Blubber thickness variation

in grey, harbour and ringed seals A proxy for seal health and an indicator of energetic constraints

Photo taken by Ken Howard. Used with permission

Andre Hermansson

Degree project for Master of Science in Biology

Animal Ecology, 30 hec, vt 2015

Department of Biological and Environmental Sciences

University of Gothenburg

Supervisor: Karin Hårding

Examiner: Lotta Kvarnemo Abstract

HELCOM has chosen seals’ nutritional status as one of several so called ‘core indicators’ that shall be monitored in all Baltic countries to document changes in the environment. The parameter measured to describe seal nutritional status is blubber thickness. The seals included in this study were grey, harbour and ringed seals either shot, caught in fishing gear or found dead along the Swedish coast of the Baltic Sea and the Swedish West coast 2002-2012. The blubber content as percentage of body mass (%fat), the so called LMD-index, was calculated. This was correlated with several other body condition indices taken from the literature, with the aim of finding out how well they could describe blubber content and thus, indirectly nutritional status. The seasonal and interannual variation in blubber thickness were also investigated. The sternum blubber thickness, the most widely used measuring site of blubber thickness in pinnipeds, was found to correlate well with %fat in all three species. Thus, suggesting that it is a good indicator of total blubber content/nutritional status. Dorsal and sternum blubber thicknesses were also found to correlate well with each other, suggesting that sternum blubber thickness can substitute the dorsal measure, by multiplication with a factor 1.114, for calculating the %fat. In agreement with previous studies, a seasonal variation in blubber thickness, with a decrease in spring and increase in fall/winter was found. Interestingly, a significant decrease in the autumn-winter %fat was found over the time period 2002-2012 for sexually mature grey seal females. This trend could pose a future problem for both individual survival and reproduction. The underlying cause is as yet unknown, but may be related to decreasing fish stocks, dietary changes and population density related factors. Total blubber content in kilograms of juvenile grey seals in autumn varied widely among individuals. Simple energetic modelling suggested that these differences corresponded to substantial differences in the amount of ingested food between fatter and leaner seals of the same cohort and these differences may prove to be insurmountable given the limited time available for feeding before their first winter. This may reflect differences in lactation and early foraging success. Thus, a good early start in the life of the juveniles, with proper lactation and good foraging success seems to be crucial.

Introduction

Study species

The Atlantic grey seal (Halichoerus grypus) is a large, fish-eating phocid seal that inhabits temperate coastal waters of the North Atlantic and the Baltic Sea (Abt et al. 2002). In the Atlantic it is a land-based colonial breeder, with large sexual dimorphism and a polygynous mating system (Worthington Wilmer et al. 1999). Female grey seals come ashore once a year to give birth to and rear a single cub (Sparling et al. 2006). The Baltic grey seal population is different from the Atlantic population in several ways, the most obvious being the ability to shift breeding habitat depending on sea ice availability, thus it is a facultative land/ice breeder. It has been shown that pup survival, weaning weight and health are significantly increased on ice as compared to land, most likely because of lower exposure to crowding and pathogens on ice (Jüssi et al. 2008). The Baltic grey seal breeds in early spring, with pupping peaking in late February to early March (Thompson and Härkönen 2008a). The lactation period is around 17 days long and during this period the female does not go to sea to feed but instead relies on stored energy reserves in the form of blubber. Thus, maternal energy reserves is a major factor determining cub survival rates and female fitness. Females forage at sea for 7 months between the moulting and breeding seasons in order to accumulate enough fat reserves for the next breeding season (Sparling et al. 2006).

After a century of bounty hunting and decades of low fertility due to environmental pollutants (Harding et al. 2007), the Baltic grey seal population has now been on the rise since 1990, with an annual rate of 5.8-8.5 %. This recent increase in population size has caused problems with local fisheries (Harding et al. 2007; Kauhala et al. 2015) which has led to the reintroduction of hunting in Sweden since 2001, with an annual quota of around 200 seals (Kauhala et al. 2015).

The harbour seal (Phoca vitulina) is a smaller phocid seal (Bowen et al. 1992). Its diet is very diverse, ranging from various fish species to cephalopods and crustaceans. It is one of the most widespread pinniped species, inhabiting coastal waters, bays, rivers, estuaries and intertidal areas of the Northern Hemisphere, ranging from temperate to polar climate. The harbour seal is considered a promiscuous or weakly polygynous species (Thompson and Härkönen 2008b). Like the grey seal and other phocids, the females give birth on land, lactation is brief and weaning abrupt. However, unlike most phocids, the new-born cubs are large relative to the mother’s size and follow their mother to sea only hours after birth, and the females often feed during lactation (Bowen et al. 1992).

The Swedish harbour seal has several distinct populations, around the island of Öland, on the Swedish West Coast and the southern Baltic (Bäcklin et al. 2013). Along the Swedish West Coast regional genetically distinct stocks can be identified (Olsen et al. 2014). The populations in the Baltic and Kattegatt have suffered several disease related crashes in the past but are now on the rise, increasing at an approximate rate of 12 % per year, however the rate has been declining in recent years (Härkönen et al. 2013).

The ringed seal (Pusa hispida or Phoca hispida) is a small phocid seal, possessing a very thick blubber layer. Its diet consists mainly of small schooling fish species, e.g. herring and sprat, although some variation exists, depending primarily on prey availability and energy content. It has a chiefly circumpolar distribution throughout the Arctic Basin and adjacent seas, but some subspecies are also found in lakes further south (e.g. the Saima seal and the Ladoga seals). The Arctic ringed seal is a typical ice-seal, breeding, moulting and resting on sea ice. The Baltic population however, use ice for breeding and moulting, but is forced to haul out on land during the summer due to a lack of sea ice. In the spring females give birth to a single pup in lairs made in the ice and snow. Lactation lasts a bit over a month on average and mating occurs towards the end of this period, similar to other phocids (Kovacs 2008).

Due to over-harvesting the Baltic ringed seal population declined drastically during the past century (Kokko et al. 1997; Kovacs 2008). With the exception of the Bothnian Bay, the populations in the rest of the Baltic have shown either no increase or even a decline since the 1990s. In addition to the past hunting, other major threats include bycatches in fishing gear, pouching, infectious diseases and the predicted future reductions in sea ice availability due to climate change (Kovacs 2008; Sundqvist et al. 2012) and various anthropogenic pollutants such as organochlorines and heavy metals (Nyman et al. 2002; Kovacs 2008).

Study area

The data used in this study is from seals shot, caught in fishing gear or found dead along the Swedish coast of the Baltic Sea and the Swedish West coast between the years 2002 and 2012. The grey seal data is considered applicable for the whole Baltic Sea population, as grey seals are very mobile across the Baltic Sea basin. The ringed seal data on the other hand may apply only to the Bothnian Bay, where most of the samples are from. The harbour seal data is applicable to the Swedish West coast.

Blubber

Blubber, a layer of lipid-rich tissue between the epidermis and the underlying muscles is a fundamental part of the mammalian adaptation to the aquatic lifestyle. It can constitute 15–55 % of total body mass in marine mammals. Its functions include not only thermoregulation but also storage of metabolic energy (Noren et al. 2014), buoyancy regulation (Webb et al. 1998) and hydrodynamic drag reduction (by modifying the streamlined shape of the body)(Fish 2000). The thickness of the blubber layer is important not only for individual survival but also for reproduction, being very important for lactation and cub survival (Harding et al. 2005; Stephens et al. 2014; Bowen et al. 2015).

Blubber thickness in adult seals has been shown to vary seasonally, with a decrease during reproduction, lactation and moulting in spring followed by a subsequent increase when autumn arrives (Nilssen et al. 1997; Hauksson 2013). Lactation in phocid seals is brief and often involves maternal fasting with concomitant drastic reductions in the mother’s energy reserves, the energy being transferred to the rapidly growing pup, and is ended with abrupt weaning when the mother departs to feed at sea. Maternal fat reserves may decline by as much as 79 % (e.g. harbour seal, P. vitulina) during the lactation period (Bowen et al. 1992).

The amount of fat stored by each animal is influenced by external conditions such as abundance of prey items, parasite load, environmental toxins and other stressors and thus could serve as an indicator of body condition that correlates with habitat quality and reproductive rate (Noren et al. 2014). HELCOM has chosen seals’ nutritional status as one of several so called ‘core indicators’ that shall be monitored in all Baltic countries to document changes in the environment. The parameter measured to describe seal nutritional status is blubber thickness (Bäcklin et al. 2013).

Blubber thickness can be measured in many ways, including destructive techniques such as chemical analysis (Reilly and Fedak 1990; Arnould et al. 1996; Oftedal et al. 1996) and non- destructive ones such as ultrasound imaging (Webb et al. 1998; Mellish et al. 2004; Noren et al. 2008). Due to recent technological advancements increasing its accuracy (up to 99.8 % when compared to blubber biopsies), ultrasound has become increasingly popular among scientists as a rapid and non-invasive alternative to measure blubber thickness in marine mammals (Mellish et al. 2004). Furthermore, specific areas of the body may be preferentially targeted for the mobilization of blubber stores (Mellish et al. 2007; Noren et al. 2014). The standard measuring site for blubber thickness in pinnipeds has historically been over the xiphoid process (sternum), although several studies have suggested that this may not be the best site for monitoring body condition in all species (Noren et al. 2014).

Study objectives

My objectives with this study were:

1) To describe the seasonal and interannual variation in blubber thickness in grey, harbour and ringed seals in the Baltic Sea. 2) Compare different indices of nutritional status and make recommendations for best practices to estimate nutritional status. 3) Discuss underlying processes that may affect the nutritional status of the populations by elaboration of a bioenergetic model.

Method

Data source

The study is based on a literature review on common body condition measurements on pinnipeds and statistical analysis of a large data set to compare the performance of the different indices. In addition a small bioenergetic model was developed. Data on biometric measurements from grey, harbour and ringed seals shot, caught or found between 2002 and 2012 were acquired by the courtesy of the Swedish Museum of Natural History (NRM) (Bäcklin et al. 2013).

Estimates of total blubber content/condition indices

The blubber content as percentage of body mass (%fat), was calculated according to the expression developed by Ryg et al (1990):

(1)

In this so called LMD-index L = the standard body length in cm, M = the body weight in kilograms and d = the blubber thickness in meters at 60 % of the standard body length (starting at the nose). LMD-index has previously been shown to be a reliable estimate of true blubber content in phocid seals (r2=0.88, standard deviation=3.02, for harp, grey and ringed seals pooled together) when compared to dissection data (Ryg et al. 1990). It has even proved to work relatively well with Steller sea lion, Eumetopias jubatus (Pitcher et al. 2000) and extremely well with Baltic seals (Bäcklin et al. in prep.). The original LMD-index (Ryg et al. 1990) used dorsal blubber thickness (henceforth denoted as BL (dorsal)), however the sternum blubber thickness (BL (sternum)) is the one that has been most widely used historically, and also the one used for the majority of the seals in the data set available for this study. A smaller portion of the seals in the data set used here had both the dorsal and sternum measurements, thus allowing for a comparison between them. The LMD-index was then correlated with several other indices of body condition traditionally used (taken from the literature), as well as with standard body length (SL). The goal was to see how well they could describe blubber content (as estimated through the LMD-index). Of particular interest in this regard were the BL (sternum) and the 100 x axillary girth/standard body length (100 x AG/SL, AG=axillary girth, i.e. the greatest circumference of the chest, just behind the front flippers), as these have been the two most common condition indices used for seals (Ryg et al. 1990). The traditionally used indices along with their respective sources and abbreviations are shown in Table 1.

Table 1 The different measures used as condition indices taken from the literature are listed with their respective sources and the abbreviations used herein.

Body measurements used as condition indices in the Abbreviation Source literature

Standard body length (cm) SL (Kjellqwist et al. 1995)

Body weight (kg) BW (Pitcher et al. 2000)

BW/SL (Arnould 1995; Noren et al. Body weight (kg)/standard body length (cm) 2014) Sternum blubber thickness (mm) BL (sternum) (Pitcher et al. 2000)

Dorsal blubber thickness (mm) BL (dorsal) (Ryg et al. 1990)

Sternum blubber thickness (mm)/standard body length (cm) BL (sternum)/SL (Pitcher et al. 2000)

BL Sternum blubber thickness (mm)/body weight (kg) (Pitcher et al. 2000) (sternum)/BW

100 x AG/SL (Ryg et al. 1990; Nilssen et al. 100 x axillary girth (cm)/standard body length (cm) 1997)

When looking at the seasonal variation within years, and the long term variation among years, in BL (sternum) and %fat, only healthy individuals caught by hunting (excluding bycatch) were included in the analysis so as to exclude any changes in blubber thickness due to disease, malnutrition, injuries etc. The seals were then divided into three classes, 1-3 year-olds (not sexually mature) and 4+ year-old females and 5+ year-old males (the latter two classes being sexually mature). For the variation among years, a further selection was made, only seals caught during the months September to January (non-mating and non-lactating season), when the seals are at their fattest, were included. This is because, like previously mentioned, sexually mature individuals’ blubber thickness diminishes significantly during the mating season. Thus, individuals caught during this period will have low blubber thickness regardless of any other existing trend in the population over the years caused by other factors, such as changes in hunting pressure, food stock, population density etc. This selection was also applied to non-sexually mature (1-3 year-old) individuals, as their blubber has also been shown to vary somewhat throughout the year, probably due to prey availability (Bäcklin et al. 2013). Due to the selection of only healthy individuals caught by hunting for the analysis of the variation within and among years, the sample sizes for ringed seals and most of the harbour seals became too small for statistical analysis of the trends. For this reason, only the grey seals and the 4+ year-old female harbour seals were used for the description of the seasonal variation in blubber thickness and the blubber thickness variation over time.

For the statistical analysis of the relations between the condition indices, correlation analysis (Pearson's r) was used, and for the interannual variation in BL (sternum) and %fat, linear regression was used.

Energetic comparison

With the aim to understand the potential fitness effects imposed by different body fat contents, an energetic comparison was made. The weight difference (m) between the fattest (mmax) and thinnest (mmin) healthy juvenile (1 year-old) grey seals caught by hunting September-November 2002-2012 was calculated to get a notion of how much energy reserves differ among individuals in the first critical winter. Body fat content as percentage of body mass (henceforth referred to as %fat) for each of the two seals was multiplied with the body weights in kg to transform percentage fat into absolute blubber mass in kg. Thereafter, the difference between the seal with the highest and lowest absolute blubber mass was calculated:

To find out how much the thinnest seal would need to eat to compensate for this difference several steps were taken.

The expressions for calculating the maintenance energy requirement, foraging and transport costs and growth energy requirement of body tissue, as well as the values for mean daily energy intake and metabolic efficiency that now follow, were all based on numbers used for juveniles of another closely related phocid seal species, the harbour seal (Markussen et al. 1990; Härkönen and Heide-Jørgensen 1991).

Steps to calculate how much and during how long a period the lean seal needs to eat to compensate for the low energy reserve:

The average amount of fat the thinnest juvenile seal would gain per day was calculated in the following way:

First the metabolic efficiency rate was accounted for, i.e. the process of converting food into blubber tissue was not 100 % efficient. Here it was assumed that 80 % of the energy in the ingested food was metabolized (Härkönen and Heide-Jørgensen 1991). In addition to this metabolic cost, the costs of maintenance energy (i.e. basal metabolism, thermoregulation and voluntary actions) and foraging and transport between haul-out and feeding sites were also taken into account.

The maintenance energy requirement was calculated according to the expression: (Markussen et al. 1990) and the cost of foraging and transport between haul-out and feeding sites according to the expression: (Härkönen and Heide-Jørgensen 1991). The body mass of the thinnest seal was the one inserted into both these equations.

The average amount of fat in kg the seal gained per day was calculated in the following steps:

1. The mean daily energy intake, here assumed to be 2 798 kcal based on the lowest mean daily intake for juvenile harbour seals taken from a previous experimental study (Markussen et al. 1990), was multiplied with the metabolic efficiency rate (0.80).

2. Thereafter, the costs of maintenance energy and foraging and transport between haul-out and feeding sites were subtracted from the result, the remainder being the energy left for growing blubber tissue. 3. This remainder energy was then divided by the gross energy requirement for growing one kg of body tissue: (Markussen et al. 1990), thus giving the average amount of blubber tissue in kg the seal gained per day.

Then, in order to calculate the amount of days the seal would have to feed, the ∆fat was divided by the average amount of fat the seal gained per day as calculated above.

The total amount of fish eaten during the feeding period would depend on the fish species being consumed, as each species has a different energy content. Since the main prey species of the Baltic grey seal is herring, Clupea harengus (Andersen et al. 2007; Lundström et al. 2010), and in order to simplify the calculations, the seal was assumed to eat only herring. The average energy content of herring is 2.30 kcal/g wet mass (Härkönen and Heide-Jørgensen 1991). The mean daily energy intake was divided with the energy content of herring in order to get the amount of fish eaten per day, and this was then multiplied with the length of the feeding period in days to get the total amount of fish eaten during the period.

For a schematic overview of the entire energy calculation process see Fig 1.

Figure 1 A schematic overview of the energetic calculations, beginning with the calculation of the difference in body fat content between the fattest and leanest seal followed by the steps used to estimate the average amount of fat the seal gained per day, the required duration of the feeding period to compensate for the difference in body fat content (∆fat) and the total amount of fish eaten during the feeding period. Results

Condition indices

Grey seal:

BL (dorsal) was well correlated (r=0.88) with BL (sternum), the dorsal measure being on average 11.4 % thicker than the one at the sternum (Fig. 2). Thus, sternum measurements could be transformed into dorsal measurements by multiplication with a factor 1.114, and then inserted into Ryg’s equation for %fat (LMD-index) that is based on dorsal measurements.

BL (sternum) was found to predict %fat fairly well (Fig 3) albeit with a large variation around the mean. %fat was not well correlated (r=0.39) with 100 x AG/SL (Fig. 4) nor with BL (sternum)/BW (r=0.51). However, a strong correlation (r=0.91) was found between %fat and BL (sternum)/SL (Table 2).

80 H. grypus 70 60 r=0.88 50 40 30

BL (dorsal) BL 20 10 0 10 20 30 40 50 60 70 80 BL (sternum)

Figure 2 The relationship between BL (dorsal)(measured in mm at chest height, as used in Ryg’s equation for estimating the %fat and BL (sternum) in mm (i.e. the commonly available measure in the large Swedish data base) of grey seals shot, caught as bycatch or found dead 2002-2012, p < 0.0001, n=114. The data is taken from a unique data set where both the dorsal and sternum data were available (Bäcklin and Moraeus unpublished).

70,00 H. grypus 60,00 r=0.81 50,00

40,00 index index - 30,00

20,00 LMD 10,00 0,00 5 25 45 65 85 BL (sternum)

Figure 3 This graph illustrates how variable the % fat (estimated by the LMD-index) was among individual seals for a given sternum blubber thickness (mm), of grey seals shot, caught as bycatch or found dead 2002-2012, p < 0.0001, n=1377. 60,00 H. grypus 50,00 r=0.39

40,00 index

- 30,00

LMD 20,00

10,00

0,00 55,00 60,00 65,00 70,00 75,00 80,00 85,00 100 x AG/SL

Figure 4 The relationship between the %fat and 100 x axillary girth (cm)/standard length (cm) of grey seals shot, caught as bycatch or found dead 2002-2012, p < 0.0001, n=115.

Harbour seal: As with the grey seals, a significant correlation was found between the %fat and BL (sternum), the correlation being even stronger this time (Fig. 5). Also in accord with what was found with the grey seals, %fat was more strongly correlated with BL (sternum)/SL (Fig. 6) than with BL (sternum)/BW (Table 2).

70,00 P. vitulina 60,00 r=0.91 50,00

40,00

index index - 30,00

LMD 20,00

10,00

0,00 5 15 25 35 45 55 65 BL (sternum)

Figure 5 The relationship between the % fat and sternum blubber thickness (mm) of harbour seals shot, caught as bycatch or found dead 2002-2012, p < 0.0001, n=216. Unlike the grey seals, however, the harbour seal's %fat was significantly correlated with both SL and BW, as well as with BW/SL. However, none of these correlations were particularly strong (r=0.40, r=0.33 and r=0.28 respectively)(Table 2). 70,00 P. vitulina 60,00 r=0.91

50,00

40,00

index -

30,00 LMD 20,00

10,00

0,00 0,00 0,10 0,20 0,30 0,40 0,50 BL (sternum)/SL

Figure 6 The relationship between the %fat and sternum blubber thickness (mm)/standard length (cm) of harbour seals shot, caught as bycatch or found dead 2002-2012, p < 0.0001, n=216.

Ringed seal:

60,00 r=0.94 P. hispida

50,00

40,00 index index

- 30,00

LMD 20,00

10,00

0,00 5 15 25 35 45 55 65 75 BL (sternum)

Figure 7 The relationship between the %fat and sternum blubber thickness (mm) of ringed seals shot, caught as bycatch or found dead 2002-2012, p < 0.0001, n=112. Similarly as with the grey and harbour seals, a strong correlation was found between the %fat and BL (sternum) of the ringed seals, the correlation being stronger (r=0.94) than in both the other species (Fig. 7).

Also in agreement with what was found for the other seals, a strong correlation was found between the %fat and BL (sternum)/SL. With a near perfect fit (r=0.97), it is an even stronger correlation than was the case with the other seals (Fig. 8), and in accord with what was found with the other seals it is substantially stronger than the correlation with BL (sternum)/BW (r=0.57)(Table 2).

Unlike with the grey seals but in agreement with the harbour seals, there was a significant, but weak (r=0.32), correlation between the %fat and BW, as well as BW/SL (r=0.42)(Table 2).

60,00 P. hispida 50,00

r=0.97

40,00 index index

- 30,00

LMD 20,00

10,00

0,00 0,00 0,10 0,20 0,30 0,40 0,50 BL (sternum)/SL

Figure 8 The relationship between the %fat and sternum blubber thickness (mm)/standard length (cm) of ringed seals shot, caught as bycatch or found dead 2002-2012, p < 0.0001, n=112.

Table 2 The results of correlation analysis performed on different condition indices correlated with each other, with their respective p-values, sample size (N), type of relation, Pearson's r and seal species. All seals were shot, caught as bycatch or found dead 2002-2012.

Relation Measures being P-value N (positive or Pearson's r Seal correlated (correlation) negative)

LMD-index x BL < 0.0001 1377 + 0.51 Grey (sternum)/BW

LMD-index x BL < 0.0001 1377 + 0.91 Grey (sternum)/SL

LMD-index x SL < 0.0001 216 + 0.40 Harbour

LMD-index x BW < 0.0001 216 + 0.33 Harbour

LMD-index x BW/SL < 0.0001 216 + 0.28 Harbour LMD-index x BL < 0.0001 216 + 0.91 Harbour (sternum)/SL

LMD-index x BL < 0.0001 216 + 0.34 Harbour (sternum)/BW

LMD-index x BW 0.001 112 + 0.32 Ringed

LMD-index x BW/SL < 0.0001 112 + 0.42 Ringed

LMD-index x BL < 0.0001 112 + 0.57 Ringed (sternum)/BW

Seasonal variation in blubber thickness

Grey seal:

60

50

H. grypus

40 index - 4+ year-old females N=173 30 5+ year old males N=102 1-3 year-olds N=106

20 Mean LMD Mean

10

0 Apr May Jun Jul Aug Sep Oct Nov

Figure 9 Monthly mean %fat ±SD of healthy 1-3 year-old (blue), 4+ year-old female (green) and 5 + year-old male (red) grey seals caught by hunting from April to November 2002-2012. Only data points with at least 3 individuals per month were included. The mean %fat, instead of the mean BL (sternum), was plotted against month (Fig. 9), as the trends are very similar (Appendix 1, Figs. 13-15) but the %fat has the advantage of having taken the animals’ body sizes into account. The %fat was found to vary by season, being lower in spring and peaking in fall/winter for all age and sex classes (Fig. 9). Harbour seal:

70,00

60,00

50,00

P. vitulina index - 40,00

30,00 4+ yo females N=39

20,00 Mean LMD Mean 10,00

0,00 May Sep Oct Nov Dec

Figure 10 Monthly mean %fat ±SD of healthy 4+ year-old female harbour seals caught by hunting from May to December 2002- 2012. At least 3 individuals per month were used. The harbour seals' %fat also varied seasonally according to the same pattern as the grey seals, being lower in spring and higher in fall/winter (Fig. 10). However, as previously mentioned, only the 4+ year-old females could be used for analysis due to too small sample sizes in the other age and sex classes. For this same reason the ringed seals were completely excluded from analysis.

Interannual variation in blubber thickness

Grey seal:

60,00 y = -0,8442x + 1736,1

50,00 R² = 0,1772

40,00

index index

- 30,00

LMD 20,00

10,00

0,00 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Figure 11 The %fat of healthy 4+ year-old female grey seals caught by hunting from September to December over the years 2002-2012, p=0.045, n=23. Significant negative trend. A significant negative trend was found in the %fat of the 4+ year-old females during the time period from 2002 to 2012. The average decrease was 0.84 % per year (Fig. 11). The yearly mean sternum blubber thickness also showed negative trends in all age and sex classes, albeit none of these trends proved significant (Appendix 2. Figs. 16-18). Harbour seal: A non-significant positive trend was found in the yearly mean BL (sternum) of the 4+ year-old female harbour seals during the time period 2007 to 2011.

Energetic comparison

35,00 30,00 H. grypus 25,00

20,00

15,00 (kg) 10,00 5,00 0,00 Absolute blubber mass mass blubber Absolute 0 1 2 Age (years)

Figure 12 The absolute blubber mass in kg, calculated by multiplying the %fat with the body weight, of healthy 1 year-old grey seals caught by hunting from September to November 2002-2012. The fattest seal had a blubber mass of 31.57 kg and the thinnest 14.48 kg. The absolute blubber mass in kg for the juvenile (1 year-old) grey seals varied substantially among individuals, ranging from 14.48 kg to 31.57 kg (Fig. 12). The difference in absolute blubber mass between the fattest and thinnest seals was thus 17.09 kg. The maintenance energy requirement was 1 100.48 kcal per day and the foraging and transport costs were 752.96 kcal per day (Table 3).

Table 3 The different parameters used for calculating the average amount of fat the seal gained per day, the required length of the feeding period and the required amount of herring to be eaten are listed.

Difference in blubber content between the fattest and 17.09 kg thinnest individual

Maintenance energy requirement 1 100.48 kcal * day-1

Energy costs of foraging and transport between haul- 752.96 kcal * day-1 out and feeding sites

The average amount of fat that the seal gained per day during the feeding period was estimated to 0.04 kg. The required length of the feeding period was 403.54 days. The average daily amount of herring eaten was 1.22 kg. The total amount of herring eaten during the entire length of the feeding period was 490.92 kg (Table 4). Table 4 The average amount of fat the seal gained per day, the required length of the feeding period, the average daily amount of herring eaten and the total theoretically required amount of herring to be eaten to compensate for the weight difference between a lean and a fat pup are shown with two decimal places of precision.

Average amount of fat the seal gained per day 0.04 kg

Required length of the feeding period 403.54 days

Average daily amount of herring eaten 1.22 kg

Total theoretical amount of herring eaten during the 490.92 kg entire feeding period

Discussion

Condition indices

Of particular interest to this study was to find out how well BL (sternum) and 100 x AG/SL could describe the %fat, as these have been the two most commonly used condition indices for seals historically. It is important to make historical data bases compatible with recent data. In all three species studied here BL (sternum) correlated very well with the %fat (Figs. 3, 5 and 7). This is in agreement with previous findings from Baltic ringed and grey seals (Bäcklin et al. 2013) and ringed, harp (Phoca groenlandica) and grey seals (Ryg et al. 1990). This finding, taken together with the previously published results, suggests that BL (sternum) is a good indicator of total blubber content and consequently body condition/nutritional status, at least for the species of phocid seals studied here. A positive correlation was also found between the %fat and 100 x AG/SL, but only for grey seals, and even then it was not very strong (Fig. 4). Thus, this condition index could still be used for grey seals, but it would be advisable to instead use BL (sternum) if possible, as the latter was found to correlate much more strongly with the %fat.

The original version of calculating the %fat was based on the parameters SL, BW and BL (dorsal) (Ryg et al. 1990). However, as BL (sternum) is more commonly available in databases it was of great interest to find out whether it could be used instead. In order to do this, BL (dorsal) and BL (sternum) were plotted against each other for a unique data set of 114 seals from the NRM that contained data on both the dorsal and sternum measures. A strong positive correlation was found, the dorsal measure being on average 11.4 % thicker (Fig. 2). These findings suggest that the use of BL (sternum) instead of BL (dorsal) is acceptable if that is the only measure available. However, it may be advisable to correct for the difference between them, this could be achieved by increasing all BL (sternum) values by 11.4 % before calculating the index.

In all three seal species the %fat was significantly positively correlated to BL (sternum)/BW and BL (sternum)/SL, and in all cases the latter correlation was much stronger. In the grey and harbour seals the r value for the latter correlation was 0.91, and in the ringed seal it was as high as 0.97 (Fig. 6, Table 2 and Fig. 8 respectively). In the grey and ringed seals the correlation between the %fat and BL (sternum)/SL (Table 2 and Fig. 8) was even stronger than the one between the %fat and BL (sternum)(Figs. 3 and 7), in the harbour seal it was also stronger but only negligibly so (Figs. 5 and 6). Thus, in the grey and in the ringed seals, using BL (sternum)/SL instead of just BL (sternum) alone as an indicator of body condition, might be a good option if data on body weight is missing, thus preventing the calculation of %fat.

In both the harbour and ringed seals but not the grey seal, a significant positive relationship was found between the %fat and BW, as well as between the %fat and BW/SL. In the harbour seal but not the other seals, a significant positive relationship was also found between the %fat and SL, this correlation being slightly stronger than the one between the %fat and BW (Table 2). However, since none of these correlations were particularly strong, it would be advisable not to use these indices if any of the previously mentioned, better indices are available.

It must be added here, that the aforementioned correlations between %fat (LMD-index) and the different body condition indices must be interpreted with caution since BL (sternum), BW and SL are all included in the calculation of the LMD-index, and one might thus expect a correlation for this reason alone. On the other hand, since the LMD-index has been previously shown to be a very accurate estimate of the true fat content of the body when compared to dissection data, one might argue that if the condition indices correlate well with the LMD-index, then it is not unreasonable to think that they would also correlate well with the true fat content of the body as verified by dissection. Nevertheless, this data dependency must still be considered as a possible source of error in this study.

Intra- and interannual variation

Both grey and harbour seals’ %fat was found to vary seasonally, being lower in spring and higher in fall/winter (Figs. 9 and 10 respectively). This pattern is in agreement with previous studies on seasonal variation in blubber thickness (Nilssen et al. 1997; Bäcklin et al. 2013; Hauksson 2013).

A significant decline was found in the fall/winter %fat of the healthy sexually mature female grey seals caught by hunting over the time period 2002-2012, the average decrease rate being 0.84 % per year (Fig. 11). There were also negative trends in mean BL (sternum) in all age and sex classes among the grey seals, however none of these proved significant (Appendix 2, Figs. 16- 18). Perhaps with larger sample sizes these trends would also have proved significant, as was the case in a previous study examining the variation in blubber thickness of Baltic seals (Bäcklin et al. 2013). By contrast the mean BL (sternum) in harbour seals displayed a positive trend, this was however also non-significant. A possible source of error is the choice of simple regression analysis for testing the significance of the interannual trends, more sophisticated (and perhaps more appropriate) statistical modelling was not used due to the limited time span of this study.

If there really is a declining trend in %fat, as the present study suggests for the healthy sexually mature grey seal females, this could prove to be a serious problem for both adult survival and reproductive success. This is because, like previously mentioned, blubber is important both for individual survival and reproduction, being of utmost importance for lactation and cub survival. The latter is especially true for a species like the grey seal which has maternal fasting during the whole lactation period, depending exclusively on stored fat for survival and cub rearing. Such reductions in fecundity due to thinning of the seals is not unheard of, and even increases in the ages of maturity for both sexes has been documented for Barents Sea harp seals, Phoca groenlandica (Kjellqwist et al. 1995). The fact that only healthy females were used makes this finding even more interesting since this has not been previously shown for this source material (Karin Hårding, personal communication, January 18, 2016). The underlying cause for the declining trend in %fat of the seals is unknown. It could be due to several factors, including disease, decreasing fish stocks, changes in diet or changes in the quality of diet. However, no link to disease has been found so far in the Baltic seals (Bäcklin et al. 2013). Another reason could be that the populations are reaching carrying capacity and the observed decrease is due to increased population densities with concomitant increases in food competition. This is unlikely though, as the Baltic grey seal populations are well below historical levels (Harding et al. 2013). It could be however, that due to competition with the modern fishing industry, pollutants and other factors introduced to the environment in modern times, the carrying capacity of the environment has changed compared to its historical state.

Energetic comparison

In order to get a better sense of the variability in blubber content/nutritional status of the juvenile seals and its implications, a relatively simplistic energetic comparison was done between the fattest and thinnest healthy juvenile (1 year-old) grey seal caught during fall/winter (when they are at their fattest). The absolute blubber mass in kg was found to vary substantially, with the fattest individual having as much as 31.57 kg of blubber while the thinnest one only had 14.48 kg (fig. 12).

To be able to level out this gap in absolute blubber mass, the thinnest seal would therefore have to gain 17.09 kg in additional blubber (Table 3). How much it would need to eat and over how long a time period to accomplish this was calculated by taking into account the fact that metabolism is not 100 % efficient, the costs of maintenance and foraging and transport between haul-out and foraging sites.

In order to simplify the calculations, the seal was assumed to have a diet consisting solely of herring. The daily average amount of herring eaten was 1.22 kg, as it was calculated from the average daily energy intake which was assumed to be constant since the seal reasonably would not be able to eat much more than a certain amount per day (Härkönen and Heide-Jørgensen 1991). It was found that the seal would need to eat a total amount of 490.92 kg of herring over a time period of 403.54 additional days (Table 4). These results suggest that the difference in absolute blubber mass/nutritional status between the fattest and thinnest seals corresponds to a substantial amount of ingested food. It points to the conclusion that it will be impossible for the thinnest seals to reach the same blubber content as the fattest in a cohort, thus a good early start with lactation and early foraging success during the first month is pivotal.

A caveat must be mention here, namely that these results must be interpreted with caution, since all of the calculations relied on numbers previously applied only to juvenile harbour and not grey seals, and a series of rather simplistic assumptions were made, due primarily to the limited timescale available for this study. Examples of possible sources of error include that the value for mean daily energy intake was based on a low end estimate from a previous study and that the fact that the seal’s energy intake, maintenance energy requirement as well as foraging and transport costs would likely be altered as the seal grew each consecutive day was not accounted for. Thus, the current results are likely to be an underestimate of the seal’s true feeding capacity and growth rate. However, they still illustrate the order of magnitude of energy requirements and that there exists substantial differences among healthy juvenile grey seals with regard to blubber content/nutritional status, and that these differences may reflect differences in factors such as lactation and foraging success. Furthermore, it should be noted that since only healthy seals were selected for the calculations, disease related differences in blubber content/nutritional status can be excluded.

Conclusions

The sternum blubber thickness, the most widely used measuring site of blubber thickness in pinnipeds, was found to correlate well with the %fat in all three species studied here. Thus, suggesting that it is a good indicator of total blubber content/nutritional status. Dorsal and sternum blubber thicknesses were also found to correlate well with each other, suggesting that sternum blubber thickness can substitute the dorsal measure for calculating %fat if the latter is missing. A correction to compensate for the difference between the two can be done by increasing all sternum values by 11.4 % before calculating the %fat. In agreement with previous studies, a seasonal variation in %fat, with a decrease in spring and increase in fall/winter was found. A significant decrease in the %fat was found over the years 2002-2012 for sexually mature grey seal females. This trend could pose a future problem for both individual survival and reproduction. The underlying cause is as yet unknown, but may be related to factors such as decreasing fish stocks, dietary changes and population density related factors. Absolute blubber mass of juvenile grey seals varied widely among individuals. Simple energetic modelling suggested that these differences in blubber content corresponded to substantial differences in amounts of ingested food between fatter and leaner seals of the same cohort. This may reflect differences in lactation and foraging success. Thus, a good early start in the life of the juveniles, with proper lactation and good early foraging success seems to be crucial.

Acknowledgements

I would like to thank my supervisor at the Department of Biological and Environmental Sciences (BioEnv) at the University of Gothenburg, Karin Hårding, who introduced me to the study subject, and helped me throughout the entire project. I would also like to thank Britt Marie Bäcklin, Tero Härkönen and Charlotta Moraeus at the NRM for providing me with the data, as well as Kamran Chabokdavan at BioEnv for helping me with manual input of data, interpretation of the results and general advice.

References

Abt, K. F., N. Hoyer, L. Koch and D. Adelung (2002). "The dynamics of grey seals (Halichoerus grypus) off Amrum in the south-eastern North Sea — evidence of an open population." Journal of Sea Research 47(1): 55-67. Andersen, S. M., J. Teilmann, P. B. Harders, E. H. Hansen and D. Hjøllund (2007). "Diet of harbour seals and great cormorants in Limfjord, Denmark: interspecific competition and interaction with fishery." ICES Journal of Marine Science: Journal du Conseil 64(6): 1235-1245. Arnould, J. P. (1995). "Indices of body condition and body composition in female Antarctic fur seals (Arctocephalus gazella)." Marine Mammal Science 11(3): 301-313. Arnould, J. P., I. L. Boyd and J. R. Speakman (1996). "Measuring the body composition of Antarctic fur seals (Arctocephalus gazella): validation of hydrogen isotope dilution." Physiological Zoology: 93- 116. Bowen, W., C. E. Heyer, J. I. McMillan and S. J. Iverson (2015). "Offspring size at weaning affects survival to recruitment and reproductive performance of primiparous gray seals." Ecology and evolution 5(7): 1412-1424. Bowen, W. D., O. T. Oftedal and D. J. Boness (1992). "Mass and energy transfer during lactation in a small phocid, the harbor seal (Phoca vitulina)." Physiological Zoology: 844-866. Bäcklin, B.-M., C. Moraeus, K. Kauhala and M. Isomursu (2013). "Nutritional status of seals." HELCOM Core Indicator Report. Fish, F. E. (2000). "Biomechanics and Energetics in Aquatic and Semiaquatic Mammals: Platypus to Whale*." Physiological and Biochemical Zoology 73(6): 683-698. Harding, K. C., M. Fujiwara, Y. Axberg and T. Härkönen (2005). "Mass‐dependent energetics and survival in Harbour Seal pups." Functional Ecology 19(1): 129-135. Harding, K. C., T. Härkönen, B. Helander and O. Karlsson (2007). "Status of Baltic grey seals: Population assessment and extinction risk." NAMMCO Scientific Publications 6: 33-56. Hauksson, E. (2013). "Growth and reproduction in the Icelandic grey seal." NAMMCO Scientific Publications 6: 153-162. Härkönen, T., A. Galatius, S. Bräeger, O. Karlsson and M. Ahola (2013). Population growth rate, abundance and distribution of marine mammals, Helsinki Commission-HELCOM. Härkönen, T. and M.-P. Heide-Jørgensen (1991). "The harbour seal Phoca vitulina as a predator in the Skagerrak." Ophelia 34(3): 191-207. Jüssi, M., T. Härkönen, E. Helle and I. Jüssi (2008). "Decreasing ice coverage will reduce the breeding success of Baltic grey seal (Halichoerus grypus) females." AMBIO 37(2): 80-85. Kauhala, K., M. Kurkilahti, M. P. Ahola, A. Herrero, O. Karlsson, M. Kunnasranta, R. Tiilikainen and M. Vetemaa (2015). "Age, sex and body condition of Baltic grey seals: Are problem seals a random sample of the population?" Annales Zoologici Fennici, BioOne 52(1-2):103-114 Kjellqwist, S. A., T. Haug and T. Øritsland (1995). "Trends in age-composition, growth and reproductive parameters of Barents Sea harp seals, Phoca groenlandica." ICES Journal of Marine Science: Journal du Conseil 52(2): 197-208. Kokko, H., J. Lindström and E. Ranta (1997). "Risk analysis of hunting of seal populations in the Baltic." Conservation Biology 11(4): 917-927. Kovacs, K., Lowry, L. & Härkönen, T. (2008). " Pusa hispida. The IUCN Red List of Threatened Species." Retrieved 2015-05-21, from http://www.iucnredlist.org/details/41672/0. Lundström, K., O. Hjerne, S.-G. Lunneryd and O. Karlsson (2010). "Understanding the diet composition of marine mammals: grey seals (Halichoerus grypus) in the Baltic Sea." ICES Journal of Marine Science: Journal du Conseil 67(6): 1230-1239. Markussen, N. H., M. Ryg and N. A. Øritsland (1990). "Energy requirements for maintenance and growth of captive harbour seals, Phoca vitulina." Canadian Journal of Zoology 68(3): 423-426. Mellish, J.-A. E., M. Horning and A. E. York (2007). "Seasonal and spatial blubber depth changes in captive harbor seals (Phoca vitulina) and Steller's sea lions (Eumetopias jubatus)." Journal of Mammalogy 88(2): 408-414. Mellish, J.-A. E., P. A. Tuomi and M. Horning (2004). "Assessment of ultrasound imaging as a noninvasive measure of blubber thickness in pinnipeds." Journal of Zoo and Wildlife Medicine 35(1): 116-118. Nilssen, K., T. Haug, P. Grotnes and V. Potelov (1997). "Seasonal variation in body condition of adult Barents Sea harp seals (Phoca groenlandica)." Journal of Northwest Atlantic Fishery Science 22: 17-25. Noren, S. R., L. E. Pearson, J. Davis, S. J. Trumble and S. B. Kanatous (2008). "Different thermoregulatory strategies in nearly weaned pup, yearling, and adult Weddell seals (Leptonychotes weddelli)." Physiological and Biochemical Zoology 81(6): 868-879. Noren, S. R., M. S. Udevitz, L. Triggs, J. Paschke, L. Oland and C. V. Jay (2014). "Identifying a reliable blubber measurement site to assess body condition in a marine mammal with topographically variable blubber, the Pacific walrus." Marine Mammal Science 31(2): 658-676. Nyman, M., J. Koistinen, M. L. Fant, T. Vartiainen and E. Helle (2002). "Current levels of DDT, PCB and trace elements in the Baltic ringed seals (Phoca hispida baltica) and grey seals (Halichoerus grypus)." Environmental Pollution 119(3): 399-412. Oftedal, O. T., W. D. Bowen and D. J. Boness (1996). "Lactation performance and nutrient deposition in pups of the harp seal, Phoca groenlandica, on ice floes off southeast Labrador." Physiological Zoology: 635-657. Olsen, M. T., L. W. Andersen, R. Dietz, J. Teilmann, T. Härkönen and H. R. Siegismund (2014). "Integrating genetic data and population viability analyses for the identification of harbour seal (Phoca vitulina) populations and management units." Molecular Ecology 23(4): 815-831. Pitcher, K., D. Calkins and G. Pendleton (2000). "Steller sea lion body condition indices." Marine Mammal Science 16(2): 427-436. Reilly, J. J. and M. A. Fedak (1990). "Measurement of the body composition of living gray seals by hydrogen isotope dilution." Journal of Applied Physiology 69(3): 885-891. Ryg, M., C. Lydersen, N. H. Markussen, T. G. Smith and N. A. Øritsland (1990). "Estimating the blubber content of phocid seals." Canadian Journal of Fisheries and Aquatic Sciences 47(6): 1223-1227. Sparling, C. E., J. R. Speakman and M. A. Fedak (2006). "Seasonal variation in the metabolic rate and body composition of female grey seals: fat conservation prior to high-cost reproduction in a capital breeder?" Journal of Comparative Physiology B 176(6): 505-512. Stephens, P. A., A. I. Houston, K. C. Harding, I. L. Boyd and J. M. McNamara (2014). "Capital and income breeding: the role of food supply." Ecology 95(4): 882-896. Sundqvist, L., T. Harkonen, C. J. Svensson and K. C. Harding (2012). "Linking climate trends to population dynamics in the Baltic ringed seal: Impacts of historical and future winter temperatures." Ambio 41(8): 865-872. Thompson, D. and T. Härkönen. (2008a). "Halichoerus grypus. The IUCN Red List of Threatened Species. ." Retrieved 2015-09-02, from http://www.iucnredlist.org/details/9660/0. Thompson, D. and T. Härkönen. (2008b). "Phoca vitulina. The IUCN Red List of Threatened Species. ." Retrieved 2015-05-19, from http://www.iucnredlist.org/details/17013/0. Webb, P., D. Crocker, S. Blackwell, D. Costa and B. Boeuf (1998). "Effects of buoyancy on the diving behavior of northern elephant seals." The Journal of experimental biology 201(16): 2349-2358. Worthington Wilmer, J., P. Allen, P. Pomeroy, S. Twiss and W. Amos (1999). "Where have all the fathers gone? An extensive microsatellite analysis of paternity in the grey seal (Halichoerus grypus)." Molecular Ecology 8(9): 1417-1429. Appendix

1. Seasonal variation in blubber thickness

60

50

H. grypus 40

30 1-3 year-olds N=126

20 Mean BL (sternum) BL Mean

10

0 Apr May Jun Jul Aug Sep Oct Nov

Figure 13 Monthly mean sternum blubber thickness (mm) ±SD of healthy 1-3 year-old grey seals caught by hunting from April to November 2002-2012. At least 3 individuals per month were used.

90

80

70

H. grypus 60

50 4+ year-old females N=210 40

30 Mean BL (sternum) BL Mean 20

10

0 Apr May Jun Jul Aug Sep Oct Nov Dec

Figure 14 Monthly mean sternum blubber thickness (mm) ±SD of healthy 4+ year-old female grey seals caught by hunting from April to December 2002-2012. At least 3 individuals per month were used.

80

70

60 H. grypus

50

40 5+ year-old males N=132

30 Mean BL (sternum) BL Mean 20

10

0 Apr May Jun Jul Aug Sep Oct Nov

Figure 15 Monthly mean sternum blubber thickness (mm) ±SD of healthy 5+ year-old male grey seals caught by hunting from April to November 2002-2012. At least 3 individuals per month were used. 2. Interannual variation in blubber thickness

60

50

40 H. grypus

30 1-3 year-olds N=21

20 Mean BL (sternum) BL Mean

10

0 2005 2006 2007 2008 2009

Figure 16 Yearly mean sternum blubber thickness (mm) ±SD of healthy 1-3 year-old Grey seals caught by hunting from September to November over the years 2005-2009, p=0.069, R2=0.719. At least 3 individuals per year were used, the years excluded did not meet this minimal sample size. Non-significant negative trend.

90,00

80,00

70,00

H. grypus 60,00

50,00

40,00 4+ year-old females N=33

30,00 Mean BL (sternum) BL Mean 20,00

10,00

0,00 2002 2003 2007 2008 2009 2010 2011

Figure 17 Yearly mean sternum blubber thickness ±SD of healthy 4+ year-old female Grey seals caught by hunting from September to December over the years 2002-2011, p=0.211, R2=0.292. At least 3 individuals per year were used, the years excluded did not meet this minimal sample size. Non-significant negative trend.

90

80

70

60 H. grypus

50

40 5+ year-old males N=51

30 Mean BL (sternum) BL Mean 20

10

0 2002 2003 2005 2006 2007 2008 2009 2010 2011

Figure 18 Yearly mean sternum blubber thickness ±SD of healthy 5+ year-old male Grey seals caught by hunting from September to December over the years 2002-2011, p=0.702, R2=0.022. At least 3 individuals per year were used, the years excluded did not meet this minimal sample size. Non-significant negative trend.