- Email: [email protected]
The vitamin K requirements of wild brown rats (Rattus norvegicus) resistant to warfarin
Comp. Biochem. Physiol. Vol. 66A. pp. 83 to 87 © Pergamon Press Ltd 1980. Printed in Great Britain
0300-9629/80/0501-0083502.00/0
THE VITAMIN K REQUIREMENTS OF WILD BROWN RATS (RATTUS NORVEGICUS) RESISTANT TO WARFARIN G. G. PARTRIDGE Department of Genetics. University of Liverpool, Liverpool L69 3BX* (Received 19 July 1979) Abstract--l. Resistance to the anticoagulant rodenticide warfarin and an increased vitamin K requirement appear to be pleiotropic effects of the same allele R w 2 (Rw t being the normal warfarin susceptible allele). The vitamin K requirements of resistant ( R w 2 R w 2, R w t R w 2) and susceptible ( R w l R w t) rats were
determined. 2. Estimated requirements were (/~g vitamin K t/100 g body weight): R w 2 R w 2 males 5-7 #g; R w Z R w 2 females 3-5 #g; R w t R w 2 males and females 1 #g; R w t R w I males 0.5 #g; R w t R w ! females <0.5 pg. 3. Vitamin K2 produced by the gut microtlora and made available by coprophagy can satisfy the requirements of R w t R w z and R w l Rw t rats. 4. The fitness of the R w Z R w 2 individual will be dependent upon the dietary environment.
this trait maps at approximately the same site as the warfarin resistance allele (Greaves & Ayres, 1969). It seems likely therefore that resistance to warfarin and an increased vitamin K requirement are pleiotropic effects of the same gene. The vast majority of work on vitamin K requirement and warfarin resistance has been carried out on laboratory strains of rat (e.g. Hermodson et al., 1969; Greaves & Ayres, 1973; Martin, 1973). We required basic data on the vitamin K requirement of wild rats to supplement our studies on the relative fitnesses of the three possible genotypes in a population of rats containing resistant individuals (Bishop, Hartley & Partridge, 1977; Partridge, 1978). To extrapolate requirements from the domesticated rat studies was unacceptable because differences between laboratory strains themselves can be marked (Mellette & Leone, 1960). Vitamin K2 produced by the gut microflora is made available to the rat by coprophagy. The importance of coprophagy in providing adequate amounts of dietary vitamin K was also investigated.
INTRODUCTION
Brown rats ( R a t t u s norvegicus) resistant to the anticoagulant rodenticide warfarin were first detected in populations from Scotland and mid Wales between 1958 and 1960. Since then resistant populations have been found elsewhere in Europe and in the U.S.A. (Drummond, 1970; Jackson & Kaukeinen, 1972). The genetic basis for warfarin resistance in the Welsh and Scottish populations has been determined (Greaves & Ayres, 1967; Greaves & Ayres, 1976). Resistance is a dominant character controlled by a single autosomal gene designated R w 2 ( R w 1 being the normal susceptible allele). The Scottish and Welsh resistance phenotypes differ in that the former shows incomplete penetrance in the heterozygote, while the latter is a completely dominant trait. These differences suggest that the resistance genes controlling the trait are allelic in the Scottish and Welsh populations (Greaves & Ayres, 1976). The incorporation of the Welsh resistance allele Rw 2 into laboratory strains of rat (Pool, O'Reilly, Schneiderman & Alexander, 1968) enabled studies into the mechanism of resistance at the biochemical level. Uptake and excretion of warfarin were similar in resistant and susceptible rats but the vitamin K requirements of the three possible genotypes were markedly different. When compared with warfarin susceptible animals, homozygous and heterozygous resistant rats were found to have a twenty-fold and two-fold increase in vitamin K requirement respectively (Hermodson, Suttie & Link, 1969). Vitamin K is essential for the synthesis of at least four blood clotting factors (Biggs & MacFarlane, 1962). A deficiency of dietary vitamin K therefore results in hemorrhaging. A recessive hemorrhagic trait has been described in a laboratory strain of rats (Dunning & Curtis, 1939) and the allele controlling
MATERIALS A N D M E T H O D S
Animals
The wild rats used in this study were live captured from the population in mid Wales described by Bishop & Hartley {1976). Animals were phenotyped by injecting a mixed solution of vitamin K oxide and warfarin (Bishop et al., 1977; Greaves, Redfern, Ayres & Gill, 1977). Genotyping of resistant animals was by the method of Greaves et al. (1977). This method had been tested independently at Liverpool and was found to give adequate separation of R w l R w 2 and R w 2 R w 2 genotypes (Partridge, 1978). Diets
All rats were maintained on either a commercial cubed diet (41B, Dixon's, Ware, Herts.) or a vitamin K deficient semi-synthetic diet. The vitamin K deficient diet used in these studies was recommended by Martin A. D. (pers.
* Present address: Department of Applied Nutrition, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB 83
84
G . G . PARTRIDGE Table 1. The composition of the vitamin K deficient diet used during experiments 1 and 2 Oo in diet Sucrose Casein Vitamin mixt Mineral mix* Linoleic acid Mineral oil
66.3 24.0 0,4 4.3 1.0 4.0 100.0
"l'Calciferol 0.001 g, Vitamin Bl2 0.002g, Biotin 0.012g, Pyridoxine 0.15 g, Folic acid 0.20 g, Thiamine 0.30 g, Riboflavin 0.30 g, PABA 0.76 g, Nicotinic acid 1.0g, Pantothenate 1.2g, Inositol 10g, Choline 23g, Vitamin A 0.075 g, Vitamin E 3.0 g. * KI 0.02g, CuSO4.5H20 0.30g, ZnCI2 0.80 g, FeSO4.7H20 2.24 g, M n S O 4 ' 4 H 2 0 3.04g, NaCI 19.10g, KzHPO4 60.3g, MgSO,L" 7H20 61.7 g, CaCO3 66.5 g, CaHPQ4 216 g. comm. 1975) and it was a modification of the diet described by Greaves & Ayres (1973). Its composition is shown in Table 1.
Experiment 1: Determination o]'the daily ritamin K t requirements of wtoJdrin resistant and susceptible rats In order to prevent coprophagy rats were housed individually in specially designed tubular cages similar to those described by Metta, Nash & Johnson (1961). These cages were square in cross section (6 cm.) and made from 19 mm. square Weldmesh. Food was provided in a metal cup placed at the entrance to the cage. A hinged door allowed a cloth bag to be placed over the end of the cage to facilitate routine handling of the wild rats. Heterozygous resistant [Rw~Rw'-), homozygous resistant [Rw2Rw 2) and susceptible (RwlRw ~) rats of both sexes were blood-sampled (day 0) prior to receiving an injection {i.p.) of vitamin K~ CKonakion", Roche Products Ltd.)at the required concentration in sterile saline. The blood was obtained while the rats were under light ether anaesthesia by snipping the tail tip and collecting into tubes containing sodium citrate anticoagulant. The vitamin K dependent blood clotting factors were assayed using the citrated whole blood and Diagen 2-7-10 reagent (Diagnostic Reagents Ltd., Thame, Oxon.). The clotting time was expressed as log percentage clotting activity (log PCA) after reference to standard curves prepared beforehand {Bishop et al., 1977). Following injection of vitamin K~ the rats were transferred to the coprophagy preventing cages and supplied with vitamin K deficient diet and water ad lib. The animals were injected daily for the next 3 days with the same amount of vitamin K~ as they received on day 0. They were blood sampled again 24hr after their last injection {day 4). The day 0 and day 4 log PCA values were then compared. Seven concentrations of vitamin K~ were used and controls received injections of saline alone. Experiment 2: The effect oJ" coprophagy on vitamin K requirement in heterozyyous resistant rats [RwlRw 2) Four groups, each of three adult male Rw ~Rw z rats were used. Each group of animals had been housed together since immaturity and consequently had established dominance relationships at an early age. Aggression within each group was therefore minimal. Two groups of rats (Groups I and 2) were transferred to two standard cages 132 × 18 × 45cm.) fitted with 19mm
square Weldmesh bottoms to minimise coprophagy. The remaining two groups (Groups 3 and 4) were housed in standard cages modified to maximise coprophagy. Their normal mesh bottoms were covered by zinc gauze. Urine build-up was prevented by laying several thicknesses of absorbant paper towels between the gauze and the cage bottom. Vitamin K deficient diet and water were provided ad lib to the four groups of animals. Blood samples were taken for the determination of log PCA before allocation (day 0), day 4 and day 8 by the method previously described.
Experiment 3: Estimation of the degree of coprophagy induloed by warfarin resistant (RwZRw 2, RwlRw 2) and susceptible (Rw I Rw 1) rats Adult male rats of known genotype were used. Tubular coprophagy preventing cages were used, as well as modified tubular cages which allowed access to faeces. The laboratory diet fed (41 B) had been analysed chemically and no detectable phylloquinone had been found (Partridge. 1978). The natural components of diet 41B had a theoretical vitamin KI content of 0.84 mg/kg (data kindly supplied by Dixon's Ltd.).It has been shown, however, that the pelleting process and subsequent storage can reduce the expected quantity of phylloquinone in a diet (Charles & Huston, 1972). Batches of six animals of the same genotype were taken from stocks. Three rats were randomly allocated to individual coprophagy preventing cages and the remaining three animals were allowed access to their faeces in the modified cages. Food consumption was measured over a 24 hr period ( ¢ 4 p.m.) and the unconsumed faeces were collected from all animals. The faeces were dried overnight (100°C) and after cooling were weighed to the nearest rag. A significant relationship was found between the dry weight of faeces produced and the amount of food consumed over the 24 hr period for the rats prevented from coprophaging. The line of best fit was described by the equation y = 0.28 + 0.26x [P < 0.001), where y = dry faecal weight produced and x = food consumption (g). Using this equation it was possible to estimate the quantity of faeces which an animal in a faecal access cage should have produced from a known food consumption. An estimate of percentage coprophagy could then be derived from: wt of expected faeces - wt of observed faeces wt of expected faeces
x
100°;,
RESULTS
Experiment I Table 2 shows the mean day 0 and day 4 log PCA values for the six sex/genotypes. Both male and female homozygous resistant rats (RwZRw 2) had a higher daily requirement for vitamin K than susceptible ( R w l R w 1) and heterozygous resistant ( R w t R w ~) animals. This concurs with other studies using laboratory strains (Hermodson et al., 1969; Greaves & Ayres, 1973). In addition, homozygous resistant males required more vitamin K than their female counterparts to attain "'normal" clotting levels ("normal" log PCA values being usually > 4.00). The greater vitamin K requirement of these males compared to other genotypes was reflected in their lower day 0 log PCA values. It was found necessary to maintain homozygous resistant animals on vitamin Ka supplemented water ( 1 0 m g m e n a d i o n e sodium bisulphite/l) when not required for experiments, to prevent hemorrhagic deaths. Heterozygous resistant ( R w t R w z) and susceptible
Vitamin K requirements of rats
85
Table 2. Experiment 1: Mean log PCA values (+SE) before (Day 0) and after (Day 4) daily intraperitoneal injections of vitamin K~ at various concentrations. Five rats per group, animals fed on a vitamin K deficient diet with coprophagy prevented Concentration Rw2Rw 2 c~ (/tgKt/100g) Day0 Day4 0
--
0.5
--
1.0
--
2.0
--
3.0
--
5.0
2.79 -+0.27 2,96 -+0.35 2.78 +0.31
7.0 10.0
RwlRw I 3 Day0 D a y 4
RwtRw 2 c~ Day0 Day4
--
4.30 +0.19 -4.18 +0.09 -3.79 -+0.15 -4.16 -+0.12 -3.75 _+0.09 3.34 . . +0.13 4.51 -+0.07 4.04 -+0.12 .
2.09 +0.53 3.57 +0,25 3,76 4-0.10 4.12 -+0.16 4.11 _+0.14 . .
.
.
.
.
.
.
4.06 4-0.10 4.17 +0.09 4.02 -+0.10 4.28 +0.10 4.19 -+0.11 . .
.
2.76 --+0.36 4.05 --_+0.20 4.28 ---+0.14 4.47 ---+0.09 4.31 "4.01 3.10 ___0.10 ___0.25 -+0.10 3.42 4.27 +0.36 -+0.11
.
.
Rw2Rw z 9 Day0 Day4
.
.
.
.
.
.
.
.
RwlRw 2 ~ Day0 Day4
RwtRw 1 Day0 Day4
4.33 +0.11 4.38 -+0.16 4.29 +0.13 4.11 -+0.14 4.39 -+0.13 . .
4.14 +0.08 4.00 -+0.17 4.05 -+0.09 4.12 -+0.13 4.41 -+0.09 . .
1.89 +0.15 3.59 +0.15 4.42 4-0.10 4.29 x0.20 4.56 -+0.05 . . .
3.93 +0.18 4.40 +0.10 4.51 +0.05 4.12 -+0,11 4,55 +0.03
.
.
.
* Only four rats in this group. ( R w l R w 1) rats were dosed with vitamin Kt in the range 0-3 yg/100g body weight. Differences in response of the two genotypes are shown in Fig. 1 expressed as the difference (A) between log PCA on day 0 and day 4. The results indicate that both male and female heterozygotes required a daily quantity of 1 #g/100 g to maintain 'normal' clotting levels. Susceptible males, however, required between 0.5 and 1/~g/100g and susceptible females <0.5/tg/100 g. Experiment 2
Table 3 shows the log PCA values obtained over the 8 day period for male heterozygous resistant rats maintained throughout the trial period with coprophagy prevented (Groups 1 and 2), or coprophagy allowed (Groups 3 and 4). The three animals in G r o u p 2 died on day 5, all showing signs of internal hemorrhage. An analysis of variance over the first 4 days revealed highly significant effects between groups and treatments as well as a significant interaction
o.o
{
0.5
~
~ ~JgK,1100g
between the two. The significant interaction component possibly reflects incomplete prevention of coprophagy when rats are housed in groups on a mesh floor. Single tubular cages as used in Experiment 1 may have reduced the variability within treatments. By day 8 the remaining animals prevented from coprophaging (Group 1) had very low clotting levels. Rats with access to their faeces showed a variable response but in general they maintained similar levels to those recorded on day 0. These results indicate that in the absence of dietary vitamin K the heterozygous resistant male rat can obtain adequate amounts by coprophagy. Experiment 3
Table 4 gives the estimates of percentage coprophagy obtained for the three genotypes. The mean percentage cophrophagy value for homozygous resistant animals was significantly lower than that of heterozygotes (P < 0.01). The variability of response
o.o
I;o
z~:o
3;o ,ug K,1100g
0.5
I I.o + 1.5
i.o MALES
1.5
o resistont 2.0
2.5
I
+
•
susceptible
FEMALES ~ resistont
2.0
• susceptible
23
Fig. 1. The response of heterozygous resistant and susceptible wild rats of each sex to daily injections of vitamin KI for 4 days, where A = log PCA day 0 - l o g PCA day 4. Each point represents the mean of five animals _+SE.
G. G. PARTRIDGE
86
rats in this study are much lower than those described for the warfarin resistant Wistar strain used by Greaves & Ayres (1973). These workers, using a similar experimental design, estimated the requirement of both male R w l R w 1 and R w l R w 2 genotypes to be Coprophagy minimised (standard cages fitted with 19 mm 6 pg/lO0 g body weight and that homozygous resistsquare mesh floors) ant males (RwERw 2) required approximately 80#g/ Mean log PCA I+SE) 100g. The corresponding estimates in the present Group No. Day 0 Day 4 Day 8 study were: 0.5 #g; 1 #g; and 5-7 pg vitamin K1/100 g body weight respectively, although the latter estimates 1 4.61 + 0.00 3.68 +_0.52 2.09+ 0.92 2* 4.20 _ 0.36 1.24 + 0.11 .... should be considered approximate since only a *All animals died day 5, hemorrhaging. limited range of doses between 0 and 1/lg was tested. Coprophaoy maximised (standard cages fitted with solid Mellette & Leone (1960) noted that different strains floors) of laboratory rat have widely varying tolerances to 3 4.16 + 0.0l 4.19 + 0,03 4.27 + 0.10 low levels of dietary vitamin K. The large differences 4 4.26 + 0.25 3.63 + 0.43 3.58 + 0.76 found between this study and that of Greaves & Analysis of variance testing for differences between groups Ayres (1973) could be due to the presence of different and treatments, day 0 minus day 4 values. modifier genes in the two strains of rat used. Whether Groups p < 0.001 similar variability is present naturally in other wild Treatments p < 0.001 rat populations has yet to be established. Of particuInteraction p < 0.05 lar interest would be the vitamin K requirements of rats in populations from areas in which warfarin had in R w 2 R w 2 animals was particularly large, three not been used extensively as a rodenticide i.e. where animals giving negative values and two having values there had been no selection for genes modifying the effects of the Rw2 allele. The major gene controlling > 20%. warfarin resistance in the house mouse ( M u s musculus) is known to be influenced significantly by such DISCUSSION modifying genes (Wallace & MacSwinney, 1976; Hermodson et al. (1969) estimated that the vitamin MacSwinney & Wallace, 1978). K requirement of heterozygous and homozygous reThe higher vitamin K requirement of the male rat sistant rats was twice and twenty times respectively compared with its female counterpart has been prethat of a warfarin susceptible animal. Greaves & viously described in laboratory strains (Johnson, Ayres (1973) found that heterozygous resistant and Mameesh, Metta & Rama Rao, 1960; Bell & Theiss, susceptible rats had a similar requirement, with 1972) although this is the first report of a similar sex homozygous resistant rats having a requirement difference in the wild rat. The sex difference is under thirteen times higher. Martin (1973) working with the the control of the hormonal system; estradiol relievsame strain of rats estimated that homozygous resist- ing and testosterone enhancing the hypothrombinaeant rats required approximately thirty times more mic state (Mellette & Leone, 1960). Estradiol itself does not have vitamin K-like activity indicating that vitamin K than susceptibles, This variation in relative requirements is probably the hormone must be operating indirectly (Matsaccounted for by the different methods of estimation chiner & Bell, 1973). The mean percentage coprophagy values obtained used. Hermodson et al. (1969) and Martin (1973) used a method of curative dosing of vitamin K depleted for the R w l R w 2 and R w l R w 1 genotypes (26 and 19% animals, while Greaves & Ayres (1973) measured the respectively) are similar to those reported by Johnson response to daily doses of the vitamin over a 4 day et al. (1960) who used a variety of diets They found period, as in this study. Preliminary observations values were frequently between 20 and 30~o, although indicated that wild susceptible female rats showed no the protein source in the diet appeared to influence hypothrombinaemic response, even after feeding on a the degree of coprophagy. Mameesh, Metta, Rama vitamin K free diet for three weeks (Partridge, 1978). Rao & Johnson (1962) estimated that the amount of In view of this observation it was not possible to deplete vitamin K active substance produced in the intestines the blood clotting levels equally in the different was 8-1! mg of vitamin K equivalents per kg of dried caecal contents. Consumption of dry faeces in the groups prior to curative dosing with vitamin K. The vitamin K requirement estimates for the wild present study was frequently between 1.0 and 1.6g Table 3. Experiment 2: The effect of coprophagy on blood clotting levels (log PCA) of male heterozygous resistant rats (Rw~Rw 2) fed on a vitamin K deficient diet for 8 days; three rats per group
Table 4. Experiment 3: Percentage coprophagy indulged by the three genotypes feeding on a standard laboratory diet (41 B) with access to their faeces
Genetype RwZRw 2
Significance of difference between means
Mean body weight Mean percentage +SE coprophagy + SE (n) 338 + 59
6.53 + 4.74(8) ]
] p < 0.01
Rw1Rw 2
339 + 89
26.33 + 4.47 (9)
0.01 > p > 0.05 n.s.
RwIRw I
344 _ 71
18.70 4- 4.04 (111
Vitamin K requirements of rats
87
DUNNING W. F. & CURTIS M. R. (1939) Linkage in rats between factors determining a pathological condition and a coat colour. Genetics 24, 70. GREAVESJ. H. & AYRES P. (1967) Resistance to warfarin in rats. Nat,re 215, 877-878. GREAVES J. H. & AYRES P. (1969) Linkages between genes for coat colour and resistance to warfarin in Rattus norvegicus. Nature 224, 284-285. GREAVES J. H. & AYRES P. (1973) Warfarin resistance and vitamin K requirement in the rat. Lab. Anita. 7, 141-148. GREAVES J. H. & AYRES P. (1976) Inheritance of Scottishtype resistance to warfarin in the Norway rat. Genet. Res., Camb. 28, 231-239. GREAVES J. H., REDFERN R., AYRES P. & GILL J. E. (1977) Warfarin resistance: a balanced polymorphism in the Norway rat. Genet. Res., Camb. 30, 257-263. HERMODSON M. A., SUTTtE J. W. & LINK K. P. (1969) Warfarin metabolism and vitamin K requirement in the warfarin resistant rat. Am. J. Physiol. 217, 1316-1319. JACKSON W. B. & KAUKEINEN n. E. (1972) Resistance of wild Norway rats in North Carolina to warfarin rodenticide. Science N.Y. 176, 1343-I 344. JOHNSON B. C., MAMEESHM. S., METTA V. C. & RAMA RAO P. B. (1960) Vitamin K nutrition and irradiation sterilization. Fedn. Proc. Fedn. Am. Soc. exp. Biol. 19, 1038-1044. MACSWINNEY F. J. & WALLACE M. E. (1978) Genetics of warfarin' resistance in house mice of three separate localities, d. Hyg. 80, 69-76. Acknowledgements--I would like to thank Dr. N. T. Davies, Dr. J. A. Bishop and Dr. J. H. Greaves for critical MAMEESH M. S., MErTA V. C., RAMA RAO P. B. & JOHNSON B. C. (1962) On the cause of vitamin K deficiency in reading of the manuscript. This work was completed while male rats fed irradiated beef and the production of I was in receipt of an S.R.C. postgraduate studentship. vitamin K deficiency on an amino acid synthetic diet. J. Nutr. 77, 165. MARTIN A. D, (1973) Vitamin K requirement and antiREFERENCES coagulant response in the warfarin resistant rat. Biochem. Soc. Trans. i, 1206-1208. BELL R. G. & THElSS J. W. (1972) Effects of estrogens on vitamin K and prothrombin. Fedn. Proc. Fedn. Am. Soc. MATSCHINER J. T. & BELL R. G. (1973) Effect of sex and sex hormones on plasma proth.rombin and vitamin K defiexp. Biol. 31,714 (ABS). ciency. Proc. Soc. exp. Biol. Med. 144, 316-320. BIGGS R. P. & MACFARLANE R. G. (1962) Human Blood MELLETTE S. J. & LEONE L. A. (1960) Influence of age, sex, Coagulation and its Disorders. Oxford, Blackwell Scienstrain of rat and fat soluble vitamins on hemorrhagic tific Publications. syndromes in rats fed irradiated beef. Fedn. Proc. Fedn. BISHOP J. A. & HARTLEY D. J. (1976) The size and age Am, Soc. exp. Biol. 19, 1045-1049. structure of rural populations of Rattus norveaicus conMETTA V. C., NASH L. & JOHNSON B. C. (1961) A tubular taining individuals resistant to the anticoagulant poison coprophagy-preventing cage for the rat. J. Nutr. 74, warfarin. J. anita. Ecol. 45, 623-646. 473--476. BISHOP J. A., HARTLEY D. J. & PARTRIDGEG. G. (1977) The population dynamics of genetically determined resist- PAR'rRID~E G. G. (1978) The pleiotropic effects of the gene conferring warfarin resistance in the brown rat (Rattus ance to warfarin in Rattus norveoicus from mid Wales. norvegicus). Ph.D. Thesis, University of Liverpool. Heredity 39, 389-398. CHARLES O. W. & HUSTON T, M. (1972) The biological POOL J. G., O'REILLY R. A., SCHNEIDERMANL. J. & ALEXANDER M. (1968) Warfarin resistance in the rat. Am. J. activity of vitamin K material following storage and pelPhysiol. 215, 627-631. leting. Pouhry Sci. 51, 1421-1427. DRUMMOND D. C. (1970) Variation in rodent populations WALLACE M. E. & MACSWINNEY F. J. (1976) A major gene controlling warfarin resistance in the house mouse. J. in response to control measures. Syrup. Zool. Soc. Lurid. Hyg. 76, 173-181. 26, 351-367.
(dry weight). Using the above estimates m a n y animals could therefore obtain in excess of 8/~g of vitamin K equivalents from coprophagy. Expressed in relation to mature body weight (say 350 g) this corresponds to an intake of approximately 2/tg/100 g. Experiment l showed that this quantity of vitamin K is sufficient for b o t h R w t R w I and Rw~Rw 2 genotypes, but it may be inadequate for RwZRw 2 animals. Partridge (1978) calculated the relative fitnesses of the three possible genotypes in the Welsh farm building population (Rwt R w I 1.00; Rwl R w 2 0.77; Rw2Rw 2 0.46). These estimates were based on the observed decline in the frequency of resistance in the farm buildings over an 18 m o n t h period when warfarin was withheld. Some rats on the study farm had access to a b u n d a n t cereal grain which was a poor source of vitamin K (Partridge, 1978). In contrast, other animals were feeding in the poultry sheds on a vitamin supplemented laying hen diet containing approximately 0.23/~g of vitamin K3 equivalents/g of diet (data kindly supplied by Seemeel Ltd., L o n d o n E.15). Selection pressure on resistant rats could therefore vary markedly depending on the food sources available to resistant populations.
0300-9629/80/0501-0083502.00/0
THE VITAMIN K REQUIREMENTS OF WILD BROWN RATS (RATTUS NORVEGICUS) RESISTANT TO WARFARIN G. G. PARTRIDGE Department of Genetics. University of Liverpool, Liverpool L69 3BX* (Received 19 July 1979) Abstract--l. Resistance to the anticoagulant rodenticide warfarin and an increased vitamin K requirement appear to be pleiotropic effects of the same allele R w 2 (Rw t being the normal warfarin susceptible allele). The vitamin K requirements of resistant ( R w 2 R w 2, R w t R w 2) and susceptible ( R w l R w t) rats were
determined. 2. Estimated requirements were (/~g vitamin K t/100 g body weight): R w 2 R w 2 males 5-7 #g; R w Z R w 2 females 3-5 #g; R w t R w 2 males and females 1 #g; R w t R w I males 0.5 #g; R w t R w ! females <0.5 pg. 3. Vitamin K2 produced by the gut microtlora and made available by coprophagy can satisfy the requirements of R w t R w z and R w l Rw t rats. 4. The fitness of the R w Z R w 2 individual will be dependent upon the dietary environment.
this trait maps at approximately the same site as the warfarin resistance allele (Greaves & Ayres, 1969). It seems likely therefore that resistance to warfarin and an increased vitamin K requirement are pleiotropic effects of the same gene. The vast majority of work on vitamin K requirement and warfarin resistance has been carried out on laboratory strains of rat (e.g. Hermodson et al., 1969; Greaves & Ayres, 1973; Martin, 1973). We required basic data on the vitamin K requirement of wild rats to supplement our studies on the relative fitnesses of the three possible genotypes in a population of rats containing resistant individuals (Bishop, Hartley & Partridge, 1977; Partridge, 1978). To extrapolate requirements from the domesticated rat studies was unacceptable because differences between laboratory strains themselves can be marked (Mellette & Leone, 1960). Vitamin K2 produced by the gut microflora is made available to the rat by coprophagy. The importance of coprophagy in providing adequate amounts of dietary vitamin K was also investigated.
INTRODUCTION
Brown rats ( R a t t u s norvegicus) resistant to the anticoagulant rodenticide warfarin were first detected in populations from Scotland and mid Wales between 1958 and 1960. Since then resistant populations have been found elsewhere in Europe and in the U.S.A. (Drummond, 1970; Jackson & Kaukeinen, 1972). The genetic basis for warfarin resistance in the Welsh and Scottish populations has been determined (Greaves & Ayres, 1967; Greaves & Ayres, 1976). Resistance is a dominant character controlled by a single autosomal gene designated R w 2 ( R w 1 being the normal susceptible allele). The Scottish and Welsh resistance phenotypes differ in that the former shows incomplete penetrance in the heterozygote, while the latter is a completely dominant trait. These differences suggest that the resistance genes controlling the trait are allelic in the Scottish and Welsh populations (Greaves & Ayres, 1976). The incorporation of the Welsh resistance allele Rw 2 into laboratory strains of rat (Pool, O'Reilly, Schneiderman & Alexander, 1968) enabled studies into the mechanism of resistance at the biochemical level. Uptake and excretion of warfarin were similar in resistant and susceptible rats but the vitamin K requirements of the three possible genotypes were markedly different. When compared with warfarin susceptible animals, homozygous and heterozygous resistant rats were found to have a twenty-fold and two-fold increase in vitamin K requirement respectively (Hermodson, Suttie & Link, 1969). Vitamin K is essential for the synthesis of at least four blood clotting factors (Biggs & MacFarlane, 1962). A deficiency of dietary vitamin K therefore results in hemorrhaging. A recessive hemorrhagic trait has been described in a laboratory strain of rats (Dunning & Curtis, 1939) and the allele controlling
MATERIALS A N D M E T H O D S
Animals
The wild rats used in this study were live captured from the population in mid Wales described by Bishop & Hartley {1976). Animals were phenotyped by injecting a mixed solution of vitamin K oxide and warfarin (Bishop et al., 1977; Greaves, Redfern, Ayres & Gill, 1977). Genotyping of resistant animals was by the method of Greaves et al. (1977). This method had been tested independently at Liverpool and was found to give adequate separation of R w l R w 2 and R w 2 R w 2 genotypes (Partridge, 1978). Diets
All rats were maintained on either a commercial cubed diet (41B, Dixon's, Ware, Herts.) or a vitamin K deficient semi-synthetic diet. The vitamin K deficient diet used in these studies was recommended by Martin A. D. (pers.
* Present address: Department of Applied Nutrition, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB 83
84
G . G . PARTRIDGE Table 1. The composition of the vitamin K deficient diet used during experiments 1 and 2 Oo in diet Sucrose Casein Vitamin mixt Mineral mix* Linoleic acid Mineral oil
66.3 24.0 0,4 4.3 1.0 4.0 100.0
"l'Calciferol 0.001 g, Vitamin Bl2 0.002g, Biotin 0.012g, Pyridoxine 0.15 g, Folic acid 0.20 g, Thiamine 0.30 g, Riboflavin 0.30 g, PABA 0.76 g, Nicotinic acid 1.0g, Pantothenate 1.2g, Inositol 10g, Choline 23g, Vitamin A 0.075 g, Vitamin E 3.0 g. * KI 0.02g, CuSO4.5H20 0.30g, ZnCI2 0.80 g, FeSO4.7H20 2.24 g, M n S O 4 ' 4 H 2 0 3.04g, NaCI 19.10g, KzHPO4 60.3g, MgSO,L" 7H20 61.7 g, CaCO3 66.5 g, CaHPQ4 216 g. comm. 1975) and it was a modification of the diet described by Greaves & Ayres (1973). Its composition is shown in Table 1.
Experiment 1: Determination o]'the daily ritamin K t requirements of wtoJdrin resistant and susceptible rats In order to prevent coprophagy rats were housed individually in specially designed tubular cages similar to those described by Metta, Nash & Johnson (1961). These cages were square in cross section (6 cm.) and made from 19 mm. square Weldmesh. Food was provided in a metal cup placed at the entrance to the cage. A hinged door allowed a cloth bag to be placed over the end of the cage to facilitate routine handling of the wild rats. Heterozygous resistant [Rw~Rw'-), homozygous resistant [Rw2Rw 2) and susceptible (RwlRw ~) rats of both sexes were blood-sampled (day 0) prior to receiving an injection {i.p.) of vitamin K~ CKonakion", Roche Products Ltd.)at the required concentration in sterile saline. The blood was obtained while the rats were under light ether anaesthesia by snipping the tail tip and collecting into tubes containing sodium citrate anticoagulant. The vitamin K dependent blood clotting factors were assayed using the citrated whole blood and Diagen 2-7-10 reagent (Diagnostic Reagents Ltd., Thame, Oxon.). The clotting time was expressed as log percentage clotting activity (log PCA) after reference to standard curves prepared beforehand {Bishop et al., 1977). Following injection of vitamin K~ the rats were transferred to the coprophagy preventing cages and supplied with vitamin K deficient diet and water ad lib. The animals were injected daily for the next 3 days with the same amount of vitamin K~ as they received on day 0. They were blood sampled again 24hr after their last injection {day 4). The day 0 and day 4 log PCA values were then compared. Seven concentrations of vitamin K~ were used and controls received injections of saline alone. Experiment 2: The effect oJ" coprophagy on vitamin K requirement in heterozyyous resistant rats [RwlRw 2) Four groups, each of three adult male Rw ~Rw z rats were used. Each group of animals had been housed together since immaturity and consequently had established dominance relationships at an early age. Aggression within each group was therefore minimal. Two groups of rats (Groups I and 2) were transferred to two standard cages 132 × 18 × 45cm.) fitted with 19mm
square Weldmesh bottoms to minimise coprophagy. The remaining two groups (Groups 3 and 4) were housed in standard cages modified to maximise coprophagy. Their normal mesh bottoms were covered by zinc gauze. Urine build-up was prevented by laying several thicknesses of absorbant paper towels between the gauze and the cage bottom. Vitamin K deficient diet and water were provided ad lib to the four groups of animals. Blood samples were taken for the determination of log PCA before allocation (day 0), day 4 and day 8 by the method previously described.
Experiment 3: Estimation of the degree of coprophagy induloed by warfarin resistant (RwZRw 2, RwlRw 2) and susceptible (Rw I Rw 1) rats Adult male rats of known genotype were used. Tubular coprophagy preventing cages were used, as well as modified tubular cages which allowed access to faeces. The laboratory diet fed (41 B) had been analysed chemically and no detectable phylloquinone had been found (Partridge. 1978). The natural components of diet 41B had a theoretical vitamin KI content of 0.84 mg/kg (data kindly supplied by Dixon's Ltd.).It has been shown, however, that the pelleting process and subsequent storage can reduce the expected quantity of phylloquinone in a diet (Charles & Huston, 1972). Batches of six animals of the same genotype were taken from stocks. Three rats were randomly allocated to individual coprophagy preventing cages and the remaining three animals were allowed access to their faeces in the modified cages. Food consumption was measured over a 24 hr period ( ¢ 4 p.m.) and the unconsumed faeces were collected from all animals. The faeces were dried overnight (100°C) and after cooling were weighed to the nearest rag. A significant relationship was found between the dry weight of faeces produced and the amount of food consumed over the 24 hr period for the rats prevented from coprophaging. The line of best fit was described by the equation y = 0.28 + 0.26x [P < 0.001), where y = dry faecal weight produced and x = food consumption (g). Using this equation it was possible to estimate the quantity of faeces which an animal in a faecal access cage should have produced from a known food consumption. An estimate of percentage coprophagy could then be derived from: wt of expected faeces - wt of observed faeces wt of expected faeces
x
100°;,
RESULTS
Experiment I Table 2 shows the mean day 0 and day 4 log PCA values for the six sex/genotypes. Both male and female homozygous resistant rats (RwZRw 2) had a higher daily requirement for vitamin K than susceptible ( R w l R w 1) and heterozygous resistant ( R w t R w ~) animals. This concurs with other studies using laboratory strains (Hermodson et al., 1969; Greaves & Ayres, 1973). In addition, homozygous resistant males required more vitamin K than their female counterparts to attain "'normal" clotting levels ("normal" log PCA values being usually > 4.00). The greater vitamin K requirement of these males compared to other genotypes was reflected in their lower day 0 log PCA values. It was found necessary to maintain homozygous resistant animals on vitamin Ka supplemented water ( 1 0 m g m e n a d i o n e sodium bisulphite/l) when not required for experiments, to prevent hemorrhagic deaths. Heterozygous resistant ( R w t R w z) and susceptible
Vitamin K requirements of rats
85
Table 2. Experiment 1: Mean log PCA values (+SE) before (Day 0) and after (Day 4) daily intraperitoneal injections of vitamin K~ at various concentrations. Five rats per group, animals fed on a vitamin K deficient diet with coprophagy prevented Concentration Rw2Rw 2 c~ (/tgKt/100g) Day0 Day4 0
--
0.5
--
1.0
--
2.0
--
3.0
--
5.0
2.79 -+0.27 2,96 -+0.35 2.78 +0.31
7.0 10.0
RwlRw I 3 Day0 D a y 4
RwtRw 2 c~ Day0 Day4
--
4.30 +0.19 -4.18 +0.09 -3.79 -+0.15 -4.16 -+0.12 -3.75 _+0.09 3.34 . . +0.13 4.51 -+0.07 4.04 -+0.12 .
2.09 +0.53 3.57 +0,25 3,76 4-0.10 4.12 -+0.16 4.11 _+0.14 . .
.
.
.
.
.
.
4.06 4-0.10 4.17 +0.09 4.02 -+0.10 4.28 +0.10 4.19 -+0.11 . .
.
2.76 --+0.36 4.05 --_+0.20 4.28 ---+0.14 4.47 ---+0.09 4.31 "4.01 3.10 ___0.10 ___0.25 -+0.10 3.42 4.27 +0.36 -+0.11
.
.
Rw2Rw z 9 Day0 Day4
.
.
.
.
.
.
.
.
RwlRw 2 ~ Day0 Day4
RwtRw 1 Day0 Day4
4.33 +0.11 4.38 -+0.16 4.29 +0.13 4.11 -+0.14 4.39 -+0.13 . .
4.14 +0.08 4.00 -+0.17 4.05 -+0.09 4.12 -+0.13 4.41 -+0.09 . .
1.89 +0.15 3.59 +0.15 4.42 4-0.10 4.29 x0.20 4.56 -+0.05 . . .
3.93 +0.18 4.40 +0.10 4.51 +0.05 4.12 -+0,11 4,55 +0.03
.
.
.
* Only four rats in this group. ( R w l R w 1) rats were dosed with vitamin Kt in the range 0-3 yg/100g body weight. Differences in response of the two genotypes are shown in Fig. 1 expressed as the difference (A) between log PCA on day 0 and day 4. The results indicate that both male and female heterozygotes required a daily quantity of 1 #g/100 g to maintain 'normal' clotting levels. Susceptible males, however, required between 0.5 and 1/~g/100g and susceptible females <0.5/tg/100 g. Experiment 2
Table 3 shows the log PCA values obtained over the 8 day period for male heterozygous resistant rats maintained throughout the trial period with coprophagy prevented (Groups 1 and 2), or coprophagy allowed (Groups 3 and 4). The three animals in G r o u p 2 died on day 5, all showing signs of internal hemorrhage. An analysis of variance over the first 4 days revealed highly significant effects between groups and treatments as well as a significant interaction
o.o
{
0.5
~
~ ~JgK,1100g
between the two. The significant interaction component possibly reflects incomplete prevention of coprophagy when rats are housed in groups on a mesh floor. Single tubular cages as used in Experiment 1 may have reduced the variability within treatments. By day 8 the remaining animals prevented from coprophaging (Group 1) had very low clotting levels. Rats with access to their faeces showed a variable response but in general they maintained similar levels to those recorded on day 0. These results indicate that in the absence of dietary vitamin K the heterozygous resistant male rat can obtain adequate amounts by coprophagy. Experiment 3
Table 4 gives the estimates of percentage coprophagy obtained for the three genotypes. The mean percentage cophrophagy value for homozygous resistant animals was significantly lower than that of heterozygotes (P < 0.01). The variability of response
o.o
I;o
z~:o
3;o ,ug K,1100g
0.5
I I.o + 1.5
i.o MALES
1.5
o resistont 2.0
2.5
I
+
•
susceptible
FEMALES ~ resistont
2.0
• susceptible
23
Fig. 1. The response of heterozygous resistant and susceptible wild rats of each sex to daily injections of vitamin KI for 4 days, where A = log PCA day 0 - l o g PCA day 4. Each point represents the mean of five animals _+SE.
G. G. PARTRIDGE
86
rats in this study are much lower than those described for the warfarin resistant Wistar strain used by Greaves & Ayres (1973). These workers, using a similar experimental design, estimated the requirement of both male R w l R w 1 and R w l R w 2 genotypes to be Coprophagy minimised (standard cages fitted with 19 mm 6 pg/lO0 g body weight and that homozygous resistsquare mesh floors) ant males (RwERw 2) required approximately 80#g/ Mean log PCA I+SE) 100g. The corresponding estimates in the present Group No. Day 0 Day 4 Day 8 study were: 0.5 #g; 1 #g; and 5-7 pg vitamin K1/100 g body weight respectively, although the latter estimates 1 4.61 + 0.00 3.68 +_0.52 2.09+ 0.92 2* 4.20 _ 0.36 1.24 + 0.11 .... should be considered approximate since only a *All animals died day 5, hemorrhaging. limited range of doses between 0 and 1/lg was tested. Coprophaoy maximised (standard cages fitted with solid Mellette & Leone (1960) noted that different strains floors) of laboratory rat have widely varying tolerances to 3 4.16 + 0.0l 4.19 + 0,03 4.27 + 0.10 low levels of dietary vitamin K. The large differences 4 4.26 + 0.25 3.63 + 0.43 3.58 + 0.76 found between this study and that of Greaves & Analysis of variance testing for differences between groups Ayres (1973) could be due to the presence of different and treatments, day 0 minus day 4 values. modifier genes in the two strains of rat used. Whether Groups p < 0.001 similar variability is present naturally in other wild Treatments p < 0.001 rat populations has yet to be established. Of particuInteraction p < 0.05 lar interest would be the vitamin K requirements of rats in populations from areas in which warfarin had in R w 2 R w 2 animals was particularly large, three not been used extensively as a rodenticide i.e. where animals giving negative values and two having values there had been no selection for genes modifying the effects of the Rw2 allele. The major gene controlling > 20%. warfarin resistance in the house mouse ( M u s musculus) is known to be influenced significantly by such DISCUSSION modifying genes (Wallace & MacSwinney, 1976; Hermodson et al. (1969) estimated that the vitamin MacSwinney & Wallace, 1978). K requirement of heterozygous and homozygous reThe higher vitamin K requirement of the male rat sistant rats was twice and twenty times respectively compared with its female counterpart has been prethat of a warfarin susceptible animal. Greaves & viously described in laboratory strains (Johnson, Ayres (1973) found that heterozygous resistant and Mameesh, Metta & Rama Rao, 1960; Bell & Theiss, susceptible rats had a similar requirement, with 1972) although this is the first report of a similar sex homozygous resistant rats having a requirement difference in the wild rat. The sex difference is under thirteen times higher. Martin (1973) working with the the control of the hormonal system; estradiol relievsame strain of rats estimated that homozygous resist- ing and testosterone enhancing the hypothrombinaeant rats required approximately thirty times more mic state (Mellette & Leone, 1960). Estradiol itself does not have vitamin K-like activity indicating that vitamin K than susceptibles, This variation in relative requirements is probably the hormone must be operating indirectly (Matsaccounted for by the different methods of estimation chiner & Bell, 1973). The mean percentage coprophagy values obtained used. Hermodson et al. (1969) and Martin (1973) used a method of curative dosing of vitamin K depleted for the R w l R w 2 and R w l R w 1 genotypes (26 and 19% animals, while Greaves & Ayres (1973) measured the respectively) are similar to those reported by Johnson response to daily doses of the vitamin over a 4 day et al. (1960) who used a variety of diets They found period, as in this study. Preliminary observations values were frequently between 20 and 30~o, although indicated that wild susceptible female rats showed no the protein source in the diet appeared to influence hypothrombinaemic response, even after feeding on a the degree of coprophagy. Mameesh, Metta, Rama vitamin K free diet for three weeks (Partridge, 1978). Rao & Johnson (1962) estimated that the amount of In view of this observation it was not possible to deplete vitamin K active substance produced in the intestines the blood clotting levels equally in the different was 8-1! mg of vitamin K equivalents per kg of dried caecal contents. Consumption of dry faeces in the groups prior to curative dosing with vitamin K. The vitamin K requirement estimates for the wild present study was frequently between 1.0 and 1.6g Table 3. Experiment 2: The effect of coprophagy on blood clotting levels (log PCA) of male heterozygous resistant rats (Rw~Rw 2) fed on a vitamin K deficient diet for 8 days; three rats per group
Table 4. Experiment 3: Percentage coprophagy indulged by the three genotypes feeding on a standard laboratory diet (41 B) with access to their faeces
Genetype RwZRw 2
Significance of difference between means
Mean body weight Mean percentage +SE coprophagy + SE (n) 338 + 59
6.53 + 4.74(8) ]
] p < 0.01
Rw1Rw 2
339 + 89
26.33 + 4.47 (9)
0.01 > p > 0.05 n.s.
RwIRw I
344 _ 71
18.70 4- 4.04 (111
Vitamin K requirements of rats
87
DUNNING W. F. & CURTIS M. R. (1939) Linkage in rats between factors determining a pathological condition and a coat colour. Genetics 24, 70. GREAVESJ. H. & AYRES P. (1967) Resistance to warfarin in rats. Nat,re 215, 877-878. GREAVES J. H. & AYRES P. (1969) Linkages between genes for coat colour and resistance to warfarin in Rattus norvegicus. Nature 224, 284-285. GREAVES J. H. & AYRES P. (1973) Warfarin resistance and vitamin K requirement in the rat. Lab. Anita. 7, 141-148. GREAVES J. H. & AYRES P. (1976) Inheritance of Scottishtype resistance to warfarin in the Norway rat. Genet. Res., Camb. 28, 231-239. GREAVES J. H., REDFERN R., AYRES P. & GILL J. E. (1977) Warfarin resistance: a balanced polymorphism in the Norway rat. Genet. Res., Camb. 30, 257-263. HERMODSON M. A., SUTTtE J. W. & LINK K. P. (1969) Warfarin metabolism and vitamin K requirement in the warfarin resistant rat. Am. J. Physiol. 217, 1316-1319. JACKSON W. B. & KAUKEINEN n. E. (1972) Resistance of wild Norway rats in North Carolina to warfarin rodenticide. Science N.Y. 176, 1343-I 344. JOHNSON B. C., MAMEESHM. S., METTA V. C. & RAMA RAO P. B. (1960) Vitamin K nutrition and irradiation sterilization. Fedn. Proc. Fedn. Am. Soc. exp. Biol. 19, 1038-1044. MACSWINNEY F. J. & WALLACE M. E. (1978) Genetics of warfarin' resistance in house mice of three separate localities, d. Hyg. 80, 69-76. Acknowledgements--I would like to thank Dr. N. T. Davies, Dr. J. A. Bishop and Dr. J. H. Greaves for critical MAMEESH M. S., MErTA V. C., RAMA RAO P. B. & JOHNSON B. C. (1962) On the cause of vitamin K deficiency in reading of the manuscript. This work was completed while male rats fed irradiated beef and the production of I was in receipt of an S.R.C. postgraduate studentship. vitamin K deficiency on an amino acid synthetic diet. J. Nutr. 77, 165. MARTIN A. D, (1973) Vitamin K requirement and antiREFERENCES coagulant response in the warfarin resistant rat. Biochem. Soc. Trans. i, 1206-1208. BELL R. G. & THElSS J. W. (1972) Effects of estrogens on vitamin K and prothrombin. Fedn. Proc. Fedn. Am. Soc. MATSCHINER J. T. & BELL R. G. (1973) Effect of sex and sex hormones on plasma proth.rombin and vitamin K defiexp. Biol. 31,714 (ABS). ciency. Proc. Soc. exp. Biol. Med. 144, 316-320. BIGGS R. P. & MACFARLANE R. G. (1962) Human Blood MELLETTE S. J. & LEONE L. A. (1960) Influence of age, sex, Coagulation and its Disorders. Oxford, Blackwell Scienstrain of rat and fat soluble vitamins on hemorrhagic tific Publications. syndromes in rats fed irradiated beef. Fedn. Proc. Fedn. BISHOP J. A. & HARTLEY D. J. (1976) The size and age Am, Soc. exp. Biol. 19, 1045-1049. structure of rural populations of Rattus norveaicus conMETTA V. C., NASH L. & JOHNSON B. C. (1961) A tubular taining individuals resistant to the anticoagulant poison coprophagy-preventing cage for the rat. J. Nutr. 74, warfarin. J. anita. Ecol. 45, 623-646. 473--476. BISHOP J. A., HARTLEY D. J. & PARTRIDGEG. G. (1977) The population dynamics of genetically determined resist- PAR'rRID~E G. G. (1978) The pleiotropic effects of the gene conferring warfarin resistance in the brown rat (Rattus ance to warfarin in Rattus norveoicus from mid Wales. norvegicus). Ph.D. Thesis, University of Liverpool. Heredity 39, 389-398. CHARLES O. W. & HUSTON T, M. (1972) The biological POOL J. G., O'REILLY R. A., SCHNEIDERMANL. J. & ALEXANDER M. (1968) Warfarin resistance in the rat. Am. J. activity of vitamin K material following storage and pelPhysiol. 215, 627-631. leting. Pouhry Sci. 51, 1421-1427. DRUMMOND D. C. (1970) Variation in rodent populations WALLACE M. E. & MACSWINNEY F. J. (1976) A major gene controlling warfarin resistance in the house mouse. J. in response to control measures. Syrup. Zool. Soc. Lurid. Hyg. 76, 173-181. 26, 351-367.
(dry weight). Using the above estimates m a n y animals could therefore obtain in excess of 8/~g of vitamin K equivalents from coprophagy. Expressed in relation to mature body weight (say 350 g) this corresponds to an intake of approximately 2/tg/100 g. Experiment l showed that this quantity of vitamin K is sufficient for b o t h R w t R w I and Rw~Rw 2 genotypes, but it may be inadequate for RwZRw 2 animals. Partridge (1978) calculated the relative fitnesses of the three possible genotypes in the Welsh farm building population (Rwt R w I 1.00; Rwl R w 2 0.77; Rw2Rw 2 0.46). These estimates were based on the observed decline in the frequency of resistance in the farm buildings over an 18 m o n t h period when warfarin was withheld. Some rats on the study farm had access to a b u n d a n t cereal grain which was a poor source of vitamin K (Partridge, 1978). In contrast, other animals were feeding in the poultry sheds on a vitamin supplemented laying hen diet containing approximately 0.23/~g of vitamin K3 equivalents/g of diet (data kindly supplied by Seemeel Ltd., L o n d o n E.15). Selection pressure on resistant rats could therefore vary markedly depending on the food sources available to resistant populations.