Evaluating suitability of saliva to measure steroid concentrations in grey seal pups.

Measurement of steroids in wild pinnipeds can facilitate assessment of breeding, nutritional and stress status, and is useful in understanding behavioral responses. Even in young animals, sex steroids may be important in behavioral interactions and immune modulation. Use of saliva can avoid the large fluctuations seen in some steroids in plasma, and can negate the need for venipuncture, making it a potentially useful matrix in the wildlife. However, its utility in estimating steroid levels in wild young pinnipeds has not been evaluated. Here, we investigated the suitability of saliva for steroid hormone analysis in wild grey seal pups during their suckling and post-weaning fast periods. We collected saliva (n = 38) and plasma (n = 71) samples during the breeding season on the Isle of May, Scotland, 2012. We investigated success of sample collection, ease of preparation, accuracy and precision of analysis, and, where possible, comparability of measurements (n = 27) from saliva and plasma. Plasma sampling was rapid, whereas sampling saliva took up to five times longer. Analytical performance criteria (parallelism, accuracy, and precision (intra and inter assay co-efficient of variation (% CV)) of commercial ELISA kits to measure estradiol, testosterone and cortisol in both matrices were assessed. Estradiol and cortisol assays performed well and can be used in plasma and saliva. However, we could not confidently validate testosterone for either matrix. Saliva estradiol correlated with levels in plasma. Saliva sample preparation was faster and simpler than plasma preparation because it did not require extraction. However, given the additional time taken to obtain saliva in the wild, the possibility of blood contamination from oral damage and the lower success rate in obtaining sufficient sample for analysis, we recommend that this matrix only be used as an alternative to plasma sampling measurement in pinnipeds when animals are anaesthetized, tolerate mouth swabbing, or have been trained to accept saliva sampling in captivity.

Rapid changes in Atlantic grey seal milk from birth to weaning - immune factors and indicators of metabolic strain.

True seals have the shortest lactation periods of any group of placental mammal. Most are capital breeders that undergo short, intense lactations, during which they fast while transferring substantial proportions of their body reserves to their pups, which they then abruptly wean. Milk was collected from Atlantic grey seals (Halichoerus grypus) periodically from birth until near weaning. Milk protein profiles matured within 24 hours or less, indicating the most rapid transition from colostrum to mature phase lactation yet observed. There was an unexpected persistence of immunoglobulin G almost until weaning, potentially indicating prolonged trans-intestinal transfer of IgG. Among components of innate immune protection were found fucosyllactose and siallylactose that are thought to impede colonisation by pathogens and encourage an appropriate milk-digestive and protective gut microbiome. These oligosaccharides decreased from early lactation to almost undetectable levels by weaning. Taurine levels were initially high, then fell, possibly indicative of taurine dependency in seals, and progressive depletion of maternal reserves. Metabolites that signal changes in the mother's metabolism of fats, such as nicotinamide and derivatives, rose from virtual absence, and acetylcarnitines fell. It is therefore possible that indicators of maternal metabolic strain exist that signal the imminence of weaning.

Adiponectin and Insulin in Gray Seals during Suckling and Fasting: Relationship with Nutritional State and Body Mass during Nursing in Mothers and Pups.

Animals that fast during breeding and/or development, such as phocids, must regulate energy balance carefully to maximize reproductive fitness and survival probability. Adiponectin, produced by adipose tissue, contributes to metabolic regulation by modulating sensitivity to insulin, increasing fatty acid oxidation by liver and muscle, and promoting adipogenesis and lipid storage in fat tissue. We tested the hypotheses that (1) circulating adiponectin, insulin, or relative adiponectin gene expression is related to nutritional state, body mass, and mass gain in wild gray seal pups; (2) plasma adiponectin or insulin is related to maternal lactation duration, body mass, percentage milk fat, or free fatty acid (FFA) concentration; and (3) plasma adiponectin and insulin are correlated with circulating FFA in females and pups. In pups, plasma adiponectin decreased during suckling (linear mixed-effects model [LME]: T = 4.49; P < 0.001) and the early postweaning fast (LME: T = 3.39; P = 0.004). In contrast, their blubber adiponectin gene expression was higher during the early postweaning fast than early in suckling (LME: T = 2.11; P = 0.046). Insulin levels were significantly higher in early (LME: T = 3.52; P = 0.004) and late (LME: T = 6.99; P < 0.001) suckling than in fasting and, given the effect of nutritional state, were also positively related to body mass (LME: T = 3.58; P = 0.004). Adiponectin and insulin levels did not change during lactation and were unrelated to milk FFA or percentage milk fat in adult females. Our data suggest that adiponectin, in conjunction with insulin, may facilitate fat storage in seals and is likely to be particularly important in the development of blubber reserves in pups.

The role of glucocorticoids in naturally fasting grey seal (Halichoerus grypus) pups: dexamethasone stimulates mass loss and protein utilisation, but not departure from the colony.

Seals must manage their energy reserves carefully while they fast on land to ensure that they go to sea with sufficient fuel to sustain them until they find food. Glucocorticoids (GCs) have been implicated in the control of fuel metabolism and termination of fasting in pinnipeds. Here we tested the hypothesis that dexamethasone, an artificial GC, increases fat and protein catabolism, and induces departure from the breeding colony in wild, fasting grey seal pups. A single intramuscular dose of dexamethasone completely suppressed cortisol production for 24-72 h, demonstrating activation of GC receptors. In experiment 1, we compared the effects of a single dose of dexamethasone or saline administered 10 days after weaning on fasting mass and body composition changes, cortisol, blood urea nitrogen (BUN) and glucose levels, and timing of departure from the colony. In experiment 2, we investigated the effects of dexamethasone on short-term (5 days) changes in mass loss, body composition and BUN levels. In experiment 1, dexamethasone induced a short-lived increase in mass loss, but there was no difference in timing of departure between dexamethasone- and saline-treated pups (N=10). In experiment 2, dexamethasone increased protein and water loss and prevented a decrease in BUN levels (N=11). Our data suggest changes in cortisol contribute to regulation of protein catabolism in fasting seal pups, irrespective of the sex of the animal, but do not terminate fasting. By affecting the rate of protein depletion, lasting changes in cortisol levels could influence the amount of time seal pups have to find food, and thus may have important consequences for their survival.

Effect of induction dose, lactation stage and body condition on tiletamine-zolazepam anaesthesia in adult female grey seals (Halichoerus grypus) under field conditions.

Tiletamine-zolazepam (TZ) was used at a mean (sd) dose of 1.18 (0.15) mg/kg administered intramuscularly to anaesthetise adult female grey seals (Halichoerus grypus) under field conditions at three different stages during their lactation period. A significant correlation was observed between the induction dose and time to induction (r=-0.582, P=0.011). Stage of lactation had a significant effect on condition index (CI), calculated as axial girth divided by length (P<0.001), and time to induction (P=0.009). No effect of CI on induction or recovery time was demonstrated. Respiratory rate decreased during induction and increased significantly (P<0.001) during surgical biopsy of blubber. Recovery occurred after 32.5 (11.9) minutes. Minor complications (tremor, vocalisation and mild dyspnoea) were observed in a small number of cases, none of which required treatment.

Estimating metabolic heat loss in birds and mammals by combining infrared thermography with biophysical modelling.

Infrared thermography (IRT) is a technique that determines surface temperature based on physical laws of radiative transfer. Thermal imaging cameras have been used since the 1960s to determine the surface temperature patterns of a wide range of birds and mammals and how species regulate their surface temperature in response to different environmental conditions. As a large proportion of metabolic energy is transferred from the body to the environment as heat, biophysical models have been formulated to determine metabolic heat loss. These models are based on heat transfer equations for radiation, convection, conduction and evaporation and therefore surface temperature recorded by IRT can be used to calculate heat loss from different body regions. This approach has successfully demonstrated that in birds and mammals heat loss is regulated from poorly insulated regions of the body which are seen to be thermal windows for the dissipation of body heat. Rather than absolute measurement of metabolic heat loss, IRT and biophysical models have been most useful in estimating the relative heat loss from different body regions. Further calibration studies will improve the accuracy of models but the strength of this approach is that it is a non-invasive method of measuring the relative energy cost of an animal in response to different environments, behaviours and physiological states. It is likely that the increasing availability and portability of thermal imaging systems will lead to many new insights into the thermal physiology of endotherms.

Effects of age and body mass on development of diving capabilities of gray seal pups: costs and benefits of the postweaning fast.

Development of adequate diving capabilities is crucial for survival of seal pups and may depend on age and body size. We tracked the diving behavior of 20 gray seal pups during their first 3 mo at sea using satellite relay data loggers. We employed quantile analysis to track upper limits of dive duration and percentage time spent diving, and lower limits of surface intervals. When pups first left the breeding colony, extreme (ninety-fifth percentile) dive duration and percentage time spent diving were positively correlated with age, but not mass, at departure. Extreme dive durations and percentage time spent diving peaked at [Formula: see text] d of age at values comparable with those of adults, but were not sustained. Greater peaks in extreme percentage time spent diving occurred in pups that had higher initial values, were older at their peak, and were heavier at departure. Pups that were smaller and less capable divers when they left the colony improved extreme dive durations and percentage time spent diving more rapidly, once they were at sea. Minimum survival time correlated positively with departure mass. Pups that were heavier at weaning thus benefitted from being both larger and older at departure, but smaller pups faced a trade-off. While age at departure had a positive effect on early dive performance, departure mass impacted on peak percentage time spent diving and longer-term survival. We speculate that once small pups have attained a minimum degree of physiological development to support diving, they would benefit by leaving the colony when younger but larger to maximize limited fuel reserves, rather than undergoing further maturation on land away from potential food resources, because poor divers may be able to "catch up" once at sea.

Factors influencing the radiative surface temperature of grey seal (Halichoerus grypus) pups during early and late lactation.

The aim of this study was to examine the variation in body surface temperature of grey seal (Halichoerus grypus) pups throughout lactation in response to different environmental conditions. Radiative surface temperatures (T (r), degrees C) of pups were measured on the Isle of May (56 degrees 11'N, 02 degrees 33'W), southeast Scotland from 29 October to 25 November 2003. Records were obtained from a total of 60 pups (32 female and 28 male) from three different pupping sites during early and late lactation. Pups were sheltered from high wind speeds but air temperature, humidity and solar radiation at pupping sites were similar to general meteorological conditions. The mean T (r) of all pups was 15.8 degrees C (range 7.7-29.7 degrees C) at an average air temperature of 10.2 degrees C (range 6.5-13.8 degrees C). There was no difference in the mean T (r) of pups between early and late lactation. However, the T (r) varied between different regions of the body with hind flippers on average 2-6 degrees C warmer than all other areas measured. There was no difference in mean T (r) of male and female pups and pup body mass did not account for the variation in T (r) during early or late lactation. Throughout the day there was an increase in the T (r) of pups and this explained 20-28% of the variation in T (r) depending on stage of lactation. There was no difference in the mean T (r) of pups between pupping sites or associated with different substrate types. Wind speed and substrate temperature had no effect on the T (r) of pups. However, solar radiation, air temperature and relative humidity accounted for 48% of the variation in mean T (r) of pups during early lactation. During late lactation air temperature and solar radiation alone accounted for 43% of the variation in T (r). These results indicate that environmental conditions explain only some of the variation in T (r) of grey seal pups in natural conditions. Differences in T (r) however indicate that the cost of thermoregulation for pups will vary throughout lactation. Further studies examining intrinsic factors such as blubber thickness and activity levels are necessary before developing reliable biophysical models for grey seals.

Patterns of parental relatedness and pup survival in the grey seal (Halichoerus grypus).

Levels of parental relatedness can affect offspring survival and susceptibility to disease. We investigated parental relatedness of live and dead Halichoerus grypus pups between and within island populations and between possible causes of mortality. Nine microsatellites were used to calculate internal relatedness (IR) and standardized mean d2. We find that pups with higher than average levels of IR have significantly lower survival and that this varied between island populations and that certain loci contributed to the effect more than others. Although, there were no significant differences between causes of mortality, peritonitis, infection and stillborn had the highest levels of IR. These results provide evidence that parental relatedness is an important determinant of pre-weaning pup survival in the grey seal and that this may vary with cause of mortality given a larger sample size.

An unexpected parallelism between Vitamin A and PCBs in seal milk.

Bioaccumulating pollutants such as polychlorinated biphenyls (PCBs) induce a range of adverse effects in mammals. Vitamin A metabolism is prone to such pollutant disruption which may be particularly harmful for young animals. During lactation, maternal PCBs are transferred to the offspring through the milk. Seal milk is very fatty and consequently contains large amounts of these persistent lipophilic contaminants. In the present study, we investigated the relationships between PCBs and Vitamins A and E during lactation, in free-ranging grey seals, using longitudinal samples of milk and blubber. We discovered that, unlike Vitamin E or triglycerides, the dynamics of Vitamin A matches closely those of PCBs in milk throughout lactation. Levels of Vitamin A and PCBs remain constant during the first half of lactation and then increase at late lactation, indicating that pups ingesting higher levels of PCBs also ingest higher amounts of Vitamin A.

Reproductive performance links to fine-scale spatial patterns of female grey seal relatedness.

Fine-scale spatial patterns of female relatedness throughout the established grey seal breeding colony of North Rona, Scotland, were investigated by accurate mapping and spatially explicit analyses of a large sample (n = 262) of mothers using variation at nine microsatellite DNA loci. Local spatial autocorrelation analyses identified locations where seals were more highly related to the colony than average. These locations were also areas where the more successful females bred, were occupied first during each breeding season, were centrally placed locations of preferred habitat types and were likely to be the locations which were the first to be colonized historically. Mothers occupying such sites achieved higher than average pup growth rates, suggesting a founder fitness benefit.

Patterns of paternal relatedness in British grey seal colonies.

Previous studies of breeding behaviour in the grey seal, Halichoerus grypus, have painted conflicting pictures. Behavioural observations suggest a classical polygynous system with a small number of dominant males fathering most of the offspring. However, genetic analysis suggests that many potential fathers spend little time ashore, that some pairs of seals show partner fidelity and that the dominant males are not as successful as their behaviour would suggest. Here we used paternal relatedness between pups with known mothers, sampled over an 11-year period, to show that behavioural dominance leading to enhanced fitness is a feature of only a handful of males located near the centre of the breeding colony. The vast majority of pups are fathered by any of a large number of males who all share approximately equal success, including virtually all those males who have previously escaped our best sampling efforts. As expected, the frequency of full-sibs is reduced in this longer time series relative to the original study. However, absolute estimates of the frequency of full-sibs seem to be confounded by a tendency for females who produce paternally unrelated pups to have conceived to males who are more genetically dissimilar from each other than expected by chance alone. Together, these elements of breeding behaviour would help to maintain maximum genetic diversity and to minimize the effects of inbreeding.

Where have all the fathers gone? An extensive microsatellite analysis of paternity in the grey seal (Halichoerus grypus).

Microsatellites were used to conduct an extensive analysis of paternity of grey seals from two Scottish breeding colonies at North Rona (n = 1189) and the Isle of May (n = 694), spanning more than a decade. A maximum of 46% of pups at North Rona and 29% of pups at the Isle of May could be allocated a father, even though the majority of candidate males for specific study sites within each colony were believed to have been sampled. Based on the paternities which could be assigned, both colonies showed evidence of reproductive skew, apparently due to the presence of approximately five males who were exceptionally successful. Some males were assigned paternities at least 10 years before, and colleagues 10 years after, being sampled, implying a reproductive lifespan of at least 10 years, and there are indications that the real maximum lies in the range 15-20 years. Male grey seals appear to have at least two breeding strategies they can adopt. On land, some males benefit from a traditionally polygynous system. However, between 50 and 70% of grey seal pups born at a particular colony are not fathered by males who are likely to be sampled by us, implying that these males seldom venture ashore here. We conclude that aquatic mating may play a much larger role in the grey seal than has previously been thought.

Microsatellite variation in grey seals (Halichoerus grypus) shows evidence of genetic differentiation between two British breeding colonies.

Eight highly variable microsatellite loci were used to examine the genetic variability and differentiation of grey seals (Halichoerus grypus) at two widely spaced British breeding colonies. Samples were collected from adults and pups on the island of North Rona, off the north-west coast of Scotland, and on the Isle of May, situated at the mouth of the Firth of Forth on the east coast. Highly significant differences in allele frequencies between these two sites were found for all eight loci, indicating considerable genetic differentiation. Thus, although grey seals are known to range over very large areas outside the breeding season, site fidelity of adults and philopatry of pups for these breeding colonies must be sufficiently common to have effects, through genetic drift, at the sub-population level. Migration rate was estimated using Wright's fixation index (FST), Slatkin's private alleles model and the new statistic, RST, which is analogous to FST but which takes into account the process of microsatellite mutation. An almost 8-fold discrepancy between the values we obtained provides cautionary evidence that microsatellite loci may contravene one or more of the assumptions on which these methods are based.

Male mating success and paternity in the grey seal, Halichoerus grypus: a study using DNA fingerprinting.

Grey seals breed colonially on remote coastal sites. Within the colony, males compete aggressively for access to the females. We compare field observations of breeding behaviour with paternity, as determined by DNA fingerprinting, in the breeding colony on the island of North Rona. In 89% of cases where paternity was assigned, the father was observed near the mother during her perioestrous period, although in some cases this was discovered retrospectively. However, the most likely candidate male, judged on the basis of behavioural criteria, was shown not to be the father in 36% of cases. Overall, DNA typed males were more dominant, maintained positions amongst the females for longer, and accounted for disproportionately more paternities than untyped males. However, the reproductive success of the typed males is not as great as their behavioural domination of copulatory opportunities would suggest. Possible contributory factors which could explain this include: (i) imprecision in the estimates of copulatory opportunity due to mobility of males or topographical influences on individual behaviour; and (ii) opportunities for subordinate males to copulate with receptive females, either sneakily within the colony or in the water.

Plasma thymulin concentrations, the thymus and organochlorine contaminant levels in seals infected with phocine distemper virus.

Blood samples collected from live common seals (Phoca vitulina) and grey seals (Halichoerus grypus) around the coast of Scotland and Northern Ireland during, and immediately after, an epizootic caused by phocid distemper virus (PDV) were analysed for thymulin content. Thymulin levels were compared with neutralization titres and concentrations of organochlorine contaminants (DDT and its metabolites, seven chlorinated biphenyl congeners) derived from blood and blubber samples collected from the same animals and analysed independently. Thymulin levels in grey seals (mean 2827 +/- 1355 fg/ml) were negatively correlated with the logarithm of virus neutralization titre. In common seals they varied significantly between age classes. There was no direct relationship between thymulin levels and contaminant levels in either species. However, when an estimate of time since exposure was included in the regression analysis for common seals, there was a highly significant relationship between thymulin and the two chlorinated biphenyl congeners with the highest concentrations in blubber.

Organochlorine levels in common seals (Phoca vitulina) which were victims and survivors of the 1988 phocine distemper epizootic.

We compared concentrations of organochlorines in the blubber of common seals (Phoca vitulina) found dead during the 1988 phocine distemper epizootic with levels in animals which survived it. There were highly significant differences between the live and dead animals, and between sample sites. These were not fully accounted for by seasonal and condition-related changes in blubber thickness.

The descriptive epizootiology of phocine distemper in the UK during 1988/89.

The 'time, place, individual' approach, widely used in characterising human epidemics, was applied to the 1988 phocine distemper virus (PDV) epizootic affecting North Sea seals. Estimates of time of death from 157 (69%) of the 228 dead seals necropsied in 1988 indicated that the number of carcasses which were found more than 14 days post mortem increased as the epizootic progressed. Although information provided by epizootic curves based on when and where carcasses were reported are affected by the accuracy of such data, the PDV epizootic curves were characteristic of a propagative epidemic. The individual characteristics of the carcasses provided more useful information. 1. Common seals were more susceptible than grey seals. 2. Males and older seals had a greater exposure to the virus than females and younger animals. 3. The likelihood of viral transmission was greater on land. Seasonal, sex and age related variation in haul-out behaviour affected transmission probabilities and rates. 4. The risk of infection for a susceptible individual during August was higher for seals in England and Northern Ireland than for those in Scotland. These findings illustrate the importance of population characteristics including behaviour and social organisation in determining the pattern and spread of wildlife epizootics.

The serological response of the common seal (Phoca vitulina) and the grey seal (Halichoerus grypus) to phocine distemper virus as measured by a canine distemper virus neutralisation test.

The application of a canine distemper virus (CDV) neutralisation test revealed that antibody titres of up to 11,620 were present in the sera of common seals during the epizootic of phocine distemper in British waters. However, during the later stages of the epizootic and afterwards, a surprisingly high proportion of animals were still seronegative. Titres of from 90 to 183 were common in diseased animals and were probably indicative of partial immunosuppression. A few animals with proven phocine distemper had titres of less than 64, this being evidence of a more profound immunosuppression. Recovered animals tended to have moderate to high titres and in some cases these persisted for at least 6.5 months. By contrast, the administration of an inactivated CDV vaccine produced antibody which declined rapidly to very low levels within the same period. Infection also occurred in grey seals in 1988 but without severe mortality. Antibody titres in this species nearly always lay in the range 128-734 and persisted at or around this level for at least 12 months. In both common and grey seals, antibody was transferred by suckling from mother to pup but absorption by the pup seemed to take place more slowly than in domestic mammals.