Br. vet.j. (1 977 ), 133,41 2
EFFECT OF FEEDING OCHRATOXIN TO PIGS DURING EARLY PREGNANCY BYB.]. SHREEVE,
D. S.
P . PATIERSON,
G . A.
P EP IN,
B. A . ROBERTS AND A. E. WRATHALL
Ministry of Agriculture, Fisheries and Food, Centrai Veterinary Laboratory, Weybridge, Surrey
SUMMARY
O chratoxin (equivalent to 0·38 mg and 0·13 mg ochratoxins A. and B/kg body weight respectively) was fed to pregnant sows from 21 to 28 days of pregnancy. When killed at 30 days, the reproductive tracts showed no obvious abnormalities either in the adult or the embryo. This may be due to the fact that while ochratoxin A was detected in maternal tissues it had not crossed the placenta into the foetus . Ochratoxin B was not found in either maternal or foetal tissues.
INTROD UC TION
O chratoxin A has been isolated from cultures of Aspergillus ochraceus Wilhelm (Van der Merwe et ai., 1965), several other members of the A. ochraceus group (Lai , Semeniuk & Hesseltine, 1968 ; Hesseltine et ai., 1972), Penicillium viridicatum Westling (Van Walbeek et ai., 1969) and other Penicillium species (Ciegler et ai., 1972; Scott et ai., 1972 ). Representatives of the A. ochraceus group and P. viridicatum are widely distributed in nature (Raper & Fennell, 1965; Raper & Thorn, 1949), and the natural occurrence of ochratoxin A in cereals has been reported from Canada (Scott et ai., 1970), Denmark (Krogh, Hald & Pedersen, 1973), Sweden (Krogh et ai., 1974b), the U.S.A. (Shotwell, H esseltine & Goulden, 1969 ) and Yugoslavia (Shotwell & Bothast, 1973). Reproductive failure in pigs presents a serious economic problem in Britain and during a recent investigation by Shreeve, Patterson & Roberts (1975 ), abortion, stillbirths and expulsion of mummified foetuses were found to have an association (though not necessarily a causal relationship ) with the consumption of mouldy feed. Potential ochratoxin-producing fungi were also isolated on many occasions and ochratoxin A was detected in some animal feeds . Renal disease in pigs has been associated with the presence of ochratoxin A in fodder (Krogh, Hald & Pedersen, 1973) and foetal death in rats has been produced experimentally by feeding the toxin (Still et ai., 1971). The present study was undertaken to determine whether ochratoxin A would interrupt pregnancy, increase embryonic mortality or produce teratogenic effects when fed to gilts during early pregnancy.
OCHRATOXIN
41 3
MATERIAL S AND METHODS
Preparation of crude ochratoxins A crude extract containing ochratoxin A was prepared from cultures of Aspergillus ochraceus grown on cereal substrates. Wheat and rice grains were inoculated with two strains (NRRL 3519 and NRRL 5175 ) known to produce ochratoxin A (H esseltine et al., 1972) and agitated during incubation at 28°C as described by Hesseltine (1972 ). These and static cultures on the same substrates and on shredded wheat were incubated at 21°C and 28°C (Nesheim, 1969) and harvested after five , 12 and 20 days. Individual cultures were homogenized with 150 ml distilled water and 5 ml 2N-HCI (fi nal pH 2·0) and the homogenizer washed out with 50 ml water. After extraction with 100 ml chloroform, the presence of ochratoxins A and B was confirmed by thin layer chromatography and their concentrations estimated by visual matching with solutions of pure toxins (Scott, Kennedy & Van Walbeek, 197 I ). Extracts containing toxin were bulked, evaporated to dryness in a rotary evaporator and the residue fe-dissolved in ethanol to give a stock solution of crude toxin. Concentrations of ochratoxins A a nd B determined by thin-layer chromatography and ultra-violet absorptiometry (Steyn , 19 71 ; Chu, Noh & Chang, 1972) were 0 ·19 mg/ml and 0 ·06 mg/ml respectively. Animals and procedures Three healthy Landrace-cross gilts, aged eight to ten months and weighing 87 to 142 kg were used . They were housed in adjacent pens and mated naturally to the same boar on the first day of oestrus and again on subsequent days until the end of oestrus. A commercial brand of sow and weaner meal (BO CM -Silcock: Sowcare meal) was fed throughout. Two gilts were fed ochratoxin in the food (equivalent to 0 ·38 mg and 0· 13 mg ochratoxin A and B/kg body wt./day respectively) from 21 to 28 days of gestation inclusive (last day of mating = day 0). An appropriate volume of the ethanolic stock solution of mixed ochratoxins was suspended in distilled water (final concentration of ethanol less than 0 ·1%) immediately before use and thoroughly m ixed with approximately 0·5 kg dry meal. Food was withheld overnight so that the resultant toxic feed was eaten completely at the early morning feed . During the re mainder of the day, a wet mash of normal meal was fed to appetite. One gilt was used as a control and fed in an identical manner except that no ochratoxin was added to the wet meal. Each gilt was weighed weekly from mating, examined clinically each day and slaughtered at 30 ± I days of gestation using intravenous sodium pentobarbitone. At post-mortem examination samples of liver, kidney, spleen, lung and heart were fixed in 10% phosphate buffered neutral formalin (Lillie, 1965 ). Paraffin-embedded sections were stained with haematoxylin and eosin for histopathological examination. Examination of reproductive tracts Reproductive tracts were removed immediately after death and mesometria and mesovaria trimmed off close to the uterus. Embryos were exposed by opening the uterus carefully along the antimesometrial border. Intrauterine positions of all embryos were recorded and those which were dead or otherwise abnormal were noted . Each embryo was weighed, measured (crown-anus length ) and fixed in 10% p hosphate buffered formalin. A period of at least 24 h fixation was allowed to enable
414
BRITISH VETERINARY JO U RNAL, 133, 4
emb ryos to be dissected more easily. The following organs were carefully separated and weighed to the nearest 0·0 1 g after removing excess fluid : whole brain, spinal cord, heart, liver, gut, mesonephros/metanephros complex and eyes. Placentae were p eeled off the endometrial surface, weighed and the area of occupied endometrium m easured .
Analysis oj tissues Jor ochratoxin A Samples of muscle, kidney, liver, placenta and whole foetu s were homogenized with five ~o lumes phosphoric acid (H 3 P0 4 ) a nd extracted with ch loroform. The extracts were then examined for the presence of ochratoxin A by thin layer chromatography using proced ures described by Krogh et al. (197 4a). Assay oj urinary leucine amino-peptidase Fresh urine samples taken from the gi lts during the experiment and at post mortem were assayed for enzyme activity using a Boehringer test kit. To allow for varying urine co ncentratio ns, results were expressed as milli-units (mU) per mg of creatinine, the latter being determined by the method of King (1951 ). RESULTS
All gilts remained clinically normal throughout the experimental p eriod although those exposed to ochratoxin became increasingly reluctant to eat meal contaminated with this compound . No gross lesions were apparent at post-mortem examination and no microscopical changes were detected in the organs examined . Results of examination of the reproductive tracts are shown in Tables I and II. The proportion of surviving embryos was lower in the two experimental litters than in the con trol although embryonic survival rates of a ll li tters were sti ll at or above the average figure for pigs at this stage of pregnancy (Wrathall , 1971). Mean body weights of embryos and lengths of placentae were also signi fican tly different from the controls. In one sow, the mean placental weight and area also differed significantly from the contro l. Organ weights were determined on embryos not required for chemical analysis and the data for both li tters were pooled. Mean heart and liver weights were found to be significantly lower than the corresponding control values. Residues of ochratoxin A but no ochratoxin B were found in muscle, liver and kidney of both experimental gilts but none was present in the control. The toxin was
TABLE I EFFECT OF OCHRATOXIN A ON REPRODUCTIVE T RA CTS OF PREGNANT GILTS
Experimentalliller 1
Experimentalliller 2
Control
29 II 7 64
30 12
31 16 16 100
Day of gestation at slaughter Corpora lutea Normal embryos Embryo survival" rate (%) " No dead or abnormal embryos were found .
11 92
OCHRATOXIN
415
TABLE 11 MEA
BODY, PLACE
TAL A 0
ORGAN MEASUREMENTS OF PIG EMB RYO S FROM DAMS FED OCHRATOXIN A (± SEM )
Experimentalliller 1 Body weight g Body length mm Placental weight g Placental length cm Placental area cm 2
Organ weight mg
"" 1·8± 29·0± "3 7·0± " ""22 · 1± ""23 1·0±
0·039 0·44 3·6 1·6 17 ·2
Experimentalliller 2
Control
"" 1·9± 0·019 27·6± 0·31 3 1· 7± 2·4 " 16·9± 1·5 148·4± 11 ·3
2· 0 ± 0 ·035 28·3± 0 ·21 25·8± 2·4 12·8± 1·2 12 1·8±1O·1
6 embryos
8 embryos
+3 embryos
Whole brain Spinal cord Heart Liver Gut Kid ney/Gonads Eyes
65·6±7·2 43·6±3 ·4 "44·4±1 ·4 "240 ·6±7 ·3 26 ·8 ± 2·3 69·1±2 ·8 5·8±0 · 72
71·5± 47 · 1± 50·9± 278·4± 26 ·9 ± 72 ·6± 7·0±
6 ·2 4·1 2·6 12·5 2·2 4 ·5 0·53
t
Re ma ining embryos u sed for chemical ana lysis. Significant diffe rence from mean of untreated control litter. "P=0·05; *** P =O· OOI.
TABLE III OCHRATOXIN A R ES ID UES IN MATERNAL AN D FOETAL TISSUES ' (I'g/g fresh wI: mean of 3 determinations)
Muscle Liver Kidney Placenta Whole foetus
Experimentalliller 1
Experimentalliller 2
Control
0·15 0·30 0 ·43 0·04 n .d.
0·13 0·34 0 ·70 0·06 n.d.
n.d. n .d. n.d . n.d . n.d .
n.d. = none de tected. * No ochratoxin B could be detected .
a lso present in the placentae of gilts receiving the toxic rations but none could be detected in foetal tissues (Table III). At the conclusion of the feeding period, leucine amino-peptidase activity was higher in urine samples from experimental gilts (both 12 m U/mg creatinine) than in the control (5 mU/mg creatinine) but this may not be significant in view of considerable day-to-day variation (5-33 mU/mg creatinine) and differences in pre-feedi ng a nd control values for enzyme activity. DISC USSI ON
In the present study no embryonic abnormalities occurred in gilts following oral administration of ochratoxin during early pregnancy. Exposure to toxin corresponded
416
BRITISH VETERINARY JOURNAL, 133,4
to a time of rapid morphological development of the porcine embryo (Patten, 1948 ) so that any teratogenic effect might have been expected to occur during this period. Since no dead or abnormal embryos were found , the reduced rates of embryonic survival probably relate to embryonic losses occurring prior to feeding the experimental diet. Although there were significant differences between embryos from experimental and control gilts in relation to body weights and placental parameters this may have been due to small but important differences in the stage of gestation and litter size. Thus the control litter was both older and contained more embryos than either of the litters from the treated sows. Differences in heart and liver weights can probably be attributed to differences in body weight. There were no clinical signs of ochratoxin poisoning (Krogh et ai., 1974a) which is consistent with the lack of gross or microscopical lesions in the sows. Urinary leucine amino-peptidase activity is considered to be a useful diagnostic guide in suspected cases of porcine ochratoxicosis (Hyldgaard-Jensen, 1973) but in the present experiment there was no indication of kidney damage in the gilts receiving the toxic ration. This agrees with the findings of Szczech, Carlton & Tuite (1973 ) who were unable to detect a significant increase in urinary leucine amino-peptidase concentration in SPF swine fed 1 mg/kg ochratoxin A for six days . Although present in maternal kidney, liver, muscle and placenta ochratoxin A was not found in foetal tissues, a result which indicates that, as in the case of the sheep (Munro et ai., 1973), ochratoxin A does not readily cross placental membranes. O chratoxin B was not detected in maternal or foetal tissues. Up to 1% of pigs slaughtered in Denmark may exhibit lesions of ochratoxin induced nephropathy but as far as we are aware the presence of ochratoxin A in fodder has never been associated with reproductive failure in pigs in that country. Although a sufficiently high dose of the toxin may cause foetal resorption in the rat (Still et ai. , 1971) and embryonic mortality in mice (Hayes & Hood, 1973), present experimental evidence suggests that a similar effect is not readily reproduced in the pig. A daily intake of about 40 mg ochratoxin A in this experiment is approximately equivalent to feed ing a diet containing 17 parts/l0 6 of toxin, which is over four times higher than the highest level used by Krogh et ai. (I974a) to induce experimental porcine nephropathy and of the same order as levels detected in cereals (Hesseltine, 1974). For these reasons it seems that o chratoxin A cannot seriously be regarded as a potential cause of reproductive failure in the sow. ACKNOWLEDGEMENTS
We are indebted to Miss S. M. Eades for setting up mycological cultures, Mr P. M. D. Foster for preparing the crude toxic extract, Mrs B. Small for ochratoxin analyses, Mr J . Bailey for dissection and Miss C. N. Hebert for statistical analysis. REFERENC ES
CHU, F. S., NOH, I. & CHANG, C. C. ( 1972). Life Sci. 11,503. CIEGLER, A., FENNELL, D. J., MINTZLAFF, H .-J. & L EISTNE R, L . ( 1972). Naturwissenschaflen 59,365. HAYES, A . W . & HOOD, R . D. (1973). Toxic. appl. Pharmac. 25, 457. H ESSELTINE, C . W . ( 1972). Biotechnol. & Bioeng. 14,517.
OCHRATOXIN
417
HESSELTINE, C . W . (]974). Mycopath. Mycol. appl. 53, 141. H ESSELTINE, C . W ., VANDEGRAFT, E . E. , FENNELL, D. l. , SMITH, M . L. &SH01WELL, O . L. (]972). Mycologia, 64, 539. HYLDGAA RD-J E SE ,j. (]973 ). In Aaarsberetning, p. 11 5. Copenhagen: Inst. Sterilite tsforskning. KING, E . j. (]95 I). Micro-analysis in Medical Biochemistry, 2 nd ed. London : Churchill. KROGH , P., HALO, B. & PED ERSEN , E.j. (]973). Acta path. microbiol. scand. Sect. B. III, 689. KROGH, P ., AX ELSEN, N . H ., ELLING, F., GYRo-HANSEN, N ., HALO, B., HVLD GAARD· JENSEN, j. , LARSEN, A . E., MADSEN, A., MORTENSEN, H . P., MO LLER, T., P ETERSEN, O. K. , RAVNSKOY, U., ROSTGAA RD, M . & AALUND, O . (]9 74a). Acta path. microbiol. scand. Sect. A , Supp. No. 246 . KROGH, P., H ALO, B., ENGLUND, P., R UTQU IST, L. & SWAHN, O . (]9 74b ). Acta path. microbiol. scand. Sect. B . 82, 30 1. LAI, M ., SEMENIUK, G . & HESSELTIN E, C . W . (1968 ). Phytopathology, 58,1056. LILLIE, R. D. (]965 ). Histopathologic Technic and Practical Histochemistry, 3rd edn. New York: McGraw Hill. MUNRO, 1. C ., SCOTT, P. M ., MOODI E, C . A. & WILLES, R . F . ( 197 3). ). Am. vet. med. Ass. 163, 1269. NESHEIM, S. (]969) .j. Ass. off. analyt. Chem. 52, 975 . PATTEN, B. M. ( 1948 ). Embryology of the Pig, 3 rd e dn. Philadelphia: The Blakiston Co. RAPER, K. B. & FENNELL, D. 1. ( 1965). The genus Aspergillus. Bal ti more: Williams & Wilkins Co. RAPER, K. B. & THOM, C . (] 949 ). A Manual of the Penicillia, Baltimore: Williams & Wilkins Co . SCOTT, P. M ., KENNEDY, B. &VANWALBEEK, W. (1971).]. Ass. off. analyt. Chem. 54,1445. SCOTT, P. M ., VANWALBEEK, W. , HARWI G, j. & FENNELL, D . 1. (1970). Can.j. Pl. Sci. 50, 583. SCOTT, P. M. , V AN W ALBEEK, W ., KENNEDY, B. &ANYETI, D . (]972 ). Agric. Fd Chem. 20,1103 . SH01WELL, O . L., H ESSELTI NE, C. W. & GO ULDEN, M . L. (] 969 ). Appl. Microbiol. 17,765 . SH01WELL, O . L. & BOTHAST, R. j. (]973 ). Cited by H esseltine, C. W . (]9 74 ). Mycopath. Mycol. appl. 53, 141. SHREEVE, B. j. , PATTERSON , D. S. P . & ROB ERTS, B. A. ( 1975). Vet . Rec. 97,275 . STEYN, P. S. (]97 I). In Microbiol Toxins, Vol. VI, p. 179 , ed. Ciegler, A ., Kadis, S. & Ajl , S. j. N ew York: Academic Press . ST ILL, P . E., MACKLI N, A. W. , RI BELIN, W. E. & SMALL EY, E. B. (197 1). Nature, Lond. 234, 563 . SZCZECH, G. M. , CARLTON, W. W . & T UITE, j. (19 73). Toxic. appl. Pharmac. 25,45 6. VAN D ER M ERWE, K. j. , STEYN, P. S. , FOUR IE, L., SCOTT, D E B. &THERON, j.j. (1965). Nature, Lond. 205,111 2. VAN W ALBEEK, W ., SCOTT, P. M ., HARWI G, j. & LAWRENCE, J. W . ( 1969 ). Can.}. Microbiol. 15, 1281. WRATHALL, A. E. (]9 7 I). Vet. Rec. 89,61.
(Accepted for publication 23 November 1976)
EFFECT OF FEEDING OCHRATOXIN TO PIGS DURING EARLY PREGNANCY BYB.]. SHREEVE,
D. S.
P . PATIERSON,
G . A.
P EP IN,
B. A . ROBERTS AND A. E. WRATHALL
Ministry of Agriculture, Fisheries and Food, Centrai Veterinary Laboratory, Weybridge, Surrey
SUMMARY
O chratoxin (equivalent to 0·38 mg and 0·13 mg ochratoxins A. and B/kg body weight respectively) was fed to pregnant sows from 21 to 28 days of pregnancy. When killed at 30 days, the reproductive tracts showed no obvious abnormalities either in the adult or the embryo. This may be due to the fact that while ochratoxin A was detected in maternal tissues it had not crossed the placenta into the foetus . Ochratoxin B was not found in either maternal or foetal tissues.
INTROD UC TION
O chratoxin A has been isolated from cultures of Aspergillus ochraceus Wilhelm (Van der Merwe et ai., 1965), several other members of the A. ochraceus group (Lai , Semeniuk & Hesseltine, 1968 ; Hesseltine et ai., 1972), Penicillium viridicatum Westling (Van Walbeek et ai., 1969) and other Penicillium species (Ciegler et ai., 1972; Scott et ai., 1972 ). Representatives of the A. ochraceus group and P. viridicatum are widely distributed in nature (Raper & Fennell, 1965; Raper & Thorn, 1949), and the natural occurrence of ochratoxin A in cereals has been reported from Canada (Scott et ai., 1970), Denmark (Krogh, Hald & Pedersen, 1973), Sweden (Krogh et ai., 1974b), the U.S.A. (Shotwell, H esseltine & Goulden, 1969 ) and Yugoslavia (Shotwell & Bothast, 1973). Reproductive failure in pigs presents a serious economic problem in Britain and during a recent investigation by Shreeve, Patterson & Roberts (1975 ), abortion, stillbirths and expulsion of mummified foetuses were found to have an association (though not necessarily a causal relationship ) with the consumption of mouldy feed. Potential ochratoxin-producing fungi were also isolated on many occasions and ochratoxin A was detected in some animal feeds . Renal disease in pigs has been associated with the presence of ochratoxin A in fodder (Krogh, Hald & Pedersen, 1973) and foetal death in rats has been produced experimentally by feeding the toxin (Still et ai., 1971). The present study was undertaken to determine whether ochratoxin A would interrupt pregnancy, increase embryonic mortality or produce teratogenic effects when fed to gilts during early pregnancy.
OCHRATOXIN
41 3
MATERIAL S AND METHODS
Preparation of crude ochratoxins A crude extract containing ochratoxin A was prepared from cultures of Aspergillus ochraceus grown on cereal substrates. Wheat and rice grains were inoculated with two strains (NRRL 3519 and NRRL 5175 ) known to produce ochratoxin A (H esseltine et al., 1972) and agitated during incubation at 28°C as described by Hesseltine (1972 ). These and static cultures on the same substrates and on shredded wheat were incubated at 21°C and 28°C (Nesheim, 1969) and harvested after five , 12 and 20 days. Individual cultures were homogenized with 150 ml distilled water and 5 ml 2N-HCI (fi nal pH 2·0) and the homogenizer washed out with 50 ml water. After extraction with 100 ml chloroform, the presence of ochratoxins A and B was confirmed by thin layer chromatography and their concentrations estimated by visual matching with solutions of pure toxins (Scott, Kennedy & Van Walbeek, 197 I ). Extracts containing toxin were bulked, evaporated to dryness in a rotary evaporator and the residue fe-dissolved in ethanol to give a stock solution of crude toxin. Concentrations of ochratoxins A a nd B determined by thin-layer chromatography and ultra-violet absorptiometry (Steyn , 19 71 ; Chu, Noh & Chang, 1972) were 0 ·19 mg/ml and 0 ·06 mg/ml respectively. Animals and procedures Three healthy Landrace-cross gilts, aged eight to ten months and weighing 87 to 142 kg were used . They were housed in adjacent pens and mated naturally to the same boar on the first day of oestrus and again on subsequent days until the end of oestrus. A commercial brand of sow and weaner meal (BO CM -Silcock: Sowcare meal) was fed throughout. Two gilts were fed ochratoxin in the food (equivalent to 0 ·38 mg and 0· 13 mg ochratoxin A and B/kg body wt./day respectively) from 21 to 28 days of gestation inclusive (last day of mating = day 0). An appropriate volume of the ethanolic stock solution of mixed ochratoxins was suspended in distilled water (final concentration of ethanol less than 0 ·1%) immediately before use and thoroughly m ixed with approximately 0·5 kg dry meal. Food was withheld overnight so that the resultant toxic feed was eaten completely at the early morning feed . During the re mainder of the day, a wet mash of normal meal was fed to appetite. One gilt was used as a control and fed in an identical manner except that no ochratoxin was added to the wet meal. Each gilt was weighed weekly from mating, examined clinically each day and slaughtered at 30 ± I days of gestation using intravenous sodium pentobarbitone. At post-mortem examination samples of liver, kidney, spleen, lung and heart were fixed in 10% phosphate buffered neutral formalin (Lillie, 1965 ). Paraffin-embedded sections were stained with haematoxylin and eosin for histopathological examination. Examination of reproductive tracts Reproductive tracts were removed immediately after death and mesometria and mesovaria trimmed off close to the uterus. Embryos were exposed by opening the uterus carefully along the antimesometrial border. Intrauterine positions of all embryos were recorded and those which were dead or otherwise abnormal were noted . Each embryo was weighed, measured (crown-anus length ) and fixed in 10% p hosphate buffered formalin. A period of at least 24 h fixation was allowed to enable
414
BRITISH VETERINARY JO U RNAL, 133, 4
emb ryos to be dissected more easily. The following organs were carefully separated and weighed to the nearest 0·0 1 g after removing excess fluid : whole brain, spinal cord, heart, liver, gut, mesonephros/metanephros complex and eyes. Placentae were p eeled off the endometrial surface, weighed and the area of occupied endometrium m easured .
Analysis oj tissues Jor ochratoxin A Samples of muscle, kidney, liver, placenta and whole foetu s were homogenized with five ~o lumes phosphoric acid (H 3 P0 4 ) a nd extracted with ch loroform. The extracts were then examined for the presence of ochratoxin A by thin layer chromatography using proced ures described by Krogh et al. (197 4a). Assay oj urinary leucine amino-peptidase Fresh urine samples taken from the gi lts during the experiment and at post mortem were assayed for enzyme activity using a Boehringer test kit. To allow for varying urine co ncentratio ns, results were expressed as milli-units (mU) per mg of creatinine, the latter being determined by the method of King (1951 ). RESULTS
All gilts remained clinically normal throughout the experimental p eriod although those exposed to ochratoxin became increasingly reluctant to eat meal contaminated with this compound . No gross lesions were apparent at post-mortem examination and no microscopical changes were detected in the organs examined . Results of examination of the reproductive tracts are shown in Tables I and II. The proportion of surviving embryos was lower in the two experimental litters than in the con trol although embryonic survival rates of a ll li tters were sti ll at or above the average figure for pigs at this stage of pregnancy (Wrathall , 1971). Mean body weights of embryos and lengths of placentae were also signi fican tly different from the controls. In one sow, the mean placental weight and area also differed significantly from the contro l. Organ weights were determined on embryos not required for chemical analysis and the data for both li tters were pooled. Mean heart and liver weights were found to be significantly lower than the corresponding control values. Residues of ochratoxin A but no ochratoxin B were found in muscle, liver and kidney of both experimental gilts but none was present in the control. The toxin was
TABLE I EFFECT OF OCHRATOXIN A ON REPRODUCTIVE T RA CTS OF PREGNANT GILTS
Experimentalliller 1
Experimentalliller 2
Control
29 II 7 64
30 12
31 16 16 100
Day of gestation at slaughter Corpora lutea Normal embryos Embryo survival" rate (%) " No dead or abnormal embryos were found .
11 92
OCHRATOXIN
415
TABLE 11 MEA
BODY, PLACE
TAL A 0
ORGAN MEASUREMENTS OF PIG EMB RYO S FROM DAMS FED OCHRATOXIN A (± SEM )
Experimentalliller 1 Body weight g Body length mm Placental weight g Placental length cm Placental area cm 2
Organ weight mg
"" 1·8± 29·0± "3 7·0± " ""22 · 1± ""23 1·0±
0·039 0·44 3·6 1·6 17 ·2
Experimentalliller 2
Control
"" 1·9± 0·019 27·6± 0·31 3 1· 7± 2·4 " 16·9± 1·5 148·4± 11 ·3
2· 0 ± 0 ·035 28·3± 0 ·21 25·8± 2·4 12·8± 1·2 12 1·8±1O·1
6 embryos
8 embryos
+3 embryos
Whole brain Spinal cord Heart Liver Gut Kid ney/Gonads Eyes
65·6±7·2 43·6±3 ·4 "44·4±1 ·4 "240 ·6±7 ·3 26 ·8 ± 2·3 69·1±2 ·8 5·8±0 · 72
71·5± 47 · 1± 50·9± 278·4± 26 ·9 ± 72 ·6± 7·0±
6 ·2 4·1 2·6 12·5 2·2 4 ·5 0·53
t
Re ma ining embryos u sed for chemical ana lysis. Significant diffe rence from mean of untreated control litter. "P=0·05; *** P =O· OOI.
TABLE III OCHRATOXIN A R ES ID UES IN MATERNAL AN D FOETAL TISSUES ' (I'g/g fresh wI: mean of 3 determinations)
Muscle Liver Kidney Placenta Whole foetus
Experimentalliller 1
Experimentalliller 2
Control
0·15 0·30 0 ·43 0·04 n .d.
0·13 0·34 0 ·70 0·06 n.d.
n.d. n .d. n.d . n.d . n.d .
n.d. = none de tected. * No ochratoxin B could be detected .
a lso present in the placentae of gilts receiving the toxic rations but none could be detected in foetal tissues (Table III). At the conclusion of the feeding period, leucine amino-peptidase activity was higher in urine samples from experimental gilts (both 12 m U/mg creatinine) than in the control (5 mU/mg creatinine) but this may not be significant in view of considerable day-to-day variation (5-33 mU/mg creatinine) and differences in pre-feedi ng a nd control values for enzyme activity. DISC USSI ON
In the present study no embryonic abnormalities occurred in gilts following oral administration of ochratoxin during early pregnancy. Exposure to toxin corresponded
416
BRITISH VETERINARY JOURNAL, 133,4
to a time of rapid morphological development of the porcine embryo (Patten, 1948 ) so that any teratogenic effect might have been expected to occur during this period. Since no dead or abnormal embryos were found , the reduced rates of embryonic survival probably relate to embryonic losses occurring prior to feeding the experimental diet. Although there were significant differences between embryos from experimental and control gilts in relation to body weights and placental parameters this may have been due to small but important differences in the stage of gestation and litter size. Thus the control litter was both older and contained more embryos than either of the litters from the treated sows. Differences in heart and liver weights can probably be attributed to differences in body weight. There were no clinical signs of ochratoxin poisoning (Krogh et ai., 1974a) which is consistent with the lack of gross or microscopical lesions in the sows. Urinary leucine amino-peptidase activity is considered to be a useful diagnostic guide in suspected cases of porcine ochratoxicosis (Hyldgaard-Jensen, 1973) but in the present experiment there was no indication of kidney damage in the gilts receiving the toxic ration. This agrees with the findings of Szczech, Carlton & Tuite (1973 ) who were unable to detect a significant increase in urinary leucine amino-peptidase concentration in SPF swine fed 1 mg/kg ochratoxin A for six days . Although present in maternal kidney, liver, muscle and placenta ochratoxin A was not found in foetal tissues, a result which indicates that, as in the case of the sheep (Munro et ai., 1973), ochratoxin A does not readily cross placental membranes. O chratoxin B was not detected in maternal or foetal tissues. Up to 1% of pigs slaughtered in Denmark may exhibit lesions of ochratoxin induced nephropathy but as far as we are aware the presence of ochratoxin A in fodder has never been associated with reproductive failure in pigs in that country. Although a sufficiently high dose of the toxin may cause foetal resorption in the rat (Still et ai. , 1971) and embryonic mortality in mice (Hayes & Hood, 1973), present experimental evidence suggests that a similar effect is not readily reproduced in the pig. A daily intake of about 40 mg ochratoxin A in this experiment is approximately equivalent to feed ing a diet containing 17 parts/l0 6 of toxin, which is over four times higher than the highest level used by Krogh et ai. (I974a) to induce experimental porcine nephropathy and of the same order as levels detected in cereals (Hesseltine, 1974). For these reasons it seems that o chratoxin A cannot seriously be regarded as a potential cause of reproductive failure in the sow. ACKNOWLEDGEMENTS
We are indebted to Miss S. M. Eades for setting up mycological cultures, Mr P. M. D. Foster for preparing the crude toxic extract, Mrs B. Small for ochratoxin analyses, Mr J . Bailey for dissection and Miss C. N. Hebert for statistical analysis. REFERENC ES
CHU, F. S., NOH, I. & CHANG, C. C. ( 1972). Life Sci. 11,503. CIEGLER, A., FENNELL, D. J., MINTZLAFF, H .-J. & L EISTNE R, L . ( 1972). Naturwissenschaflen 59,365. HAYES, A . W . & HOOD, R . D. (1973). Toxic. appl. Pharmac. 25, 457. H ESSELTINE, C . W . ( 1972). Biotechnol. & Bioeng. 14,517.
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HESSELTINE, C . W . (]974). Mycopath. Mycol. appl. 53, 141. H ESSELTINE, C . W ., VANDEGRAFT, E . E. , FENNELL, D. l. , SMITH, M . L. &SH01WELL, O . L. (]972). Mycologia, 64, 539. HYLDGAA RD-J E SE ,j. (]973 ). In Aaarsberetning, p. 11 5. Copenhagen: Inst. Sterilite tsforskning. KING, E . j. (]95 I). Micro-analysis in Medical Biochemistry, 2 nd ed. London : Churchill. KROGH , P., HALO, B. & PED ERSEN , E.j. (]973). Acta path. microbiol. scand. Sect. B. III, 689. KROGH, P ., AX ELSEN, N . H ., ELLING, F., GYRo-HANSEN, N ., HALO, B., HVLD GAARD· JENSEN, j. , LARSEN, A . E., MADSEN, A., MORTENSEN, H . P., MO LLER, T., P ETERSEN, O. K. , RAVNSKOY, U., ROSTGAA RD, M . & AALUND, O . (]9 74a). Acta path. microbiol. scand. Sect. A , Supp. No. 246 . KROGH, P., H ALO, B., ENGLUND, P., R UTQU IST, L. & SWAHN, O . (]9 74b ). Acta path. microbiol. scand. Sect. B . 82, 30 1. LAI, M ., SEMENIUK, G . & HESSELTIN E, C . W . (1968 ). Phytopathology, 58,1056. LILLIE, R. D. (]965 ). Histopathologic Technic and Practical Histochemistry, 3rd edn. New York: McGraw Hill. MUNRO, 1. C ., SCOTT, P. M ., MOODI E, C . A. & WILLES, R . F . ( 197 3). ). Am. vet. med. Ass. 163, 1269. NESHEIM, S. (]969) .j. Ass. off. analyt. Chem. 52, 975 . PATTEN, B. M. ( 1948 ). Embryology of the Pig, 3 rd e dn. Philadelphia: The Blakiston Co. RAPER, K. B. & FENNELL, D. 1. ( 1965). The genus Aspergillus. Bal ti more: Williams & Wilkins Co. RAPER, K. B. & THOM, C . (] 949 ). A Manual of the Penicillia, Baltimore: Williams & Wilkins Co . SCOTT, P. M ., KENNEDY, B. &VANWALBEEK, W. (1971).]. Ass. off. analyt. Chem. 54,1445. SCOTT, P. M ., VANWALBEEK, W. , HARWI G, j. & FENNELL, D . 1. (1970). Can.j. Pl. Sci. 50, 583. SCOTT, P. M. , V AN W ALBEEK, W ., KENNEDY, B. &ANYETI, D . (]972 ). Agric. Fd Chem. 20,1103 . SH01WELL, O . L., H ESSELTI NE, C. W. & GO ULDEN, M . L. (] 969 ). Appl. Microbiol. 17,765 . SH01WELL, O . L. & BOTHAST, R. j. (]973 ). Cited by H esseltine, C. W . (]9 74 ). Mycopath. Mycol. appl. 53, 141. SHREEVE, B. j. , PATTERSON , D. S. P . & ROB ERTS, B. A. ( 1975). Vet . Rec. 97,275 . STEYN, P. S. (]97 I). In Microbiol Toxins, Vol. VI, p. 179 , ed. Ciegler, A ., Kadis, S. & Ajl , S. j. N ew York: Academic Press . ST ILL, P . E., MACKLI N, A. W. , RI BELIN, W. E. & SMALL EY, E. B. (197 1). Nature, Lond. 234, 563 . SZCZECH, G. M. , CARLTON, W. W . & T UITE, j. (19 73). Toxic. appl. Pharmac. 25,45 6. VAN D ER M ERWE, K. j. , STEYN, P. S. , FOUR IE, L., SCOTT, D E B. &THERON, j.j. (1965). Nature, Lond. 205,111 2. VAN W ALBEEK, W ., SCOTT, P. M ., HARWI G, j. & LAWRENCE, J. W . ( 1969 ). Can.}. Microbiol. 15, 1281. WRATHALL, A. E. (]9 7 I). Vet. Rec. 89,61.
(Accepted for publication 23 November 1976)
