An external telemetry system for recording resting heart rate variability and heart rate in free-ranging large wild mammals.

Measures of heart rate variability (and heart rate more generally) are providing powerful insights into the physiological drivers of behaviour. Resting heart rate variability (HRV) can be used as an indicator of individual differences in temperament and reactivity to physical and psychological stress. There is increasing interest in deriving such measures from free ranging wild animals, where individuals are exposed to the natural and anthropogenic stressors of life. We describe a robust, externally mounted heart rate monitor for use in wild mammals, deployed here on wild breeding adult female grey seals (Halichoerus grypus), that delivers millisecond precise measures of inter beat intervals (IBIs), allowing computation of resting HRV parameters. Based on Firstbeat™ heart rate belts, our system allows for remote, continuous recording of IBI data from over 30 individuals simultaneously at ranges of up to 200m. We assessed the accuracy of the IBI data provided by the Firstbeat™ system using concurrent IBI data derived from in-field electrocardiogram (ECG) recordings. Bland-Altmann analyses demonstrated high correspondence between the two sets of IBI data, with a mean difference of 0.87±0.16ms. We used generalized additive mixed-effects models to examine the impact of the default Firstbeat™ software artefact correction procedure upon the generation of anomalous data (flats and stairs). Artefact correction and individual activity were major causes of flats and stairs. We used simulations and models to assess the impact of these errors on estimates of resting HRV and to inform criteria for subsampling relatively error free IBI traces. These analyses allowed us to establish stringent filtering procedures to remove traces with excessive numbers of artefacts, including flats and stairs. Even with strict criteria for removing potentially erroneous data, the abundance of data yielded by the Firstbeat™ system provides the potential to extract robust estimates of resting HRV. We discuss the advantages and limitations of our system for applications beyond the study system described here.

Energetic limits: Defining the bounds and trade-offs of successful energy management in a capital breeder.

Judicious management of energy can be invaluable for animal survival and reproductive success. Capital breeding mammals typically transfer energy to their young at extremely high rates while undergoing prolonged fasting, making lactation a tremendously energy demanding period. Effective management of the competing demands of the mother's energy needs and those of her offspring is presumably fundamental to maximizing lifetime reproductive success. How does the mother maximize her chances of successfully rearing her pup, by ensuring that both her pup and herself have sufficient energy during this 'energetic fast'? While energy management models were first discussed in the 1990s, application of this analytical technique is still very much in its infancy. Recent work suggests that a broad range of species exhibits 'energy compensation'; during periods when they expend more energy on activity, their bodies partially compensate by reducing background (basal) metabolic rate as an adaptation to limit overall energy expenditure. However, the value of energy management models in understanding animal ecology is presently unclear. We investigate whether energy management models provide insights into the breeding strategy of phocid seals. Not only do we expect lactating seals to display energy compensation because of their breeding strategy of high energy transfer while fasting, but we anticipate that mothers exhibiting a lack of energy compensation are less likely to rear offspring successfully. On the Isle of May in Scotland, we collected heart rate data as a proxy for energy expenditure in 52 known individual grey seal (Halichoerus grypus) mothers, repeatedly across 3 years of breeding. We provide evidence that grey seal mothers typically exhibit energy compensation during lactation by downregulating their background metabolic rate to limit daily energy expenditure during periods when other energy costs are relatively high. However, individuals that fail to energy compensate during the lactation period are more likely to end lactation earlier than expected. Our study is the first to demonstrate the importance of energy compensation to an animal's reproductive expenditure. Moreover, our multi-seasonal data indicate that environmental stressors may reduce the capacity of some individuals to follow the energy compensation strategy.

Reactive stress-coping styles show more variable reproductive expenditure and fitness outcomes.

Stress-coping styles dictate how individuals react to stimuli and can be measured by the integrative physiological parameter of resting heart-rate variability (HRV); low resting HRV indicating proactive coping styles, while high resting HRV typifies reactive individuals. Over 5 successive breeding seasons we measured resting HRV of 57 lactating grey seals. Mothers showed consistent individual differences in resting HRV across years. We asked whether proactive and reactive mothers differed in their patterns of maternal expenditure and short-term fitness outcomes within seasons, using maternal daily mass loss rate to indicate expenditure, and pup daily mass gain to indicate within season fitness outcomes. We found no difference in average rates of maternal daily mass loss or pup daily mass gain between proactive and reactive mothers. However, reactive mothers deviated more from the sample mean for maternal daily mass and pup daily mass gain than proactive mothers. Thus, while proactive mothers exhibit average expenditure strategies with average outcomes, expenditure varies much more among reactive mothers with more variable outcomes. Overall, however, mean fitness was equal across coping styles, providing a mechanism for maintaining coping style diversity within populations. Variability in reactive mothers' expenditures and success is likely a product of their attempts to match phenotype to prevailing environmental conditions, achieved with varying degrees of success.

Stockpiling by pups and self-sacrifice by their fasting mothers observed in birth to weaning serum metabolomes of Atlantic grey seals.

During the uniquely short lactations of true seals, pups acquire a greater proportion of maternal body resources, at a greater rate, than in any other group of mammals. Mothers in many species enter a period of anorexia but must preserve sufficient reserves to fuel hunting and thermoregulation for return to cold seas. Moreover, pups may undergo a period of development after weaning during which they have no maternal care or nutrition. This nutritionally closed system presents a potentially extreme case of conflict between maternal survival and adequate provisioning of offspring, likely presenting strains on their metabolisms. We examined the serum metabolomes of five mother and pup pairs of Atlantic grey seals, Halichoerus grypus, from birth to weaning. Changes with time were particularly evident in pups, with indications of strain in the fat and energy metabolisms of both. Crucially, pups accumulate certain compounds to levels that are dramatically greater than in mothers. These include compounds that pups cannot synthesise themselves, such as pyridoxine/vitamin B6, taurine, some essential amino acids, and a conditionally essential amino acid and its precursor. Fasting mothers therefore appear to mediate stockpiling of critical metabolites in their pups, potentially depleting their own reserves and prompting cessation of lactation.

High oxytocin infants gain more mass with no additional maternal energetic costs in wild grey seals (Halichoerus grypus).

Maximising infant survival requires secure attachments and appropriate behaviours between parents and offspring. Oxytocin is vital for parent-offspring bonding and behaviour. It also modulates energetic balance and neural pathways regulating feeding. However, to date the connections between these two areas of the hormone's functionality are poorly defined. We demonstrate that grey seal (Halichoerus grypus) mothers with high oxytocin levels produce pups with high oxytocin levels throughout lactation, and show for the first time a link between endogenous infant oxytocin levels and rates of mass gain prior to weaning. High oxytocin infants gained mass at a greater rate without additional energetic cost to their mothers. Increased mass gain in infants was not due to increased nursing, and there was no link between maternal mass loss rates and plasma oxytocin concentrations. Increased mass gain rates within high oxytocin infants may be due to changes in individual behaviour and energy expenditure or oxytocin impacting on tissue formation. Infancy is a crucial time for growth and development, and our findings connect the oxytocin driven mechanisms for parent-infant bonding with the energetics underlying parental care. Our study demonstrates that oxytocin release may connect optimal parental or social environments with direct physiological advantages for individual development.

Positive social behaviours are induced and retained after oxytocin manipulations mimicking endogenous concentrations in a wild mammal.

The neuropeptide hormone oxytocin modulates numerous social and parental behaviours across a wide range of species, including humans. We conducted manipulation experiments on wild grey seals (Halichoerus grypus) to determine whether oxytocin increases proximity-seeking behaviour, which has previously been correlated with endogenous oxytocin concentrations in wild seal populations. Pairs of seals that had never met previously were given intravenous injections of 0.41 µg kg-1 oxytocin or saline and were observed for 1 h post-manipulation. The dose was designed to mimic endogenous oxytocin concentrations during the observation period, and is one of the lowest doses used to manipulate behaviour to date. Seals given oxytocin spent significantly more time in close proximity to each other, confirming that oxytocin causes conspecifics to seek others out and remain close to one another. Aggressive and investigative behaviours also significantly fell after oxytocin manipulations. Despite using a minimal oxytocin dose, pro-social behavioural changes unexpectedly persisted for 2 days despite rapid dose clearance from circulation post-injection. This study verifies that oxytocin promotes individuals staying together, demonstrating how the hormone can form positive feedback loops of oxytocin release following conspecific stimuli, increased motivation to remain in close proximity and additional oxytocin release from stimuli received while in close proximity.

Maternal Oxytocin Is Linked to Close Mother-Infant Proximity in Grey Seals (Halichoerus grypus).

Maternal behaviour is a crucial component of reproduction in all mammals; however the quality of care that mothers give to infants can vary greatly. It is vital to document variation in maternal behaviour caused by the physiological processes controlling its expression. This underlying physiology should be conserved throughout reproductive events and should be replicated across all individuals of a species; therefore, any correlates to maternal care quality may be present across many individuals or contexts. Oxytocin modulates the initiation and expression of maternal behaviour in mammals; therefore we tested whether maternal plasma oxytocin concentrations correlated to key maternal behaviours in wild grey seals (Halichoerus grypus). Plasma oxytocin concentrations in non-breeding individuals (4.3 ± 0.5 pg/ml) were significantly lower than those in mothers with dependent pups in both early (8.2 ± 0.8 pg/ml) and late (6.9 ± 0.7 pg/ml) lactation. Maternal plasma oxytocin concentrations were not correlated to the amount of nursing prior to sampling, or a mother's nursing intensity throughout the dependent period. Mothers with high plasma oxytocin concentrations stayed closer to their pups, reducing the likelihood of mother-pup separation during lactation which is credited with causing starvation, the largest cause of pup mortality in grey seals. This is the first study to link endogenous oxytocin concentrations in wild mammalian mothers with any type of maternal behaviour. Oxytocin's structure and function is widely conserved across mammalian mothers, including humans. Defining the impact the oxytocin system has on maternal behaviour highlights relationships that may occur across many individuals or species, and such behaviours heavily influence infant development and an individual's lifetime reproductive success.

Conspecific recognition and aggression reduction to familiars in newly weaned, socially plastic mammals.

Recognising conspecifics and behaving appropriately towards them is a crucial ability for many species. Grey seals (Halichoerus grypus) show varying capabilities in this regard: mother-pup recognition has been demonstrated in some geographical populations but is absent in others, yet there is evidence that individuals aggregate with prior associates. The recognition capabilities of newly weaned grey seal pups were investigated using class recognition trials within the habituation/dishabituation paradigm. Trials took place in pens, using pairs of individuals that either had previously cohabited (familiar) or that had never met before (stranger). Frequencies of olfactory and visual investigative behaviours ('checks') and aggressive interactions were recorded during trials. Familiar individuals recognised each other: paired strangers showed significantly more checks and aggressive interactions than were seen in trials pairing familiars. Oxytocin concentrations in post-trial plasma samples were analysed to investigate the underlying physiology modulating recognition abilities; however, no significant differences were detected between familiar or stranger trials. This study demonstrates that at a young age, grey seals can recognise individuals they have previously encountered. Recognition abilities in this species have adaptive value by allowing the reduction of costly aggressive interactions between familiar conspecifics, which is often cited as the first step towards the evolution of sociality in a species. This study is the first with wild subjects to find conspecific recognition abilities in a pinniped species outside of reproductive contexts. It demonstrates that even largely solitary species can be capable of recognition and pro-social behaviours that benefit them during times when they must aggregate.

Validation of an enzyme-linked immunoassay (ELISA) for plasma oxytocin in a novel mammal species reveals potential errors induced by sampling procedure.

BACKGROUND: The neuropeptide oxytocin is increasingly the focus of many studies investigating human and animal social behaviours and diseases. However, interpretation and comparison of results is made difficult by a lack of consistent methodological approaches towards analysing this hormone. NEW METHOD: This study determined the sample collection and analysis protocols that cause the least amounts of protocol dependant variation in plasma oxytocin concentrations detected by ELISA. The effect of vacutainer type, sample extraction prior to analysis and capture and restraint protocol were investigated while validating an assay protocol for two novel species, grey seals (Halichoerus grypus) and harbour seals (Phoca vitulina). RESULTS: Where samples are extracted prior to analysis, vacutainer type (EDTA mean: 8.25±0.56 pg/ml, heparin mean: 8.25±0.62 pg/ml, p=0.82), time taken to obtain a sample and restraint protocol did not affect the concentration of oxytocin detected. However, concentrations of oxytocin detected in raw plasma samples were significantly higher than those in extracted samples, and varied significantly with vacutainer type (EDTA mean: 534.4±43.7 pg/ml, heparin mean: 300.9±19.6 pg/ml, p<0.001) and capture and restraint methodology. There was no relationship between oxytocin concentrations detected in raw and extracted plasma (p=0.25). COMPARISON WITH EXISTING METHOD(S): Over half the reviewed published studies analysing plasma oxytocin use raw plasma and different vacutainer types are used without consistency or justification through-out the literature. CONCLUSIONS: We caution that studies using raw plasma are likely to over estimate oxytocin concentrations, cannot be used to accurately infer true values via correlations and are susceptible to variation according vacutainer type.

Variation in female grey seal (Halichoerus grypus) reproductive performance correlates to proactive-reactive behavioural types.

Consistent individual differences (CIDs) in behaviour, indicative of behavioural types or personalities, have been shown in taxa ranging from Cnidaria to Mammalia. However, despite numerous theoretical explanations there remains limited empirical evidence for selective mechanisms that maintain such variation within natural populations. We examined behavioural types and fitness proxies in wild female grey seals at the North Rona breeding colony. Experiments in 2009 and 2010 employed a remotely-controlled vehicle to deliver a novel auditory stimulus to females to elicit changes in pup-checking behaviour. Mothers tested twice during lactation exhibited highly repeatable individual pup-checking rates within and across breeding seasons. Observations of undisturbed mothers (i.e. experiencing no disturbance from conspecifics or experimental test) also revealed CIDs in pup-checking behaviour. However, there was no correlation between an individuals' pup-checking rate during undisturbed observations with the rate in response to the auditory test, indicating plasticity across situations. The extent to which individuals changed rates of pup-checking from undisturbed to disturbed conditions revealed a continuum of behavioural types from proactive females, who maintained a similar rate throughout, to reactive females, who increased pup-checking markedly in response to the test. Variation in maternal expenditure (daily mass loss rate) was greater among more reactive mothers than proactive mothers. Consequently pups of more reactive mothers had more varied growth rates centred around the long-term population mean. These patterns could not be accounted for by other measured covariates as behavioural type was unrelated to a mother's prior experience, degree of inter-annual site fidelity, physical characteristics of their pupping habitat, pup sex or pup activity. These findings are consistent with the hypothesis that variation in behavioural types is maintained by spatial and temporal environmental variation combined with limits to phenotype-environment matching.

Identification and real-time PCR quantification of Phocine distemper virus from two colonies of Scottish grey seals in 2002.

The North Sea European harbour seal (Phoca vitulina) population has endured two phocine distemper virus (PDV) epidemics in 1988 and 2002. The grey seal (Halichoerus grypus) is a sympatric seal species that shows little or no mortality from PDV. Two Scottish grey seal breeding colonies were sampled for evidence of PDV infection approximately 2 months after the peak of the 2002 epidemic. In both colonies, a proportion of mothers (13/109) and pups (6/84) tested positive for PDV in their leukocytes. All infected animals were asymptomatic and completed the breeding season successfully. These results illustrate that grey seals come into contact with infectious seals and can become infected themselves without experiencing acute effects. In some seals the virus is able to replicate from the primary site of infection. This study provides evidence that grey seals may have an active role in the spread of PDV during an epidemic.