Influence of low-chitin krill meal on reproduction of Clethrionomys glareolus (Schreber, 1780)

Cony Bmhem. Phyd Prmted m Great Bntam

Vol. 94C. No. I. pp 313-320,

1989 c

INFLUENCE REPRODUCTION

OF LOW-CHITIN KRILL OF CLETHRIONOMYS (SCHREBER, 1780)

MEAL

0306-4492/89 $3 00 + 0.00 1989 Pergamon Pressplc

ON

GLAREOLUS

ALICJA KRA~OWSKA

Institute of Biology. Biatystok Branch of Warsaw University, Swlerkowa, 20B, 15-950 Biatystok, Poland (Received 24 April 1989) Abstract-l. Clethrionomys glare&s fed on a diet containing krill meal assimilated excessive amounts of fluoride. 2. Excess of fluoride caused disturbances in the reproduction of these animals: reduction of the number of litters. higher mortahty of offspring and degenerative changes in seminiferous tubules. 3. The disturbances m the reproduction occurred still more distinctly in the successive generation. 4. Among the bank vole offspring which received the diet with the greater amount of krill meal, both in the parent generation and in the generation F , , a changed sex ratio was found, which was evidenced by a sigmficantly higher number of males.

INTRODUCTION Research on the utilization of krill meal concentrated mainly on the question of whether krill meal can constitute full value source of protein in animal nutrition. Results of most of these studies indicate that it can be a partial or full substitute for fish meal. However, in cases where rats and laboratory mice were given diets with krill meal for a longer period of

time or when amounts of krill meal in the diet were great, it was observed that krill meal influenced the organism negatively (Pastuszewska, 1979; Heinz et al., 1981; Seidler et al., 1982; Pastuszewska et al., 1983b). These animals exhibited changes in the reproduction cycle. anatomicopathologic changes of alimentary tract and distinct overgrowth and discolouration of incisors. The aforementioned authors do not determine univocally the toxic agent responsible for originating these disturbances although disfigurement of incisors indicates the excess of fluoride ions in the organism, and it is commonly regarded as the typical symptom of toxicity of this element. _Euphausiu superba Dana contains great amounts of fluoride which is mainly situated in the shell (Soevik and Braekkan, 1979; Boone and Manthey, 1983; Buchholz, 1983; Adelung et al., 1987). After the death of krill fluoride passes from the shell into muscles. Intensity of this migration depends on the freezing temperature. on the length of time it remains in the frozen state and on the method of preparation (Christians and Leineman, 1983). Moreover, the average content of fluoride is about 50% higher in krill larvae than compared with the adults (Hempel and Manthey, 1981). That is why the amount of fluoride in various parts of krill meal is different. Thus, it seems that divergences in the opinions concerning the possibility of using krill meal as food additive may be explained mainly by different content of fluoride in the diets containing krill meal as well as by different and most often too short duration of experiments.

Krill meal also contains quite substantial amounts of chitin, which according to Seidler et al. (1982) can cause anatomicopathologic changes of alimentary tract in mice. In order to eliminate possible harmful influence of chitin C. glareolus was used (in this study) whose natural food contains certain amounts of this component. The aim of the present study was to investigate the effect of low-chitin krill meal on the reproduction of bank vole. Up till now, research on the effect of krill meal on reproduction of animals has been limited only up to the time of obtaining generation F, This period of time is too short to obtain objective assessment of the effect of krill meal in the diet on the reproduction of animals especially, if the diet does not contain much krill meal. Toxicity of fluoride depends not only on the dose quantity but also on the length of time during which this element is received. This is why it was decided in this study that breeding observations should be carried out on successive generations. MATERIALS AND METHODS

Assessment of the influence of low-chitin krill meal on the reproduction of C. glareolus was carried out on 284 animals. The bank voles came from the Mammals Research Institute, Polish Academy of Sciences in Biatowieia. Experimental diets were made on the basis of data taken from literature concerning bank vole’s food maintaining the same percentage of components as in the natural food, I.e. seeds, green feed and animal food. The control diet (A) included seeds (wheat), 65%; dried green feed (clover, alfalfa, grasses), 22% as well as meat and bone meal, 13%. Two experimental diets were made in which protein from meat and bone meal was substituted in 50% and 100% respectively by protein from the low-chitin krill meal which was provided by Sea Fishery Institute in Gdynia. As a result. the amount of krill meal in-these diets was in percentages as follows: in diet B. 6.2% in dret C, 12.4%. Other components were left in the &me amounts as in the control diet. All diets were enriched by vitamins, Polfamix (I gram/kilogram diet). In order to prevent the animals from selective eating of 313

314

ALICJA KRASOWSKA

Table

1 Chemtcal composttton of expertmental dtets. A-control dtet, B and C--expertmental duets Dtct Ash “0 Ether extract I% Crude hhrr Of0 N “0 Na 0’0 K “0 Ca “0 Mtl PPm Ztt PP”’ Cd

A

B

681

7 11

125

704

6.83

6 78

5 74

6 38

6.81

3 29

3 22

3 32

0.49

0 48

0 51

0 56

0 55

0 56

1 12

I 00

0 90

20.00

22.50

25 00

17.50

1700

19 00

c

---

RESUl.TS

ppm Pb ppm cu ppm F ppm

0 25

0 25

0.35

400

4 50

5 00

3 00

3 50

4 50

18 37

46 60

96 96

parttcular components, the diets were ground and mixed thoroughly. Food and water were provided ad ~i~iiurn once daily, in the mornmg. Chemical compositton of the diets is presented in Table 1. Two groups of bank voles were taken mto account during the breeding observations. The first group constituted of 3-4 month old animals which were given expertmental diets for stx weeks after whtch they were combined in pairs and observed for seven months being still fed on the same diets. The second group constituted bank voles from generation F, whtch were constantly fed on the experimental duets and which were taken from the parents fed on the same diets for half a year. At the age of 34 months they were combined in patrs and observed for half a year. In both groups the control constituted bank voles tested in parallel which were given diet with meat and bone meal. During the breeding observations of the two groups the following data were registered: number of litters and their stze. mortahty of offspring in the nest period, period of time from mating to producing the first litter, period of time between successive litters and changes concerning mctsors of the animals under study Testes were taken for histological examinations from males of both groups receiving diets A and C. The testes were fixed m Bourn solution, embedded m paraffin and Table 2. The mfluence of food wtth low-chttm Group Dtet Number of pat is

sectioned at 10~ The sections were stained with haematoxylin and eosin. The content of fluortde in femur and in blood, was examined in the bank voles receiving experimental duets for five months and in 8-9 month old animals from generation F,. Bank voles were etherized. The removed femurs were carefully cleaned and dried Samples of blood were taken from vein sinus. Ffouride extraction by microdiffuslon was followed by calorimetric analysts. The detailed analytical method was descrtbed in Baumler and Ghnz (1964) and Cuhk (1986). The received results were analyzed stattsttcally wtth calculatton of standard deviatton and with examination of stgmficance of differences between average values by Student’s t-test. The statistical significance of deviations of sex ratio from the norm was assessed by means of chi-square test.

Mated Dead Frrttle

Number of lrtters per one fertile female Litter stze

krtll meal on the reproductton

m CWrrronont.rs

29 2 23 2 57 + 0.69

B

C

A

B

C

30 5 21

14

15 3 IO

15 5 8

186

& 0.W

12 2 58 + 0.86

3 53 * 0 95

2.97 & 1 02

Number of days from matmg to producmg the first htter

56 70 i: 28.90

64.80 + 36 70

68 00 + 30 30

6490+

Number of days between successive litters

41 20 t 16.30

46.70 + 28 50

47 70 I 27 20

31 60 + 18.20

first expertmental group (parent generation). second expertmental group (generation F, 1. A-control duet, B and C-experimental duets. a--P 4 0.01 as compared with control diet I-the

II-the

(mean values It SE)

29 3 20 2.10~091

1.11

glareolus

II

I

-_.-l___-.-” A

3.00*

The number of litters per one fertile female was lower in the bank voles fed the experimental diets (B and C) while in the animals receiving diet C it was significantly lower (P < 0.01) compared with bank voles receiving control diet (Table 2). Regardless of the diet. the litter size m all bank voles was similar. The number of days between mating and producing the first litter was higher in the bank voles receiving food with krill meal. The C. glare&s fed on all diets produced successive litters every 4148 days on average and the differences between them were statistically insignificant (Table 2). In bank voles receiving the food with krill meal the mortality of offspring in the nest period was higher than in the control bank voles (Table 3). There were greater numbers of males (P < 0.05) among the offspring born by the bank voles receiving diet C than among the offspring born by bank voles receivmg diet B and diet A (Table 3). In order to inspect whether increase of mortality of bank voles receiving diet C during nest period can affect females to a higher degree, the sex ratio of the young was determined immediately after birth. The received results indicate that the significant (P < 0.05) deviations from the expected sex ratio I : 1 in favour of males occur already at the moment of birth. In the generation F, the bank voles receiving diets B and C exhibited fewer number of litters compared with the control animals, whereas in bank voles receiving diet C the number of litters per one fertile female was signi~cantly lower (P < 0.01, Table 2).

2 74 i 0 76 1620

i90t070

137*049a

2.84 + 0 74 72OOi

14 IO

32.70 i It 50

3 54 * 0 98” 84 70 * 8 70” 3440*

1260

Bank voles on a high fluoride diet Table 3 Mortahty

among

young voles m the nest period and sex ratm

I

Group Diet Number

of fertde pans

Number of young

Born Weaned

Mortahty of young ,I, nest period III ‘!/a Sex

rat10

;j ‘+‘.

315

II

A

B

C

A

B

23

20

21

12

10

8

167 51 52

153 48 44

116 41 2s

85 29 30

54 16 9

39 6 4

30.59

53 70

7-l 36

34 33 I IO I

39.87 109.1

l-the first ewperm~ental group (parent generation) II-the second expenmental group (generatton F, A+ontrol diet. B and C+xpertmental diets n--P < 0.05 as compared wth control diet b--sex ratlo at birth

43 II

I 64.

I”

I02.Ih

135

C

lb

I 78

IAb

)

The average litter size m C. glareolus fed on diet B was similar to the litter size in the control group. Statistically greater litters (P < 0.01) were observed in bank voles receiving diet C (Table 2). The number of days from mating to producing the first litter was higher in the animals receiving experimental diets than it was in the animals fed on the control diet, whereas in the bank voles eating more krill meal (diet C) this period was significantly longer (P < 0.01, Table 2). Also sigmficantly longer (P < 0.01) was the period between mating and producing the first Inter in the bank voles from the generation F, fed on diet C compared with the animals from parent generation (the first experimental group) fed on the same diet (Table 2). Hence also the number of litters in animals from generation F, is lower than in the bank voles from parent generation, but this difference is not significant. The period of time between producing successive litters m C. glureolus in the second experimental group was the same at the administration of three different diets and did not differ from the time between successive litters produced by the bank voles from the parent generation (Table 2). In the animals

from generation F, receiving control diet the mortality of offspring in the nest period was similar to the data received for bank voles from parent generation fed on the same diet. In animals fed on the experimental diets, the mortality of offsprmg in the nest was higher (Table 3). Significant (P < 0.05) change of sex ratio in favour of males (Table 3) was observed among the offspring of bank voles from generation F, receiving diet C as well as in the animals from parent generation fed on the same diet. The histological picture of testes from the second experimental group (generation F,) receiving diet C indicates that a large number of semimferous tubules underwent degenerative changes (Fig. 1). These tubules have a diminished surface of germinal epithelium or do not have it at all. The particular sex cells do not form concentrically arranged layers but they are, so to say, mixed. Some of the sex cells are of a very large size usually containing pyknotic nuclei (Fig. 2). Empty spaces resembling vacuoles can be seen between the cells. They might presumably have come into being after the disintegration of the cells of germinal epithelium of changed appearance the

Fig. 1. Microscopic picture of transverse section of testis of male from the second experimental group (generatlon F, ) receivmg diet C (magnified 240 x ) group of tubules with distinct retroactive changes Amount of germinal epithehum is reduced (1) or there IS hardly any at all (7).

ALICJA KRASOWSKA

316

Fig 2. Microscopic experimental group

picture of sectlon across semmiferous tubule of male testis from the second (generatlon F, ) recetvmg diet C (magnified 360 x ) dimnushed amount of cells of

germinal eptthelium. presence of distinctly enlarged cells containing vacuolated cytoplasm and pyknottc nucleus (1): there are empty spaces between cells of germinal eptthelium resemblmg vacuoles (2). resultmg probably after decomposition of cells of changed vtew. more so as in some changed sex cells a bright cytoplasm with marked slight vacuoles was observed. Beside the epithelia which underwent degenerative changes, there are epithelia having normal histological structure. The epithelia of control males had normal histological structure (Fig. 3). No markedly changed epithelia were observed in testes of C. glureolus from the first experimental group receiving diet C. Growth of incisors was found in 50% of bank voles fed on diet C and in about 30% of bank voles

receiving diet B during the breeding observations m the first experimental group. Changes of appearance of incisors of the animal fed on diet C was observed at about the 5th month of being fed on this diet and in the bank voles receiving diet B they were observed at about the 6th month. Incisors elongated gradually and distorted (Fig. 4). No changes in the shape of incisors were observed in the control bank voles (Fig. 5). Changes in the shape of inctsors in the second experimental group were observed earlier, at

Ftg. 3. Microscopic picture of the sectlon across semmiferous tubule of the male testrs from the second experimental group receivmg diet A (control duet) (magmfied 360 x )

Bank voles on a high fluoride

Fig. 4 Picture

of incisors

of bank

voles fed on low-chltm

about the 3rd month of hfe in the animals fed on diet C and at about the 4th month of life in the bank voles receiving diet B. In the final period of observation the overgrowth of inctsors was found in bank voles from generation F, in 90% of animals fed on diet C, and in about 60% of bank voles fed on diet B. In bank voles fed on experimental diets for five months the amount of fluoride accumulated in their femur was significantly higher (P < 0.001) compared with the amount of fluoride accumulated in femur of animals receiving control diet (Table 4). In males and females the amount of fluoride in bone tissue was simtlar (Table 4). In 889 month old C. glureolus from

Fig. 5. Picture

of mclsors

of bank

317

diet

krlll meal diet for a longer

period

of time

generation F, receiving experimental duets the amount of fluoride in the femur was also significantly higher (P < 0.001) compared with control animals (Table 4) and this amount was the same in males and females. The amount of fluoride m bone of 8-9 month old bank voles from generatton F, was significantly higher (P < 0.001) in all examined animals compared with the amount of this element deposited in the femur of bank voles from parent generation receiving the same diets for five months. This 1s the evidence for accumulation of fluoride in the bone tissue of the examined animals.

voles not recelvmg

low-chitin

krill meal m their food.

318

ALICJA KRASOWSKA

Bank voles receiving experimental diets for five months exhibited significantly higher contents of fluoride in blood (P < 0.001) compared with animals fed on control diet (Table 4). In 8-9 month old C. glureofus from generation F, receiving experimental diets the content of fluoride was also significantly higher (for diet B P < 0.01 and for diet C P < 0.001, Table 4). The content of fluoride in the blood of bank voles from generation F, fed on diet B and C was significantly greater than in the blood of bank voles receiving the same diets for five months (for diet B P < 0.01 and for diet C P < 0.001). The concentration of fluoride in blood was the same in males and females receiving experimental diets for five months and in 8-9 month old animals from generation F, (Table 4). DISCUSSION

The abnormal shape of incisors and changes in their pigmentation are the most visible symptoms of excessive amounts of fluoride in the body of rodents (McClure and Mitchell, 1931: Newman and Markey. 1976). Elongation of incisors in C. glareohs in this study depended on the concentration of fluoride in the diet and on the length of time durmg which the food was provided to the animals. Deformation of incisors in the bank voles appeared earlier when animals were fed on the food containing greater amount of fluoride (diet C) and in animals from generation F, compared with parent generation. The earher elongation of incisors of bank voles from generation F, resulted mainly from the fact that these animals were exposed to harmful effect of fluoride during adolescence This is so because the growing animals exhibit greater ability to accumulate fluoride in the bone than the adult animals (Savchuck and Armstrong. 1951). Moreover, the bank voles from generation F, received fluoride during prenatal development. because this element is transferred through placental barrier to skeletal system of the fetuses (Fleming and Greenfield, 1954; Brzezinski et al., 1961; Katz and Stookey, 1973). The confirmation of the excess of fluoride in the body of C. glareolus fed on the diet containing low-chitin krill meal is its presence in the blood and bone of these animals. The amount of deposited fluoride in the femur of bank voles depended on the content of this element in the diet (Table 4). Moreover, greater content of fluoride in the femur of the bank voles from generation F, (Table 4) resulted mainly from much longer time of receiving the experimental diets. These results agree with the reports of other authors according to which the amount of accumulated fluoride in the bone is positively correlated with the content of fluoride in food or in drinking water and with the time of administering (Weber and Reid, 1969; Newman and Markey, 1976; Ekstrand et al., 1981; Khalawan, 1981; Boros er al., 1984). Simon and Sutie (1968), Patz (1973) and Boros et al. (1984) found that the amount of fluoride in blood plasma depends also on the amount of fluoride in the food. It seems that slightly higher content of fluoride in the blood of the bank voles from generation F, receiving food with krill meal in comparison with the animals from parent generation was the result of lower capacity of

Bank voles on a high fluoride diet bone tissue of these animals to sequestrate fluoride. This is so because the amount of fluoride already accumulated in their osseus system was much greater than in the adult bank voles fed on the same diets for five months. The excess of fluoride in the body of the C. glareolus was undoubtedly the cause of the observed disturbances in their reproduction. The animals from the first experimental group receiving food with krill meal gave birth to a smaller number of litters than the control bank voles and the longevity of their offspring in the nest period was lower (Tables 2 and 3). The reduction of the number of litters and higher mortality of offspring occurred even more distinctly in the bank voles from the second experimental group (generation F, , Tables 2 and 3). Since the number of days between successively produced litters was similar in the experimental bank voles and in the control group ones. the smaller number of litters receiving food with krill meal resulted from the lengthening of time between the mating and giving birth to the first litter (Table 2). Pastuszewska et al. (1983b) providing food with krill shell meal to growing rats found the retarded development of seminiferous eplthelium in the testes of these animals. It may be supposed that similar phenomenon could take place in C. glareolus fed on diets with krill meal. However, it was not the only cause of decrease in the number of litters in these animals, because htstological picture of male testes from the second experimental group fed on diet C indicates that a large part of seminiferous epithelia underwent degenerative changes. In the first experimental group so clearly changed seminiferous epithelia were not observed in the testes of males receiving the same food. It may be, however, considered that in spite of the lack of distinct changes in the histological picture, the normal functioning of these males’ testes had already been disturbed and could be the cause of a slight delay in giving birth to the first litter by this group of animals. The degenerative changes of the germinal epithelium were caused by the toxic effect of fluoride. The conclusion is confirmed by the studies of Kour and Singh (1980). These authors, administering fluoride to the males of mice in drinking water at the amount of 500 and 1000 ppm, observed similar changes in the seminiferous epithelia; these changes clearly intensified alongside the lengthening of time of fluoride intake by these animals. As in these investigations bank voles were receiving much smaller amounts of fluoride than the mice in the study by the aforementioned authors, and moreover the fluoride from krill meal is assimilated to a lower degree than the fluoride from sodium fluoride (Pastuszewska et al., 1983a) clear degenerative changes were not observed in bank voles until the second generation. Kanwar and Singh (198 1) and Singh (1982) report that administering fluoride in drinking water to laboratory mice causes significant decrease of manganese and zinc in liver and kidneys of these animals. Decrease of these elements in soft tissues may have a harmful influence on their normal functioning. Among other things, both of these elements condition normal development of testes and normal spermatogenesis (Underwood, 1962). Deficiency of these elements leads to degenerative changes of seminiferous tubules and their

319

atrophy (Boyer et al., 1942; Swenerton and Hurley, 1968; Diamond et al., 1971). Thus, it is likely that administering to bank voles food with greater amount of krill meal and hence with greater amount of fluoride could decrease the level of zinc and manganese in the soft tissues of these animals and consequently it could result in the degenerative changes of seminiferous tubules. This hypothesis will be confirmed in further studies. Addition of low-chitin krill meal to the food of C. glareolus also increased the mortality of the young in their nest period. The increase of the mortality of the young obtained from the bank voles of parent generation (the first experimental group) was admittedly not great, but the increase of the mortality of the offspring of animals from the second experimental group was so great that carrying out further breeding observations became impossible (Table 3). The mortality of the young in the nest period in bank voles not receiving krill meal was approximate to the data by Droidi (1963) and Buchalczyk (1970). The observed increase of mortality of the offspring of bank voles fed on food with krill meal was thus probably caused by the toxic influence of fluoride. The size of litter can influence the longevity of offspring (Gustaffson et al., 1980). However, the litter size in this study was similar in the control of animals and in the experimental ones. Therefore, it can be said that mortality of the offspring of the bank voles fed on food with krill meal depends on the amount of fluoride in the diet, as well as on time of feeding. When the amount of fluoride in food or drinking water is great, high mortality is observed already in the first generation (Messer et al., 1973; Marks et al., 1984). In the offspring of bank voles receiving more krill meal in food (diet C) a changed sex ratio was found in both generations, evidence manifested by the greater number of males (Table 3). Investigations by Buchalczyk (1969) showed that sex ratio in young bank voles at the time of weaning amounted 1: 1. Similar data were obtained by Mazak (1962) Zejda (1968) and Gustaffson et al. (1980). Since sex ratio among the offspring of bank voles receiving the control diet was also 1: 1 in this study, it may be suggested that sex ratio disturbances in the animals fed on diet C were a result of a great amount of fluoride in this food. Acknowledgements-1 would like to thank Professor Dr A. Myrcha for her assistance and critical remarks during carrying out this study; Professor Dr D. Adelung for hts help in determining fluortde and Dr B. Sawtcki for histological verification,

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320

ALICJA KRASOWSKA

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