University of Veterinary Medicine Hannover

Analysis of the reproductive biology of harbour porpoises (Phocoena phocoena) from the North and Baltic Sea on a long-term basis

Inaugural-Dissertation in fulfillment of the requirements of the degree of - Doctor medicinae veterinariae - (Dr. med. vet.)

submitted by Tina Kesselring Rostock

Hannover 2019

Academic supervision:

1st TiHo supervisor: Prof. Prof. h. c. Dr. Ursula Siebert, Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation

2nd TiHo Supervisor: Prof. Dr. Ralph Brehm, Institute for Anatomy, University of Veterinary Medicine Hannover, Foundation

1st Referees:

Prof. Prof. h. c. Dr. Ursula Siebert, Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation

Prof. Dr. Ralph Brehm, Institute for Anatomy, University of Veterinary Medicine Hannover, Foundation

2nd Referee:

Prof. Dr. Andreas Beineke, Institute for Pathology, University of Veterinary Medicine Hannover, Foundation

Day of the oral examination: 01.04.1019

Funding statement: This project was partly funded by the Ministry of Energy, Agriculture, the Environment, Nature and Digitalization Schleswig Holstein (MELUND) and the state centre for coastal protection, national park and ocean protection Schleswig – Holstein.

To my mother and my grandparents

‘For most of history, man has had to fight nature to survive; in this century he is beginning to realize that, in order to survive, he must protect it.’

Jacques Yves Cousteau

Parts of this thesis have been published in peer reviewed journals:

Publication I

Kesselring T, Viquerat S, Brehm R, Siebert U.

Coming of age: - Do female harbour porpoises (Phocoena phocoena) from the North Sea and Baltic Sea have sufficient time to reproduce in a human influenced environment?

Published in October 2017: PLoS One 2017; 12. doi:10.1371/journal.pone.0186951.

Corrected version was published in June 20, 2018. Correction: Coming of age: - Do female harbour porpoises (Phocoena phocoena) from the North Sea and Baltic Sea have sufficient time to reproduce in a human influenced environment? PLoS ONE 13(6): e0199633. https://doi.org/10.1371/journal.pone.0199633

Publication II

Kesselring, T, Viquerat, S, IJsseldijk, LL, Langeheine, M, Wohlsein, P, Gröne, A, Bergmann, M, Siebert, U, Brehm, R.

Testicular morphology and spermatogenesis in harbour porpoises (Phocoena phocoena)

Published online in November 2018 in Theriogenology, Theriogenology 126 (2019) 177-186 DOI: 10.1016/j.theriogenology.2018.11.031

Parts of this thesis have been presented in poster presentations and an oral presentation at the following conferences:

Poster presentations:

Kesselring, T.; Viquerat, S.; Wohlsein, P.; Langeheine, M.; Gröne, A.; Ijsseldijk, L.; Bergmann, M.; Siebert, U.; Brehm, R. : Seasonal effects on testicular development of harbour porpoises from the North and Baltic Sea.

In: Reproduction in Domestic Animals, special Issue: 50th Annual Conference of Physiology and Pathology of Reproduction and 42nd Mutual Conference on Veterinary and Human Reproductive Medicine, Munich, Germany, 15th - 17th February 2017. Volume 52, Issue Supplement S1, Pp 28-29

31st Annual Conference of the European Cetacean Society, Middelfart, Denmark, 29th April - 03rd Mai, 2017

Kesselring, T.1, Viquerat, S. 1, Brehm, R. 2, Siebert, U. 1: Identifying sexual maturity in female harbour porpoises (Phocoena phocoena) from the North and Baltic Seas using ovarian characteristics

22nd Biennial Society for Marine Mammalogy Conference on the Biology of Marine Mammals, Halifax, Canada, 22nd - 27th October 2017

Kesselring, T.; Viquerat, S.; Bergmann, M.; Isseldijk, L.; Gröne, A.; Langeheine, M.; Wohlsein, P.; Brehm, R.; Siebert, U.: New insights into the reproductive biology of harbour porpoises (Phocoena phocoena) from the North and Baltic Sea. DOI: 10.13140/RG.2.2.32302.20806

Oral presentation:

Kesselring, T.; Viquerat, S.; Wohlsein, P.; Langeheine, M.; Gröne, A.; Ijsseldijk, L.; Bergmann, M.; Siebert, U.; Brehm, R. Assessment of spermatogenesis in harbour porpoises (Phocoena phocoena) from the North and Baltic Seas.

In: Reproduction in Domestic Animals, special Issue: 51th Annual Conference of Physiology and Pathology of Reproduction and 43rd Mutual Conference of Veterinary and Human Reproductive Medicine, Hanover, Germany, 21st – 23rd February 2018. Volume 53, Issue Supplement S1, P 21, DOI: 10.1111/rda.13127

List of contents

Chapter 1: Introduction ...... - 1 - 1.1. General background ...... - 1 - 1.2. First part of the thesis: study of age at sexual maturity and demography in female harbour porpoises ...... - 4 - 1.3. Second part of the thesis: study of testicular morphology and spermatogenesis in male harbour porpoises ...... - 6 - Chapter 2: Publication I: Correction: Coming of Age: - Do female harbour porpoises (Phocoena phocoena) from the North Sea and Baltic Sea have sufficient time to reproduce in a human influenced environment? ...... - 9 - 2.1. Abstract ...... - 10 - 2.2. Introduction ...... - 11 - 2.3. Materials and Methods ...... - 13 - 2.3.1. Sample collection...... - 13 - 2.3.2. Age assessment ...... - 13 - 2.3.3. Ovarian examination ...... - 14 - 2.3.4. Data analysis ...... - 14 - 2.4. Results ...... - 16 - 2.5. Discussion ...... - 24 - 2.6. Acknowledgements ...... - 29 - 2.7. References ...... - 30 - Chapter 3: Publication II: Testicular development and spermatogenesis in harbour porpoises (Phocoena phocoena) ...... - 37 - 3.1. Abstract ...... - 38 - 3.2. Introduction ...... - 39 - 3.3. Materials and Methods ...... - 41 - 3.3.1. Sample collection...... - 41 - 3.3.2. Histological preparation ...... - 44 - 3.3.3. Age determination...... - 44 - 3.3.4. Histological examination ...... - 44 - 3.3.5. Modelling the diameter of the convoluted seminiferous tubules over the Julian day of the year ...... - 45 - 3.4. Results ...... - 47 - 3.4.1. Macroscopic findings of the testes during the course of the year .. - 47 - 3.4.2. Testicular maturation and staging of the seminiferous epithelium . - 48 - 3.4.3. Morphometrical analysis and maturity classes ...... - 56 -

3.5. Discussion ...... - 60 - 3.5.1. Testicular maturation and staging of the seminiferous epithelium . - 60 - 3.5.2. Morphometrical analysis and age groups ...... - 61 - 3.6. Conclusions ...... - 63 - 3.7. Acknowledgements ...... - 64 - 3.8. References ...... - 65 - Chapter 4: Discussion ...... - 73 - 4.1. General discussion ...... - 73 - 4.2. First part of the thesis: study of age at sexual maturity and demography in female harbour porpoises ...... - 75 - 4.3. Second part of the thesis: study of testicular morphology and spermatogenesis in male harbour porpoises ...... - 80 - Chapter 5: Summary ...... - 85 - Chapter 6: Zusammenfassung ...... - 87 - References ...... - 90 - Chapter 7: Acknowledgements ...... - 101 -

List of abbreviations

ASCOBANS Agreement on the Conservation of Small Cetaceans of the Baltic, North East Atlantic, Irish and North Seas

AWZ Ausschließliche Wirtschaftszone

°E Degree East longitude

µm micrometer b_corpora binomial response describing presence or absence of corpora c1 corpora Model 1 c2 corpora Model 2

CI 95% Confidence interval cm centimeter dev explained deviance e.g. exempli gratia et al. et alii

Fig 1 Figure 1

Fig 2 Figure 2

Fig 3 Figure 3

Fig 4 Figure 4 gam Generalized additive model

GCV Generalized cross validation score

GLG Growth Layer Groups

GnRH Gonadotropin-releasing-hormone

GSI Gonadosomatic Index

HELCOM Helsinki Commission

H & E Haematoxylin and Eosin staining

IWC International whaling commission

ITAW Institute for Terrestrial and Aquatic Wildlife Research

IUCN International Union for Conservation of Nature kg kilogram m4 model 4

PAS Periodic acid-Schiff reaction

PCB polychlorinated biphenyls

Sm Seemeilen

List of figures

Chapter 1 Introduction:

Figure 1: Fresh harbour porpoise carcass, stranded in October 2016. Copyright: Institute for Terrestrial and Aquatic Wildlife Research……………………………. - 2 -

Figure 2: Figure 2: Seasonal distribution of harbour porpoises in the German exclusive economic zone of the North Sea based on survey flights 2002 – 2006 during winter (left side) and summer (right side) (Herr 2009)……………………… - 2 -

Figure 3: Testis of an adult harbour porpoise from the high mating season in July (above) and from the low mating season in December (below)…………………… - 6 -

Figure 4: Figure 4: Boxplot of testes ratio (testis weight / total body weight) of adult harbour porpoises during the course of the year (n = 83). The black line indicates the median, the blue box indicates the 95 % confidence interval and the dotted lines show minimum and maximum ratios per months, respectively. The x – axis indicates the month of stranding / bycatch of the carcass (including number of carcasses), the y – axis shows the relative testes mass in percent of total body weight.………………………………………...... - 7 -

Chapter 2 Publication I:

Figure 1: Mid-longitudinal section of a tooth from a neonate (A) and an adult (B) harbour porpoise (Phocoena phocoena)…………………………………………….. - 14 -

Figure 2: Population structure of female harbour porpoises of the North Sea (green bars) and Baltic Sea (red bars). Proportion of animals indicates the proportion of animals relative to the total number of animals within a given age bracket. Round brackets indicate exclusive values square brackets indicate inclusive values…... - 17 -

III

Histological section showing the Cumulus oophorus with the oocyte surrounded by the Zona pelucida and Corona radiata. Asterisk: Antrum folliculare. Staining: H&E. (F): Histological section through a Corpus albicans showing an increased amount of connective tissue (asterisk). Staining: Masson´s Trichrome………………………..- 19 -

Figure 4: Binomial regression model showing the functional relationship between the presence of corpora (purple polygon) in female harbour porpoises from the German North Sea and Baltic Sea in relation to their age as determined by GLG. The filled area indicates the 95% confidence interval of predicted values. The red line within that area indicates the predicted average probability of finding corpora within specimens at the given age; the blue dashed line marks a probability of 50%, with the red solid line indicating the corresponding age based on the intersection of the blue dashed line with the x-axis; samples are indicated as dark grey dots; a purple circle around data points indicates animals below the age threshold showing at least one corpus, an orange circle indicates animals above the threshold showing no corpora; background histogram (orange) shows number of animals (secondary y-axis) across tooth age classes (at 1-year intervals)……………………………………………… - 21 -

Chapter 3 Publication II:

Figure 1: Cross section through the seminiferous epithelium of a neonate (A) and juvenile (B, C and D) harbour porpoise. The arrow in (B) signals a spermatogonium and the arrow in (D) indicates a mitotic germ cell. The juvenile individual was found in April, length: 118 cm. Staining: H&E, primary magnification: X 40……………….. - 49 -

Figure 2: Cross section through the seminiferous epithelium of adult harbour porpoises found in January (A), May (B) and September (C). (A) Sexual inactive adult individual found in January: tubules contain only a single layer of spermatogonia and Sertoli cells along the basement membrane. Magnification: X 40. (B) Additional germ cells can be found in a harbour porpoise from May. Primary magnification: X 40. (C) Activity of spermatogenesis is decreasing in August and September, only some residual spermatozoa can be identified in the lumen of the tubules. Staining: H&E, primary magnification: X 40……………………………………………………………. - 50 -

Figure 3: Summary of samples from adult harbour porpoise testes from the North IV and Baltic Sea and their reproductive status based on testicular activity, that has been investigated through histological evaluation; the x-axis indicates the month the carcasses have been found (Jan1 indicates one specimen within January that was attributed with ‘decreasing activity’, Jun2 indicates there was one animal found in June that showed ‘increasing activity’); the y-axis indicates the number of specimens studied; the background colours identify the conclusion of the reproductive status drawn based on the morphological histological appearance of testes: blue / inactive: no sign of activity or only a few isolated germ cells present in the convoluted seminiferous tubules; pink / increasing activity: increase in abundance of germ cells and some spermatogenic stages can be identified; yellow / active: active reproduction with all eight stages present; green / decreasing activity: only isolated residual germ detected…………………………………………………………………………………..- 52 -

Figure 4: Cross section through the seminiferous epithelium of an adult harbour porpoise found in July, showing a multi-stage-arrangement in one cross section. Bars indicate borders between the stages. Staining: H&E, primary magnification: X 40………………………………………………………………………………………. - 52 -

Figure 5: Staging of the seminiferous epithelium as featured by morphology staining: PAS,primary magnification: X 40…………………………………………………….. - 54 -

Figure 6: Stages of spermatogenesis in the harbour porpoise (modified according to [42]……………………………………………………………………………………….. - 55 -

Figure 7: Cross section through the seminiferous epithelium of an adult harbour porpoise found in July. The arrow indicates an area of missing germ cells. Staining: H&E, primary magnification: X 20…………………………………………………….. - 56 -

Figure 8: Predicted diameter of tubules per maturity class based on model m4 from generalized additive modelling. The light green area indicates the prediction for non- adult specimens; the horizontal solid purple line indicates the average tubules diameter of all specimens across the full year; the horizontal thick red line indicates the average diameter of the convoluted seminiferous tubules of adult specimens across the full year. The light red area indicates the predicted the diameter the convoluted seminiferous tubules of adult specimen across the full year; red and green dots mark the dataset of non-adult and adult specimens, respectively. The

V dotted vertical lines indicate the time of year with predicted the convoluted seminiferous tubules diameter above the mean the convoluted seminiferous tubules diameter (in all animals; corresponding Julian days next to respective lines)…… - 59 -

List of tables

Chapter 2 Publication I:

Table 1: Quartile age distribution of harbour porpoises from the German North Sea and Baltic Sea between 1996 and 2017……………………………………………... - 16 -

Table 2: Overview of analysed specimens………………………………………...…- 20 -

Table 3: Model diagnostics for the binomial regression model of the probability of detecting corpora (models c1 and c2)………………………………………………...- 22 -

Table 4: Percentile of all animals found between 1990 and 2017 that were older than the estimated threshold age of reaching sexual maturity of the population……...- 23 -

Chapter 3 Publication II:

Supplemental table 1: Sample collection of adults, juveniles and neonate in accordance to the month of stranding or bycatch. N indicates the total number of the specimens, month indicates the month of sampling……………………………….. - 43 -

Table 1: Summary of average age, diameter of convoluted seminiferous tubules and GSI per maturity class. Subset: identifies the maturity class as assessed by experts during the dissection (all: all specimen regardless of maturity class; adults; non- adults); Ø age: mean age in respective subset using GLG (± standard error); Ø convoluted seminiferous tubules: diameter of convoluted seminiferous tubules (± standard error) [µm]; Ø GSI: gonadosomatic index (± standard error) [%] (sensu Barber and Blake 2006) [43]…………………………………………………………...- 48 -

Table 2: Model diagnostics of the generalized additive modelling analysis. Model: identifier of the model as used throughout the text; covariates: combination of smoother functions and covariates as supplied to the gam; N: sample size available used in the analysis (numbers vary due to missing values in some covariate VI combinations); dev: explained deviance; GCV: generalized cross validation score. Chosen model in bold (m4)…………………………………………………………… - 58 -

VII

Introduction

1.1. General background

Harbour porpoises (Phocoena phocoena) are the only cetacean species native to German waters (Addink and Smeenk, 1999; Benke et al., 1998; Kinze, 1990). Their natural environment comprises shallow coastal waters of the northern hemisphere (Addink and Smeenk, 1999; Benke et al., 2014; Gaskin et al., 1984; Kinze, 1990). The harbour porpoise from the central Baltic Sea has been listed as critically endangered in the IUCN (International Union for Conservation of Nature) Red List of 2008 (Hammond et al., 2008). Although commercial exploitation stopped when Germany signed the IWC moratorium in 1973, the harbour porpoise as a top predator still faces adverse anthropogenic influences that affect the populations. Due to these increasing threats that harbour porpoises are exposed to, several international agreements for the protection of the marine environment including marine mammals have been made in the last decades. Depending on the area, different countries and areas are involved. For the North and Baltic Sea, the Agreement on the Conservation of Small Cetaceans of the Baltic, North East Atlantic, Irish and North Seas (ASCOBANS) and the Baltic Marine Environment Protection Commission - Helsinki Commission (HELCOM) serve as good examples for such protection agreements. Within these conventions, several measures are implemented – for example the ascertainment of protected areas, fishery restrictions or threshold values for marine noise or toxic substances in an ecosystem. This data can only be gathered, if basic knowledge about important parameters of a population, like knowledge about the reproductive capacity is present and available.

For German waters, research about the populations of harbour porpoises relies on observations and annual population counts and as well as on post-mortem examinations of stranded or by - caught animals that have been collected after their death. In order to organise collection of strandings and bycatches, a so-called stranding network has been established along the coast of Schleswig-Holstein, which supports the documentation and pathological analysis of the majority of the carcasses (Figure 1).

- 1 -

Figure 1: Fresh harbour porpoise carcass, stranded in October 2016. Copyright: Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hanover, Foundation

During the survey flights that are conducted every year in the spring to autumn, a different distribution and abundance of individuals in the German area of the North Sea has been observed for several years (Figure 2). During summer months, a high number of individuals including recently born calves accumulate in the area of the Sylt outer reef (Gilles et al., 2009; Gilles et al., 2016) and give an idea of a strong seasonal pattern in reproduction.

Figure 2: Seasonal distribution of harbour porpoises in the German exclusive economic zone of the North Sea based on survey flights 2002 – 2006 during winter (left side) and summer (right side) (Herr 2009). AWZ = Auschließliche Wirtschaftszone; 12 sm Zone = 12 Seemeilen Zone

Furthermore, post mortem examinations of harbour porpoises from other areas reveal further indices for a seasonal reproduction cycle (Read and Hohn, 1995; - 2 -

Karakosta et al., 1999; Lockyer et al., 2001; Learmonth et al., 2014) of this species like the fact that females ovulate seasonally and show a main period for mating and calving between May and August with a peak calving period in June (Lockyer, 1995; Lockyer and Kinze, 2003; Murphy et al., 2010; Learmonth et al., 2014). The gestation period is known to last 10 to 11 months, lactation approximately eight to eleven months (Fisher and Harrison, 1970; Gaskin et al., 1984; Lockyer and Kinze, 2003). For German waters, stranding pattern confirmed these findings (Hasselmeier et al., 2004).

Corresponding to these data about seasonality in female harbour porpoises, seasonal increase in testes mass and weight with sperm present in the epididymis during the peak season have been observed in male individuals (Fontaine and Barrette, 1997; Karakosta et al., 1999; Neimanis et al., 2000).

In order to assess some essential parameters of reproduction in male and female harbour porpoises from the German area, this study aims to obtain basic and fundamental knowledge about different aspects of reproduction such as age at sexual maturity, seasonality and germ cell maturation of this species in the North and Baltic Sea. Thus, the first part of the thesis deals with the analysis of the ovaries of harbour porpoises from German waters in order to identify the exact value for the attainment of female sexual maturity. Combined with a demographic analysis of the populations, a statement for the reproductive potential for both, the North Sea and the Baltic Sea can be made.

While at least some information about the reproductive biology of female harbour porpoises exists, even less data about morphology of the male reproductive organs, the histological appearance of the testis and physiological information about spermatogenesis in male individuals is available. Therefore, the second part of the thesis aims to obtain basic knowledge about the reproductive biology of male harbour porpoises.

- 3 -

1.2. First part of the thesis: study of age at sexual maturity and demography in female harbour porpoises

While for female harbour porpoises from other oceans some knowledge about life history is available, little information about reproductive parameters is given for the North- and the Baltic Sea. Until now, only a few investigations deal with age at sexual maturity and puberty of female harbour porpoises from German waters (Bandomir et al., 1998; Kinze, 1990; Lockyer and Kinze, 2003). In general, sexual maturity in females is reached at the point an individual is capable of taking part in reproduction (Vasantha and Kona, 2016). Like in mammals in general, this coincides with the first ovulation event in marine species either (Perrin and Reilly, 1984; DeMaster, 1984; Silver, 2001). Mammalian ovaries contain large numbers of follicles, which undergo a maturing process and finally release the oocyte (Schroeder and Talbot, 1985; Morioka et al., 1989; Gonçalves et al., 2012). If not followed by an ovulation, a follicle can regress and form a corpus atreticum. In order to prove that an ovulation has definitely occurred, ovarian features like corpora lutea and corpora albicantia have to be present on the ovary. Identification and counting of these features is the most common way in marine mammals to prove puberty (Perrin and Reilly, 1984) and has been performed for some species from different regions. For harbour porpoises from areas out of Germany, the age at sexual maturity is known to occur between the age of three to six years, depending on study areas and methodology (North Sea: Van Utrecht, 1978; Gaskin et al., 1984: age of five to six years, Benke et al., 1995: four years); Gulf of Maine: Read and Hohn, 1995: age of 3 years; England and Wales: Karakosta et al., 1999: no age given, but avarage body length at sexual maturity; Greenland: Lockyer et al., 2001: three to four years; Olafsdottir et al., 2002: two to four years); Scottish waters: Learmonth et al., 2014: four to five years). In harbour porpoises from Danish waters including the North Sea and Baltic Sea (1985 – 1991), sexual maturity was determined as starting at the age of 3.63 years (Lockyer and Kinze, 2003). In Dutch waters of the North Sea, early investigations (1955 – 1978) revealed that females are considered to be mature at an age of six years (Van Utrecht, 1978), but none of these studies focused on a long-term data collection and analyses, so that this would be a new feature in our study additional to the fact, that only a few data on reproduction of harbour porpoises from German waters exist so far (Bandomir et al., 1998; Benke et al., 1998; Lockyer and Kinze, 2003). The

- 4 - analysis of long-term data might be helpful to identify shifts that are induced by human activities and thus represent an indicator of an ongoing changing environment. In other marine mammalian species, changes in the onset of sexual maturity have been identified as possible results of shifts caused by natural or anthropogenic effects (Bando et al., 2014: Minke whales; Hohn et al., 2007: Spotted dolphins; Ohsumi, 1986: Fin whales). One of the anthropogenic influences that harbour porpoises are exposed to are contaminants that might have endocrine disrupting effects. As top predators harbour porpoises are sensitive to chemical influences (contaminants) that may accumulate in their blubber and have been accused to have a certain impact on their reproductive biology (Beineke et al., 2005; Das et al., 2004; Genov et al., 2018, Siebert et al., 1999). Some contaminants can pass the blood-testes-barrier, the placental-barrier and milk – blood - barrier and might influence by the way the reproductive physiology of marine mammals (Bennett et al., 2001; Berggren et al., 2002; Borgå et al., 2004; Jepson et al., 2016; Murphy et al., 2010; Siebert et al., 2001; Wiemann et al., 2010; Wolkers et al., 1999). There have already been reports on reproductive failure including foetal death, abortion, dystocia or stillbirth in female harbour porpoises from Scottish waters that may be connected to the concentration of high toxicants in the blubber of these animals (Murphy et al., 2010), but there is no knowledge on how these contaminants influence the reproductive organs. Therefore, some more basic knowledge in this field that defines the physiological status is strongly required.

- 5 -

1.3. Second part of the thesis: study of testicular morphology and spermatogenesis in male harbour porpoises

For male harbour porpoises, investigating the age at sexual maturity would not be as simple in methodology as in females, since the basic histological analysis of the gonads is required for the evaluation of this parameter. During post-mortem examinations it is possible to distinguish juveniles from adult male individuals through body length and body weight, but no exact value for the beginning of puberty has been identified yet. Analyses of adult harbour porpoises from other regions give evidence, that testis morphology and morphometry follows a strong seasonal pattern (Karakosta et al., 1999; Neimanis et al., 2000), likewise our observations in post- mortem examinations of individuals from the North and Baltic Sea. The mass and size of the testes of adult harbour porpoises varies extremely throughout the course of the year (Figure 3 and Figure 4).

Figure 3: Testis of an adult harbour porpoise from the high mating season in July (above, megatestis) and from the low mating season in December (below).

- 6 -

Figure 4: Boxplot of testes ratio (testis weight / total body weight) of adult harbour porpoises during the course of the year (n = 83). The black line indicates the median, the blue box indicates the 95 % confidence interval and the dotted lines show minimum and maximum ratios per months, respectively. The x – axis indicates the month of stranding / bycatch of the carcass (including number of carcasses), the y – axis shows the relative testes mass in percent of total body weight.

It has been previously reported, that testicular development and spermatogenesis in harbour porpoises occurs roughly in the same pattern as in terrestrial mammals (Karakosta et al., 1999; Neimanis et al., 2000). Seasonality has been documented by macroscopic and microscopic data comparison of specimens from every month of the year (Karakosta et al., 1999; Neimanis et al., 2000; Ólafsdóttir et al., 2002; Plön and Bernard, 2007). Furthermore the presence of various germ cell associations that occur simultaneously in one tubular cross section has been noted (Holt et al., 2004; Karakosta et al., 1999; Neimanis et al., 2000; Plön and Bernard, 2007). Based on measurements of the diameter of the seminiferous tubules throughout the course of the year it has been described, that a peak mating season exists, which is characterized through a maximum in tubular diameter (Karakosta et al., 1999; Neimanis et al., 2000; Plön and Bernard, 2007). These authors focused rather on the - 7 - presence of spermatozoa in the tubular lumen or in the epididymis, than on a detailed description of the spermatogenic process itself. However, with an explicit analysis of the stages of spermatogenesis it would be possible in the future to detect changes that might be the result of anthropogenic influences on harbour porpoises like increasing stress levels due to noise, pollution, disturbance and toxic substances. As endocrine disruptors are suspected to cause reproductive failure in females (Murphy et al., 2010; Ylitalo et al., 2005), these substances might of course also evoke alterations in the male germ cell development and spermatogenesis. In other species it has been observed, that spermatogenic waves can be interrupted, resulting in so called ‘missing generations’. This link to infertility on the level of spermatogenesis is a marker for impaired efficiency of spermatogenesis in men (Johnson et al., 1992), for endocrine disruption in rodents (Gely-Pernot et al., 2015; Hogarth et al., 2015) and might be assigned to marine mammals in the future.

The aim of the second part of the thesis was to provide baseline information on reproductive key parameters like age of sexual maturity and stages of spermatogenesis and their seasonal shift for male harbour porpoises from the North and Baltic Sea. With these parameters it could be possible to detect pathological changes giving evidence for impacts of anthropogenic factors that influence the physiology of reproduction.

- 8 -

Chapter 2: Publication I: Correction: Coming of Age: - Do female harbour porpoises (Phocoena phocoena) from the North Sea and Baltic Sea have sufficient time to reproduce in a human influenced environment?

Tina Kesselring1, Sacha Viquerat1, Ralph Brehm2, Ursula Siebert1*

1Institute for Terrestrial and Aquatic Wildlife Research (ITAW), University of Veterinary Medicine Hannover Foundation, Büsum, Germany

2Institute for Anatomy, University of Veterinary Medicine Hannover Foundation, Hannover, Germany

* Corresponding author

E-mail: [email protected] (US)

- 9 -

2.1. Abstract

The harbour porpoise is the only cetacean species native to German waters. Since human pressures are suggested to shorten their reproductive lifespan, basic knowledge on reproduction is strongly required. One parameter is the onset of sexual maturity in female harbour porpoises. Therefore, we investigated the first signs of sexual maturity for a period of almost two decades (1990 – 2016). Ovaries from 111 female harbour porpoises from the German North Sea and Baltic Sea were examined for the presence and morphological structure of follicles, corpora lutea and corpora albicantia. Based on the ovarian characteristics we performed the first model-based estimation of age at sexual maturity for harbour porpoises from German waters. Additionally, we produced a demographical age structure based on all female strandings and bycatches from German coasts. Our results showed that corpora lutea and corpora albicantia as signs of former ovulation could be found in individuals at an age of 4.95 (± 0.6) years. No significant differences between specimens from the North Sea and Baltic Sea were detected. However, the average age at death differed significantly with 5.70 (± 0.27) years for North Sea animals and 3.67 (± 0.30) years for those in the Baltic Sea. Growing evidence exists that the shortened lifespan of Baltic Sea harbour porpoises is linked to an anthropogenically influenced environment with rising bycatch mortalities due to local gillnet fisheries. Thus, our findings support the idea of local management plans based on a model-based detection of age at sexual maturity and considering the anthropogenic impacts on the population for effective protection of harbour porpoises and the North Sea and Baltic Sea.

Keywords: cetacean, reproduction, life history, demography, population growth, fecundity

- 10 -

2.2. Introduction

The harbour porpoise (Phocoena phocoena) is one of the smallest cetacean species and common in shallow coastal waters of the Northern hemisphere [1-3]. The mating season is considered to occur between June and September, the birth period from June to August [4-6]. Since harbour porpoises give birth only once a year, they are considered as a slowly reproducing species [7] and therefore depend on successful mating seasons. The age of sexual maturity is an important parameter for estimating the reproductive cycle of a species and for assessing the reproductive potential in a population. In general, sexual maturity is reached when an individual is capable of taking part in reproduction. For female mammals, this coincides with the first ovulation event [8]. Female harbour porpoises from areas outside the German North Sea and Baltic Sea attain sexual maturity at the age of 3 - 6 years, depending on the methodology used and which sub-population was assessed [5, 9-14]. In harbour porpoises from Danish waters including the North Sea and Baltic Sea (1985 – 1991), sexual maturity was determined as starting at the age of 3.63 years [5]. Specimens from West Greenland (1988 – 1995) showed an average age at sexual maturity of 2.45 years [13], while porpoises from the Bay of Fundy, Canada (1985 – 1988) reach sexual maturity between the age of 3.15 and 3.44 years [11]. In Scottish waters (1992 – 2005), harbour porpoises were found to be sexually mature at 4.35 years [14]. In Dutch waters, early investigations (1955 – 1978) revealed that females are considered to be mature at an age of 6 years [9]. Examining follicular activity and the presence and consistency of structures found on the ovaries, such as corpora lutea and corpora albicantia, is the most recommended way to monitor the individual status of reproduction and sexual maturity [8, 15]. Mammalian ovaries contain large numbers of follicles, which undergo a maturing process and release the oocyte [16, 17]. If not followed by ovulation, a follicle can regress and form a corpus atreticum. For other odontocetes, like Globalicephalus macrorhynchus, Physeter macrocephalus and Stenella attenuata, it is known that corpora atretica occur regularly at every stage of the oestrus cycle [8]. After ovulation has occurred, a corpus luteum is formed from residual cells of the Graafian follicle and functions as an endocrinal gland for the production of oestrogen and progesterone [18]. The corpus luteum is a distinct feature on the surface of the ovary that signals an ovulation event and thus sexual maturity [8]. After full efflorescence, a corpus luteum

- 11 - rearranges into a smaller structure on the ovarian surface and persists as a corpus albicans. Finally, after regressing almost completely, a small flat scar on the surface of the ovary may remain discernible for an unknown time [15, 18-21]. For other odontocetes, the attainment of the age at sexual maturity ranges according to their longevity between 2-3 years (franciscana dolphin), 7 years (bottlenose dolphin), 6-9 years (hectors dolphin) and 7-12 years (short-finned pilot whale) [22-25]. Although the harbour porpoise is the most abundant cetacean found along the beaches of the German North Sea and Baltic Sea [2], only a few studies have analysed the reproduction of females so far [2, 5, 26] and none of these undertook long-term data collections, but only carried out investigations of 3-5 year intervals [11, 13, 26]. In other species, changes in the onset of sexual maturity have been identified as possible results of shifts caused by natural or anthropogenic effects [27-30]. One major anthropogenic threat is the increasing bycatch rate in the Baltic Sea, which is suspected of reducing the living stocks of the harbour porpoise in this area [31-33]. Furthermore, toxic influx into the oceans through human activities are suspected drivers of reproductive failure [34-38] and have the potential to reduce the time span of reproduction in a species. Another aspect that is considered to be related to foetal loss and reduced fecundity (at least in other areas) is the incidence of infectious diseases such as infections with Brucella ceti [39]. This present study provides the first model-based estimation of age of sexual maturity of female harbour porpoises in the German North Sea and Baltic Sea considering mortalities attributable to anthropogenic causes to estimate the total number of females contributing to the reproductively active population.

- 12 -

2.3. Materials and Methods

2.3.1. Sample collection

For determining the demographical age of subpopulations a dataset of 526 female harbour porpoises was utilised. Specimens were collected within the German stranding network, which conducts work (collect and hold carcasses and samples from European protected species) on German strandings following appropriate licenses from the relevant authorities (Ministry of Energy, Agriculture, the Environment, Nature and Digitalization, Ministry of Agriculture, Environment and Rural areas). Most of the animals were found stranded along the coast of Schleswig Holstein of the North Sea and western Baltic Sea between 1990 and 2016, only a small number of animals have been identified as bycatch (n=159 between 1990 and 2014). All specimens were sampled during necropsies at the Institute for Terrestrial and Aquatic Wildlife Research (ITAW) in Büsum, Germany. Specimens were either dissected upon arrival or stored at -20°C prior to necropsy. All necropsies followed standardised protocols for harbour porpoises [40].

2.3.2. Age assessment

The extraction, preparation, processing and age assessment followed a standard protocol [41]. At least two different readers made three independent assessments of 15 different tooth sections per individual, resulting in over 30 independent age determinations per individual (Growth Layer Groups, hereafter referred to as GLG). All readings were averaged and approximated to the nearest whole year (Fig 1).

- 13 -

Figure 1: Mid-longitudinal section of a tooth from a neonate (A) and an adult (B) harbour porpoise (Phocoena phocoena).

2.3.3. Ovarian examination

The reproductive tract of 111 animals was extracted, weighed and measured. A 4% phosphate-buffered formalin solution was used for fixating the samples. The ovaries were sliced into 1mm thick sections and all corpora and ovarian scars were counted and measured under a microscope (magnification: 4x) to the nearest 0.01mm using digital callipers. The formalin – fixed ovaries were transferred to 70% ethanol, dissected and embedded in paraffin wax using standard techniques. Sections of 5 µm thickness were stained with hematoxylin and eosin (H and E) and assessed under a light microscope. A Masson´s trichrome staining was performed for identifying connective tissue in potential corpora albicantia. A specimen was assumed to be sexually mature when at least one corpus could be detected on the ovaries. All examinations adhered to the procedures and terminology as recommended by the International Whaling Commission [8].

2.3.4. Data analysis

In a first step, the information on the number of corpora per individual was translated into a binomial response value (b_corpora), indicating the presence or absence of

- 14 - corpora in each specimen. This was irrespective of the actual number of corpora, enabling us to identify the age at which we expected more than 50% of the animals to exhibit corpora within the ovaries (and thus, according to the above definition, to have entered the reproductive cycle).

Assuming a functional relationship between the specimen’s age and the binomial response variable b_corpora, we utilised a logistic regression model (formula 1) using a logit link (formula 2) testing for an effect of the North Sea and Baltic Sea specimen on the intercept in R 3.2.2 [42]:

( | ) ∏푛 ( ) ( ) 퐿 휇 푌 = 푖=1(1푦푖=1 휇푖 + 1푦푖=0 1 − 휇푖 ) (formula 1)

where L denotes the likelihood that the predicted probabilities of the link function μi results in a success and 1yi denotes the indicator function taking the value 1 if yi occurs and 0 otherwise. In order to incorporate age as a covariate, we used the logit link function (formula 2):

푀 휇 = 푔(푥) = (formula 2) 1+푒−푘(푥−푥0) where M denotes the maximum of the sigmoid, k the steepness of the sigmoid, x0 the x-value of the sigmoid midpoint and x the observed sample values.

In a second step, we used all available data on female specimens collected between 1990 and 2016 that had undergone GLG age estimation in order to assess a demographical structure of the harbour porpoise within the study area. After grouping into age classes, we calculated the estimated proportion of animals that is part of the reproductive population at any given time, applying the result of the binomial regression model in the previous step as a threshold value. Using this model approach, covariates such as tooth age and area can be included, which would not be as trivial in an ANOVA environment e.g. due to the error distribution of the binomial response variable.

- 15 -

2.4. Results

We evaluated a demographic age structure based on stranding records of 526 female harbour porpoises that had had their age assessed by means of GLG (of which 311 had been found along the German North Sea shore and 215 on the German Baltic Sea shore between 1990 and 2016). The age structure ranged between 0 and 22 years, with an estimated mean age of 4.87 ± 0.20 years (Table 1). The distribution of age classes is given in Table 1 and in more detail in Fig 2. The average age at death of German North Sea specimens was 5.70 (± 0.27) years for North Sea animals and 3.67 (± 0.30) years for specimens from the German Baltic Sea, respectively.

Table 1: Quartile age distribution of harbour porpoises from the German North Sea and Baltic Sea between 1996 and 2017. No. indicates number of available samples; percentage indicates the respective quartile applied to the data; max. indicates maximum observed age; average indicates respective mean age including Standard Errors (SE); all values are given in years.

Area No. 25% 50% 75% 90% Max. Average (± SE)

North Sea 311 1.25 5.00 9.00 12.00 22.00 5.70 (± 0.27)

Baltic Sea 215 1.00 1.6 5.0 9.6 22.00 3.67 (± 0.30)

Total 526 1.00 3.78 8.00 11.00 22.00 4.87 (± 0.20)

- 16 -

- 17 -

Ovaries from 111 specimens were analysed in this study (Fig 3). Of these, 69 were found along the North Sea shore (41 of which displayed at least one corpus luteum or albicans) and 42 were found along the Baltic Sea shore, of which 18 specimens providing at least one corpus luteum or albicans (Table 2). Age determination was available for all 111 animals through GLG.

- 18 -

Figure 3: Ovary of an adult female harbour porpoise showing signs of former ovulation and tertiary follicles. (A): Corpus luteum (asterisk) and corpus albicans (arrow). (B): Corpus luteum as a protuberance on the ovarian surface (C), (D): Histological section through a Corpus luteum showing luteal cells. Staining: HandE. (E): Histological section showing the Cumulus oophorus with the oocyte surrounded by the Zona pelucida and Corona radiata. Asterisk: Antrum folliculare. Staining: H & E. (F): Histological section through a Corpus albicans showing an increased amount of connective tissue (asterisk). Staining: Masson´s Trichrome.

- 19 -

Table 2: Overview of analysed specimens. Area: Origin of the specimen (NS: German North Sea, BS: German Baltic Sea); Corpora: Binary value indicating the presence or absence of corpora on the ovaries; No.: Total number of specimens assessed.

Area Corpora No.

Absent 28

NS Present 41

Total 69

Absent 24

BS Present 18

Total 42

Using model c2 (Table 3), we predicted the threshold age at which more than 50% of the specimens displayed one corpus or more as a sign of former ovulation. The threshold was determined at 4.95 years or higher (95% CI: 4.15-5.83 years, Fig 4).

- 20 -

Figure 4: Binomial regression model showing the functional relationship between the presence of corpora (purple polygon) in female harbour porpoises from the German North Sea and Baltic Sea in relation to their age as determined by GLG. The filled area indicates the 95% confidence interval of predicted values. The red line within that area indicates the predicted average probability of finding corpora within specimens at the given age; the blue dashed line marks a probability of 50%, with the red solid line indicating the corresponding age based on the intersection of the blue dashed line with the x-axis; samples are indicated as dark grey dots; a purple circle around data points indicates animals below the age threshold showing at least one - 21 - corpus, an orange circle indicates animals above the threshold showing no corpora; background histogram (orange) shows number of animals (secondary y-axis) across tooth age classes (at 1-year intervals).

Table 3: Model diagnostics for the binomial regression model of the probability of detecting corpora (models c1 and c2). Response: the response variable for the model; model name: The identifier for the model as used in the text (model name in bold indicates the chosen model); AIC: Akaike Information Criterion [42] to assess model fit; covariate: Individual covariates tested in the model (covariates in bold show significance at α=0.05; Parameter: Name of the model parameter (c0 is the null model, i.e. assuming a uniform distribution of data without covariates); Sig.: Significance level (*** p-value ≤ 0.001).

Response Model name AIC Parameter Sig.

c0 155.44 intercept 0.51

intercept ***

c1 83.304 tooth age *** Presence of corpora area 0.174

intercept *** c2 83.256 tooth age ***

- 22 -

Assuming that the animals enter their reproductive cycle at 4.95 years of age (as indicated by the previous analysis step), we estimated a total of 54.66% of female harbour porpoises in the North Sea and 27.44% in the Baltic Sea to participate in reproduction (Table 4).

Table 4: Percentile of all animals found between 1990 and 2017 that were older than the estimated threshold age of reaching sexual maturity of the population. No.: Total number of samples available; above threshold: Number of animals that were older than the threshold identified in this study; P: Proportion of animals that are above the threshold.

Area No. Above threshold P

North Sea 311 170 54.66%

Baltic Sea 215 59 27.44%

Total 526 229 43.54%

- 23 -

2.5. Discussion

This study presents the first model-based estimation of sexual maturity using ovarian characteristics from female harbour porpoises collected from the German North Sea and Baltic Sea. Using a model approach, we identified the threshold at which more than 50% of all specimens qualify as mature without setting an arbitrary threshold that is biased by the observer. While data from stranding networks do not necessarily reflect the status of the entire population, these estimates have to be regarded as best available estimates rather than an absolute representation of the population. Using necropsy data from an unknown proportion of animals is always connected with a degree of uncertainty. We do not assume that animals found dead on the beaches represent the average population individual, nor that these stranded animals are spatially correlated to all population members and therefore might only represent a small fraction of the actual population. The onset of sexual maturity is very likely to be quite robust throughout individuals from stranding events across 20 years (as shown in the modelling step) and we therefore assume that the result is true for at least the proportion of animals that were found along the beaches. A data set spanning two decades is more susceptible to long term effects such as pollutants and individual life history events, which we aim to address once more data on GLG age estimates are collected. However, this is the best data set currently available and we must therefore assume that our results provide information on the average harbour porpoise (be they in perfect condition or under influence of pathological changes). The age of specimen ranged from 0 to 22 years, with a mean age of sexual maturity of 4.95 years. We could not detect any significant differences between specimens found in the Baltic Sea and those from the North Sea. It appears that specimens from the German Baltic Sea shore are slightly younger when reaching sexual maturity. Considering regional differences such as the magnitude of anthropogenic impacts, environmental settings and distinct prey availability, life parameters such as threshold age of sexual maturity, birth rates and calving intervals cannot be generalised for all harbour porpoises across (sub-) populations. It has been observed in other mammalian species that a good nutritional status can relate to an earlier onset of sexual maturity due to physiological features [43, 44]. Anthropogenic factors such as underwater noise, disturbance, bycatch, increasing amounts of marine debris and chemical pollution trigger changes in marine mammal physiology and manifest in an

- 24 - increased stress hormone level [45-47]. It has been shown that high stress levels are directly connected to the function of the hypothalamo-pituitary axis of the reproductive system of other mammals like humans, pigs and rats [48-52] and earlier onset of sexual maturity [53-55]. The direct effects on the reproductive hormonal system evoke a modified synthesis and secretion of Gonadotropin-releasing- hormone and affected responsiveness of the gonadotrophs to Gonadotropin- releasing-hormone. Furthermore, stress hormones are suspected of affecting the feedback mechanisms of steroid hormones in the hypothalamus and the pituitary gland [55]. These stress-induced effects on the reproductive system might be an evolutional value for the adaption to unfavourable environmental conditions. On the one hand, resources can be focussed on survival or improvement of an overall health status and, on the other hand, young females that might not have the potential to survive too long can be capable of producing offspring [54]. As first evidence is given on how the hormonal systems of harbour porpoises respond to stressors and in which way they might be influenced [47], a correlation between stress hormone levels and reproductive parameters in the future is strongly required in order to see whether existing knowledge from other mammals can be aligned with that of harbour porpoises. Furthermore, high levels of stress hormones are known to suppress the immune system [55], leading to an increased risk of viral and bacteriological infections [56]. These infections often cause high levels of inflammatory proteins such as cytokines that suppress fertility [57-58].

Growing evidence is given that besides the hormonal system of reproduction, also the immune system is susceptible to endocrine disrupters [59-61]. Chemical toxicants such as pesticides and plasticisers are major anthropogenic drivers of changes in the reproductive cycle of marine mammals, in particular the onset of sexual maturity [35]. PCBs (polychlorinated biphenyls) accumulating in the blubber of harbour porpoises from the North Sea could act as estrogenic as well as anti - estrogenic agents since they are capable of interacting with different hormonal mechanisms [60]. As an example, pituitary cells of laboratory rats exposed to contaminants show enhanced gonadotropin responses to GnRH and oestrogens [62], or they are able to bind to oestrogen receptors and thus inhibit or modify the cell response [63]. As such, it cannot be ruled out that changes in the onset of sexual maturity might depend on the spatial and temporal concentration of chemical pollutants within a local food web and

- 25 - might include a generation bias. Subsequent studies using larger datasets with additional information on potential factors that might influence reproductive parameters such as toxicant load, endocrine disruptors or marine debris findings would support a more explicit and spatial analysis of our findings. Regarding the poor availability of estimations for the age at sexual maturity in the study region, we consider the estimated mean age of sexual maturity to be a valid baseline for future investigations. This issue could support the calculation of reproductive lifespan, pregnancy rate and calving interval as indicators for the reproductive status of a population. Although our findings are in accordance with the range of observations reported from harbour porpoises from the North Atlantic, the age at sexual maturity appears to occur slightly later in the study area than in most of the other investigated regions. If we apply our age estimate of first signs of sexual maturity to the age structure of specimens found in the same region, we conclude that about 54% of animals in the North Sea and up to 27% of animals in the Baltic Sea might belong to the reproductively active part of the populations. Population estimates from the National German Monitoring Programme for harbour porpoises reveal that the population in the German North Sea contains about 50,000 animals [64] and that in the German Baltic Sea about 3,000 animals [personal communication with Sacha Viquerat]. Based on necropsy data, the sex ratio was assumed to be roughly 46% females. We infer that at peak population size in summer the total number of females belonging to the reproductive population amounted to 12,430 females in the German North Sea and only 372 females in the German Baltic Sea. For the Baltic Sea subpopulations this value is still very low compared to other harbour porpoise populations and might be more vulnerable to environmental factors that shorten the reproductive lifespan like increasing bycatches in the Baltic Sea [33]. Of the 4,006 individuals, 159 were directly handed over by fishermen and classified as bycatch (~3% of all animals). 159 of 548 dissected animals were suspected to be bycaught based on pathological findings (~30% of all animals). The German stranding data showed that most of the 159 recorded bycaught porpoises between the years 1990- 2014 were reported from bottom-set gillnet fisheries or stranded with characteristic net marks [46, 65]. Bycatches might affect the (sub-) populational development since female adult harbour porpoises are often bycaught due to their distribution near the shore during the reproductive period and their distinct foraging behaviours during lactation [66, 67] as well as juvenile harbour porpoises that show unestablished

- 26 - foraging habits.

In this study, the significantly lower age at death of individuals from the Baltic Sea compared to North Sea specimens supports the proposal to reduce the magnitude of bycatches to meet the conservation issue for harbour porpoises [33].We also observed a decline in the number of corpora in animals older than 8 years in samples from the German North Sea shore (but not in specimens from the German Baltic Sea, which we attribute mainly to the overall lower age of 3.67 years in Baltic Sea specimens). This decline might show, that more ancient signs of ovulation on the ovaries disappear than new ones develop as a potential sign of sexual senescence. This could indicate, that animals beyond that age do not successfully mate regularly at least once a year after the age of 8 has been reached and thus, that the lifespan of full reproductive activity amounts to only about 3 years. This is a sensitive parameter with respect to human influences that might even shorten this timeframe that is essential for maintaining a constant population size. For future studies it is paramount that more data on the individual health condition and pathological findings of the investigated animals are included in the analyses to minimise this possible bias. Despite the fact that our data were too scarce to be able to conduct an elaborate analysis of the different observations in the North Sea and Baltic Sea, it might be crucial for future investigations to consider the age at sexual maturity and corpus counts. Furthermore, other reproductive parameters such as pregnancy rate, birth rate, ovulation rate and even pathological findings in the reproductive tracts as well as toxicological analyses to gain a complete overview of the reproductive status of a population are essential. Comprehensive long-term data including geographical information could support the analysis on a smaller spatial scale. Such datasets have been initiated for collecting from all HELCOM and OSPAR countries. Combining demographical data from specimens and population census data to estimate the proportion of sexually active animals of a population would be a key tool for any population viability study and a decisive concept to be applied in management plans. We reiterate the need for monitoring the age of sexual maturity at least bi-annually to identify any potentially serious changes in the reproductive potential of the (sub-) populations and thereby to conduct a retrospective trend analysis of data. Reporting bi-annual estimates of sexual maturity per (sub) population would be a valuable contribution to understanding population dynamics in harbour porpoises and to

- 27 - providing guidelines for swift policy decisions.

- 28 -

2.6. Acknowledgements

We would like to thank all personnel who helped during the necropsies at the ITAW Büsum. Special thanks go to our lab technicians at the ITAW Büsum as well as staff at the Institute of Anatomy in Hannover, who helped us tremendously with the acquisition of samples, their handling and their age determination. We are especially grateful to Marion Langeheine for her expertise in professional histology. We would also like to thank Christina Lockyer for her great effort in helping us with the age determination technique. Furthermore, we would like to thank everyone involved in the stranding network who regularly collect, pass on specimens and report to the ITAW. We thank two anonymous referees for their comments on a previous draft of the paper and also Frances Sherwood-Brock for proofreading the article. Thanks to Julia Carlström for cross-checking the manuscript.

- 29 -

2.7. References

1. Kinze CC. On the harbour porpoise in Danish waters in Danish waters: Distribution, breeding areas, and threats. Fauna Flora 1990; 85 (3–4): 114-121.

2. Benke H, Siebert U, Lick R, Bandomir B, Weiss R. The current status of harbour porpoises (Phocoena phocoena) in German waters. Archive of Fishery and Marine Research 1998; 46: 97–123.

3. Addink MJ, Smeenk C. The harbour porpoise (Phocoena phocoena) in Dutch coastal waters: analysis of stranding records for the period 1920 – 1994. Lutra 1999; 41(1–2): 55–80.

4. Sørensen TB, Kinze C. Reproduction and reproductive seasonality in Danish harbour porpoises (Phocoena phocoena). Ophelia, 1994; 39 (3), 159. –176.

5. Lockyer C. Harbour porpoises (Phocoena phocoena) in the North Atlantic: Biological parameters. NAMMCO Scientific Publications 2003; 5: 71–89.

6. Hasselmeier I, Abt KF, Adelung D, Siebert U. Stranding patterns of Harbour Porpoises (Phocoena phocoena) in the North and Baltic Seas: when does the birth period occur? Journal of Cetacean Research and Management 2004; 6 (3): 259–263.

7. Fisher HD, Harrison RJ. Reproduction in the Common porpoise (Phocoena phocoena) of the North Atlantic. Journal of Zoology London 1970; 161: 471–486.

8. Perrin WF, Reilly SB. Reproductive parameters of dolphins and small whales of the family delphinidae. Report of the Workshop. Report of the International Whaling Commission 1984: Special Issue 6: 97–133.

9. Van Utrecht WL. Age and growth in Phocoena phocoena Linnaeus, 1758 (Cetacea, Odontoceti) from the North Sea. Bijdragen tot de Dierkunde 1978; 48: 16– 26.

10. Gaskin DE, Smith GJD, Watson AP, Yasui WY, Yurick DB. Reproduction in porpoises (Phocoenidae): implications for management. Report of the International Whaling Commission 1984: (Special Issue 6): 135–148. - 30 -

11. Read AJ, Hohn, AA. Life in the fast lane: the life history of harbour porpoises from the Gulf of Maine. Marine Mammal Science 1995: 11: 423–440.

12. Karakosta C, Jepson P, Ohira H, Moore A, Bennett P, Holt W. Testicular and ovarian development in the harbour porpoise (Phocoena phocoena). Journal of Zoology, London 1999; 249: 111–121.

13. Lockyer C, Heide-Jørgensen M, Jensen J, Kinze C, Sørensen T. Age, length and reproductive parameters of harbour porpoises (Phocoena phocoena) from West Greenland. ICES Journal of Marine Science 2001; 58: 154–162. 13

14. Learmonth JA. Life history of harbor porpoises (Phocoena phocoena) in Scottish (UK) waters. Marine Mammal Science 2014: 30 (4): 1427–1455.

15. Perrin WF, Brownell RL Jr, DeMaster DP. Reproduction in Whales, Dolphins and Porpoises. Report of the Workshop. Report of the International Whaling Commission 1984: Special Issue 6: 1–24.

16. Smollich A and Michel G. Mikroskopische Anatomie der Haustiere. 1992; 2: 283– 292.

17. Eckert R. Tierphysiologie. 1993 2nd Edition: p. 358 – 359. ISBN 3–13-664002–0

18. Halldórsson SD, Víkingsson GA. Analysis of seasonal changes in reproductive organs from Icelandic harbour porpoises (Phocoena phocoena). NAMMCO Science Publications 2001; 5: 121–142.

19. Lockyer C. A review of factors involved in zonation in odontocete teeth, and an investigation of the likely impact of environmental factors and major life events on harbour porpoise tooth structure. Rep. Mtg Int. Whal. Commn 1995: special issue 16: 511–529

20. Ólafsdóttir D, Víkingsson GA, Halldórsson DS, Sigurjónsson J. Growth and reproduction in harbour porpoises (Phocoena phocoena) in Icelandic waters. NAMMCO Scientific Publications 2002; 5, 121–142.

21. Takahashi Y, Ohwada S, Watanabe K, Ropert-Coudert Y, Zenitani R, Naito Y et al. Does elastin contribute to the persistence of corpora albicantia in the ovary of the

- 31 - common dolphin (Delphinus delphis)? Marine Mammal Science 2006; 22: 819–830.

22. Kasuya T, Brownell RL. Age determination, reproduction and growth of the Franziscana dolphin, Pontoporia blainvillei. Nōgyō Doboku Shikenjō gihō. F, Sōgō 1965

23. Slooten E. Age, growth, and reproduction in Hector's dolphins. Canadian Journal of Zoology. 1999; 69(6): 1689-1700

24. March H, Kasuya T. Changes in the ovaries of the Short-Finned Pilot Whale, Globicepha macrorhynchus with Age and Reproductivet activity, Rep.Int.Whal.Commn. 1984 (special issue 6)

25. Sergeant DE, Caldwell DK, Caldwell MC. Age, Growth, and Maturity of Bottlenosed Dolphin (Tursiops truncatus) from Northeast Florida. Journal of the Fisheries Research Board of Canada, 1973; 30(7): 1009-1011

26. Bandomir B, Chavez-Lisambart L, Siebert U. Reproduction In Harbour Porpoises (Phocoena Phocoena), From German Waters. Proceedings of the Twelfth Annual Conference of the European Cetacean Society. 1998. 2pp

27. Lockyer C. The age at sexual maturity of the southern fin whale (Balaenoptera physalus) using annual layer counts in the ear plug. Journal du Conseil International d´Exploration de Mer. 1972; 34(2): 276-294.

28. Ohsumi S. Yearly change in age and body length at sexual maturity of a fin whale stock in the eastern north pacific. Scientific Reports of the Whale Research Institute 1986; No. 37: 1-16 28

29. Bando T, Kishiro T, Kato H. Yearly trend in the age at sexual maturity of Antarctic minke whales examined by transition phase in earplugs collected during JARPA and JARPAII surveys. Scientific paper submitted to the JARPAII review meeting called by the IWC 2014. F14/J8 2014. 29

30. Hohn A, Ewing RY, Zaias J. Reproduction in: Debra Lee Miller. Relation to conservation and commercial exploitation. In: Reproductive Biology and Phylogeny of Cetacea: Whales, Porpoises and Dolphins 2007: 7th edition: chapter 14

- 32 -

31. Berggren P. Bycatches of the Harbour Porpoise (Phocoena phocoena) in the Swedish Skagerrak, Kattegat and Baltic Seas 1973–1993. Reports of the International Whaling Commission 1994: Spec Issue 15:211–215

32. Berggren P, Wade PR, Carlström J, Read AJ. Potential limits to anthropogenic mortality for harbour porpoises in the Baltic region. Biological Conservation 2002; 103: 313–322 32

33. Koschinski S, Pfander A. By-catch of harbour porpoises (Phocoena phocoena) in the Baltic coastal waters of Angeln and Schwansen (Schleswig-Holstein, Germany). 16th ASCOBANS Advisory Committee Meeting Document 2009: AC16/Doc.60. Dist. 8

34. Jepson PD, Deaville R, Barber JL, Aguilar A, Borrell A, Murphy S et al. PCB pollution continues to impact populations of orcas and other dolphins in European waters. Scientific Reports 2016; 6: doi: 10.1038. 34

35. Murphy S, Barber JL, Learmonth JA, Read FL, Deaville R et al. Reproductive Failure in UK Harbour Porpoises Phocoena phocoena: Legacy of Pollutant Exposure? PLoS ONE 2015; 10 (7): doi:10.1371.

36. Addison RF. Organochlorines and marine mammal reproduction. Canadian Journal of Fishery and Aquatic Science 1989; 46 (2): 360–368

37. Aguilar A, Borrell A: Pollution and harbour porpoises in the eastern North Atlantic: A review. Reports of the International Whaling Commission 1995: special issue 16: 231–242,

38. Reijnders PJH. Reproductive failure in common seals feeding on fish from polluted coastal waters. Nature, London 1986; 324: 556–557

39. Jauniaux T, Brenez C, Fretin D, Godfroid J, Haelters J, Jacques T, et al. Brucella ceti Infection in Harbor Porpoise (Phocoena phocoena). Emerg Infect Dis. 2010 16(12): 1966–1968

40. Siebert U, Wünschmann A, Weiss R, Frank H, Benke H and Frese K. Post- mortem Findings in Harbour Porpoises (Phocoena phocoena) from the German North and Baltic Seas. Journal of Comparative Pathology 2001; 124: 102–114. - 33 -

41. Lockyer C. A review of factors involved in zonation in odontocete teeth, and an investigation of the likely impact of environmental factors and major life events on harbour porpoise tooth structure. Rep. Mtg Int. Whal. Commn 1995b: special issue 16: 511–529.

42. Akaike H. Information theory and an extension of the maximum likelihood principle. 2nd International Symposium on Information Theory 1974; 267–281.

43. Baker ER. Body Weight and the Initiation of Puberty. Clinical Obstetrics and Gynecology 1985; 28 (3): 573–579.

44. Kaplowitz PB. Link Between Body Fat and the Timing of Puberty. Pediatrics 2008; 121: 208–217.

45. Romano TA, Keogh MJ, Kelly C and Finneran JJ. Anthropogenic sound and marine mammal health: Measures of the nervous and immune systems before and after intense sound exposure. Canadian Journal of Fisheries and Aquatic Sciences 2004; 61 (7): 1124–1134.

46. Unger B, Herr H, Benke H, Böhmert M, Burkhardt-Holm P, Dähne M, et al. Marine debris in harbour porpoises and seals from German waters. Marine Environmental research 2017. Doi: 10.1016

47. Müller S, Lehnert K, Seibel H, Driver J, Ronnenberg K, Teilmann J, et al. Evaluation of immune and stress status in harbour porpoises (Phocoena phocoena): can hormones and mRNA expression levels serve as indicators to assess stress? BMC Veterinary Research 2013 (9): 145.

48. Hennessy DP, Williamson P. The effects of stress and of ACTH administration in hormone profiles, oestrus ans ovulation in pigs. Theriogenology 1983; 20 (1): 13–26.

49. Rivier C, Vale W. Cytokines Act within the Brain to Inhibit Luteinizing Hormone Secretion and Ovulation in the Rat. Endocrinology 2016; 127 (2): 849–856.

50. Roozendaal MM, Swarts HJM, Wiegant VM and Mattheij JAM. Effect of restraint stress on the preovulatory luteinizing hormone profile and ovulation in the rat. European Journal of Endocrinology 1995; 133: 347-353.

- 34 -

51. Nakamura K, Sheps S, Clara Arck P. Stress and reproductive failure: past notions, present insights and future directions. Journal of Assisted Reproduction and Genetics 2008; 25 (2–3): 47–62.

52. Tilbrook AJ, Turner AI, Clarke IJ. Effects of stress on reproduction in non-rodent mammals: The role of glucocorticoids and sex differences. Reviews of Reproduction 2000; 5:105–113

53. Ellis BJ, Essex MJ. Family Environments, Adrenarche, and Sexual Maturation: A Longitudinal Test of a Life History Model, Child Development 2007; 78(6):1799-817

54. Ellis BJ. Timing of pubertal maturation in girls: an integrated life history approach., Psychol Bull. 2004; 130(6):920-58.

55. Glaser D, Kiecolt-Glaser JK. Stress-induced immune dysfunction: implications for health. Nature Reviews Immunology 2005; 5: 243–251

56. Siebert U and Weiss R. Regional differences in bacteria flora in harbour porpoises from the North Atlantic: environmental effects. Journal of Applied Microbiology 2009; 106: 329–337

57. Mikuni M. Effect of interleukin-2 and interleukin-6 on ovary in the ovulatory period--establishment of the new ovarian perfusion system and influence of interleukins on ovulation rate and steroid secretion. The Hokkaido Journal of Medical Science 1995; 70 (4): 561–572

58. Ahmed SA. The immune system as a potential target for environmental estrogens (endocrine disrupters): a new emerging field. Toxicology 2000; 150 (1–3): 191–216.

59. Beineke A, Siebert U, MacLachlan M, Bruhn R, Thron K, Failing K et al. Investigations of the potential influence of environmental contaminants on the thymus and spleen of harbor porpoises (Phocoena phocoena). Environmental Science and Technology 2005; 39: 3933–3938.

60. Das K, Vossen A, Tolley K, Vikingsson G, Thron K, Müller G. Interfollicular fibrosis on the thyroid glands of the harbour porpoise (Phocoena phocoena): An endocrine disruption? Archive of Environmental Contamination and Toxicology 2006; 51: 720-729 - 35 -

61. Imazaki PH, Brose F, Jauniaux T, Das K, Muller M, Scippo ML. Estrogenic Evaluation and Organochlorine Identification in Blubber of North Sea Harbour Porpoise (Phocoena phocoena) Stranded on the North Sea Coast. BioMed Research International 2015. doi: 10.1155/2015/438295

62. Jansen HT, Cooke PS, Porcelli J, Liu TC, Hansen LG. Estrogenic and antiestrogenenic actions of PCBs in the female rat: In vitro and in vivo studies. Reproductive Toxicology. 1993; 7, pp. 237-248

63. Lind PM, Eriksen EF, Sahlin L, Edlund M, Orberg J. Effects of the Antiestrogenic Environmental Pollutant 3,39,4,49,5-Pentachlorobiphenyl (PCB #126) in Rat Bone and Uterus: Diverging Effects in Ovariectomized and Intact Animals.Toxicology and Applied Pharmacology 1999; 154: 236–244

64. Gilles A, Scheidat M, Siebert U. Seasonal distribution of harbour porpoises and possible interference of offshore wind farms in the German North Sea. Mar Ecol Prog Ser. 2009;383:295-307

65. Siebert U, Tolley K, Vikingsson GA, Baumgärtner W. Pathological Findings in Harbour Porpoises (Phocoena phocoena) from Norwegian and Icelandic Waters. Journal of Comparative Pathology 2006 ; 134(2-3):134-42

66. Yasui WY and Gaskin DE. Energy budget of a small cetacean, the harbour porpoise, Phocoena phocoena. Ophelia 1986 ; 25(3) : 183-197

67. Sveegaard S, Teilmann J, Tougaard J, Dietz R, Mouritsen KN, Desportes G et al. High-density areas for harbor porpoises (Phocoena phocoena) identified by satellite tracking. Marine Mammals Science 2010; 27(1): 230-246

- 36 -

Chapter 3: Publication II: Testicular development and spermatogenesis in harbour porpoises (Phocoena phocoena)

Kesselring, Ta, Viquerat, Sa, IJsseldijk, L.L.b, Langeheine, Mc, Wohlsein, Pd, Gröne, Ab, Bergmann, Me, Siebert, Ua, Brehm, Rc. aInstitute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hanover Foundation, Werftstr. 6, 25761 Büsum, Germany bDepartment of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands cInstitute for Anatomy, University of Veterinary Medicine Hannover Foundation, Bischofsholer Damm 15, 30173 Hanover, Germany dDepartment of Pathology, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hanover, Germany eInstitute of Veterinary Anatomy, Histology and Embryology, University of Giessen, Germany

Corresponding Author: [email protected]

- 37 -

3.1. Abstract

Knowledge about reproductive parameters in male harbour porpoises such as testicular histology and germ cell maturation as well as seasonal changes in spermatogenesis is scarce. Thus, the aim of the present study was to report changes in the histological appearance of the testicular morphology of neonatal and juvenile harbour porpoises during maturation, to identify stages of spermatogenesis in adult males and to detect seasonal modifications. The identification of these stages can be used to assess the developmental profile of gene expression during spermatogenesis and to identify defects in spermatogenesis arising in pathological conditions. Testes of adult male harbour porpoises from the North and Baltic Sea that became stranded or by-caught in the years 1998 to 2016 were histologically examined using Haematoxylin and Eosin – staining. The Periodic Acid Schiff (PAS) staining was used for spermatogenic staging and the evaluation of the development of the acrosomic cap. For the identification of changes in testes morphology and morphometry during the course of the year, histological characteristics like germ cell associations and diameter of the convoluted seminiferous tubules were noted for each month. The analysis showed that in adult males more than one stage of spermatogenesis could be found per cross section of the convoluted seminiferous tubules similar to findings in men and some ape species. This rare phenomenon is called multi-stage-arrangement. In sexually active males from the peak breeding season (June and July) eight stages of spermatogenesis were identified and all stages occurred simultaneously, while during the low breeding season (August to May) only residual spermatogenesis or constituent germ cell populations were found. Missing germ cell generations were recorded in specimens from July to September. Our investigations provide a detailed staging of spermatogenesis and give new insight into the reproductive biology of male harbour porpoises. With these new basic parameters, indicators for endocrine disruptors can be developed in the future, aiming to detect how environmental factors could affect male fertility in wildlife.

Key words: cetacean, reproduction, testes, seasonality, fertility

- 38 -

3.2. Introduction

The harbour porpoise (Phocoena phocoena) is the smallest cetacean species native to German and Dutch waters and exhibits a seasonal pattern in its annual reproductive cycle [1–3]. The conservation status of this species especially in the Baltic area has been of concern for many years, since anthropogenic impacts such as climate change, noise emissions during fishery activities and pile driving, bycatch and pollutants within the aquatic food chain have increased substantially in the North and Baltic Sea [4–8].

As top predators harbour porpoises are sensitive to contaminants [9–12]. Some contaminants might be capable of passing the blood-testes-barrier [12], the placental- barrier and milk – blood - barrier and may harm the development of the living stocks in an yet unpredictable way [7,13–19]. There have been reports on reproductive failure including foetal death, abortion and dystocia in harbour porpoises from Scottish waters that may be connected to the concentration of high toxicants in the blubber of these animals [7], but there is no knowledge on how these contaminants influence the reproductive organs of the population. In order to detect changes in reproductive organs due to human impacts, basic knowledge on a cellular level is considered to be a first step towards a better understanding of physiological processes in the reproductive system [20]. Therefore, information on testicular development and histology of male harbour porpoises on a cellular level is urgently required.

It is known from other mammalian species, that spermatogenesis is a very sensitive process and vulnerable to environmental disturbances. It has been shown in humans and rats, that cytotoxic effects and hormonal changes can be a result of toxicants that have entered an individual’s physiological endocrine system [21,22]. Additionally, knowledge about the general process of spermatogenesis and seasonal effects in harbour porpoises is restricted to a few studies [23–26] and detailed staging of spermatogenesis based on germ cell morphology and their specific associations within the seminiferous epithelium have not been reported. It has been noted, that various cell associations (stages) can be observed in one tubular cross-section [26], but no specific number of stages has been given. Staging is a common approach to assess the cycle of the seminiferous epithelium [27]. Based on this procedure,

- 39 - alterations in the testicular development due to environmental changes or disturbances can be recognised. These changes can occur seasonally and thus be considered as a physiologic phenomenon or have a pathological origin. With a detailed staging of spermatogenesis in harbour porpoises, the detection of potential influences of endocrine disruptors on male reproductive biology can be possible [28]. It is known from other species, that the influence of certain noxae can cause alterations in the development of germ cells and might be capable of paralyzing the germ cell differentiation. This failure in the spermatogenic process leads to the presence of missing generations of germ cells within one cross-section of a convoluted seminiferous tubule [29] and might lead to reduced fertility of the individual.

To define a baseline for investigations on testicular structures as an indicator for an individual´s reproductive status, the histological arrangement of the seminiferous epithelium of juvenile and adult individuals has to be considered. It has been observed in harbour porpoises from other waters that more than one spermatogenic stage can be found in one cross section of a seminiferous tubule [24–26], but no fundamental staging has been performed yet.

Furthermore, macroscopic and microscopic morphometric parameters of the testes and the diameter of seminiferous tubules undergo a seasonal cycle [24,25,30–34].

Seasonal breeding is efficient as it conserves energy and benefits natural selection [35]. Male harbour porpoises from different regions show full sexual activity from June to July (North Sea and Baltic Sea, Gulf of Maine, Bay of Fundy) [25,33,34]. These periods coincide with the time of ovulation in female harbour porpoises [33,35–39]. Thereby, the parturition occurs under the best climatic and nutritional circumstances and offers good environmental conditions for raising the neonates.

The testes of harbour porpoises appear to be ‘inactive’ from October – April [24,25,30]. Investigations on this testicular cycle including testes mass and diameter of convoluted seminiferous tubules reveal a maximum in the peak breeding season (Bay of Fundy, North and Baltic Sea: July) and might be slightly earlier or later in our study areas depending on the ovulation time of female individuals reflecting environmental differences. Furthermore, the examination of testicular tissue might have the potential to add information on the status of sexual maturity of an individual - 40 - to the tooth age determination.

The aim of this study was to generate fundamental data on testicular morphology, time of initiation of spermatogenesis and seasonal changes with histological methods as a baseline for further studies that might focus on the effects of environmental influences on male reproduction in harbour porpoises.

3.3. Materials and Methods

3.3.1. Sample collection

A total of 127 testes of male harbour porpoises stranded and bycaught along German and Dutch coasts between 1998 and 2016 were sampled during necropsies. The study was supervised in Hanover by Prof. Dr. Ralph Brehm (Department for Anatomy, University of Veterinary Medicine Hanover, Germany) and in Büsum by Prof. Dr. Ursula Siebert (Institute for Terrestrial and Aquatic Wildlife Research (ITAW), Büsum, Germany). Specimens were collected within the German stranding network, which conducts work (collect and hold carcasses and samples from European protected species) on German strandings following appropriate licenses from the relevant authorities (Ministry of Energy, Agriculture, the Environment, Nature and Digitalization, Ministry of Agriculture, Environment and Rural areas). Most of the animals were found stranded along the coast of Schleswig Holstein of the North Sea and western Baltic Sea, only a small number of animals has been identified as bycatch. Most specimens were sampled during necropsies in Büsum, Germany and Utrecht, The Netherlands. The necropsies in The Netherlands are commissioned by the Dutch Ministry of Agriculture, Nature and Food Quality. An additional approval for animal experiments by German or Dutch authorities was not necessary. Based on the initial classification from the necropsy protocol (Supplemental table 1, [40]), we grouped specimens into two maturity classes: juveniles and neonates as non-adult (n = 93) and adult specimens (n = 34). Depending on organisational and logistical conditions, the porpoises were stored at -20 °C until necropsy or were dissected immediately after death. Necropsies were performed according to a standardised necropsy protocol for harbour porpoises [40]. Tissue preservation status of samples was assessed using a scale from 1 (very fresh) to 4 (decomposed). Only samples originating from carcasses with a decomposition status of 3 or better were included into the analysis. Testes morphometry (n = 127) was measured and their weight - 41 -

(without epididymis) was noted. All testes were then fixed in 4% buffered formalin or Bouin‘s solution and prepared for histological examination by two sections of 1 cm in diameter - one section of the testes surface and one from the centre. Only one section covering the centre and the periphery was taken from individuals with smaller testes (neonates and juveniles).

- 42 -

Supplemental table 1: Sample collection of adults, juveniles and neonate in acordance to the month of stranding or bycatch. N indicates the total number of the specimens, month indicates the month of sampling

Month n adult n juvenile n neonate

January 3 3 1

February 1 4 1

March 3 3 0

April 1 9 0

May 3 3 1

June 3 0 10

July 7 5 9

August 4 5 7

September 4 5 5

October 1 6 11

November 2 0 1

December 2 3 1

- 43 -

3.3.2. Histological preparation

Tissue samples were dehydrated and embedded in paraffin. Using a Leitz 1512 microtome (Techno Med GmbH Bielefeld), three 3 – 5 µm slices of each sample were produced. The slices were mounted on glass layers and stained with Haematoxylin and Eosin (HE) (n = 127).

3.3.3. Age determination

Six teeth from 86 animals of these specimens (34 adults, 52 non-adults) were removed from the lower jaw and preserved in 4% buffered formalin. Tooth preparation was performed using a standard protocol [31].

3.3.4. Histological examination

For the qualitative assessment of the testes, a staging of the seminiferous epithelium of adult harbour porpoises from the peak breeding season was performed with a microscope (Olympus BH2, Olympus optical Co., Ltd). Based on acrosomal development after Periodic Acid Schiff (PAS) staining [41] (from 3 specimens), the staging was analysed according to [42]. For this, specimens that showed no tissue alterations due to freezing or decomposition were chosen (n = 3).

In order to detect seasonal changes during the course of the year, at least one individual of each maturity class was examined per month. At least 10 tubular cross sections per histological slice and specimen were selected for the investigation. For the assessment of the reproductive status of adult males only specimens with a sufficient tissue preservation status and no tissue alteration due to freezing or decomposition were chosen (n=25). Individuals that displayed all stages of spermatogenesis were classified as ‘active’. If only some stages of spermatogenesis or single germ cell types were present within the tubules, the individual was classified as ‘inactive’. Within this ‘inactive’ group, specimens with abundant germ cells that indicate the beginning of the breeding season were attributed with ‘increasing activity’, while specimens that show residual germ cells that remained from former spermatogenic waves of the breeding season were attributed with ‘decreasing activity’. Based on the histological examinations, the reproductive status of the - 44 - individual was then determined.

For morphometric analysis of the testes, the diameters of 20 seminiferous tubules per individual were measured in transverse section using a Zeiss Axioskop (Carl Zeiss Microscopy GmbH, Jena, Germany) with an Olympus DP 70 camera and the Olympus DP Soft software (Olympus Deutschland GmbH, Hamburg, Germany). The tubules were randomly selected out of those with a circular profile to minimise measuring errors. In samples of overall poor quality, the best available tubules were selected with a minimum of 10 tubules. The shortest distances between the peritubular membranes were measured and the diameter in each individual sample was averaged.

3.3.5. Modelling the diameter of the convoluted seminiferous tubules over the Julian day of the year

In a first step, the Gonadosomatic Index (GSI) [43] as the percentile ratio of testes weight versus body weight of each specimen was calculated. Using the age determination from growth layer group analysis (GLG) and the post-mortem examinations, the dataset was classified into two maturity classes comprising adults (specimen older than or equal to 4 years of age) and non-adults (specimen younger than 4 years of age). As each of the 127 specimen includes an assessment of age class by the experts present during the dissection (neonate, juvenile and adult), we used this data to assess the validity of our post necropsy age classification. Only carcasses with a decomposition status of 1-3 were used in the analysis to minimise bias from the unknown time difference between the time of death and finding the carcass (for lack of better measure, we therefore assumed the date of the stranding to be near equivalent to date the animal died).

Using a set of 68 samples, we modelled the (log-transformed) diameter of the convoluted seminiferous tubules (assessed according to 2.4.) using a generalized additive model. The included covariates were body length (as measured from rostrum to peduncle) as proxy for developmental age, using a thin plate smoother restricted to 5 knots available to smoothing. We tested for an effect of the Julian day of the stranding event / report date (with the 1st of January defined as the 1st day of the year, i.e. the 31st of December being the 366th Julian day of the year) for each specimen as a cyclic covariate smooth with 4 knots as a proxy for seasonality. We - 45 - tested each covariate individually and as interaction terms. We also tested for group effects on smoothing parameters using the maturity class as a ‘by’ condition. A regular smoothing function was used for single variables and a tensor smooth was used for interaction terms. Models selection was performed using generalised cross validation (gcv) scores and deviance explained measures. All analysis was conducted in R [44] using mgcv [45–47].

- 46 -

3.4. Results

3.4.1. Macroscopic findings of the testes during the course of the year

Our classification based on GLG was matched by both the average diameter of seminiferous tubules as well as the mean testis mass in relation to the overall body weight (Gonadosomatic Index, GSI). We found a clear distinction between specimens classified as adults and non-adult specimens. Adult specimen (GLG ≥ 4 years) did not include any juvenile or neonate specimen, whereas non-adult (neonate and juvenile) specimen (GLG < 4 years) did not include any specimen previously assessed as adults (Table 1) during the necropsies. The average age determined from GLG was 5.73 (± 1.11) years in all (n = 86), 9.56 (± 1.14) years in adults (n = 34) and 0.5 (± 1.16) years in non-adult specimen (n = 52). The average GSI was 0.951 (± 0.179) % in adult specimens, 0.048 (± 0.004) % in non-adult specimens and 0.282 (± 0.064) % over all age classes (Table 1). The mean testis mass in relation to the overall body weight (GSI) reached a peak in July, while the lowest values were found in September to February. Non-adult individuals showed no significant variation in the course of the year (Table 1).

- 47 -

Table 1: Summary of each age class. Subset: identifies the age class (all: all specimen regardless of age class; adult: specimen older or equal to 4 years of age; non-adult: specimen younger than 4 years of age); age: age structure in respective subset; tubuli seminiferi contorti: diameter of tubuli seminiferi contorti [µm]; GSI: gonadosomatic index (sensu Barber and Blake 2006) [7] [%]; Ø: indicates the averaged value (± standard error).

Subset Ø age (n = 86) Ø convoluted Ø GSI (n = 127) seminiferous tubules (n = 68)

All 5.73 (± 1.11) years 73.9 (± 5.66) µm 0.282 (± 0.064) %

Adults 9.56 (± 1.14) years 125.27 (± 10.17) µm 0.951 (± 0.179) % non-adults 0.5 (± 0.16) years 43.08 (± 1.67) µm 0.048 (± 0.004) %

3.4.2. Testicular maturation and staging of the seminiferous epithelium

Histological examination indicated that in seminiferous cords of neonatal harbour porpoises only Sertoli cells and gonocytes, which mostly have not reached the basement membrane, can be found. The seminiferous cords displayed no lumen. There was no evidence of mitosis and no other cell types of spermatogenesis were present (Figure 1A). In juvenile harbour porpoises aged older than one year, a lumen in the seminiferous tubules was identified and spermatogonia were abutting the basement membrane (exemplified in Figure 1B). In animals older than three years, first signs of germ cell activity like cell divisions were found (exemplified in Figure 1C, D). In individuals older than four years, the seminiferous epithelium showed a seasonal cycle (Figure 2, seasonal cycle shown in Figure 3).

- 48 -

Fully active males that showed all stages of spermatogenesis were found only in the months June and July. During this time, one cross-section contained several stages of spermatogenesis at once (Figure 4). One specimen from mid of June showed an increasing activity, but no elongated spermatids in the lumen of the convoluted seminiferous tubules. In individuals that have been found dead between September and April, only some stages or residual germ cells were identified (Figure 2C and 3). In individuals that were classified as inactive adults (Figure 2A and 3), either only the epithelial layer with spermatogonia and Sertoli cells was found, or in the case of one specimen, the epithelial layer and some residual germ cells were found. In individuals that were listed with an increasing activity, abundant germ cell types were observed, but no elongated spermatids or stages of spermatogenesis were present (Figure 2B and 3). Individuals listed with ‘decreasing activity’ showed some residual germ cells from former spermatogenic waves like spermatids in the lumen of the convoluted

- 49 - seminiferous tubules (Figure 2C and 3).

In specimens from the breeding season, different germ cell associations within one cross section of a convoluted seminiferous tubule were detected with a maximum of eight stages during June and July (Figure 3).

- 50 -

Figure 3: Summary of samples from adult harbour porpoise testes from the North and Baltic Sea and their reproductive status based on testicular activity, that has been investigated through histological evaluation; the x-axis indicates the month the carcasses have been found (Jan1 indicates one specimen within January that was attributed with ‘decreasing activity’, Jun2 indicates there was one animal found in June that showed ‘increasing activity’); the y-axis indicates the number of specimens studied; the background colours identify the conclusion of the reproductive status drawn based on the morphological histological appearance of testes: blue / inactive: no sign of activity or only a few isolated germ cells present in the convoluted seminiferous tubules; pink / increasing activity: increase in abundance of germ cells and some spermatogenic stages can be identified; yellow / active: active reproduction with all eight stages present; green / decreasing activity: only isolated residual germ detected.

- 51 -

The PAS staining of three representative specimens that showed no tissue alterations due to freezing or decomposition confirmed eight different and recurrent germ cell associations during the peak breeding season as follows (Figure 5, based on classification shown in Figure 6): Stage I was featured by the presence of two generations of spermatids, such as round and elongated spermatids. Elongated spermatid bundles were more packed and some were located deep within the epithelium. Additionally, young pachytene spermatocytes were located between round spermatids and spermatids with an acrosomal vesicle in which only occasional proacrosomal granules were identified. At the end of this stage, the small proacrosomal vesicles are located towards the basal lamina. Type A spermatogonia were observed in the HE-staining. The second stage (stage II) of spermatogenesis showed early round spermatids containing single acrosome granule in contact with the nucleus. The elongated spermatids were lined along the tubular lumen prior to - 52 - spermiation and Pachytene spermatocytes, intermediate- and Type A spermatogonia were observed. Stage III of the cycle showed round spermatids with an acrosome that was spread over the nucleus and a round acrosome vesicle. The elongated spermatids were located very close to the tubular lumen and residual bodies were present. A number of type A spermatogonia and intermediate spermatogonia have been identified, along with pachytene spermatocytes with larger nuclei. In the following stage IV of the seminiferous cycle, the round spermatids show an extensive acrosomal vesicle. Elongated spermatids were undergoing spermiation towards the tubular lumen and large residual bodies were observed. Type B spermatogonia were present near to the basal lamina. Type A spermatogonia and preleptotene spermatocytes have been observed. Stage V showed preleptotene, leptotene and pachytene spermatocytes as well as spermatogonia of the type A and spermatids that line the apical parts of the Sertoli cells (sperm release). The following stage VI is featured by a generation of elongating spermatids, since it is the stage immediately after the sperm release. Pachytene spermatocytes that enter the diplotene level of the meiosis and spermatogonia type A were identified. Stage VII of the cycle shows bundles of elongated spermatids that were deeply inserted in the epithelium, head orientated to the basement membrane. Diplotene spermatocytes and primary leptotene spermatocytes were present. Finally, stage VIII of the seminiferous cycle showed meiotic figures of the first and second divisions and secondary spermatocytes. Additionally, zygotene spermatocytes, spermatogonia type A and spermatids still located in the epithelium were found in this stage. Depending on the seasonal state, a certain number of stages or germ cell types are apparent. Our results showed, that re-initiation of spermatogenesis proceeds in May, while in August and September the spermatogenic processes are ceasing. A number of missing germ cell generations from July to September as a sign of decreasing efficiency of spermatogenesis has been encountered (example in Figure 7). While in July only a few areas with missing germ cell generations within one convoluted seminiferous tubule were found, specimens from August and September showed more absent germ cell populations, leading to tubules containing only some residual spermatids in the tubular lumen and Sertoli cells. This indicates that spermatogenic waves are interrupted and only residual cells from former waves can be observed (Figure 2C).

- 53 -

Figure 5: Staging of the seminiferous epithelium as featured by morphology (staining: PAS, primary magnification: X 40).

- 54 -

Figure 6: Stages of spermatogenesis in the harbour porpoise (modified according to [42].

- 55 -

3.4.3. Morphometrical analysis and maturity classes

Average diameter of the convoluted seminiferous tubules in adult animals was 125.27 (± 10.17) µm, in non-adult animals 43.08 (± 1.67) µm and the average across all age classes was 73.90 (± 5.66) µm. In non-adult animals, the average mean tubular diameter corresponds to a body length of at least 114 cm. The average diameter of the convoluted seminiferous tubules during April to August was 171.03 µm, while during the remaining months of the year, the average diameter was found to be 110.02 µm. In non-adults, the tubular diameter averaged at 43.98 µm with no significant changes throughout the year.

- 56 -

A total of 5 models were tested, each using a different combination of body length (M1_cm), day of the year (yday) and maturity class (maturity_class) and age derived from GLG (age_glg). A model including a tensor smooth of yday (cyclic smoother) and M1_cm (thin plate smoother) was identified as the best model (m4) (Table 2). While model m5 technically yielded a better overall score, the inclusion of the by variable introduced an artificial prior classification of data. Model m4 performed slightly worse, but represented a purely numerical solution not requiring prior clustering of data and was therefore preferred over m5. Using model m4, the diameter of the convoluted seminiferous tubules across a range of a whole year was predicted (with Julian days spanning 1-366) and across ages ranging between 0 and 12 years of age. The maximum predicted age was restricted to 12 years as 75% of all samples were identified by the GLG as 12 years of age or younger. Using the average predicted tubular diameter for adult specimens (≥ 4 years), the predicted average tubular diameter is above the average of specimen classified as adults between early April and late July and always above the average of all non-adult specimens. Peak tubular diameters are predicted for mid-May to late June (Figure 8).

- 57 -

Table 2: Model diagnostics of the generalized additive modelling analysis. Model: identifier of the model as used throughout the text; covariates: combination of smoother functions and covariates as supplied to the gam; N: sample size available used in the analysis (numbers vary due to missing values in some covariate combinations); dev: explained deviance; GCV: generalized cross validation score. Chosen model in bold (m4).

Model Covariates N dev GCV m5 yday, M1_cm, by: maturity_class 63 0.9123 0.05 m4 yday, M1_cm 63 0.8846 0.07 m3 yday, age_glg 19 0.9216 0.09 m2 yday, by: maturity_class 68 0.2152 0.34 m1 Yday 68 0.1405 0.38

- 58 -

- 59 -

3.5. Discussion

3.5.1. Testicular maturation and staging of the seminiferous epithelium

Our study provides the first detailed histological description of the seminiferous epithelial cycle in adult male harbour porpoises. The observation of a various number of spermatogenic stages within one seminiferous tubule confirms the findings of former investigations in cetaceans performed by other authors [24–26,48,49] and is called multi-stage-arrangement [50]. Although other authors that examined spermatogenesis did not mention a clear definition for a spermatogenic stage, they gave hints for the presence of this phenomenon [23–26,48,51].

Our results are similar to findings in testes from new world monkeys (Platyrrhini), old world monkeys (Cercopithecoidea) and humans (Homo sapiens) [50,52]. Until now, this phenomenon was unique to these species [50,52]. In humans, 6 stages of spermatogenesis are identified and have been described in detail [50,53]. The appearance, number and abundance of the different morphological stages depend on the mammalian species. Other mammals show species-specific numbers of stages (Dog: VIII stages, Cat: VIII, Mouse: XV, Rat: XIV stages, Horse: VIII stages, Cow VIII stages) [54–59] according to the association of distinct germ cells defined by the differentiation of the spermatid acrosome. Regarding the number of stages of spermatogenesis, the harbour porpoise can be compared with dogs and bovines, which show also eight stages of spermatogenesis. In the case of cattle, this might link to the phylogenetic relation of odontocetes with artiodactyls.

While in peak breeding season in summer, all generations of germ cells and thus all stages of spermatogenesis can be detected; only residual germ cells are observed in the autumn and winter, which conforms to findings in other waters [24–26,35]. This can be affiliated to the fact that as season progresses, more missing generations of germ cells are found in the cross sections resulting in the level of spermatozoa as the last remaining germ cells of the season. Our results show that re-initiation of spermatogenesis starts in May, while in August and September the spermatogenic processes are ceasing. This seasonal cycle, which is enforced mainly by photoperiod, supports energy balance in terms of seasonal foraging pattern, is a

- 60 - common regulatory mode in mammalian species [60–62]. In wildlife species, a seasonal focus in sperm production is suspected to support sperm competition. This mechanism targets the female ovulation period as a strategy for successful mating and may support behavioural mating strategies in different ways [20,63–65]. The fact that missing germ cell generations occurred already during the peak breeding season could be documented both as a physiological or pathological process. In other mammalian species, evidence is given that missing generations are a sign of reduced fertility [66] due to environmental disturbances. Whether this fact is transferable to harbour porpoises or whether it might be a physiological phenomenon as a sign of low efficiency has to be proven in further studies that are supported by a larger sample size.

3.5.2. Morphometrical analysis and age groups

Based on GSI measurements and histological features of the testes, no seasonal shift can be found in animals classified as non-adults, which coincided with a maximum age > 4 years and thus no sexual activity can be assumed for these individuals. Specimens classified as adults were consistently older than 4 years and showed a seasonal variation in the GSI and in histological features. Based on these observations, we conclude a sexual maturity in individuals older than 4 years.

Our results regarding the tubular diameter findings in adult specimens are in accordance to findings in harbour porpoises from other regions. Karakosta et al. 1999 [24] estimate the average tubular diameter in adult harbour porpoises from the Coasts of England and Wales at 117.5 µm (SD: 38.0). This value is slightly lower compared to our study (125.27 µm (± 10.17), but is within confidence limits. Neimanis et al. 2000 [25] investigated harbour porpoises from the bay of Fundy and Gulf of Maine and reported a mean diameter of the convoluted seminiferous tubules of 161.6 µm (SD: 4.07) at the end of July and a maximum diameter of convoluted seminiferous tubules (225 µm) in an individual found in early July. This higher value might be the result of a different sample composition, since the study of Neimanis et al. 2000 includes individuals from June to December. These months include the peak breeding season (June-July, [25]) but only a part of the low-breeding season. Generally, there are some factors that might have an influence on these values like - 61 - sample size, sample handling and processing as well as the software used to measure the values. Our data predict a peak in the diameter of the convoluted seminiferous tubules in mid-May to June, while our data including macroscopic (GSI) and microscopic findings (stages of spermatogenesis) indicate a breeding season from late June to July. Additionally, both methods document a peak of spermatogenesis including the macroskopic analysis and the microscopic morphology in July. Although the majority of our findings comply with this season, some outliers like mentioned in 3.2. and Figure 3 might indicate a shift in seasonality depending on the area the animal originates from and the subpopulation it belongs to. We cannot exclude that specimens found in German and Dutch areas might have migrated from other areas that show different seasonal pattern. Another possible reason for a shift in seasonality would be changes in the endocrine system due to various reasons like pathological disorders or endocrine disruption we just can suggest at this point of the analysis and with this small sample size. These issues could be further investigated in the future using a larger dataset including information about blood hormone levels and genetics.

Our observations suggest that adult male harbour porpoises synchronise their spermatogenesis with the ovulation time of females, thus maximising their chance at reproductive success which supports the hypothesis of sperm competition [65].

For other wildlife mammals it is known, that they synchronise their energetically demanding sperm production to the oestrus of female individuals and thus to the most favourable time of the year [67]. Environmental factors regulate spermatogenesis through the hypothalamic–pituitary–gonadal axis and trigger the onset of testicular function in spring and the testicular regression in autumn [67]. Since the exact point of time when juvenile males step into puberty cannot be observed macroscopically, the histological analysis of the testes would give additional evidence on the developmental stage of the individual. A combination of assessing the existing germ cell types with measuring the diameter of the seminiferous tubules draws a reliable picture on the sexual status of a juvenile/pubertal animal. As a next step, more samples with morphological records are required to study the transition period between immature and mature individuals in detail. This would complement a recent analysis of female harbour porpoises of the area [20]. In addition, more studies are needed to assess the correlation between the - 62 - variability in testes diameter observed in this study and morphometric features such as body length, body weight and indicators of the endocrine system.

Due to the fact, that our study provides first evidence of the stages of spermatogenesis with a small number of samples, further analyses on this topic are strongly required to confirm our findings. Using more samples of good quality, various other parameters that improve the understanding of male reproduction like efficiency of spermatogenesis or duration of the stages can be described on a higher level. Furthermore, immunohistochemistry could reveal information about proliferation and apoptosis within the spermatogenic stages, as well as knowledge about the cellular mechanisms during puberty.

3.6. Conclusions

Prior studies on testicular activity of harbour porpoises of the German North and Baltic Sea were restricted to small sample sizes and short periods of time. We here present long-term dataset on reproductive parameters in male harbour porpoises in accordance to age classes and across seasons. With the detailed description of spermatogenesis and stages of spermatogenesis, a baseline is given for further studies on the reproductive biology of male harbour porpoises and can be used to develop an indicator for the effects of environmental impacts on the reproductive system such as impaired spermatogenesis due to various factors. It has been observed in other domesticated and wildlife mammals that stress (influence of toxicants) has a negative impact on spermatogenesis [67,68] as well as a wide variety of endocrine disruptors that are capable of compromising male infertility [69– 72]. Studies on cetaceans in human care may not reveal representative results for wild individuals and therefore a long-term stranding monitoring programme is a favourable method in order to achieve a sufficient sample size as well as a seasonal average of specimen. With that, information on the reproductive system of harbour porpoises in the North and Baltic Sea could be provided and build an important basis for management plans for endangered species that are no longer capable of reaching the Good Environmental Status like suggested by the Marine Strategy Framework Directive of the European Union. This would be essential for the evaluation of genetic and evolutionary studies and thus essential for conservation biology.

- 63 -

3.7. Acknowledgements

We would like to thank all personnel helping during the necropsies at the Institute for terrestrial and aquatic wildlife research (ITAW Büsum) Büsum. Special thanks go to our lab technicians at the Institute for terrestrial and aquatic wildlife research (ITAW Büsum) and the Institute of Anatomy from the University of Veterinary Medicine Hanover, Germany as well as at the Department of Pathology, Faculty of Veterinary Medicine, Utrecht University, the Netherlands, who helped us tremendously with the acquisition of samples, their handling and age determination. We are especially grateful to our lab technicians at the Institute of Anatomy, University of Veterinary Medicine Hanover, Germany and at the Institute of Veterinary Anatomy, Histology and Embryology, University of Giessen, Germany for professional histology. We also thank Christina Lockyer for her great effort to help us with the age determination technique. We thank Abbo Van Neer for proof-reading the article. Furthermore, we would like to thank everyone participating in the stranding network who regularly collect, report and transfer specimen to the ITAW. Special thanks belong to Caren Imme von Stemm, Institute of Anatomy from the University of Veterinary Medicine Hanover, Germany, for her depictions of germ cell stages. This project was partly funded by the Ministry of Energy, Agriculture, the Environment and Rural Areas Schleswig Holstein (MELUR) and the state centre for coastal protection, national park and ocean protection Schleswig - Holstein (LKN).

- 64 -

3.8. References

[1] Kinze CC. On the harbour porpoise in Danish waters: Distribution, breeding areas, and threats. Fauna Flora Stock 1990;85:3–4.

[2] Benke H, Bräger S, Dähne M, Gallus A, Hansen S, Honnef CG, et al. Baltic Sea harbour porpoise populations: Status and conservation needs derived from recent survey results. Mar Ecol Prog Ser 2014;495:275–90. doi:10.3354/meps10538.

[3] Addink MJ, Sørensen TB, García Hartmann M. Aspects of reproduction and seasonality in the harbour porpoise from dutch waters. Dev. Mar. Biol., 1995, p. 459– 64. doi:10.1016/S0163-6995(06)80046-6.

[4] Heide-Jørgensen MP, Iversen M, Nielsen NH, Lockyer C, Stern H, Ribergaard MH. Harbour porpoises respond to climate change. Ecol Evol 2011;1:579–85. doi:10.1002/ece3.51.

[5] Dähne M, Adler S, Krügel K, Siebert U, Gilles A, Lucke K, et al. Effects of pile- driving on harbour porpoises ( Phocoena phocoena ) at the first offshore wind farm in Germany. Environ Res Lett 2013;8:025002. doi:10.1088/1748-9326/8/2/025002.

[6] Nabe-Nielsen J, Sibly RM, Tougaard J, Teilmann J, Sveegaard S. Effects of noise and by-catch on a Danish harbour porpoise population. Ecol Modell 2014;272:242– 51. doi:10.1016/j.ecolmodel.2013.09.025.

[7] Murphy S, Pierce GJ, Law RJ, Bersuder P, Jepson PD, Learmonth JA, et al. Assessing the effect of persistent organic pollutants on reproductive activity in common dolphins and harbour porpoises. J Northwest Atl Fish Sci 2010;42:153–73. doi:10.2960/J.v42.m658.

[8] von Benda-Beckmann AM, Aarts G, Sertlek HÖ, Lucke K, Verboom WC, Kastelein RA, et al. Assessing the impact of underwater clearance of unexploded ordnance on harbour porpoises (Phocoena phocoena) in the Southern North Sea. Aquat Mamm 2015;41:503–23. doi:10.1578/AM.41.4.2015.503.

[9] Siebert U, Joiris CR, Holsbeek L, Benke H, Failing K, Frese K, et al. Potential

- 65 - relation between mercury concentrations and necropsy findings in cetaceans from German waters of the North and Baltic Seas. Mar Pollut Bull 1999;38:285–95. doi:10.1016/S0025-326X(98)00147-7.

[10] Beineke A, Siebert U, McLachlan M, Bruhn R, Thron K, Failing K, et al. Investigations of the potential influence of environmental contaminants on the thymus and spleen of harbor porpoises (Phocoena phocoena). Environ Sci Technol 2005;39:3933–8. doi:10.1021/es048709j.

[11] Das K, Siebert U, Fontaine M, Jauniaux T, Holsbeek L, Bouquegneau J-M. Ecological and pathological factors related to trace metal concentrations in harbour porpoises Phocoena phocoena from the North Sea and adjacent areas. Mar Ecol Prog Ser 2004;281:283–95. doi:10.3354/meps281283.

[12] Mruk DD, Cheng CY. Environmental contaminants: Is male reproductive health at risk? Spermatogenesis 2011;1:283–90. doi:10.4161/spmg.1.4.18328.

[13] Berggren P, Wade PR, Carlström J, Read AJ. Potential limits to anthropogenic mortality for harbour porpoises in the Baltic region. Biol Conserv 2002;103:313–22. doi:10.1016/S0006-3207(01)00142-2.

[14] Bennett PM, Jepson PD, Law RJ, Jones BR, Kuiken T, Baker JR, et al. Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environ Pollut 2001;112:33–40. doi:10.1016/S0269-7491(00)00105-6.

[15] Jepson PD, Deaville R, Barber JL, Aguilar À, Borrell A, Murphy S, et al. PCB pollution continues to impact populations of orcas and other dolphins in European waters. Sci Rep 2016:1–17. doi:10.1038/srep18573.

[16] Borgå K, Fisk AT, Hoekstra PE, Muir DCG. Biological and chemical factors of importance in the bioaccumulation and trophic transfer of persistent organochlorine contaminants in Arctic marine food webs. Environ Toxicol Chem 2004;23:2367–85. doi:10.1897/03-518.

[17] Wolkers J, Burkow IC, Monshouwer M, Lydersen C, Dahle S, Witkamp RF. Cytochrome P450-mediated enzyme activities and polychlorinated biphenyl accumulation in harp seal (Phoca groenlandica). Mar Environ Res 1999;48:59–72.

- 66 - doi:10.1016/S0141-1136(99)00032-X.

[18] Siebert U, Wünschmann A, Weiss R, Frank H, Benke H, Frese K. Post-mortem findings in harbour porpoises (phocoena phocoena) from the German North and Baltic Seas. J Comp Pathol 2001;124:102–14. doi:10.1053/jcpa.2000.0436.

[19] Wiemann A, Andersen LW, Berggren P, Siebert U, Benke H, Teilmann J, et al. Mitochondrial Control Region and microsatellite analyses on harbour porpoise (Phocoena phocoena) unravel population differentiation in the Baltic Sea and adjacent waters. Conserv Genet 2010;11:195–211. doi:10.1007/s10592-009-0023-x.

[20] Kesselring T, Viquerat S, Brehm R, Siebert U. Correction: Coming of age: - Do female harbour porpoises (Phocoena phocoena) from the North Sea and Baltic Sea have sufficient time to reproduce in a human influenced environment? (PLoS One (2017) 12:10 (e0186951) DOI: 10.1371/journal.pone.0186951). PLoS One 2018;13:1–14. doi:10.1371/journal.pone.0199633.

[21] Henriksen K, Hakovirta H, Parvinen M. Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: In situ quantification in squash preparations after administration of ethane dimethane sulfonate. Endocrinology 1995;136:3285–91. doi:10.1210/endo.136.8.7628362.

[22] Li D, Han X. Evaluation of toxicity of methyl tert-butyl ether (MTBE) on mouse spermatogenic cells in vitro. Toxicol Ind Health 2006;22:291–9. doi:10.1177/0748233706070310.

[23] Holt WV, Waller J, Moore A, Jepson PD, Deaville R, Bennett PM. Smooth muscle actin and vimentin as markers of testis development in the harbour porpoise (Phocoena phocoena). J Anat 2004;205:201–11. doi:10.1111/j.0021- 8782.2004.00328.x.

[24] Karakosta CV, Jepson PD, Ohira H, Moore A, Bennett PM, Holt WV. Testicular and ovarian development in the harbour porpoise (Phocoena phocoena). J Zool London 1999;249:111–21.

[25] Neimanis AS, Read AJ, Foster R a, Gaskin DE. Seasonal regression in testicular size and histology in harbour porpoises (Phocoena phocoena, L.) from the Bay of

- 67 -

Fundy and Gulf of Maine. J Zool 2000;250:221–9. doi:10.1017/S0952836900002077.

[26] Plön S, Bernard R. Testis, spermatogenesis and testiculat cycles. In: Miller DL, editor. Reprod. Biol. Phylogeny Cetacea Whales, Dolphins Porpoises. 1st ed., Boca Raton, Fl: CRC Press; 2007, p. 116–35.

[27] Hess RA, De Franca LR. Spermatogenesis and cycle of the seminiferous epithelium. Adv Exp Med Biol 2008;636:1–15. doi:10.1007/978-0-387-09597-4_1.

[28] Meistrich ML, Hess RA. Assessment of Spermatogenesis Through Staging of Seminiferous Tubules. In: Carrell DT, Aston KI, editors. Methods Mol. Biol., vol. 927, Totowa, NJ: Humana Press; 2013, p. 299–307. doi:10.1007/978-1-62703-038-0_27.

[29] Hogarth CA, Evans E, Onken J, Kent T, Mitchell D, Petkovich M, et al. CYP26 Enzymes Are Necessary Within the Postnatal Seminiferous Epithelium for Normal Murine Spermatogenesis1. Biol Reprod 2015;93:19. doi:10.1095/biolreprod.115.129718.

[30] Learmonth JA, Murphy S, Luque PL, Reid RJ, Patterson IAP, Brownlow A, et al. Life history of harbor porpoises (Phocoena phocoena) in Scottish (UK) waters. Mar Mammal Sci 2014;30:1427–55. doi:10.1111/mms.12130.

[31] Lockyer C, Heide-Jørgensen MP, Jensen J, Kinze CC, Buus Sørensen T. Age, length and reproductive parameters of harbour porpoises Phocoena phocoena (L.) from West Greenland. ICES J Mar Sci 2001;58:154–62. doi:10.1006/jmsc.2000.0998.

[32] Ólafsdóttir D, Víkingsson GA, Halldórsson SD, Sigurjónsson J. Growth and reproduction in harbour porpoises ( Phocoena phocoena ) in Icelandic waters. NAMMCO Sci Publ 2003;5:195–210. doi:10.7557/3.2747.

[33] Sørensen TB, Kinze CC. Reproduction and reproductive seasonality in Danish harbour porpoises, Phocoena Phocoena. Ophelia 1994;39:159–76. doi:10.1080/00785326.1994.10429541.

[34] Read AJ, Hohn AA. Life in the fast lane: the life history of horbor porpoises from the Gulf of Maine. Mar Mammal Sci 1995;11:423–40.

[35] Dardente H, Lomet D, Robert V, Decourt C, Beltramo M, Pellicer-Rubio MT.

- 68 -

Seasonal breeding in mammals: From basic science to applications and back. Theriogenology 2016;86:324–32. doi:10.1016/j.theriogenology.2016.04.045.

[36] Benke H, Siebert U, Lick R, Bandomir B, Weiss R. The current status of harbour porpoises (Phocoena phocoena) in German waters. Arch Fish Mar Res 1998;46:97– 123.

[37] Lockyer C, Kinze C. Status, ecology and life history of harbour porpoise (Phocoena phocoena), in Danish waters. NAMMCO Sci Publ 2003;5:143–75. doi:10.7557/3.2745.

[38] Lockyer C. Harbour porpoises (Phocoena phocoena) in the North Atlantic: Biological parameters. NAMMCO Sci Publ 2003;5:71–89. doi:10.7557/3.2740.

[39] Hasselmeier I, Abt KF, Adelung D, Siebert U. Stranding patterns of harbour porpoises ( Phocoena phocoena ) in the German North and Baltic Seas : when does the birth period occur ? J Cetacean Res Manag 2004;6:259–63.

[40] Kuiken T, Garcia Hartmann M, European Cetacean Society. Proceedings of the First ECS Workshop on Cetacean Pathology : dissection techniques and tissue sampling : Leiden, the Netherlands, 13-14 September 1991. Proc. First ECS Work. Cetacean Pathol., Stralsund, Germany: European Cetacean Society; 1991, p. 1–39.

[41] Aescht E, Büchl-Zimmermann S, Burmester A, Dänhardt-Pfeiffer S, Desel C, Hamers C, et al. Romeis Mikroskopische Technik. 2010. doi:10.1007/978-3-8274- 2254-5.

[42] Russell LD, Ettlin RA. Mammalian spermatogenesis. In: Sinha Hikim AP, Clegg ED, editors. Histol. Histopathol. Eval. Testis. 1st ed., Clearwater, Fl: Cache River Press; 1990, p. 1–40.

[43] Barber BJ, Blake NJ. Chapter 6 Reproductive Physiology. Dev Aquac Fish Sci 2006;35:357–416. doi:10.1016/S0167-9309(06)80033-5.

[44] R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: 2018.

[45] Wood SN, Pya N, Säfken B. Smoothing Parameter and Model Selection for

- 69 -

General Smooth Models. J Am Stat Assoc 2016;111:1548–63. doi:10.1080/01621459.2016.1180986.

[46] Wood SN. Thin-plate regression splines. J R Stat Soc 2003;65:95–114.

[47] Wood SN. Fast stable restricted maximum likelihood and marginal likelihood estimation of semiparametric generalized linear models. J R Stat Soc 2011;73:3–36.

[48] Hohn AA, Read AJ, Fernandez S, Vidal O, Findley LT. Life history of the vaquita, Phocoena sinus (Phocoenidae, Cetacea). J Zool 1996;239:235–51. doi:10.1111/j.1469-7998.1996.tb05450.x.

[49] Neuenhagen C, García Hartmann M, Greven H. Histology and morphometrics of testes of the white-sided dolphin (Lagenorhynchus acutus) in bycatch samples from the northeastern Atlantic. Mamm Biol 2007;72:283–98. doi:10.1016/j.mambio.2006.10.008.

[50] Bergmann M. Spermatogenese. Urologe 2005;44:1131–8. doi:10.1007/s00120- 005-0909-2.

[51] Learmonth JA, Murphy S, Luque PL, Reid RJ, Patterson IAP, Brownlow A, et al. Life history of harbor porpoises (Phocoena phocoena) in Scottish (UK) waters. Mar Mammal Sci 2014;30:1427–55. doi:10.1111/mms.12130.

[52] Luetjens CM, Weinbauer GF, Wistuba J. Primate spermatogenesis: New insights into comparative testicular organisation, spermatogenic efficiency and endocrine control. Biol Rev Camb Philos Soc 2005;80:475–88. doi:10.1017/S1464793105006755.

[53] Clermont Y. The cycle of the seminiferous epithelium in man. Am J Anat 1963;112:35–51. doi:10.1002/aja.1001120103.

[54] Foote RH, Swierstra EE, Hunt WL. Spermatogenesis in the dog. Anat Rec 1972;173:341–51. doi:10.1002/ar.1091730309.

[55] Böhme G, Pier C. [Classification criteria for the epithelium cycle of the seminiferous tubule, based on studies in the cat (Felis catus)]. Berl Munch Tierarztl Wochenschr 1986;99:232–6.

- 70 -

[56] Oakberg EF. A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am J Anat 1956;99:391–413. doi:10.1002/aja.1000990303.

[57] Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann N Y Acad Sci 1952;55:548–73. doi:10.1111/j.1749- 6632.1952.tb26576.x.

[58] Johnson L, Kattansaid AF, Hardy VB, Scrutchfield WL. Isolation and Staging of Horse Seminiferous Tubules by Transillumination. J Reprod Fertil 1990;89:689–96. doi:10.1530/jrf.0.0890689.

[59] Ekstedt E, Söderquist L, Plöen L. Fine Structure of Spermatogenesis and Sertoli Cells ( Epitheliocytus sustentans ) in the Bull. Anat Histol Embryol 1986;15:23–48. doi:10.1111/j.1439-0264.186.tb00529.x.

[60] Takalhashi S., Turek F. W. MRY. Circannual rhythms. Handb. Behav. Neurobiol., 2001, p. 509–28.

[61] Bronson FH. Climate change and seasonal reproduction in mammals. Philos Trans R Soc B Biol Sci 2009;364:3331–40. doi:10.1098/rstb.2009.0140.

[62] Vasantha I. Physiology of Seasonal Breeding: A Review. J Vet Sci Technol 2015;07:1–4. doi:10.4172/2157-7579.1000331.

[63] Stockley P. Sperm competition in mammals. Hum Fertil 2004;7:91–7. doi:10.1080/14647270410001699054.

[64] Gomendio M, Roldan ERS. Mechanisms of sperm competition: Linking physiology and behavioural ecology. Trends Ecol Evol 1993;8:95–100. doi:10.1016/0169-5347(93)90059-X.

[65] Fontaine PM, Barrette C. Megatestes - anatomical evidence for sperm competition in the harbor porpoise. Mammalia 1997;61:65–71.

[66] Gely-Pernot A, Raverdeau M, Teletin M, Vernet N, Féret B, Klopfenstein M, et al. Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor. PLOS Genet 2015;11:e1005501.

- 71 - doi:10.1371/journal.pgen.1005501.

[67] Wingfield JC. Organization of vertebrate annual cycles: implications for control mechanisms. Philos Trans R Soc B Biol Sci 2008;363:425–41. doi:10.1098/rstb.2007.2149.

[68] Potemina TE. Impairment of spermatogenesis in male rats during stress. Bull Exp Biol Med 2008;145:700–2. doi:10.1007/s10517-008-0173-8.

[69] Reijnders PJH. Reproductive failure in common seals feeding on fish from polluted coastal waters. Nature 1986;324:456–7. doi:10.1038/324456a0.

[70] Sedha S, Kumar S, Shukla S. Role of oxidative stress in male reproductive dysfunctions with reference to phthalate compounds. Urol J 2015;12:2304–16.

[71] Vos JG, Dybing E, Greim HA, Ladefoged O, Lambré C, Tarazona J V., et al. Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol 2000;30:71–133. doi:10.1080/10408440091159176.

[72] Yeung BH, Wan HT, Law AY, Wong CK. Endocrine disrupting chemicals: Multiple effects on testicular signaling and spermatogenesis. Spermatogenesis 2011;1: 231–9. doi:10.4161/spmg.1.3.18019.

- 72 -

Chapter 4: Discussion

4.1. General discussion

This study provides new and basic data about the reproductive biology of female and male harbour porpoises from the North and Baltic Sea. Besides the data that was available for harbour porpoise populations from other areas like age at sexual maturity, seasonal trends or pregnancy rates, some basic aspects like the detailed description of spermatogenesis in males were completely missing until now. For the evaluation of the reproductive potential of wild populations it is essential to have a dataset available that is as complete as possible, with information about reproduction in female and male individuals, since different environmental impacts might influence both, the female or the male reproductive system in yet unknown ways. For another marine mammal species it has been shown, that the introduction of contaminants leads populations to extinction (Desforges et al., 2018), but these investigations require invasive methods such as biopsy sampling. For the harbour porpoise as only cetacean species in German waters, it would not be possible in the near future to conduct invasive sampling, so it is more and more important to make use of the material that is accessible for investigations. The advantage of post-mortem examination and sampling is definitely the availability and variety of any material that might be of interest. With that, a detailed dedicated analysis on a cellular basis like the histological investigation of the gonads like in the present study is possible, even though some difficulties emerged regarding quality of the tissue that might have been compromised and been frozen or decomposed before examination. Besides the sample composition itself, the bias that occurs due to population – specific differences are hard to quantify. Diverse populations of harbour porpoises from distinct marine areas of the world are influenced by various abiotic and biotic factors. So, distinctions between populations may reflect their various environmental conditions and might lead to conclusions regarding these ecosystems. For the future it would be interesting to know, which differences between harbour porpoises from arctic regions and populations from the North and Baltic regions can be noticed. It might be expected, that stress and human influences and disturbances are reduced to a minimum in arctic regions, but the question still remains, whether individuals from polar environments reflect the ‘real’ baseline for reproductive physiology and morphology. - 73 -

At least, the results of this study may serve as essential data reflecting the current status of the North Sea- and Baltic Sea environment and could serve as an important basis for management plans especially for the critically endangered harbour porpoise from the central Baltic Sea, that is no longer capable of reaching the good environmental status as suggested by the Marine Strategy Framework Directive of the European Union.

To summarise, the present study complements and extends the current state of knowledge of reproductive biology of harbour porpoises and can serve as a baseline for further studies that establish the methods used in these analyses and furthermore arrange a wider perspective for science in the field of wildlife reproduction and in particular for the harbour porpoise of German waters.

- 74 -

4.2. First part of the thesis: study of age at sexual maturity and demography in female harbour porpoises

The results of this study present the first model-based estimation of sexual maturity (n = 111) from the German North Sea and Baltic Sea combined with the evaluation of the demographic age structure (n = 526) of harbour porpoises that became stranded or by - caught in the period of 1990 to 2016. Using a binomial regression model it was possible to find a threshold, at which 50 % of the females were sexually mature – estimated at the age of 4.95 years. This approach enables the inclusion of variables and error structures and can be used as a robust estimator of an observed advance in female life stages. As such, the model suffers from inaccurate measurements and most of all from small sample sizes, which limited the number of linear combinations of covariates available in the modelling process. At the same time, model metrics proved robustness and thus strongly indicate that the true mean is centred on 4.95 years with a 95% CI of 4.15-5.83 years. Future studies with larger sample sizes, at different locations, using different predictors can use this estimate to compare their study results. Furthermore, the analysis of the datasets of the age determination revealed an age structure between zero and 22 years, with a mean age at death of 5.70 (± 0.27) years for animals from the North Sea and 3.67 (± 0.30) years for specimens from the German Baltic Sea.

The value for the age at sexual maturity differs from findings in harbour porpoises that other authors analysed and shows that many factors could influence the outcome of studies (Van Utrecht, 1978; Gaskin et al., 1984; Read and Hohn, 1984; Karakosta et al., 1999; Lockyer et al, 2001; Lockyer and Kinze, 2003; Learmonth et al., 2014). The method used is a new approach that assumes only specimens with signs of former ovulation as ‘sexually mature’. When including tertiary follicles as ‘first signs of sexually maturity’ into the method, a lower value of age at sexual maturity was detected, but this was not verified by the international reviewers as an acceptable method, because no scientists had investigated ovaries of harbour porpoises on a histological basis before and thus there were no values to compare to. Consequently, the method that was used might produce a bias by itself and furthermore, the person that analyses the specimens might miss something or make mistakes in the process of evaluating the structures found on the ovaries. The other point that might correspond with the bias produced by the method is the true - 75 - difference between the populations and the areas they live in. This might be hard to distinguish and requires the utilisation of the exact same method in all areas the harbour porpoise occurs.

It is known from populations from other waters, that every regional population can show different trends in reproductive seasonal parameters according to external and environmental factors they are exposed to (Berggren et al., 2002; Galatius et al., 2012; Lockyer et al., 2001).

Assuming this case, different values in age of sexual maturity can be caused by environmental factors that have impact on the individuals and their reproductive and endocrine system. One factor is the condition of their feeding habitat and prey availability. It has been shown in humans, that a good nutritional status can initiate an earlier onset of sexual maturity (Baker, 1985; Kaplowitz, 2008). Furthermore, anthropogenic factors such as underwater noise, disturbance, bycatch, increasing amounts of marine debris and chemical pollution might trigger changes in marine mammal physiology (Reijnders, 1986; Aguilar and Borrell, 1995; Berggren et al., 2002; Romano et al., 2004; Jepson et al., 2016; Unger et al., 2017) and manifest in an increased stress hormone level (Müller et al., 2013). It has been shown that high stress levels are connected to the function of the hypothalamic-pituitary axis and thus of the reproductive system of other mammals like humans, pigs and rats (Ellis and Essex, 2007; Hennessy and Williamson, 1983; Nakamura et al., 2008; Rivier and Vale, 1990; Roozendaal et al., 1995; Tilbrook et al., 2000) and thus e.g. to an earlier onset of sexual maturity (Ellis, 2004; Ellis and Essex, 2007). The direct effects on the reproductive hormonal system evoke a modified synthesis and secretion of Gonadotropin-releasing-hormone (GnRH). Furthermore, stress hormones are suspected of affecting the feedback mechanisms of steroid hormones in the hypothalamus and the pituitary gland (Glaser and Kiecolt-Glaser, 2005). These stress-induced changes of the reproductive system might be an evolutional adaption to unfavourable environmental conditions, under which resources can be focussed on survival or improvement of an overall health status and young females can be capable of producing offspring (Ellis, 2004). Since stress hormones are known to supress the immune system (Ellis and Essex, 2007) and consequently leading to an increased risk of viral and bacteriological infections with the expression of cytokines (Siebert et al., 2009), they are capable to induce fertility-suppression effects (Mikuni, - 76 -

1995; Ahmed, 2000). For future studies, a correlation between stress hormone levels and reproductive parameters is strongly required, since the existing knowledge from other mammals gives indices for this close relationship.

The immune system itself is also susceptible to endocrine disrupters (Beineke et al., 2005; Das et al., 2006; Imazaki et al., 2015), since e.g. pesticides and plasticisers are major anthropogenic disruptors of the reproductive cycle and the onset of sexual maturity (Murphy et al., 2015) of marine mammals. PCBs (polychlorinated biphenyls) can accumulate in the blubber of harbour porpoises and might act as estrogenic as well as anti - estrogenic agents. (Das et al., 2006) and thus, it cannot be ruled out that changes in the onset of sexual maturity might depend on the spatial and temporal concentration of chemical pollutants within a local food web and might include a generation bias.

Another bias that might arise in this context is the discrepancy between specimens collected in a stranding network and the real living stock of an area. Although data from stranding networks do not necessarily reflect the status of the entire population, these estimates have to be regarded as best available estimates rather than an absolute representation of the population. Using necropsy data from an unknown proportion of animals is always connected with a degree of uncertainty. It cannot be assumed that animals found dead on the beaches represent the average population individual, nor that these stranded animals are spatially correlated to all population members and therefore might only represent a small fraction of the actual population.

With consideration of this issue, an approach of calculation of the proportion of sexually mature individuals of all investigated individuals and the transfer to actual population counts has been performed in this study.

Using the estimated age at sexual maturity (4.95 years) and the respective age structure of the North – and Baltic Sea populations, the conclusion can be made, that 54% of the North Sea and up to 27% of animals in the Baltic Sea belong to the reproductively active part of the populations. Population estimates from the National German Monitoring Programme for harbour porpoises reveal that the population in the German North Sea contains up to 50,000 animals (Gilles et al., 2009) and in the German Baltic Sea about 3,000 animals during the summer months (personal communication with Sacha Viquerat, Institute for Terrestrial and Aquatic Wildlife - 77 -

Research; further information can provide the following maps provided by https://geodienste.bfn.de/schweinswalverbreitung). Based on necropsy data, the sex ratio was assumed to be roughly 46% females. This would imply that the total number of females within the reproductive population is 12,430 females in the German North Sea and only 372 females in the German Baltic Sea. These values are rough estimates and do not reflect the exact status of the populations. In particular since no information is available about the dynamics of population boundary shifts throughout the seasons. For the Baltic Sea subpopulations this value is very low compared to other harbour porpoise populations like the one in the North Sea and these animals might thus be more vulnerable to environmental factors that shorten the reproductive lifespan like an increase in bycatches in the Baltic Sea (Koschinski and Pfander, 2009). Bycatches might affect the population development since female adult harbour porpoises are often by-caught due to their distribution near the shore during the reproductive period and their distinct foraging behaviours during lactation (Yasui and Gaskin, 1986; Sveegaard et al., 2011) as well as juvenile harbour porpoises that show unestablished foraging habits. The significantly lower age at death of individuals from the Baltic Sea compared to North Sea specimens supports the proposal to reduce the magnitude of bycatches (Koschinski and Pfander, 2009).

For future studies on life history in harbour porpoises it would be essential to include more data on the individual health condition and pathological findings of the investigated animals into the analyses to minimise possible bias. Furthermore, other reproductive parameters such as pregnancy rate, birth rate, ovulation rate and even pathological findings in the reproductive tracts should be included to gain a complete overview of the reproductive status of a population. Subsequent investigations that use datasets including information about potential factors that might influence reproductive parameters such as toxicant load, endocrine disruptors or marine debris findings would support more explicit and spatial results that represent the reproductive status of the female part of a population.

Furthermore, a larger dataset would be required in order to make more precise statements that have a smaller error due to statistical inaccuracies that arise from a small sample size. For example, a separation between Baltic and North Sea specimen would have reduced some of the error presented in the manuscript ‘Coming of Age: - Do female harbour porpoises (Phocoena phocoena) from the North - 78 -

Sea and Baltic Sea have sufficient time to reproduce in a human influenced environment?’.

- 79 -

4.3. Second part of the thesis: study of testicular morphology and spermatogenesis in male harbour porpoises

For male harbour porpoises it is more complex to make a statement about the reproductive status and to find parameters that have the potential to answer this question. While for female harbour porpoises, the age at sexual maturity, seasonal aspects and fertility are easier to access and thus, scientists from other areas have already given those investigations a trial. The analysis of male gonads in this species is restricted to only a few studies (Sørensen and Kinze, 1994; Karakosta et al., 1999; Neimanis et al., 2000; Lockyer et al., 2001; Ólafsdóttir et al., 2003; Holt et al., 2004; Plön and Bernard, 2007) and therefore the main goal of the second part of the study was to complete the knowledge about postnatal testicular development, spermatogenesis and seasonal changes in this species. Results reveal that first signs of spermatogenesis can be observed in animals older than three years and seasonal changes of the seminiferous epithelium occur from the age of four years. Furthermore, results show that the peak breeding season occurs in June and July, while the low breeding season was found during August to May.

Results of the present study provide the first detailed histological description of the seminiferous epithelial cycle in adult harbour porpoises and the observation of eight different spermatogenic stages within one tubular cross-section confirms the initial findings of former investigations (Neimanis et al., 2000; Plön and Bernard, 2007) and is called multi-stage-arrangement (Bergmann, 2005). While other authors that examined spermatogenesis did not define spermatogenic stages, findings in this study confirm results from findings in testes from new world monkeys (Platyrrhini), old world monkeys (Cercopithecoidea) and humans (Homo sapiens) (Bergmann, 2005). Until now, this ‘multi-stage-arrangement’ (Luetjens et al., 2005) was unique to these species. According to the association of distinct cell differentiation types, mammalian species show different specific appearances and numbers of the spermatogenic stages (Man: VII stages, dog: VIII stages, tomcat: VIII stages, mouse: XV stages, rat: XIV stages, bull: VIII stages, stallion: VIII stages) (Clermont, 1963; Böhme, 1986; Foote et al., 1972; Johnson et al., 1990; Leblond and Clermont, 1952; Oakberg, 1956, Johnson et al., 1990).

Eight stages of spermatogenesis have been found in adult individuals during the

- 80 - peak breeding season in summer, while only spermatocytes and a few remaining spermatozoa are observed in autumn, winter and spring (low breeding season, concomitant with smaller testes), which could be linked to the fact that as season progresses, more missing generations are found in the cross sections resulting in the level of spermatozoa as the last remaining germ cells of the season. During the peak breeding season, samples have been found that showed also some missing germ cell generations in the seminiferous epithelium, which could right now be documented as either a physiological or pathological condition. In other mammalian species, evidence is given that missing generations are a sign of reduced fertility (Gely-Pernot et al., 2015) due to environmental disturbances. For harbour porpoises it would be interesting to compare samples from oceans with low contaminant levels with samples from areas with high contaminant influx.

The seasonal aspects in terms of the main breeding months are similar to findings in harbour porpoises from the English and Welsh coast, the Bay of Fundy and Gulf of Maine and Greenland (Karakosta et al., 1999; Neimanis et al., 2000, Lockyer et al., 2001). The methods used in these studies are slightly different from this study, since no staging of spermatogenesis has been previously performed, but testes mass, presence of spermatozoa in the epididymides and diameter of the seminiferous tubules lead to similar results in the regions mentioned above.

In order to analyse a parameter that is more common in the scientific community of cetacean scientists, a measurement of the tubular diameter of all specimens was performed and compared to findings in harbour porpoises from other areas. Our results in adult specimen are in accordance to findings in harbour porpoises from other regions. Regarding to Karakosta et al., (1999), the average tubular diameter in adult harbour porpoises is 117.5 µm (SD: 38.0). For porpoises from Canada, Neimanis et al., (2000) reported a mean diameter of the seminiferous tubules of 161.6 µm (SD: 4.07) at the end of July and a maximum diameter of the seminiferous tubules (225 µm) in an individual found in early July. This result from the morphometrical analysis matches the observation of full spermatogenic activity with all stages present in June and July in the present study and confirms a high-breeding season in late June and July for harbour porpoises of these areas. These observations further suggest that adult male harbour porpoises maximise their chance at reproductive success by synchronising their spermatogenic activity with - 81 - the ovulation time of females, which supports the hypothesis of sperm competition (Fontaine and Barrette, 1997). For other wild life mammals it is known, that they synchronise their energetically demanding sperm production to the oestrus of female individuals and thus to the most favourable time of the year (Wingfield, 2008). Environmental factors regulate spermatogenesis through the hypothalamic–pituitary– gonadal axis and trigger the onset of testicular function in spring and the testicular regression in autumn (Wingfield, 2008). This seasonal cycle supports energy balance in terms of seasonal foraging pattern, is a common regulatory mode in mammalian species (Bronson, 2009; Takalhashi & Turek, 2001; Dardente et al., 2016). In harbour porpoises, a seasonal focus in sperm production is suspected to support sperm competition (Fontaine and Barrette, 1997). This mechanism targets the female ovulation period as a strategy for successful mating and may support behavioural mating strategies in different ways (Gomendio and Roldan, 1993; Fontaine and Barrette, 1997; Stockley, 2004; Keener et al., 2018). In order to specify seasonal changes and detect possible shifts and pathological changes, an implementation of immunohistochemical methods would complete the present findings in the future. While it is known, that in some other species proliferation of germ cells occurs during the initiation of spermatogenesis in spring and germ cell apoptosis occurs during the downregulation of spermatogenesis in the end of the mating season (Young and Nelson, 2001), there is no information about these processes in harbour porpoises so far. Therefore it would be interesting to concept future studies that focus on immunohistochemical methods that might have the potential to haul in this information about specific processes in the testicular tissue of harbour porpoises. Holt et al., (2004) successfully tested some immunohistochemical markers of testicular maturation in juvenile harbour porpoises, but markers for possible endocrine disruption (for example Anti-Muellerian Hormone and Androgen Receptor) have not been investigated yet. So, immunohistochemistry might be a next step in the future in order to identify and measure possible influences that endocrine disruptors might have on the male endocrine system in general and on spermatogenesis in particular.

Like all other methods of life sciences, the histological analysis of gonadal tissues is prone to methodical bias. Potential biases can stem from poor tissue preservation, freezing, fixation, the process of staining and finally misinterpretation of the

- 82 - microscopic image of specimens, for example when examining the qualitative and morphometric properties of the testicular tissue. Although only the diameter of completely round tubules was measured, it cannot be excluded that some of the tubules were not perfectly round, so these might bias the value. Furthermore, in some cases the basal membrane of the tubules disconnected from the epithelium, making it difficult to find the exact starting point for the measurements. For the staging of the seminiferous epithelium, only specimens with an appropriate tissue preservation status were included, but there were differences present (due to factors that might influence the tissue like freezing prior to post-mortem examination), even between these well preserved and fixated specimens, what might have produced some bias too. Some of the individuals have been found dead and stored in previous years and thus, the histological staining did not take place during the same process, which would be desirable in the perfect case. Specimens stained in the same passage would exclude the bias that might occur due to slightly different chemicals and their composition. The same issue arises regarding the tooth preparation for the tooth age determination. This method of age determination has already been optimised by the same technicians, but still some biases cannot be fully excluded either. Another error that is hard to detect is the data collection and data handling that follows after the necropsy and laboratory work is finished. Although the system of data preparation and transfer to the database has been steadily improved over the last years, it is still possible that some findings in a specimen are not documented well or confused with another one, even during the necropsy. All these errors would reproduce in ongoing steps of the study and might influence our results, but nevertheless all steps and methods of the present work have been performed with a maximum of effort and carefulness. The limited number of samples available for the statistical analysis rendered some analysis not viable. As such, differences between specimens collected on the North Sea versus the Baltic Sea coasts could not be assessed due to sparse data from the Baltic Sea. All model estimates include error measurements, such as standard errors or confidence intervals, where applicable. The width of the error structure is inherent to the modelling process and reflects some unresolved statistical noise that could be avoided using more data.

Whether those individuals, that showed missing generations in their histological specimens might have been exposed to stressors and contaminants and prove

- 83 - infertility as a result of these influences, or if this phenomenon is a physiological situation during the course of the year, has to be verified in further studies.

- 84 -

Chapter 5: Summary

Tina Kesselring

Langzeitdaten zur Reproduktionsbiologie des Schweinswals (Phocoena phocoena) aus der deutschen Nord- und Ostsee

As top predators and the only native cetacean species, harbour porpoises (Phocoena phocoena) play an important role in the German oceanic ecosystems. Due to the fact, that harbour porpoises are already exposed to numerous anthropogenic influences, the reproductive lifespan is a sensitive time frame and reproductive health might be the key to a stabile population size in the German ocean areas. This study focussed on the macroscopic and microscopic analysis of the reproductive organs of both, female and male individuals from the North and Baltic Seas. With these analyses it was possible to obtain data on life history events like postnatal gonadal development, age at sexual maturity and seasonality.

For female harbour porpoises, we investigated the first signs of sexual maturity for a period of almost two decades (1990 – 2016). Ovaries from 111 female harbour porpoises from the German North and Baltic Sea were examined for the presence and morphological structure of follicles, corpora lutea and corpora albicantia. These specimens were selected by the criteria of status of decomposition in order to exclude biases due to poor tissue preservation. Based on the ovarian characteristics we performed the first model-based estimation of age at sexual maturity for harbour porpoises from German waters. Additionally, we produced a demographical age structure based on all female strandings and bycatches from German coasts.

For males, the testes of 127 harbour porpoises from the North and Baltic Sea that became stranded or by-caught in the years 1998 to 2016 were histologically examined. For the identification of changes in testes morphology and morphometry of adult harbour porpoises during the course of the year, special characteristics like germ cell associations (stages) and diameter of seminiferous tubules were noted for each month. - 85 -

Our results of the examination of female harbour porpoises showed that corpora lutea and corpora albicantia as signs of former ovulation events could be found in individuals at an age of 4.95 (± 0.6) years. No significant differences between specimens from the North and Baltic Sea were detected. However, the average age at death differed significantly with 5.70 (± 0.27) years for North Sea animals and 3.67 (± 0.30) years for those in the Baltic Sea.

The histological analysis of the testes showed that first signs of sexual maturity could be detected in animals older than three years, while individuals older than four years could be listed as ‘adults’, showing a staging of spermatogenesis and a seasonal circle. Since more than one stage of spermatogenesis could be found per cross section of the seminiferous tubules of adult harbour porpoises, these results are similar to findings in men and some ape species. This rare phenomenon is called multi-stage-arrangement. In sexually active males from the high breeding season (June and July) eight stages of spermatogenesis were identified and all stages occurred simultaneously, while during the non-breeding season (August to May) only residual spermatogenesis or constituent germ cell populations were found. In addition, an increasing number of missing germ cell generations from August to September was encountered.

Our studies provide a detailed description and analysis of the functional anatomy and histology of the female and male reproductive organs of harbour porpoises and might serve as baseline studies for further investigations that deal with effects of anthropogenic influences on the reproductive system of this top predator in the German North and Baltic Sea, the harbour porpoise.

- 86 -

Chapter 6: Zusammenfassung

Tina Kesselring

Analysis of the reproductive biology of harbour porpoises (Phocoena phocoena) from the North and Baltic Sea on a long-term basis

Der Schweinswal ist die einzige in der deutschen Nord- und Ostsee beheimatete Walart und trägt als eine von wenigen Topprädatoren in diesen Gewässern eine hohe Bedeutung für das Gleichgewicht des Ökosystems dieser Meere. Da Schweinswale zahlreichen anthropogenen Einflüssen ausgesetzt sind, ist die mögliche Zeitspanne der Reproduktionsfähigkeit eine sensible Phase für ein Individuum und somit auch für die Populationsstabilität.

In dieser Arbeit wurden die Gonaden von weiblichen und männlichen Schweinswalen der Nord- und Ostsee auf makroskopischer und mikroskopischer Ebene untersucht, um reproduktionsbezogene Parameter wie postnatale Entwicklung, saisonale Veränderungen und Alter der Geschlechtsreife dokumentieren und analysieren zu können.

Von den weiblichen Schweinswalen konnte anhand von 111 Ovarien aus den Jahren 1990 bis 2016 das Alter der Geschlechtsreife durch die morphologische Untersuchung von Funktionskörpern wie Follikeln und Gelbkörpern bestimmt werden. Die Exemplare und Proben wurden aufgrund von Parametern ausgewählt, welche sich negativ auf die Qualität der Präparate auswirken könnte, wie zum Beispiel Verwesungsgrad der Gewebe oder Einfrieren der Proben vor Untersuchung.

Auf dieser Analyse der Ovarien basierend wurde erstmalig eine Modellierung für das Alter der Geschlechtsreife von Schweinswalen aus deutschen Gewässern durchgeführt. Diese Analyse wurde mit der Berechnung der demographischen Altersstruktur ergänzt, um dann den Anteil geschlechtsreifer Weibchen an der Gesamtpopulation in der Nord- und Ostsee schätzen zu können.

Für die Analyse der männlichen Gonaden wurden die Hoden von 127 Schweinwalen der deutschen Nord- und Ostsee aus dem Zeitraum 1998 bis 2016 makroskopisch

- 87 - und histologisch untersucht und ausgewertet. Um Veränderungen der Hodenmorphologie und –morphometrie im Jahresverlauf messen zu können, wurden für jeden Monat des Jahres Merkmale im histologischen Bild, wie zum Beispiel Keimzellassoziationen dokumentiert und die Durchmesser der Tubuli seminiferi contorti gemessen.

Die Ergebnisse der Analysen der weiblichen Gonaden zeigen, dass Weibchen in einem durchschnittlichen Alter von 4.95 (± 0.6) Zeichen vorheriger Ovulation wie Gelbkörper oder ältere Narben (Corpora albicantia oder Corpora atretica) auf dem Ovar zeigen. Es konnten keine Unterschiede zwischen Tieren der Nord- und Ostsee festgestellt werden. Das durchschnittliche Alter, in dem die Tiere gefunden wurden, unterschied sich jedoch signifikant: Die Schweinswale der Nordsee waren zum Zeitpunkt ihres Todes durchschnittlich 5.70 (± 0.27) Jahre alt, während Tiere aus der Ostsee im durchschnittlichen Alter von 3.67 (± 0.30) Jahren gestorben sind.

Die Analyse der männlichen Gonaden ergab, dass erste Anzeichen der Pubertät bereits ab einem Alter von drei Jahren erkennbar sind und Tiere ab einem Alter von vier Jahren Spermatogenesestadien, einen saisonalen Verlauf des Keimepithels und somit volle Geschlechtsreife zeigen. Die Hauptpaarungszeit findet in den Monaten Juni und Juli statt. Während dieser Zeit sind im histologischen Schnitt des Hodengewebes acht verschiedene Keimzellassoziationen gleichzeitig in einem Tubulus seminiferus contortus auffindbar. Dieses Phänomen, welches bisher nur beim Menschen und einigen Affenarten gefunden worden ist, wird „multi-stage- arrangement“ genannt. Es konnten acht verschiedene Stadien der Spermatogenese identifiziert werden. In den restlichen Monaten des Jahres konnten vereinzelte Keimzellpopulationen oder die völlige Abwesenheit von Spermatogenese in adulten Tieren dokumentiert und somit der Schluss gezogen werden, dass die Reproduktionsruhe von August bis Mai stattfindet. In den Monaten August und September konnten vermehrt fehlende Keimzellgenerationen in den Tubulusquerschnitten gefunden werden.

Die vorliegenden Studien liefern eine detaillierte Beschreibung und Analyse der funktionellen Anatomie und Histologie der weiblichen und männlichen Gonaden des heimischen Schweinswales und können als Grundlage für weitergehende Untersuchungen zu Effekten von äußeren Einflüssen auf das Reproduktionspotential

- 88 - der Schweinswalpopulationen herangezogen werden.

- 89 -

References

Aguilar, A. and Borrell A. (1995): Pollution and harbour porpoises in the eastern North Atlantic: A review. Reports of the International Whaling Commission 1995: special issue 16: 231–242

Addink, M.J. and Smeenk, C. (1999). The harbour porpoise Phocoena phocoena in Dutch coastal waters: Analysis of stranding records for the period 1920-1994. Lutra, 41, 55–80.

Ahmed, S.A. (2000). The immune system as a potential target for environmental estrogens (endocrine disrupters): a new emerging field. Toxicology 2000; 150 (1–3): 191–216.

Bando, T., Kishiro, T. and Kato, H. (2014). Yearly trend in the age at sexual maturity of Antarctic minke whales examined by transition phase in earplugs collected during JARPA and JARPAII surveys. In Scientific paper submitted to the JARPAII review meeting called by the IWC 2014 (pp. 1–10).

Bandomir, B., Chavez-Lisambart, L. and Siebert, U. (1998). Reproduction In Harbour Porpoises (Phocoena Phocoena), From German Waters. In Proceedings of the Twelfth Annual Conference of the European Cetacean Society.

Baker, E.R. (1985). Body weight and the initiation of puberty. Clinical Obstetrics and Gynecology. doi:10.1097/00003081-198528030-00013

Beineke, A., Siebert, U., McLachlan, M., Bruhn, R., Thron, K., Failing, K., Müller, G. and Baumgärtner, W. (2005). Investigations of the potential influence of environmental contaminants on the thymus and spleen of harbor porpoises (Phocoena phocoena). Environmental science and technology, 39(11), 3933–3938. doi:10.1021/es048709j

Benke, H., Bräger, S., Dähne, M., Gallus, A., Hansen, S., Honnef, C. G., Jabbusch, M., Koblitz, J. C., Krügel, K., Liebschner, A., Narberhaus, I. and Verfuß, U. K. (2014). Baltic Sea harbour porpoise populations: Status and conservation needs derived from recent survey results. Marine Ecology Progress Series, 495, 275–290. doi:10.3354/meps10538

- 90 -

Benke, H., Siebert, U., Lick, R., Bandomir, B. and Weiss, R. (1998). The current status of harbour porpoises (Phocoena phocoena) in German waters. Archive of Fishery and Marine Research, 46(2), 97–123.

Bennett, P.M., Jepson, P.D., Law, R.J., Jones, B.R., Kuiken, T., Baker, J.R., Rogan, E. and Kirkwood, J.K. (2001). Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environmental Pollution, 112(1), 33–40. doi:10.1016/S0269-7491(00)00105-6

Berggren, P., Wade, P.R., Carlström, J. and Read, A.J. (2002). Potential limits to anthropogenic mortality for harbour porpoises in the Baltic region. Biological Conservation, 103(3), 313–322. doi:10.1016/S0006-3207(01)00142-2

Bergmann, M. (2005). Spermatogenese. Der Urologe, 44(10), 1131–1138. doi:10.1007/s00120-005-0909-2

Böhme G. and Pier, C. (1986). Ordnungskriterien für den Zyklus des Epithels der Tubuli seminiferi, nach Untersuchungen beim Kater (Felis catus). Berliner und Münchener Tierärztliche Wochenschriften, 1(1), 232–236.

Borgå, K., Fisk, A.T., Hoekstra, P.E., and Muir, D.C.G. (2004). Biological and chemical factors of importance in the bioaccumulation and trophic transfer of persistent organochlorine contaminants in Arctic marine food webs. Environmental toxicology and chemistry / SETAC, 23(10), 2367–2385. doi:10.1897/03-518

Bronson, F.H. (2009). Climate change and seasonal reproduction in mammals. Philosophical Transactions of the Royal Society B: Biological Sciences, 364(1534), 3331–3340. doi:10.1098/rstb.2009.0140

Clermont Y. (1963). The cycle of the seminiferous epithelium in man. American Journal of Anatomy 1963;112:35–51. doi:10.1002/aja.1001120103.

Dardente, H., Lomet, D., Robert, V., Decourt, C., Beltramo, M. and Pellicer-Rubio, M.T. (2016). Seasonal breeding in mammals: From basic science to applications and back. Theriogenology 2016;86:324–32. doi:10.1016/j.theriogenology.2016.04.045.

Das, K., Siebert, U., Fontaine, M., Jauniaux, T., Holsbeek, L. and Bouquegneau, J.M. (2004). Ecological and pathological factors related to trace metal concentrations in - 91 - harbour porpoises Phocoena phocoena from the North Sea and adjacent areas. Marine Ecology Progress Series, 281, 283–295. doi:10.3354/meps281283

DeMaster, D.P. (1984). Review of techniques used to estimate the average age at attainment of sexual maturity in marine mammals. Report of the International Whaling Commission, Special Issue, 6: 175-179.

Desforges, J.P., Hall, A., McConnell, B., Rosing-Asvid, A., Barber, J.L., Brownlow, A., De Guise, S., Eulaers, I., Jepson, P.D., Letcher, R.J., Levin, M., Ross, P.S., Samarra, F., Víkingson, G., Sonne, C. and Dietz, R. (2018). Predicting global killer whale population collapse from PCB pollution. Science, 361(6409), 1373–1376. doi:10.1126/science.aat1953

Ellis B.J. (2004). Timing of pubertal maturation in girls: an integrated life history approach., Psychol Bull. 2004; 130(6):920-58.

Ellis, B.J. and Essex, M.J. (2007). Family Environments, Adrenarche, and Sexual Maturation: A Longitudinal Test of a Life History Model, Child Development 2007; 78(6):1799-817

Foote, R.H., Swierstra, E.E. and Hunt, W.L. (1972). Spermatogenesis in the dog. The Anatomical Record, 173(3), 341–351. doi:10.1002/ar.1091730309

Fisher, H.D. and Harrison R.J. (1970). Reproduction in the Common porpoise (Phocoena phocoena) of the North Atlantic. Journal of Zoology London 1970; 161: 471–486.

Fontaine P.M. and Barrette C. (1997). Megatestes - anatomical evidence for sperm competition in the harbor porpoise. Mammalia 1997;61:65–71.

Galatius, A., Kinze, C.C. and Teilmann, J. (2012). Population structure of harbour porpoises in the Baltic region: evidence of separation based on geometric morphometric comparisons. Journal of the Marine Biological Association of the UK, 92(8), 1669–1676. doi:10.1017/S0025315412000513

Gaskin D.E., Smith G.J.D., Watson A.P., Yasui W.Y. and Yurick, D. B. (1984). Reproduction in porpoises (Phocoenidae): implications for management. In Report of the International Whaling Commission 1984 (pp. 135–148). - 92 -

Gely-Pernot, A., Raverdeau, M., Teletin, M., Vernet, N., Féret, B., Klopfenstein, M., Dennefeld, C., Davidson, I., Benoit, G., Mark, M. and Ghyselinck, N.B. (2015). Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor. PLoS Genetics, 11(10). doi:10.1371/journal.pgen.1005501

Genov, T., Jepson, P.D., Barber, J.L., Hace, A., Gaspari, S., Centrih, T., Lesjak, J. and Kotnjek, P. (2018). Linking organochlorine contaminants with demographic parameters in free-ranging common bottlenose dolphins from the northern Adriatic Sea. Science of The Total Environment 657. DOI: 10.1016/j.scitotenv.2018.12.025

Gilles. A., Scheidat, M. and Siebert, U: (2009). Seasonal distribution of harbour porpoises and possible interference of offshore wind farms in the German North Sea. Marine ecology progress series 383 (1), 296-307

Gilles, A., Viquerat, S., Becker, E.A., Forney, K.A., Geelhoed, S.C.V., Haelters, J., Nabe-Nielsen, J., Scheidat, M., Siebert, U., Sveegard, S., Van Beest, F.M., Van Bemmelen, M. and Aarts, M. (2016). Seasonal habitat-based density models for a marine top predator, the harbour porpoise, in a dynamic environment. Ecosphere 7(6): e01367. 10.1002/ecs2.1367

Glaser, R. and Kiecolt-Glaser, J.K. (2005). Stress-induced immune dysfunction: Implications for health. Nature Reviews Immunology. doi:10.1038/nri1571

Gomendio, M. and Roldan, E.R.S. (1993). Mechanisms of sperm competition: Linking physiology and behavioural ecology. Trends in Ecology and Evolution. doi:10.1016/0169-5347(93)90059-X

Gonçalves, P.B.D., Gasperin, B.G., Ferreira, R. and Santos, J.T. (2012). Control of ovulation in mammals. Animal Reproduction, v.9, n.3, p.354-361, Jul./Sept. 2012

Hammond, P.S., Bearzi, G., Bjørge, A., Forney, K.A., Karczmarski, L., Kasuya, T., Perrin, W., Scott, M.D., Wang, J.Y., Wells, R.S. and Wilson, B. (2008). Phocoena phocoena Baltic Sea subpopulation (errata version published in 2016). The IUCN Red List of Threatened Species 2008

Hasselmeier, I., Abt, K.F., Adelung, D. and Siebert, U. (2004) . Stranding patterns of

- 93 -

Harbour Porpoises (Phocoena phocoena) in the North and Baltic Seas: when does the birth period occur? Journal of Cetacean Research and Management 6 (3): 259– 263.

Hennessy, D.P. and Williamson, P. (1983). The effects of stress and of ACTH administration in hormone profiles, oestrus and ovulation in pigs. Theriogenology. doi:10.1016/0093-691X(83)90019-5

Herr, H. (2009). Vorkommen von Schweinswalen (Phocoena phocoena) in Nord- und Ostsee - im Konflikt mit Schifffahrt und Fischerei? Dissertation zur Erlangung des Doktorgrades des Departments der Fakultät für Mathematik, Informatik und Naturwissenschaften der Universität Hamburg, April 2009

Hogarth, C.A., Evans, E., Onken, J., Kent, T., Mitchell, D., Petkovich, M., and Griswold, M.D. (2015). CYP26 Enzymes Are Necessary Within the Postnatal Seminiferous Epithelium for Normal Murine Spermatogenesis1. Biology of Reproduction, 93(1), 19. doi:10.1095/biolreprod.115.129718

Hohn, A.A., Ewing, R.Y. and Zaias, J. (2007). Reproduction in Relation to Conservation and Commercial Exploitation. Reproductive Biology and Phylogeny of Cetacea population, (January), 371–389. doi:10.1201/b11001-15

Holt, W.V., Waller, J., Moore, A., Jepson, P.D., Deaville, R. and Bennett, P.M. (2004). Smooth muscle actin and vimentin as markers of testis development in the harbour porpoise (Phocoena phocoena). Journal of Anatomy, 205(3), 201–211. doi:10.1111/j.0021-8782.2004.00328.x

Imazaki P.H., Brose F., Jauniaux T., Das K., Muller M. and Scippo M.L. (2015). Estrogenic Evaluation and Organochlorine Identification in Blubber of North Sea Harbour Porpoise (Phocoena phocoena) Stranded on the North Sea Coast. BioMed Research International 2015. doi: 10.1155/2015/438295

Jepson, P.D., Deaville, R., Barber, J.L., Aguilar, À., Borrell, A., Murphy, S., Barry, J., Brownlow, A., Barnett, J., Berrow, S., Cunningham, A.A., Davison, N.J., Doeschate, M., Esteban, R., Penrose, R., Perkins, M.W., Smith, B., Stephanis, Tregenza, N., Verborgh, P., Fernández, A. and Law, R.J. (2016). PCB pollution continues to impact populations of orcas and other dolphins in European waters. Scientific Reports, - 94 -

(November 2015), 1–17. doi:10.1038/srep18573

Johnson, L., Hardy, V.B. and Martin, M.T. (1990). Staging equine seminiferous tubules by Nomarski optics in unstained histologic sections and in tubules mounted in toto to reveal the spermatogenic wave. Anatomical Records 1990; 227(2): 167-74.

Johnson, L., Chaturvedi, P.K. and Williams, J.D. (1992). Missing generations of spermatocytes and spermatids in seminiferous epithelium contribute to low efficiency of spermatogenesis in humans. Biology of Reproduction 1992 Dec;47(6):1091-8.

Kaplowitz, P.B. (2008). Link Between Body Fat and the Timing of Puberty. Pediatrics. doi:10.1542/peds.2007-1813F

Karakosta, C.V., Jepson, P.D., Ohira, H., Moore, A., Bennett, P.M. and Holt, W.V. (1999). Testicular and ovarian development in the harbour porpoise (Phocoena phocoena). Journal of Zoology, London, 249(1), 111–121.

Keener, W., Webber, M., Szczepaniak, I.D., Markowitz, T.M. and Orbach, D.N. (2018). The Sex Life of Harbor Porpoises (Phocoena phocoena): Lateralized and Aerial Behavior. Aquatic Mammals 44(6):620-632 DOI: 10.1578/AM.44.6.2018.620

Kinze, C.C. (1990). On the harbour porpoise in Danish waters: Distribution, breeding areas, and threats. Fauna Flora Stockh, 85(3), 3–4.

Koschinski S. and Pfander A. (2009). By-catch of harbour porpoises (Phocoena phocoena) in the Baltic coastal waters of Angeln and Schwansen (Schleswig- Holstein, Germany). 16th ASCOBANS Advisory Committee Meeting Document 2009: AC16/Doc.60. Dist. 8

Learmonth, J.A., Murphy, S., Luque, P.L., Reid, R.J., Patterson, I.A.P., Brownlow, A., Ross, H.M., Barley, J.P., Santos, M.B. and Pierce, G.J. (2014). Life history of harbor porpoises (Phocoena phocoena) in Scottish (UK) waters. Marine Mammal Science, 30(4), 1427–1455. doi:10.1111/mms.12130

Leblond, C.P. and Clermont, Y. (1952). Definition of the stages of the cycle of the seminiferous epithelium in the rat. Annals of the New York Academy of Sciences, 55(4), 548–573. doi:10.1111/j.1749-6632.1952.tb26576.x

- 95 -

Lockyer, C. (1995). Investigations of aspects of the life history of the Harbour Porpoise, Phocoena phocoena, in British waters. In: BJØRGE A. and DONOVAN G.P.(eds) Biology of the Phocoenids: 189-197. Special Issue 16, International Whaling Commission, Cambridge.

Lockyer, C., Heide-Jørgensen, M.P., Jensen, J., Kinze, C.C. and Sørensen, T. (2001). Age, length and reproductive parameters of harbour porpoises Phocoena phocoena (L.) from West greenland. ICES Journal of Marine Science, 58(1), 154– 162. doi:10.1006/jmsc.2000.0998

Lockyer, C. and Kinze, C. (2003). Status, ecology and life history of harbour porpoise (Phocoena phocoena), in Danish waters. NAMMCO Scientific Publications, 5(Kinze), 143–175. doi:10.7557/3.2745

Luetjens C.M., Weinbauer, G.F. and Wistuba J. (2005). Primate spermatogenesis: New insights into comparative testicular organisation, spermatogenic efficiency and endocrine control. Biol Rev Camb Philos Soc 2005; 80:475–88. doi:10.1017/S1464793105006755.

Mikuni M. (1995). Effect of interleukin-2 and interleukin-6 on ovary in the ovulatory period--establishment of the new ovarian perfusion system and influence of interleukins on ovulation rate and steroid secretion. The Hokkaido Journal of Medical Science 1995; 70 (4): 561–572

Morioka, N., Zhu, C., Brännström, M., Woessner, J.F. and LeMaire, W.J. (1989). Mechanism of mammalian ovulation. Prog Clin Biol Res. 1989;294:65-85. Progress in clinical and biological research 1989; 294:65-85

Müller S., Lehnert K., Seibel H., Driver J., Ronnenberg K., Teilmann J., Van Elk, C., Kristenen, J.H., Everaats, E. and Siebert, U. ( 2013). Evaluation of immune and stress status in harbour porpoises (Phocoena phocoena): can hormones and mRNA expression levels serve as indicators to assess stress? BMC Veterinary Research 2013 (9): 145.

Murphy, S., Pierce, G.J., Law, R.J., Bersuder, P., Jepson, P.D., Learmonth, J.A., Addink, M.J., Dabin, W., Santos, M.B., Deaville, R., Zegers, B.N., Mets, A., Rogan, E., Ridoux, V., Reid, R.J., Smeenk, C., Jauniaux, T., López, A., Alonso Farré, J.M., - 96 -

González, A.F., Guerra, A., García-Hartmann, M., Lockyer, C. and Boon, J.P. (2010). Assessing the effect of persistent organic pollutants on reproductive activity in common dolphins and harbour porpoises. Journal of Northwest Atlantic Fishery Science, 42(42), 153–173. doi:10.2960/J.v42.m658

Murphy, S., Barber, J.L., Learmonth, J.A., Read, F.L., Deaville, R., Perkins, M.W., Brownlow, A., Davison, N., Penrose, R., Pierce, G.J., Law, R.J. and Jepson, P.D. (2015). Reproductive failure in UK harbour porpoises phocoena phocoena: Legacy of pollutant exposure? PLoS ONE, 10(7), 1–32. doi:10.1371/journal.pone.0131085

Nakamura, K., Sheps, S., and Clara Arck, P. (2008). Stress and reproductive failure: Past notions, present insights and future directions. Journal of Assisted Reproduction and Genetics. doi:10.1007/s10815-008-9206-5

Neimanis, A.S., Read, A.J., Foster, R. and Gaskin, D.E. (2000). Seasonal regression in testicular size and histology in harbour porpoises (Phocoena phocoena, L.) from the Bay of Fundy and Gulf of Maine. Journal of Zoology, 250(2), 221–229. doi:10.1017/S0952836900002077

Oakberg, E.F. (1956). A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. American Journal of Anatomy, 99(3), 391–413. doi:10.1002/aja.1000990303

Ólafsdóttir, D., Víkingsson, G.A., Halldórsson, S.D. and Sigurjónsson, J. (2003). Growth and reproduction in harbour porpoises (Phocoena phocoena) in Icelandic waters. NAMMCO Sci Publ 2003;5:195–210. doi:10.7557/3.2747.

Ohsumi, S. (1986). Yearly Change in Age and Body Length at Sexual Maturity of a Fin Whale Balaenoptera-Physalus in the Eastern North Pacific. Scientific Reports of the Whales Research Institute Tokyo, 1983(37), 1–16.

Perrin, W.F. and Reilly, S.B. (1984). Reproductive Parameters of Dolphins and Small Whales of the Family Deiphinidae. Reports of the International Whaling Commission, Special Is(6), 97–133. Retrieved from http://swfsc.noaa.gov/publications/CR/1984/8470.PDF

Plön S. and Bernard R. (2007). Testis, spermatogenesis and testiculat cycles. In:

- 97 -

Miller DL, editor. Reprod. Biol. Phylogeny Cetacea Whales, Dolphins Porpoises. 1st ed., Boca Raton, Fl: CRC Press; 2007, p. 116–35.

Read, A.J. and Hohn, A.A. (1995). Life in the fast lane: the life history of harbour porpoises from the Gulf of Maine. Marine Mammal Science, 11(4), 423–440. doi:10.1111/j.1748-7692.1995.tb00667.x

Reijnders P.J.H. (1986). Reproductive failure in common seals feeding on fish from polluted coastal waters. Nature, London 1986; 324: 556–557

Rivier, C. and Vale, W. (1990). Cytokines act within the brain to inhibit luteinizing hormone secretion and ovulation in the rat. Endocrinology. doi:10.1210/endo-127-2- 849

Roozendaal, M.M., Swarts, H.J.M., Wiegant, V.M. and Mattheij, J.A.M. (1995). Effect of restraint stress on the preovulatory luteinizing hormone profile and ovulation in the rat. European Journal of Endocrinology. doi:10.1530/eje.0.1330347

Romano T.A., Keogh M.J., Kelly C. and Finneran, J.J. (2004). Anthropogenic sound and marine mammal health: Measures of the nervous and immune systems before and after intense sound exposure. Canadian Journal of Fisheries and Aquatic Sciences 2004; 61 (7): 1124–1134.

Russell, L.D. and Ettlin, R.A. (1990). Mammalian spermatogenesis. In A. P. Sinha Hikim and E. D. Clegg (Eds.), Histological and Histopathological Evaluation of the Testis (1st ed., pp. 1–40). Clearwater, Fl: Cache River Press.

Schroeder, P.C. and Talbot, P. (1984). Ovulation in the Animal Kingdom: A Review with an Emphasis on the Role of Contractile Processes. Gamete Research 11: 191- 221 (1985)

Siebert, U., Joiris, C.R., Holsbeek, L., Benke, H., Failing, K., Frese, K. and Petzinger, E. (1999). Potential relation between mercury concentrations and necropsy findings in cetaceans from German waters of the North and Baltic Seas. Marine Pollution Bulletin, 38(4), 285–295. doi:10.1016/S0025-326X(98)00147-7

Siebert, U., Wünschmann, A., Weiss, R., Frank, H., Benke, H. and Frese, K. (2001). Post-mortem findings in harbour porpoises (phocoena phocoena) from the German - 98 -

North and Baltic Seas. Journal of Comparative Pathology, 124(2–3), 102–114. doi:10.1053/jcpa.2000.0436

Siebert, U., Prenger-Berninghoff, E. and Weiss, R. (2009). Regional differences in bacterial flora in harbour porpoises from the North Atlantic: Environmental effects? Journal of Applied Microbiology. doi:10.1111/j.1365-2672.2008.04006.x

Silver, L. (2001). Ovulation. In: Encyclopedia of Genetics 2001: 1401-1402, Elsevier Science Ltd. https://doi.org/10.1006/rwgn.2001.0945

Sørensen T.B. and Kinze C.C. (1994)- Reproduction and reproductive seasonality in Danish harbour porpoises, Phocoena Phocoena. Ophelia 1994;39:159–76. doi:10.1080/00785326.1994.10429541.

Stockley, P. (2004). Sperm competition in mammals. Human Fertility. doi:10.1080/14647270410001699054

Sveegaard S., Teilmann J., Tougaard J., Dietz R., Mouritsen K.N., Desportes G. and Siebert, U. (2010). High-density areas for harbor porpoises (Phocoena phocoena) identified by satellite tracking. Marine Mammals Science 2010; 27(1): 230-246

Takahashi S., Turek F.W. and Moore, R.Y. (2001). Circannual rhythms. In Handbook of behavioural neurobiology vol. 12 (pp. 509–528).

Tilbrook, A.J., Turner, A.I. and Clarke, I.J. (2000). Effects of stress on reproduction in non-rodent mammals: the role of glucocorticoids and sex differences. Reproduction, 105–113.

Unger B., Herr H., Benke H., Böhmert M., Burkhardt-Holm P., Dähne M., Hillmann, M., Wolff-Schmidt, K., Wohlsein, P. and Siebert, U. (2017). Marine debris in harbour porpoises and seals from German waters. Marine Environmental research 2017. Doi: 10.1016

Van Utrecht, W.L. (1978). Age and growth in Phocoena phocoena Linnaeus, 1758 (Cetacea, Odontoceti) from the North Sea. Bijdragen tot de Dierkunde, 48(1), 16–28.

Vasantha, S.K.I. and Kona, S.S.R. (2016). Physiology of puberty in females - A review. International Journal of Veterinary Sciences and Animal Husbandry 2016;

- 99 -

1(2): 23-26

Wiemann, A., Andersen, L.W., Berggren, P., Siebert, U., Benke, H., Teilmann, J., Lockyer, C., Pawliczka, I., Skóra, K., Roos, A., Lyrholm, T., Paulus, K. B., Ketmaier, V. and Tiedemann, R. (2010). Mitochondrial Control Region and microsatellite analyses on harbour porpoise (Phocoena phocoena) unravel population differentiation in the Baltic Sea and adjacent waters. Conservation Genetics, 11(1), 195–211. doi:10.1007/s10592-009-0023-x

Wingfield, J.C. (2008). Organization of vertebrate annual cycles: implications for control mechanisms. Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1490), 425–441. doi:10.1098/rstb.2007.2149

Wolkers, J., Burkow, I.C., Monshouwer, M., Lydersen, C., Dahle, S. and Witkamp, R. F. (1999). Cytochrome P450-mediated enzyme activities and polychlorinated biphenyl accumulation in harp seal (Phoca groenlandica). Marine Environmental Research, 48(1), 59–72. doi:10.1016/S0141-1136(99)00032-X

Yasui W.Y. and Gaskin D.E. (1986) Energy budget of a small Cetacean, the harbour Porpoise, Phocoena Phocoena. Ophelia 1986; 25(3) : 183-197

Ylitalo, G.M., Stein, J.E., Hom, T., Johnson, L.L., Tilbury, K.L., Hall, A.J., Rowles, T., Greig, D., Lowenstine, L.J., and Gulland, F.M.D. (2005). The role of organochlorines in cancer-associated mortality in California sea lions (Zalophus californianus). Marine Pollution Bulletin, 50(1), 30–39. doi:10.1016/j.marpolbul.2004.08.005

Young, K.A. and Nelson, R.J. (2001). Mediation of seasonal testicular regression by apoptosis. Reproduction, 122(5), 677–685. doi:10.1530/reprod/122.5.677

- 100 -

Chapter 7: Acknowledgements

First of all, I thank Prof. Prof. Dr. Ursula Siebert and Prof. Dr. Ralph Brehm for giving me the opportunity to work on this unique topic that challenged me as a biologist and veterinarian. Thank you for your supervision, support, patience, time and for sharing your extensive knowledge with me.

I thank the Ministry of Energy, Agriculture, the Environment and Rural Areas Schleswig Holstein (MELUR) and the state centre for coastal protection, national park and ocean protection Schleswig - Holstein (LKN), who partly funded this work.

Furthermore, I thank the lab technicians of the Institute for Terrestrial and Aquatic Wildlife Research (ITAW) Kornelia Wolff-Schmidt and Miriam Hillmann for the help during necropsies, sampling and handling of the data, as well as the lab technicians of the Institute for Anatomy, Marion Langeheine and Doris Voigtländer, for friendly teaching me histological techniques and advising me in photo – sessions. This work would not have been possible without you!

I would like to thank Prof. Dr. Martin Bergmann and his lab team for their work and the interesting meetings in Giessen. These sessions gave me a lot of motivation.

Many thanks go to my co-authors of the scientific manuscripts for great inputs and critical annotations at any point of the work.

I am very thankful for sharing an intense and memorable time – Kathi, Abbo, Marco, Susanne, Bianca, Johannes, Miguel, Eva, Helena, Steph, Anja, Jan, Vailett, Krissi, Julia – thanks for being great colleagues and friends. Sacha, thank you for your work and friendship during all these years.

My particular thanks go to my colleague and friend Lucie Grimm for always inspiring and encouraging me.

Finally I thank my mother Dr. Petra Kesselring and my grandma Inge Kesselring for the love and support during my whole education and their never-ending encouragement and help in any part of life. - 101 -