New Data on the Blood Cell Composition of Bearded Seal

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genus stands apart from all of the true seals [1]. There are 34 chromosomes in karyotypes of the bearded seal, monk seal, and hooded seal, while the other true ...
ISSN 00124966, Doklady Biological Sciences, 2015, Vol. 462, pp. 152–154. © Pleiades Publishing, Ltd., 2015. Original Russian Text © T.V. Minzyuk, N.N. Kavtsevich, V.N. Svetochev, 2015, published in Doklady Akademii Nauk, 2015, Vol. 462, No. 6, pp. 727–729.

GENERAL BIOLOGY

New Data on the Blood Cell Composition of Bearded Seal T. V. Minzyuk, N. N. Kavtsevich, and V. N. Svetochev Presented by Academician G.G. Matishov June 5, 2014 Received November 17, 2014

DOI: 10.1134/S0012496615030138

The Atlantic subspecies of the bearded seal or squareflipper (Erignathus barbatus barbatus Erxleben, 1777) inhabit mainly the shallow Arctic Ocean. It is one of the largest members of the true seal family. Ship hunting for the bearded seal in Russia has been banned since 1972. However, in the Barents Sea, it is an important commercial object for local inhabitants, while in the White Sea, the bearded seal may be a by catch in hunting for ringed seal. According to karyological data, the Erignathus genus stands apart from all of the true seals [1]. There are 34 chromosomes in karyotypes of the bearded seal, monk seal, and hooded seal, while the other true seals (family Pocidae) have only 32 chromosomes. This sug gests that the genus Erignathus is relatively more ancient. Taking into account such a possibility, as well as specific geographical distribution of the bearded seal, we may suggest that the search for some special features of this animal is promising [2]. This study is a preliminary comparative analysis of the hematological parameters of the bearded seals liv ing in nature and of those living under oceanarium conditions. The material studied was from fiveyearold bearded seals caught during an expedition into Onega Bay of the White Sea in July 2013 (n = 5) and from the bearded seals maintained in the oceanarium of the Murmansk Marine Biological Institute (n = 3). The blood was obtained from the extradural vein into a heparincontaining syringe. Smears were prepared and stained according to the Romanowsky–Giemsa method using conventional techniques. In addition, fast green staining was used to detect cationic proteins (KPs). Myeloperoxidase (MPO) activity was detected in a reaction with benzidine. Proteins of the nucleolus organizer regions (NOR) were stained with silver

Murmansk Marine Biological Institute, Kola Scientific Center, Russian Academy of Sciences, ul. Vladimirskaya 17, Murmansk, 183010 Russia email: [email protected]

nitrate (AgNOR). Preparations were examined using immersion microscopy (lens, ×100; eyepiece, ×10). The cell quantitative parameters were measured using a video system and the Axio Vision 4.5 software from Carl Zeiss (Germany). To evaluate the NOR activity, the ratio of the NOR to the nucleus area in a lymphocyte was determined (AgNOR/Nucleus), as well as the number of AgNOR per lymphocyte and the diameter, area, and rotundity index (RI) of both nucleus and AgNOR. MPO activity and KP were determined in 200 granulocytes of each blood smear examined. We also estimated the mean cytochemical coefficient (MCC) [3], index of cell fill ing (ICF), and integrated cytochemical index (ICI) [4]. ICF is the proportion of the measured objects per cell area; ICI is the product of the total area of the product of cytochemical reaction in a cell and the optical density of this product, which corresponds to the amount of the visualized (stained) substance. The results are presented as M ± m. To evaluate sig nificance of differences, Student’s t test was used. The differences were considered significant at p < 0.05. In the bearded seal blood, the neutrophil nuclei are segmented as a rule (three to five segments). Stab neu trophils are also encountered (8.0 ± 3.0% in captivity and 1.7 ± 0.4% in wild animals). Among eosinophils, low differentiated cells with a rodshaped nucleus pre dominated. The relative number of these cells in the oceanarium seals and in wild animals are 5.7 ± 3.1% and 13.2 ± 4.1%, respectively. As in the harp and grey seals [5, 6], the bearded seal leukocytes contain nuclei of an unusual shape: the chromatin filaments connect ing nuclear segments converge in a single point instead of connecting the nuclear segments sequentially. This indicates a specific differentiation pattern of a part of myeloid cells in the seals. Various monocytes of the bearded seals proved to be significantly different in size and in the nuclear area, as well as in shape, density, and the number of vacuoles in the cytoplasm. In the bearded seals kept in captivity, small lym phocytes predominate, while large lymphocytes with a broad cytoplasm were in the wild animals. Large gran

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NEW DATA ON THE BLOOD CELL COMPOSITION OF BEARDED SEAL

ular lymphocytes (LGLs) with azurophilic granules in the cytoplasm were not found in the bearded seal blood. At the same time, LGLs are often encountered in the Romanowsky–Giemsa smears of the bottlenose dolphin (Tursiops truncates), harbor porpoise (Phoc oena phocoena), beluga (Delphinapterus leucas), harp seals (Phoca groenlandica), grey seals (Halichoerus grypus), hooded seal (Cystophora crystata), ringed seal (Phoca hispida), and northern fur seal (Callorhinus ursinus) [7]. This is probably an important fact, because LGLs are responsible for innate noninduc ible immunity against tumor and virusinfected cells. The wild bearded seals have a high content of lym phocytes, exceeding the number of neutrophilic leu kocytes (figure), while the situation is inverse in other species of adult true seals, as well as in most terrestrial mammals studied in this respect. The relative numbers of lymphocytes in the wild seals and in those kept in captivity were 45.50 ± 5.29% and 19.17 ± 1.83%, respectively (p < 0.05). In certain periods of the early postembryonic development of the harp, grey, and hooded seals, the number of lymphocytes reaches the number of neutro phils and even exceeds it [5–8]. This phenomenon referred to as “physiological decussation” of the leu kocyte blood formula is also observed in terrestrial ani mals and humans during the early postnatal ontogeny; it is caused by an intense development of the immune system. The absence of this phenomenon is, probably, sign of a low viability of the seal pups [9]. The seals were studied at the age corresponding to puberty; therefore, it can be assumed that either the extremely high number of lymphocytes is a physiological speci ficity of the bearded seal blood or these animals have a longer period of the lymphoid system formation than other true seals. At the same time, the lymphoid cell metabolic activity in the wild seals is lower than in ani mals kept in captivity, which is confirmed by the rela tive NOR areas (Table 1). In nature, bearded seals have AgNOR smaller in the absolute size (p < 0.05) and mostly “polymorphic” (AgNOR of irregular shape and with rough edges). Thus, in the wild bearded seals, the lymphoid system activity is lower, while the indices of nonspecific bactericidal function are higher than in animals kept in captivity.

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% 80 Wild In captivity

40

0

I

St

S

E

B

M

L

Leukograms of bearded seals in nature and in captivity. I, immature cells (metamyelocytes); St, stab neutrophils; S, segmented neutrophils; E, eosinophiles; B, basophiles; M, monocytes; L, lymphocytes.

KPs and MPO are important components of the antimicrobial defense of the organism. KPs and MPO of blood granulocytes are indices of nonspecific resis tance [10]. In bearded seals, a bactericidal KP is located in cytoplasmic granules. In nature, the animal leukocytes have a larger number of large granules, which are more intensely stainable (Table 2). Like harp seals, [11], the bearded seals kept in captivity have a lower level of KPs than the wild animals, which suggests an inhibited bactericidal function of leuko cytes because of the influence of unfavorable factors (food composition different from the natural one, hypodynamia, and stressful situations). Like some other true seals and bottlenose dolphin, bearded seals have a lower amount of KPs in leuko cytes than other animals and humans. In bearded seals, the KP amount (MCC in arbitrary units, a.u.) is 0.63 ± 0.18. For comparison these indices in other animals are the following: 1.60 ± 0.03 (mouse) [13], 1.36 ± 0.01 (chicken) [14], 0.29 ± 0.01 (grey seal), 0.45 ± 0.06 (harp seal), 0.77 ± 0.07 (dolphin) [11], 1.50 ± 0.02 (humans) [12]. The maximum bactericidal effect is a result of a combined action of MPO and KPs [10]. Note that

Table 1. The number, shape, and size of the lymphocyte nuclei and AgNOR in bearded seals. Here, in Tables 2 and 3, and in the figure, M ± m, n = 5 (wild) and n = 3 (in captivity) Animal group

Number of AgNOR in a lymphocyte

Wild In captivity

1.09 ± 0.03 1.10 ± 0.03

Nucleus AgNOR/ Nucleus

diameter, µm

rotundity index

AgNOR edge roughness

diameter, µm

rotundity index

edge roughness

0.066 ± 0.001 7.79 ± 0.15 0.803 ± 0.040 1.65 ± 0.02 1.86 ± 0.40 0.812 ± 0.004 0.39 ± 0.01 0.094 ± 0.002 8.66 ± 0.37 0.756 ± 0.003 1.78 ± 0.08 2.47 ± 0.11 0.825 ± 0.019 0.53 ± 0.02

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Table 2. KP staining of bearded seal granulocytes Animal group

Granule size, µm

Mean number of granules

KP+leukocytes, %

MCC

ICI

ICF

0.28 ± 0.08 0.21 ± 0.05

160.6 ± 9.4 78.3 ± 4.3

28.33 ± 5.85 5.92 ± 1.74

0.63 ± 0.18 0.09 ± 0.03

11.44 ± 0.80 2.04 ± 1.58

29.44 ± 3.37 13.34 ± 1.58

Wild In captivity

Table 3. MPO activity in bearded seal and human (data from [4]) granulocytes Animal group Bearded seal Humans

MPO+leukocytes, %

MCC

ICI

ICF

88.8 ± 2.1 90.4 ± 2.0

1.99 ± 0.15 1.84 ± 0.10

34.03 ± 2.77 5.25 ± 0.19

56.91 ± 2.51 24.70 ± 0.84

MPO is also an enzyme of the antioxidant system involved in the regulation of the free radical level to protect the body against oxidative stress. MPO demonstrates diffusegranular distribution by staining of blood granulocytes of bearded seal. Visual MCC analysis shows that the enzyme activity in these animals is comparable with that in healthy humans. Nevertheless, the morphometric indices tes tify to a significantly higher MPO activity in bearded seal leukocytes (Table 3). We have earlier detected [15] a high MPO activity in grey seal pups. MPO staining yields a high number of MPOpositive cells (90–100%) during postnatal development of grey seals, but the level of MPO is sig nificantly reduced with aging from 2.51 in newborns to 1.91 a.u. in adult animals. Over the seal lifetime, the bactericidal function of leukocyte appears to be exe cuted mainly with involvement of MPO, while KPs serve as an additional component of the bactericidal system. The results of our study suggest that the bearded seal blood system have specific morphological and functional features. In captivity, the indices of specific and nonspecific resistances of this member of the fam ily Phocidae are changed noticeably. Further studies would help to evaluate the degree of environmental physoilogical differences of the bearded seal from other true seal species. REFERENCES 1. Arnason, U., Hereditas, 1981, vol. 94, no. 1, pp. 29–34.

2. Anbinder, E.M., Kariologiya i evolyutsiya lastonogikh (Karyology and Evolution of Pinnipeds), Moscow: Nauka, 1980. 3. Astaldi, G. and Verga, L., Acta Haemat., 1957, vol. 17, pp. 1–29. 4. Slavinskii, A.A. and Nikitina, G.V., Klin. Lab. Diagn., 2000, no. 1, pp. 21–23. 5. Kavtsevich, N.N., Zool. Zh., 2003, vol. 82, no. 6, pp. 758–761. 6. Kavtsevich, N.N. and Minzyuk, T.V., Zool. Zh., 2011, vol. 90, no. 9, pp. 1122–1126. 7. Kavtsevich, N.N., The morphological and cytochemi cal characteristics of blood cells of marine mammals related to the adaptation to the environment, Extended Abstract of Doctoral (Biol.) Dissertation, Petrozavodsk, 2011. 8. Minzyuk, T.V. and Kavtsevich, N.N., Dokl. Biol. Sci., 2010, vol. 435, no. 5, pp. 444–447. 9. Kavtsevich, N.N. and Erokhina, I.A., Akt. Vopr. Vet. Biol., 2009, vol. 3, no. 3, pp. 3–8. 10. Pigarevskii, V.E., Zernistye leikotsity i ikh svoistva (Granular Leukocytes and Their Specific Characteris tics), Moscow: Meditsina, 1978. 11. Minzyuk, T.V. and Kavtsevich, N.N., Vestn. MGTU, 2013, vol. 16, no. 3, pp. 506–511. 12. Stoiko, Yu.M. and Ermakov, N.A., Khirurgiya Pril. Consilium Medicum., 2004, vol. 6, no. 2, pp. 23–26. 13. Budyka, D.A., Abzaeva, N.A., and Rudnev, S.M., Probl. Osobo Opasn. Inf., 2009, vol. 100, pp. 50–56. 14. Kletikova, L.V., Estestvozn. Guman., 2010, vol. 6, no. 1, pp. 51–52. 15. Minzyuk, T.V., Vestn. YuNTs, 2011, vol. 7, no. 4, pp. 70–73.

Translated by A. Nikolaeva

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