Urinary glucocorticoids in harbour seal (Phoca vitulina) pups during rehabilitation
Introduction
Rescue, captive care, and subsequent release of stranded young phocid seals has become increasingly common and routine practice in North America and western Europe, most often of harbour seals, Phoca vitulina, grey seals, Halichoerus grypus, and northern elephant seals, Mirounga angustirostris, with numbers now running into hundreds along some coastlines (e.g. Lander et al., 2002; Osinga and ‘t Hart, 2010, MacRae et al., 2011). During the pupping seasons of these species, neonates that have been permanently separated from their mothers have been usually termed “orphans” (e.g. Riedman and LeBoeuf, 1982; MacRae et al., 2011, Wilson and Jones, 2021) or “healthy but abandoned” (Dailey et al., 2020). These orphan pups are typically taken to “seal sanctuaries” for mother-substitute care, usually termed “rehabilitation” (rehab) centres, until they are considered well-enough grown to be released back into the sea, after a period usually varying between seven weeks to four months.
Rehab centres are only able to provide the pups with an artificial environment, which differs in many key aspects from the natural physical and social environment of mother-dependent, free-ranging pups. Pups in the wild spend about 60% of their time in the water from birth, follow their mother closely in the water, and are continually mobile around the nursery site (Venables and Venables, 1955, Wilson, 1974, Bowen et al., 1999, Skinner, 2006, Wilson and Jones, 2018). The mothers usually attend their pups full-time for at least the first 10 days, after which they often leave their pups in the colony for a few hours while they make offshore foraging trips (Wilson, 1978, Boness et al., 1994, Wilson and Jones, 2018, Wilson and Jones, 2020). Thus reproducing the natural environment of free-ranging harbour seal pups for orphans in rehab is challenging.
Rehab facilities in the British Isles and North America are usually designed to maintain the pups in isolation and in dry pens for some weeks after admission, a principal reason for this being to prevent potential transmission of infections (Larmour, 1989, Robinson, 1995, Osinga and ‘t Hart, 2010, MacRae et al., 2011). Less often, centres house the pups with free access to a pool, either in pairs (Wilson, 1999) or in small groups (Müller et al., 2003).
Harbour seal pups are born at about 11 kg mass and, when nursing from their mothers, achieve a net mass gain of about 0.4 to 0.6 kg/day (Skinner, 2006, Muelbert and Bowen, 1993, Cottrell et al., 2002). However, this mass gain is rarely reached during the first weeks in rehab, with published mass gains of 0.11 kg/day (MacRae et al., 2011), 0.10–0.16 (Richmond et al., 2010), 0.13 kg/day (Dailey et al., 2020), 0.21 kg/day (Trumble et al., 2013), and 0.30 kg/day (Wilson, 1999).
Therefore, in most cases of orphan harbour seal pup rehab, the physical and social environment and weight gain may be very different from that of a free-living pup with its mother. These differences may be expected to result in psychological and nutritional stress, affecting the dynamics of the “stress” hypothalamus–pituitary-adrenal (HPA) axis. This, in turn, will modulate the release of glucocorticoid (GC) stress hormones, such as cortisol (CL) (Atkinson et al., 2015).
When an infant mammal – such as a harbour seal, squirrel monkey (Saimiri sciureus) or guinea pig (Cavia porcellus) is initially separated from its mother, it usually emits distress calls and makes physical effort to regain contact (Perry and Renouf, 1988, Coe et al., 1983, Yusko et al., 2012). This has been termed the “acute” phase of maternal separation, when a dramatic increase in levels of CL has been recorded in squirrel monkeys (Coe et al., 1983), guinea pigs (Yusko et al., 2012) and harbour seals (di Poi et al., 2015).
CL is transported in plasma, with 80–90% bound to CL-binding globulin, 5–10% bound to albumin, while only 3–10% of CL is free and biologically active (Holst et al., 2004). At the target tissue, the free hormone diffuses across the cell membranes and binds to the glucocorticoid receptor; this complex migrates to the nucleus where it regulates gene expression (McWhinney et al., 2010). The principal effects of CL are to increase cardiac output and increase circulating glucose concentrations (Sapolsky et al., 2000), thereby facilitating an appropriate energetic response by a separated infant attempting to reunite with its mother. However, if the reunion fails, the initial acute phase of the stress response will eventually give way to the next “depressive” phase of maternal separation, when the infant may be expected to display more passive behaviours (Spencer-Booth and Hinde, 1971), accompanied by reduced circulating CL (Yusko et al, 2012). By the time an orphan seal pup enters rehab, it may already be in the depressive phase of separation, as well as being in poor body condition.
When harbour seal orphan pups enter rehab, therefore, circulating CL levels may be a dynamic function of time since maternal separation, time spent alone since separation, and other factors of an artificial environment. In pinnipeds, CL levels have been reported to increase during periods of fasting or nutritional stress (e.g. Ortiz et al., 2001, Guinet et al., 2004, Du Dot et al., 2009; Bennett et al., 2013; Kershaw and Hall, 2016). Orphan pups will be experiencing nutritional stress when rescued, and this may not be alleviated fully in the first weeks due to the difficulty of achieving a species-typical growth rate.
CL levels in the blood of harbour seal pups in rehab have been measured, by immunoassay, to investigate possible stress levels. Blood samples have been taken from the extradural intervertebral vein following restraint of the pup (e.g. Gulland et al., 1999; Trumble et al., 2013, Dailey et al., 2020). However, blood sampling can measure the GC for just that moment in time, with the pup restraint possibly affecting circulating CL levels (e.g. Engelhard et al., 2002; Johnstone et al., 2012; Kershaw and Hall, 2016). These studies have not reached a consensus on how CL levels may reflect orphan pups’ presumed stress level during rehab. Gulland et al. (1999) found that baseline plasma CL levels decreased with increasing time in rehab and suggested that the stress of the captive environment was reduced as the pups gradually adapted. Dailey et al. (2020) found that plasma CL concentrations were highly variable and showed no trends with increasing time in rehab. Trumble et al. (2013) found that plasma CL levels increased in pups while they were being tube-fed formula, stabilising at a lower level thereafter; they suggested that the pups might have a stress response to the tube-feeding (stomach gavage) procedure.
Urine may be sampled instead of blood to assess circulating CL levels in seals (Constable et al., 2006). Because protein-bound steroids, present in blood, are excluded from the kidney filtrate, only free hormone is excreted in the urine. Urinary free CL concentration correlates well with the concentration of plasma free CL (Lupo et al., 2018) and therefore reflects concentrations of the free hormone in blood (Cook, 2012). Urinary CL collects in the bladder since the previous voiding, and therefore reflect the stress response over a longer period than concentrations in a blood sample.
Previous studies of urinary CL concentrations in mammals have suggested that a rise in urinary CL is likely to occur only following a severe stressor, such as cows being isolated from their herd (Higashiyama et al., 2009), or anaesthesia of chimpanzees, Pan troglodytes (Anestis, 2009).
Studies have been carried out on stress hormones in the urine of domestic cattle and equines, using ultra performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). This has enabled simultaneous detection and measurement of different steroids, including CL and its most closely related GC hormones cortisone (CN), prednisolone (PL), and prednisone (PN) (McWhinney et al., 2010, Pompa et al., 2011, Vincenti et al., 2012, Fidani et al., 2012, Bertocchi et al., 2013, Ferranti et al., 2013, Keenan, 2015, Chiesa et al., 2017). Urinary concentrations of both CL and CN have been found to be elevated in stressful conditions, while PL and PN have been detected in low concentrations associated with extreme stress (such as transport and slaughterhouse (Pompa et al., 2011, Vincenti et al., 2012, Keenan, 2015, Chiesa et al., 2017).
The chemical structure of these four GCs is very similar (e.g. Arioli et al., 2012, Vincenti et al., 2012, De Clercq et al., 2013, Chiesa et al., 2017). CL and PL have the same molecular weight, so also do CN and PN, the difference being only that PL and PN have a double bond between two carbon atoms in the 1st carbon ring (Supplementary Fig. S1.1). Oxidation of the HO group in the 3rd carbon ring of the biologically active CL and PL yields the inactive CN and PN respectively with a double-bonded oxygen atom, while the reverse reduction process yields CL and PL respectively. CL can be converted to PL by the introduction of a double bond in the 1st carbon ring (Supplementary Fig.S1.1) These conversions are catalysed in the liver by enzymes known as 11β-HSDs (Chiesa et al., 2017, Timmermans et al., 2019). Conversion of CL and PL to the inactive forms CN and PN occurs to protect the tissues from excessive CL and PL action, whereas the reverse can occur when further activity is required (McWhinney et al., 2010, Chiesa et al., 2017; Timmermans et al., 2019). These four GCs are therefore interconnected within a dynamic system.
Because the psychological and physical traumas associated with maternal separation and transfer to a rehab environment are assumed to be extreme stressors to new-born harbour seal pups, we believed a study of levels of all four of these GCs as they occurred in different rehab conditions could be informative. We hypothesised that rehab conditions which depart the furthest from the natural conditions of pups in the wild would result in relatively high levels of some or all of the four GCs, and vice versa. Since the occurrence of urinary PL and PN were believed to indicate circumstances of extreme stress in cattle, we hypothesised that CL and CN would be highest, and PL and PN would most likely be detected, in pups newly admitted to rehab.
The present study intended to analyse pup urine for all four GCs by UPLC-MS/MS. The aim was to investigate how social isolation, dry pen, low growth rate and low body mass in rehab might be reflected in urinary levels of each of these GCs.
Section snippets
Pup background
This study focused on 32 harbour seal pups, Phoca vitulina vitulina, that had been admitted to four participating rehabilitation centres, three in Ireland (Centres I, II and IV) and one in N. Germany (Centre III). All these pups had been found alone, stranded on the shoreline during the pup birthing season in three separate years (2014, 2015 and 2016). The criteria, normally applied by centres I and II in north-east Ireland, for identifying a pup as being permanently separated from its mother
Ranges, medians, and correlation matrix of GC concentrations
Detectable urinary concentrations of CL and PL were found in 89 of the 96 samples, while PN was detected in 95 of the samples and CN in all samples. The ranges, mean and median values for the Cr content and the four GCs concentrations in all the samples analysed are given in Table 2 and Supplementary Fig. S1.3. The median values for CN were about ten times higher than for CL, PN and PL. However, CL values were highly variable, with three of the samples (all taken between rehab days 0–2) having
Endogenous levels of four GCs in the harbour seal pups of this study and in other species
The present study of seal pups in rehab is the first, to our knowledge, to analyse any seal samples for PL and PN. We found substantial urinary concentrations of both PL and PN in most samples, with CL:PL and CL:PN ratios of approximately 1.2:1. The overall ratio of PL:PN was approximately 1:1, suggesting continuous recycling of PN to PL, possibly indicative of a continuous need for PL glucogenesis throughout rehab. Detectable levels of endogenous urinary PL have previously been reported in
Conclusions
This study has found levels of four GCs in the urine of rehab harbour seal pups to be elevated in circumstances where rehab conditions depart substantially from the pups’ natural environment in the wild. The results suggest that the stress hormone levels were elevated in two circumstances, i.e. where pups were held in a dry pen with no free water access, and where they had body mass and growth rate lower than the species-typical range for nursing pups. The implication of these results is that
Funding
This study has not received any grant from any funding agency.
Ethics statement
The urine sampling in this study was carried out with the permission and collaboration of each of the four seal centres. Three of the four centres (II, III and IV) were in the public domain and acting within relevant government guidelines. Centre I was a voluntary private centre operating with local government (N. Ireland) permission. The sampling did not cause additional disturbance to the pups beyond their routine care and handling and did not impact their welfare. No specific license for
CRediT authorship contribution statement
Susan C. Wilson: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration. Stella Villanueva: Conceptualization, Methodology, Investigation, Writing – review & editing. Kayleigh A. Jones: Methodology, Formal analysis, Writing – original draft, Writing – review & editing, Visualization. Lilia Dmitrieva: Formal analysis, Writing – review & editing. Wesley Smyth: Methodology,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We would like to thank Dr Robert McCracken at the Agri-Food & Biosciences institute (AFBI) and Dr William Hayes at the University of Lincoln for their support during the initial phase of this study. We are indebted to all the personnel at the seal sanctuaries participating in this study for their generosity in permitting and facilitating the urine sampling. In particular, we would like to acknowledge the invaluable support of Fleur Brochut and Dominic Ijichi at Tara Seal Research, N. Ireland,
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