Chapter 2 Liver histopathology of Baltic grey seals (Halichoerus grypus) over three
4 Discussion
4.2 Contaminant exposure
According to SONNE ET AL.(2020A), the blubber threshold concentrations in seals in which the disease complex arose was around 100 g/g lw (∑PCB17 range: 27-390 g/g lw; DDT [p,p’-DDE, p,p’-DDD and p,p’-DDT] range: 12-970 g/g lw) being among the highest ever measured in wild phocids (Table 2) (JENSEN ET AL.1979;LETCHER ET AL.2010;ROOS ET AL. 2012)). The concentrations of these seals in the present study ranged from 8.5-1848 mg/kg
lw for PCBs and 2.3-61.1 mg/kg lw for DDT. These are above the stated thresholds and therefore it cannot be excluded that the present pathological findings are to some extent ascribed to contaminant exposure as well as age and re-current infections being the main factors.
Liver histology in relation to environmental levels of PCBs and DDTs has previously been reported for marine and terrestrial mammals (HORI ET AL.1982; PRUNESCU ET AL.2003, SONNE ET AL.2005,2007B,2008). In the present study, the visibility of hepatic stellate cells showed a positive trend with PCB concentrations, whereas mild multifocal bile duct hyperplasia accompanied by portal fibrosis showed a negative trend with PCB. In terrestrial animals, like Cynomolgus monkeys (Macaca fascicularis) bile duct proliferation has also been associated with PCB exposure (HORI ET AL.1982). In Arctic animals, this lesion was found in animals with higher concentrations of organohalogen contaminants (SONNE ET AL. 2005,2008). These results can give an insight into the possible effects of PCBs on the liver.
They also indicate that animals exposed to a higher contaminant concentration may show significant results in the relationships between contaminants and liver lesions.
In the present study, a decrease in the correlation between the prevalence of portal mononuclear cell infiltration (Lesion 1) and collection year was found. In the same period, contaminant concentrations of PCBs and DDT likewise decreased, this suggesting that the decrease may be linked to the decrease in contaminant loads.
Conclusions
The study showed high prevalence of liver lesions in Baltic grey seals. The liver lesions can be related to age and in the case of Hepatic stellate cells and mild multifocal bile duct hyperplasia also to PCB contamination. These may be important factors in pathology development together with bacterial and viral infections.
Acknowledgement
This study was financed by BONUS BaltHealth. The BaltHealth project received funding from BONUS (Art. 185), funded jointly by the EU, Innovation Fund Denmark (grants 6180-00001B and 6180-00002B), Forschungszentrum Jülich GmbH, Jülich, Germany, German Federal Ministry of Education and Research (grant number FKZ 03F0767A), Academy of Finland (decision #311966) and Swedish Foundation for Strategic Environmental Research
(MISTRA). The long-term monitoring of seals in Sweden and the Swedish Environmental Specimen Bank are funded by the Swedish Environmental Protection Agency (EPA).
We are grateful to the Swedish hunters and fishermen for their contribution to the Swedish environmental specimen bank at the Swedish Museum of Natural History, from which the livers were provided on loan. Our thanks go to Anna Roos who provided the contaminant data, to Carolin Philipp for designing the maps, to Britt-Marie Bäcklin who performed necropsies and sampled the animals as well as providing the age of some of the individual, and to all helpers for taking samples during the field trips.
Appendix Lesion 2: Random mononuclear (intralobular) cell infiltration
Adult ♀ 43.5 (10) 34.8 (8) 21.7 (5) 0 56.5 (13)
Adult ♀ 78.3 (18) 17.4 (4) 0 4.3 (1) 21.7 (5)
Adult ♂ 76.7 (23) 20.0 (6) 3.3 (1) 0 23.3 (7)
Subadult 61.8 (21) 32.4 (11) 5.9 (2) 0 38.2 (13)
Juvenile 59.6 (62) 34.6 (36) 5.8 (6) 0 40.4 (42)
All 64.9 (124) 29.8 (57) 4.7 (9) 0.5 (1) 35.1 (67) Lesion 6: Mild multifocal bile duct hyperplasia accompanied by portal fibrosis
Adult ♀ 78.3 (18) 21.7 (5) 0 0 21.7 (5)
Adult ♂ 66.7 (20) 33.3 (10) 0 0 33.3 (10)
Subadult 85.3 (29) 14.7 (5) 0 0 14.7 (5)
Juvenile 89.4 (93) 10.6 (11) 0 0 10.6 (11)
All 83.8 (160) 16.2 (31) 0 0 16.2 (31)
8. The sPCB concentrations for all 34 examined individuals with all known concentrations of the 10 congeners. Sex AgeEstimated sPCB (mg/kg lw) CB-52 (lw ppm)
CB-101 (lw ppm) CB-105 (lw ppm) CB-118 (lw ppm) CB- 138+163 (lw ppm) CB-153 (lw ppm) CB-156 (lw ppm) CB-180 (lw ppm)
CB-28 (lw ppm) M0.5 48 F1 77.2 F1 101 M3 110.4 0.9 0.2 0.3 15240.5 7 M0.5 49 F41432 1.4 0.2 0.4 51930.7 36 M1 61.3 M17176.4 1 0.2 0.4 21390.3 13.5 F4018483.3 0.4 200 420 150 M1 36.1 F35895.2 0.9 0.2 100 200 73 M3 74.90.5 0.1 0.2 9.8 170.3 4.4 M2 41.30.4 0.1 0.2 5.2 8.9 0.3 3.1 M1 30.20.3 0.1 0.2 4 6.8 0.2 1.8 Mu.42 Mu.33.1 M6 20.80.080.1 -0.12.6 4.8 1.3 -0.06 F1114.90.050.1 E.D. 0.0 1.9 3.4 0.1 0.9 <0.01
04M11-0.06 0.2 0.1 8.1 15.26 -0.0 05Fu.14.40.060.1 E.D. 0.0 2 3.3 0.1 0.8 <0.0 05F1 9.1 0.040.2 E.D. 0.1 1.3 2 0.1 0.6 <0.0 05M5 52.90.2 0.7 0.2 7.1 11.83.2 u. 05M28203.3 0.090.5 0.2 18.249.217.3u. 05F8 25.7-0.07 0.2 0.1 3.1 5.5 2.1 -0.0 06Fu.10.4-0.08 0.1 -0.11.4 2.5 0.4 -0.0 06F9 14.8-0.07 0.2 0.1 1.9 3.4 0.8 -0.0 06F8 45.90.070.2 0.1 5.5 9.5 4.1 -0.0 06F8 410.070.2 0.1 4.8 8.9 3.4 -0.0 07Mu.15.5-0.06 0.2 E.D. 0.1 2.3 3.6 0.7 -0.0 07F1 11.8-0.07 0.1 E.D. -0.11.4 2.8 0.7 -0.0 07F1 9.1 -0.07 0.2 E.D. 0.1 1.1 2.1 0.6 -0.0 08M1 8.5 -0.06 0.1 -0.11.1 1.9 0.6 -0.0 10Mu.19.2-0.07 0.2 E.D. 0.1 2.7 4.2 1.1 -0.0 10M2 9 -0.07 0.2 E.D. -0.11.1 2 0.6 -0.0 nknown; E.D.: not detected, u.d.: under limit of detection
Table 9. The sDDT concentration for all 34 examined individuals with all known
2007 F 1 2.7 2.4 0.1 0.3
2008 M 1 2.4 2 -0.1 0.2
2010 M u. 5.6 4.9 0.1 0.6
2010 M 2 2.6 2.2 0.1 0.3
u.: unknown
Chapter 3
Number of primordial follicles in juvenile ringed seals (Phoca
hispida) from the Gulf of Bothnia and West Greenland
Manuscript:
Schmidt, B., Hollnebach, J., Mühlfeld, C., Pfarrer, C., Persson, S., Kauhala, K., Siebert, U. (in prep.). Number of primordial follicles in juvenile ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland.
Number of primordial follicles in juvenile ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland
Britta Schmidt1, Julia Hollenbach2, Christian Mühlfeld3, Christiane Pfarrer2, Sara Persson4, Kaarina Kauhala5, Ursula Siebert1,*
1Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Werftstr. 6, 25761 D-Büsum, Germany
2Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 D-Hannover, Germany
3Institute for Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str.
1, 30625 D-Hannover, Germany
4Department of Environmental Research and Monitoring, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05 Stockholm, Sweden
5Natural Resources Institute Finland, Luke, Itäinen Pitkäkatu 4 A, FI-20520 Turku, Finland
*Address correspondence to U. Siebert, Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Werfstr. 6, 25761 D-Büsum, Germany. Telephone: +49511-856-8158. E-mail: ursula.siebert@tiho-hannover.de
Abstract
Primordial follicles are important for the reproduction cycle and, therefore, also for the survival of the whole population. Mammals have a large pool of primordial follicles and it is thought that this pool represents the total number of oocytes. The aim of the present study was to determine the total primordial follicle number of juvenile ringed seals (Phoca hispida) from the Baltic Sea and Greenland. Overall, 52 ovaries from two ringed seal populations (West Greenland (N = 6), Gulf of Bothnia (N = 46)) were examined. All ovaries were cut into 2 mm thick slices and every slice was embedded in paraffin. Out of each embedded tissue block, a 5 µm thick section was cut and stained with haematoxylin-eosin. The mean volume of the follicles and the total volume of primordial follicles per ovary were estimated by design-based stereology and used to calculate the total number of primordial follicles. No statistically significant differences could be found between the total primordial follicle number of the Baltic and Greenland ringed seals. However, the mean number of primordial follicles is higher in Baltic individuals than in Greenland individuals (Baltic Sea = 716,883 ± 100,909; Greenland Sea = 250.280 ± 149,527). The total number of primordial follicles differs significantly between Sweden and Greenland, with higher numbers in Swedish individuals (LM, p = 0.044). The examinations also revealed a higher number in Finnish individuals in relation to Greenlandic individuals (Finland = 646,266 ± 555,819; Greenland
= 250,280 ± 366,265). In conclusion, the number of primordial follicles differs between the three countries and makes it difficult to calculate one total range of primordial follicles in ringed seals. However, this study revealed a method for ovary examination and showed how to store the samples to preserve the follicles in the best way and, thus reduce storage induced variations in follicle numbers.
Keywords: Ringed Seal, Primordial follicles, Stereology, Baltic Sea, Greenland
1 Introduction
In the beginning of reproduction, mammalian ovaries have a large pool of primordial follicles (FORTUNE 1994,SKINNER 2005). It is thought that this pool represents the total number of oocytes for each individual (SKINNER 2005), because it decreases throughout life (KEZELE AND SKINNER 2003). Different species develop this pool at different stages of life. In primates and ruminants, the development starts during the early neonatal period, whereas in rodents and rabbits, it develops during the early postnatal period (MARION AND GIER 1971, HIRSHFIELD 1991, WEBB ET AL. 2003, SKINNER 2005). A primordial follicle consists of an oocyte arrested in prophase 1 of meiosis (FORTUNE 1994), which will not be finished until ovulation (HIRSHFIELD 1991). Moreover, primordial follicle development also depends on other cell types, like granulosa and precursor theca cells (SKINNER 2005). Granulosa cells form the microenvironment for the oocytes and together with the theca cells, they synthesise a number of hormones, which control follicular development (SKINNER 2005). Once follicles start to grow, only a few successfully ovulate, the majority dies (FORTUNE 1994). The developmental stages of the oocyte begin with primordial follicle, followed by primary and secondary follicle and eventually tertiary follicle, which are mature oocytes (EPPIG ET AL. 2001). Only tertiary follicles, which can record specific signals, reach the stadium of the Graafian follicle (EPPIG ET AL.2001) and ovulate, for the ringed seals one in every normal cycle. The complex development of the different follicle stadiums shows that there is a cell-to-cell interaction, which coordinates the whole development. Until now mechanisms for developing and activating follicles are not known (FORTUNE 1994, EPPIG ET AL. 2001).
Different environmental factors can influence the development of primordial follicles, as well as the oocyte quality and fertility (WEBB ET AL. 1999A,B). Furthermore, physiological parameters, like body weight, reproductive system ageing or body length, can also influence the follicle pool (GOSDEN AND TEFLER 1987). Some physiological parameters are determined by adaptions of the species to certain factors, for example the increased number of primordial follicles in larger species, which can be an adaption to a longer “adult life span”
(GOSDEN AND TEFLER 1987).
In this study, the focus is on ringed seals (Phoca hispida), for which nothing is known about the pool of primordial follicles. The ringed seal is distributed all over the northern hemisphere (REEVES 1998). In the Baltic Sea, a subspecies named Pusa hispida botnica occurs (RIEDMAN 1990). This population is mostly found in the Gulf of Bothnia, Finland and Riga (HÄRKÖNEN AND HEIDE-JØRGENSEN 1990, KINGSLEY AND BYERS 1998). In the 1970s, the ringed seal population decreased as a result of anthropogenic pollutants, mostly polychlorinated biphenyls (PCB) and dichlorodiphenyltrichloroethane (DDT) in the Baltic
Sea (HELLE 1981). PCB and DDT have negative effects on the health status of the ringed seals (JEPSON ET AL.2016), resulting in endocrine disruption, reproductive failures, bone lesions, reduced immune competence or tumour incidence (NYMAN ET AL.2002,OLSSON ET AL.1994,ROUTTI ET AL.2008,SCHMIDT ET AL.2020,2020). All this affects the overall health status of the seals, which plays an important role for a successful reproduction.
Nevertheless, not only pollutants can have a negative effect on the reproduction success, also low blubber reserves, stress and environmental changes can influence the reproduction (KANNAN ET AL.2000,KINGSLEY AND BYERS 1998).
The aim of this study is to determine the total number of primordial follicles in ringed seals, which are given at birth. Such a study of total numbers can provide important information about ovarian function (MYERS ET AL.2004) and will help assessing management plans to protect small populations. Information about the total numbers can show the effect of different environmental factors, such as climate and habitat change. Two ringed seal populations, from the Gulf of Bothnia and from Greenland, were examined to determine possible differences between these populations. It is also the first time that stereology is used for analysing marine mammal ovaries.
2 Materials and Methods
2.1 Samples
The samples used in this study originated from ringed seals that either died through stranding or were legally hunted by local hunters in Sweden, Finland and Greenland. The animals were shot according to the existing hunting law in the respective countries. A total of 52 juvenile ringed seal ovaries from the Gulf of Bothnia and the west coast of Greenland were examined (Figure 16).
Figure 16. Examined Area. The collection of the samples originates from the Gulf of Bothnia in the Baltic Sea and in West Greenland. The samples were predominantly collected in the locations marked with a circle in the Gulf of Bothnia and with a triangle at the west coast of Greenland.
The 46 ovaries from the Gulf of Bothnia were provided from the Swedish Museum of Natural History in Stockholm (SMNH) and from the Natural Resources Institute in Finland (LUKE), the six ovaries from Greenland were provided from the Arhus University in Denmark. All ovaries were collected in the period between 2018 and 2019. The age of 43 individuals was determined based on the pupping season in the different areas (Baltic Sea: between February and March (HÄRKÖNEN ET AL.2006), Greenland: between March and early April (REEVES 1998)). All individuals were between 1.5 and 2.5 months old. The ovaries were stored in a 4% phosphate-buffered formalin solution, whereas beforehand most of the ovaries were stored frozen at -20 °C. For microscopic examination, one ovary per animal, mostly the left (in one case the right ovary), was chosen for further investigations. The total volume of each ovary was determined by fluid displacement method (Principle of Archimedes) (SCHERLE 1970) (Table 12, Figure 17A). Measurements were repeated three times for each ovary.
2.2 Histology preparation
Each ovary was cut into 2 mm thick slices(Figure 17B) and every slice was embedded in paraffin using standard methods (Leica Paraplast Standard). One 5 µm thick section was cut of each embedded tissue slice and stained with haematoxylin-eosin (HE) (N = 170).
Figure 17. Setup for the volume determination of each ovary (A). All ovaries were weighted, from which the volume could be determined. The histological preparation for each ovary is shown in B. Every tissue slice was stored in another capsule and these capsules were labelled.
2.3 Stereology - Total primordial follicle number
The aim of stereology is to obtain quantitative information about the three-dimensional architecture of an organ by analysing plane, two-dimensional tissue sections (OCHS AND
MÜHLFELD 2013). In this study, the total volume of primordial follicles per ovary and the mean volume of the follicles were estimated by design-based stereology. The total number of primordial follicles was calculated from these two parameters. Four to nine tissue slices per ovary were digitized using a slide scanner (Axio Scan.Z1, ZEISS, Oberkochen, Germany). Images were subsampled using the newCast software (Visiopharm, Hørsholm, Denmark) and analysed with the newCast software (Visiopharm, Hørsholm, Denmark) and the STEPanizer online tool (TSCHANZ ET AL.2010). The total volume of primordial follicles per ovary was determined by point counting. The sampling fraction was set as 20% and a magnification of 20x was used. The test grid consisted of 64 points (Figure 18). All points hitting primordial follicles (P(follicle)) and all points hitting the ovary (P(ovary)) were counted.
In the first step the volume density of the follicles (VV(follicle/ovary)) was calculated by dividing the sum of points hitting the follicles by the sum of points hitting the ovary (Formula 1).
Formula 1: 𝑉 (𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒/𝑜𝑣𝑎𝑟𝑦) = ∑ ( )
∑ ( )
Figure 18. A part of the stained ovary slice with the test grid of 64 points. Points hitting the primordial follicles as well as the four green coloured points hitting the reference space (ovary) were counted and afterwards multiplied with 16 (coarse grid).
The total volume of follicles (𝑉(𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒, 𝑜𝑣𝑎𝑟𝑦)), which could be found in each ovary was computed by multiplication of the volume density by the total volume of the ovary (Formula 2).
Formula 2: 𝑉(𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒, 𝑜𝑣𝑎𝑟𝑦) = 𝑉 (𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒, 𝑜𝑣𝑎𝑟𝑦) × 𝑉(𝑜𝑣𝑎𝑟𝑦)
Figure 19. Volume determination of different primordial follicles. All primordial follicle, which cross the green line or are inside the marked area are taken into account for volume measurements.
In the next step the number-weighted mean volume of the follicles (𝑉 (𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒)) was estimated using the rotator (JENSEN AND GUNDERSEN 1993; TANDRUP ET AL. 1997). A measurement was done for each follicle crossing the green lines or within the counting frame. The computer program determined the 𝑉 in µm3.
In the last step, the total number of primordial follicles was calculated by dividing the result of formula 2 (𝑉(𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒, 𝑜𝑣𝑎𝑟𝑦)) by the number-weighted mean volume of primordial follicles (𝑉 (𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒), Figure 19). N (follicle, ovary) describes the total number of follicles per ovary.
Formula 3: 𝑁(𝑓𝑜𝑙𝑙𝑖𝑐𝑙𝑒, 𝑜𝑣𝑎𝑟𝑦) = ( , )
( )
2.4 Statistical analyses
The statistical analyses were performed with R (version 3.4.3, R Core Team 2014). The number of primordial follicles was tested for normality by applying the Shapiro-Wilk test.
Furthermore, a linear regression analysis (LM) was made, where the relationship of the number of primordial follicles versus water and country was tested, as well as the relationship of the ovary and follicle volume versus countries. For the comparison of Sweden and Finland, the relationship of the number of primordial follicles versus both areas was tested within a t-test. Level of significance was set at p < 0.05.
3 Results
The Shapiro Wilk Test shows no standard distribution (p < 0.05). The linear regression model and a t-test were used for calculations. Table 10 shows all variables needed to apply formulas 1 to 3 as well as the resulting values for V(ovary), VV(follicle/ovary), V(follicle/ovary) and 𝑉 (follicle).
10. All points hitting primordial follicles P(follicle) and all points hitting the ovary P P(ovary) were counted for the ovaries of ringed seals fr different countries. As a result of Formula 1, the volume density Vv was calculated with P(follicle) and P(ovary). Vv and V(ovary), the volum ry, were used to calculate the total volume of follicles in the ovary V(follicle, ovary) based on Formula 2. Using this value and number-weigh volume of follicles VN(follicle), the total number of follicles in the ovary was determined. iduals AgeCountry P (follicle) P (ovary)VvV (ovary) [cm3]
V(follicle, ovary) [cm3]
VN (follicle) [cm3]
N(follicle, ovary) 1Sweden3765440,005654030,990,005597490,000000007580738455,658 2Sweden20105760,001891071,620,00306354NANA 3Sweden1253760,002232140,650,001450890,000000005410268187,219 4Sweden9 93600,000961541,090,00104808NANA 5Sweden53117760,004500681,040,004680710,0000000013603441695,97 6Sweden60117280,005115961,080,005525240,000000009900558104,924 7Sweden2081440,00245581,040,002554030,0000000013601877961,4 8Sweden2863040,004441620,920,004086290,000000006630616333,999 9Sweden16154400,001036271,010,001046630,000000007580138078,117 102.5 Finland143133120,010742191,240,013320310,000000023310571441,978 112.5Sweden226141760,015942441,370,021841140,000000029100750554,638 122.5 Sweden18487520,021023770,770,01618830,000000018200889467,023 132.5 Sweden7085120,008223680,660,005427630,000000024630220366,69 141.5Finland24167680,00143131,740,00249046NANA
. 151.5Finland2872640,003854631,010,003893170,000000007580513611,056 . 161.5Finland3068960,004350350,940,004089330,000000006380640960,368 . 171.5Finland83278240,002983043,070,009157920,000000012900709916,374 . 181.5Finland1153280,002064560,550,001135510,000000003600315419,586 . 191.5Finland39170720,002284442,120,004843020,000000013750352219,477 . 202 Finland4464800,006790120,800,00543210,000000015630347543,107 . 212.5Finland3167680,004580380,800,00366430,0000000015002442868,4 . 222 Finland2556480,004426350,650,002877120,000000005410531816,016 . 232 Finland58109760,005284261,360,007186590,000000009380766160,866 . 242 Finland8 43360,001845020,510,000940960,000000005200180953,733 . 252 Finland5841440,013996140,490,006858110,000000010120677678,667 . 261.5Finland10381440,012647351,060,013406190,0000000099001354160,47 . 272 Finland2273120,003008750,910,002737960,000000006630412966,062 . 281.5Finland4368320,006293910,760,004783370,000000011670409886,235 . 292 Finland2838240,007322180,380,002782430,000000011000252947,889 . 302 Finland61119040,005124331,410,00722530,000000010440692078,776 . 312.5Finland3255360,005780350,580,00335260,000000009060370044,278 . 322.5Finland3066720,00449640,710,003192450,000000005110624744,823 . 332.5Finland1380640,00161211,050,001692710,000000010270164820,675 . 342 Finland6180800,00754950,810,00611510,000000011340539250,354
.h. 352.5Finland2895040,002946131,050,003093430,0000000013602274584,08 .h. 362.5Finland4143840,009352190,600,005611310,000000009920565656,64 .h. 372 Finland1458720,00238420,700,001668940,000000010960152275,304 .h. 382 Finland1 24000,000416670,360,000150,0000000000000 .h. 392.5Finland0 47680 0,560 0,0000000000000 .h. 402.5Finland2755680,004849140,690,003345910,000000005160648431,235 .h. 412 Finland4651200,008984380,700,006289060,000000008690723712,601 .h. 422 Finland1251680,002321980,540,001253870,000000004130303600,477 .h. 432 Finland6855520,012247840,750,009185880,0000000054301691690,42 .h. 442.5Finland8982080,010843080,950,010300930,000000012190845030,839 .h. 451.5Finland3545280,007729680,700,005410780,000000007500721436,985 .h. 461.5Finland2745280,00596291,020,006082160,000000011500528883,085 .h. 471.5Greenland14495040,015151520,750,011363640,000000011670973747,76 .h. 481.5Greenland4897280,004934210,870,004292760,000000031300137148,983 .h. 491.5Greenland56118560,004723350,740,003495280,000000013460259678,8 .h. 501.5Greenland1584160,001782320,60,001069390,000000009920107801,576 .h. 511.5Greenland7 39200,001785710,330,000589290,0000000000000 .h. 521.5Greenland3 50880,000589620,40,000235850,00000001012023305,2427 A = not available, too small P(follicle) values
3.1 Different follicle and ovary volumes (V(ovary), Vv(follicle/ovary), V(follicle/ovary), VN(follicle))
The mean ovary volume (V(ovary)) differs between the both waters, but no significant correlation was shown. The minimal mean volume in individuals of the Baltic Sea is 0.36 cm3 and in Greenland individuals 0.33 cm3. The individuals differ more between maximum (max) mean volumes (maxBaltic Sea = 3.07 cm3, maxGreenland Sea = 0.87 cm3). Whereas the Baltic Sea range is very high, the minimum and maximum mean volume of the individuals of Finland and Sweden were separate considered. The volume minimum was 0.65 cm3 and maximum was 1.62 cm3 of Sweden (1.02 ± 0.08 cm3), the minimum was 0.36 and maximum was 3.07 cm3 of Finland (0.93 ± 0.09 cm3). It can be seen, that the volume span of Finish individuals is larger, than the span of the Swedish individuals.
The Volume density of the follicles (Vv(follicle/ovary)) in the individuals of the Baltic Sea is higher than from the individuals of Greenland, but no relationship was found.
Greenland individuals have a lower total volume of follicles (V(follicle/ovary)), than the Baltic Sea individuals have. Taken a closer look the Swedish individuals have the highest total volume of follicles compared to all other ringed seal individuals.
The number-weighted mean volume of follicles do not differ significantly between both areas. In Greenland individuals, the number-weighted mean volume of follicles is higher than in individuals of the Baltic Sea.
3.2 Volume differences
A positive correlation between mean volume of follicles and ovaries was found (LM, p = 0.065) (Figure 20). The mean volume of follicles shows a slightly positive trend with the mean volume of ovaries. The mean values of both volumes differ a lot (Vfollicle = 9.494694e-09 ± 6.79287e-9.494694e-09 cm³, Vovary = 0.9134615 ± 0.4771451 cm³).
Figure 20. Correlation between mean follicle and ovary volume in juvenile ringed seals. The mean volume of follicles show a slightly positive trend with the mean volume of ovary (LM, p > 0.05).
3.3 Number of primordial follicles
The total primordial follicle number was analysed in 46 ovaries from ringed seals from the Baltic Sea, divided into 13 Swedish individuals, 33 Finish individuals and 6 ovaries from Greenland individuals. The linear regression shows no significant differences between the total number of primordial follicle in both waters (Baltic Sea and Greenland Sea), although the mean primordial follicle number is higher in the Baltic than in the Greenland Sea (mean:
Baltic Sea = 716,883 ± 684,397; Greenland Sea = 250,280 ± 366,265; Figure 21). The t-test showed a significant difference between the number of primordial follicles in Baltic individuals, than in Greenlandic individuals (p = 0.03). The Baltic ringed seals has higher number of primordial follicles.
Figure 21. Total primordial follicle number of Baltic and Greenland ringed seals. It is shown that the quantity in the Baltic Sea varies more in number of individuals.
3.4 Number of primordial follicle and differences between countries
Determination of the total primordial follicle number was available for 13 Swedish, 33 Finish and six Greenlandic individuals. Ringed seals from Finland had a higher number than Greenland ringed seals. Individuals from Sweden had significantly higher numbers in relation to Greenland individuals (LM, pGreenland = 0.044). The mean numbers of primordial follicles of the three countries show that Swedish individuals have the highest number, followed by Finnish and Greenlandic individuals (mean: Sweden = 949,920 ± 1,004,391;
Finland = 646,266 ± 555,819; Greenland = 250,280 ± 366,265). Greenland had the lowest mean value compared to Sweden and Finland (Figure 22).
Figure 22. Total primordial follicle number of Baltic ringed seals from Sweden and Finland and of ringed seals from Greenland. It is shown that the quantity varies a lot between the countries.
4 Discussion
4.1 Identification of primordial follicle number
The total primordial follicle number of ringed seals from Greenland and the Baltic Sea varies between the waters. In the Baltic Sea higher primordial follicle numbers were found, compared with the area around Greenland. Furthermore, the number of ringed seal follicles in total is markedly higher (659,272.9 in one ovary) than the primordial follicle numbers of other species, like cows (Bos taurus), humans (Homo sapiens), dogs (Canis lupus familiaris), pigs (Sus scrofa domesticus) or mice (Mus musculus) (DÄMMRICH ET AL.1991;
MOORE AND PERSAUD 2007;WALLNER S.E.2007;GUTHRIE AND GARRETT 2000;JONES AND
KROHN 1961; Table 11).
Table 11. Primordial follicle number of different species directly after birth in both ovaries.
Table 11. Primordial follicle number of different species directly after birth in both ovaries.