respectively, and a DMD of krill (Thysanoessa sp.) of 83.4 (SD 4.9)% (n 6). The DMD of krill was reduced to 73.8 (SD 7.3)% (n 8) while the DE was 70.6 (SD 10.4)% (n 7) when inoculum from whales which had recently eaten cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) was used. These results indicate a high digestibility of the most common species of prey in these animals, and also that the whales have little difficulty in changing from one prey species to another.