Mineral intake and hair analysis of horses in arizona

Mineral intake and hair analysis of horses in arizona

S ' ~ ' ~ N ~ .~ ~ ~ ~,~ ~ : ~#" '~,~ ~ ~ L~ Refereed MINERAL INTAKEAND HAIRANALYSISOF HORSES IN ARIZONA Leslie A. Wells/Raymond LeRoy, M.S.;I ...

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MINERAL INTAKEAND HAIRANALYSISOF HORSES IN ARIZONA Leslie A. Wells/Raymond LeRoy, M.S.;I Sarah L. Ralston, MS, VMD, Ph.D, ACVN 2

SUMMARY To evaluate the effectofdiet on hair mineral content, 61 feeds, 31 pasture grasses, 29 mineral supplements, 31 water samples and hair samples from 391 horses from 31 ranches in Arizona were analyzed for fifteen minerals. Based on dietary histories and estimated intakes when free choice access was involved, mineral intake of the horses was calculated and compared to mineral content of the hair. Data were evaluated by regression analysis for correlations between hair and dietary minerals and variations in hair mineral content attributable to coat color, age, sex and breed.~l.~2.~3.~4 There were differences (P<.05) in hair mineral content of horses of different breeds, coat colors, ages and sex, regardless of dietary intake. However, there were some significant correlations between feed mineral intake and hair mineral content. A few weak simple correlations (Ca:Ca, P:P, Na:Na, K:K) were found, but complex mineral correlations (Ca:P, Ca:Na, Zn:K, Fe:Zn etc.) were more common and had higher correlation coefficients (Table 7). Variations in mineral intake explained only 30% of the variation for a given hair mineral, with the exception of manganese. The multiple relationships between minerals and their effect on metabolism appear to have a greater effect on hair mineral

content than a single mineral deficiency or e x c e s s 17'182"°'~ and may offer better insight to the interpretation of mineral nutrition than simple one to one correlations.

INTRODUCTION Despite the lack of scientific data to prove reliability, hair analysis has been advertised as a prognostic and diagnostic test for dietary mineral imbalances in horses. 1-3 Mineral content of animal hair apparently varies not only with mineral intake, but also with the season, breed, sex, age, hair color and body location. There have been studies on mineral content of horse hair but those published were based on relatively small sample sizes (<50 animals) 4"7and no significant reference ranges were reported. There have been several reported attempts to use hair analysis as an aid in determining mineral status of the horse? ,z3,~Unfortunately, the lack of published "normal" or comparative values render the data difficult to interpret. This study was undertaken to: 1) determine a set of reasonable reference ranges of the commonly analyzed minerals in horse hair and 2) identify variations due to age, sex, color, and breed which might influence hair analysis interpretation, and 3 ) determine which, if any, relationships are valid for identifying mineral imbalances.

Authors' addresses: 1LAW Enterprises and Accutrace Laboratories, Phoenix, Arizona. 2Department of Animal Sciences Rutgers University, New Jersey, 08903 Acknowledgements: We are indebted to Dr. Paul Eck for his financial and technical support of this project and to Sandy Rolain, Julie A]tavilla and Joyce Farkas for the;r patience arid expert typing s~;l/s in the preparation of th;s manuscript, Drs. Jim Zumbrunnen (Colorado State University) and Charles Curtis (University of Pennsylvania) for assistance with statistical analysis and interpretations.

412

METHODS AND MATERIALS Thirty-one ranches in Arizona co-operated in this study, which included 391 horses and eleven breeds. Individual hair samples were taken from the abdomen of the

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Table 1. Average mineral intakes of horses sampled. Cal culated from reported intake and actual analysis of forages, grains, supplements and w a t e r consumed.

Table 2. Comparative values a of hair mineral content (PPM) from horses in Arizona. Mineral Mean

Mineral

Ca (gm) P (gm) Mg (gin) Na (gm) K (gin) Mn (nag) Fe (mg) Cu (nag) Zn (mg) Cr (mg) Pb (rag) Hg (mg) Cd (mg) AL (mg) Ni (rag)

SD

90%Confidence Interval

Daily Intake _+.SD Recommended Intake

18.15 + 14.03 8.55 + 3.50* 5.50 + 4.98 4.76 + 3.49 24.46 + 9.44 52.1 + 59.60* 284.40 + 222.90 21.10 + 18.60" 156.9 + 220.0* 7.08 +7.59 8.02 +2.80 8.17 +0.19 1.47 +1.25 0.51 +0.55 6.8 +4.44

16-20 11-14 6- 7.5 8-10 20- 25 320-400 320-400 80-100 320-400

National ResearchCouncil (1989) recommendationfor 400-500 kg adult horse. Most horses in the study were in this weight range. * Mean value is more than one stahdard deviation (SD) from the minimumrange recommendedindicatingthat a largeportionof the horsesin the study had, based on NRC recommendations,inadequateintakes.

animals in early winter, October, 1984 to January, 1985. All of the animals had been considered to be healthy within the last three months by veterinarian exam. Sex, age, breed and color as well as detailed feeding schedules were recorded for each animal at the time of sampling. Samples of all feeds, pasture grasses, mineral supplements and water on the premises were also collected. If on pasture or fed hay free choice, it was assumed the horses consumed 1.75% of their body weight in forage daily.21 Water intake was assumed to be 2.47 1/45 kg BW.21 Otherwise amounts fed were weighed to calculate intake. Previously published data have shown significant losses of calcium, sodium, and potassium when hair is washed with aqueous solutions,s therefore hair to be sampled was rinsed with methyl alcohol and allowed to air dry. At least 500 mg of hair then were stored in plastic bags pending analysis. Hair, feed, pasture grass, mineral supplements and water were all analyzedfor calcium (Ca), magnesium (Mg), sodium (Na), potassium (K), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), chromium (Cr), aluminum (A1), lead (Pb), mercury (Hg), cadmium (Cd), and nickel (Ni) by atomic absorption. To prepare the samples for mineral analyses, 300 milligrams of previously rinsed and sectioned hair were placed in an acid washed 16 X 125 mm tube. Perchloric acid (0.5 ml) and nitric acid (1.5 ml) were added to each tube and placed under an acid fume hood for 25 min. The tubes were then vortexed for 15 seconds and placed in 95 ° C blocks for 25 rain. The tubes were then removed from the 95 ° C blocks andplaced in 185° C blocks for 14 hrs. Six ml of 0.9% HCI diluent was added to each tube and vortexed. This solution was analyzed for the trace minerals Volume 10, Number6, 1990

Ca 1780 680 Mg 690 160 Na 1460 960 K 3360 1600 Fe 380 180 Cu 6.8 2.6 Mn 11.0 5.5 Zn 100 20 Cr 2.4 .90 P 290 120 Pb 3.0 1.3 Hg .20 .10 Cd .30 .20 AI 490 260 Ni 2,1 .80 aValuesare for horses > two (2) years old.

1100-2460 530-850 500-2420 1760-4960 200-560 4.2-9.4 5.5-16.5 80-120 1.5-3.3 170-410 1.7-4.3 .10-.30 .10-.50 230-750 1.3-2.9

Cu, Fe, Mn, Ni, Pb, Cd, A1, and Cr. Four tenths ml of the trace solution was transferred to another acid washed tube and diluted to 4.0 ml with 0.2% cesium chloride solution. This solution was analyzed for Ca, Mg, Na, K and Zn. Four tenths ml of the trace solution was analyzed for phosphorus by the vanadomolybdophosphoric acid method ? Mercury was determined using the sodium borohydride method. Thirty mg of hair was dissolved in (0.3 ml) nitric acid at 95°C for eight minutes followed by dilution to 10.0 ml with 0.9% HCI diluent. National Bureau of Standards SRM 1577a, (Bovine liver), and SRM 164 lb, (mercury in water), were used as controls with all analytical procedures. Ten grams of the feeds, pasture grasses and mineral supplements were dry washed at 450 ° C for four hours before representative samples were taken and acid digested preceding analysis),1°Water samples were analyzed without pretreatm ent.10 Data were analyzed by multiple regression analysis (statistical package for the social science version 8.3) H factoring the effects of age, sex, breed, color and mineral intake on hair mineral content. Both LSD and Durbin Watson D ~2multiple range tests were employed to determine relationships between hair minerals and the various recorded parameters. 13Ninety- five percent confidence intervals were calculated for mineral content of hair for each age group, breed, sex, and color. The Pearson correlation coefficients between hair and feed mineral content (Log 10 transformations) were calculated. TM

RESULTS

The estimated average daily intake of most minerals except Ca and K were below the NRC recommendations for 400-500 kg horses (the average weight range for horses sampled) (Table 1). Sodium,P, Mn,Zn and Cuintakes were

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Table 3 . Selected~ hair minerals (PPM) 95% confidence intervals for seven different breeds of horses in Arizona. n

Ca

Mg

P

Cu

Zn

Fe

Mn

Pb

~iiiiiiiii~i~iiiii~i::;iiiiiiii::i~iii::i~i~i~iii:: ~~i: i~i i~!~ii~!~i ~iiii~i~ii i~:i: ii~i~ii::i::i~i::ii~!~ii iii~iiiii!ili::ii!iiiiiiiiii~!iiiiii!ili ii~i iii!;iiiii ~iiiiiiiiiiii::i::!ii~::ii ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: if::::~:0~i~::iii~i::i~!~i~i!i::i:::i~ i~i~i::i:~i i:=i::i!i::ii!i~iiiiiiiiiiii Thoroughbred

46 Mean SD

1770 210 b,c

850 100 bc

340 40 b

6.7 0.7 a,b,

100 5 a

470 120 b,c

16 4 ab

3.0 0.5 b

iiiiiiiiii i i! iii iii::iiiiii~ili::iiiiiiiiiii~:i::i!ii!iiii::iiiiiiiii !!i!iiiiiiiiiiiiiiiiiiiiiiiliiiii!iii iil :~ i:iii i ii ~i~ ii :: i::i::~:::::::i::i::i::::::::::::::::::::::::::::::::::::::!iii::!~i::i::~ii::~::ii!iiii::~iii::i::ii::i::~::~iiiiii~ii~i!iiiiii::ii~iiiiiii?:i;::ii!iii~iii SD

150 b,c

60 a,b

20 b

0.5 ab

10 a

50 bc

2 ab

0.2 b

1320

650

210

6.5

120

500

14

2.5

350 a

150 a

50 a

0.7 a,b

40 b

260 b,c

7 ab

0.7 ab

ii~iiiiiii;iiliiiiiiiiii!!iiiiii~~iiii!iiiiiiiiii i~i~i~iiiiiii!iiiiiiiill i~iii!::~ii i~iiiii~i::i~i::!::iii::i::ii::iii::i~i~!ii::i::!ili::!i~i~i::i~ill!::i::iiiiii~~ii!iii!!iiiii~iiiiiiiiiiii~i~iiiiiiiiiii !ilil~iiiiiiill iiiiiiiiiiiiii:~iiiiiiiii i!ii!iiii~iiiiiiiiiii~i!iiiii!iiiiii!iiiiiiiiiiii!iiiiii!!ii!ii;~iii~ iiil !ii::~!!i i~i~ii::ili~ii~iii~!~iii::i::ii~!iiii~ iiii::::~i!i::::::i::::~i~ii:: !iiii::!i~!~i;::i::!ii!~!iii::iiii~!ii~iiiii!iiii~ii!iiii!!i!!i~;~i~!ii~!iiii!iii~!iii!iii Appaloosa

21 Mean

SD

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~ii!i

i~i~i i i~i ~i !ii~!::ii!iiiif:~iii::!iiii~iii~!i!ii~if:~::i::!::i::i~i~i::i::i:: l! ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ::::::::::::::::::::::::::::::: !~::!i! i::ii!i!il~ii!iiiiiiiiiii ~!~iiiiiiiiiiiii iiiiiiiiiiii!i

•i.iiiiil.i.i.i.il.iii.iii.l.ii.iii.liii.ii!i.iiiiiiiiiiiii.iiiiiii.iii::i::i.iii ~il.ii.~.~iliii~!iiii!iiii.i!i!iii!iiij ~!~ ili.iglj!ii~i.i.iii.!!iiii.ii.iiii~iiii::i.i!ij.i.i.~.iliiiii!!i!i!iil~iiii.il.!iiiiiijii~i.!!ili.ijii.ijjiii~i!!iiiiii!ii::ii!iii::i~i!iii::i a!~ii~i::i::i::ili::Si~i ii~i~i!i ii~i~i! a,b,c,d Ranges within a column with different subscripts differ (p<.05) by multiple regression analysi with LSD multiple range test.

1Not all breeds sampled or minerals* analyzed are included. Only breeds represented by >10 individuals and minerals which differed (p<.05) between breeds were included.

Table 4. Selected1 hair minerals (PPM) means _+ S for different coat colors in Arizona horses. Color

n

Ca

Cu

A

Chestnut 153

Mean SD

1890 810 a,b

6.4 2.4 a

660 580 a,b

Bay

Mean SD

2060 950 b

7.6 2.8 a,b,c

168 58 a,b

7.3 2.8 a,b,c

470 470 a

8.4

920 990

124

Black

36

Mean SD

1950 1020 a,b

Gray

63

Mean SD

1630 1250 a

6.1 c

b

1Only those mineralswhich differed (p<.05) betweencoat colors are included, a.b.cValues in columnswith differentsubscripts differ (p<.05) by multipleregressionanalysisand LSD multiplerangetest.

more than one standard deviation (std dev) below the lowest NRC recommended intake, whereas, Ca and K intakes were withinrecommended ranges for most of the horses sampled. None of the horses had grossly apparent clinical signs of the deficiencies. Intakes of the potentially toxic metals, Cr, Pb, Hg, Cd, AI and Ni were below toxic levels in all horses. The ranges of hair mineral content found (Tables 2-6) are comparable to previously published reports. 4~.7.8.~s Table 2 presents the mean, standard deviation and confidenceinterval for 15 minerals in horse hair. The values

414

in Table 2 do not include data from ponies or animals less than two years old. Breed, age, color and sex influenced (P<.O5) hair content of most minerals (Tables 3,4,5,6). There were significant correlations between certain hair minerals and dietary mineral intake (Table 7) but few one-to-one correlations were found, even with log 10 transformation of the data to allow for nonlinear relationships.

DISCUSSION The original intent of this study was to establish equine hair mineral reference values and to document the intermineral relationships which are essential to the interpretation of the laboratory results. Hair mineral content appears to be affected by dietary intake, (Table 7), but less than 30% of the variation recorded for a given mineral in horse hair can be attributed to that specific minerals' intake, which leaves more than 70% oftheobserved variations correlating with other minerals or metabolic factors as yet undetermined (Table 7), at least in the population studied. These findings were not surprising as similar results have been reported in humans. ~8~°It was assumed that the rations sampled were representative of mineral intake over the past 3 months during which time the horses were growing their winter coat. It is of interest that, despite wide ranges of intakes only hair Ca, P, Na and K revealed even weak (<10% total variation explained) correlations with dietary intake of that mineral and these were all non-linear, negative correlations. Sodium intake may have been underestimated since voluntary consumption of salt was not measured and most horses had access to free choice salt but

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Table 5 . Selected 1 hair minerals (PPM) mean + SD for open mares by age groups in Arizona. Mg

Na

K

Fe

Cu

Mn

Zn

Cr

P

AI

41 Mean 1670 SD 690 a

660 220 a

1870 900 a

3550 1280 a

330 200 a

6.6 1.9 a

8.5 5.0 a

100 20 a

2.2 2.1 a

280 80

340 220

a

a

21Mean 1910 SD 710 b

760 250 b

1920 1110 c

3560 1290 a

400 260 b

6.7 2.0 a

11.8 6.6 b

110 20 a,b

2.6 1.0 b

300 120 a

430 230 b

16Yrs& 16Mean 2100 Older SD 920 c

940 360 c

1170 1020 a

2930 1440 b

460 250 o

7.8 3.3 b

12.9 7.2 c

120 30 b

3.0 2.3 c

350 120 b

620 320 c

Age

n

<8Yrs

<15Yrs

Ca

1Only those minerals which differed (p<.05) between-the-3 age groups are included. .,b,¢values in columns with different subscripts differ (p<.05) by multiple regression analysis and LSD m ultiple Range test.

Table

6. Selected 1 hair minerals (PPM)

Age

n

2-8 Yrs

9 Yrs & Older

Ca

mean + SD for stallions by age group in Arizona.

Mg

Na

K

Fe

Cu

Mn

Zn

Cr

P

AI

26 Mean 1760 SD 780 a

790 330 a

1370 830 a

3000 1760 a

330 220 a

5.6 1.2 a

- 9.2 8.1 a

120 80 a

1.9 1.2 a

270 80 a

370 290 a

17 Mean 1940 SD 710 b

890 250 b

1520 1110 b

3960 1290 b

480 260 b

6.6 2.0 b

13.6 6.6 b

100 20 b

3.0 1.0 b

310 120 b

520 230 b

1Only those mineralswhich differed (p<.05) are included. "= values in columnswith differentsubscriptsdiffer(p<.05) by multipleregression analysis and LSD multipleRange test.

intake estimates of other minerals are assumed to be fairly accurate. It would appear that phosphorus deficiency is not reflected in hair P content, since the horses were, on the average, deficient in this mineral but the hair P did not fall below previously published values or vary strongly with P intake. The lack of clinical signs of P, Cu or Zn deficiency in these horses suggest that either the NRC2~recommendations for the minerals may be high or that sub-clinical syndromes existed that were not detected by the horse owners or investigator taking the samples (LAW). Heavy metals such as Hg, Ni, Pb did not vary with age, sex, breed or coat color and were minimally affected by dietary mineral content. However of the heavy metals none were ingested in toxic amounts by the horses sampled and therefore validity of hair samples to determine heavy metal toxicity~5has not been disproven in horses by this study. Many authors have voiced concern regarding variations in mineral content of hair with regard to age, breed, s e x and coat color. 7J6,17 Our data provides further support for this concern. However, deviations from the normal ranges established for Ca, Na, K, Cu and AI in this study may indicate mineral imbalances regardless of age, sex, breed or coat color, since means for these minerals grouped

Volume 10, Number6, 1990

according to the various factors fell within the 10% confidence intervals (Table 2). Positive, linear, one-to-one relationships (feed-to-hair) were not found for any of the minerals evaluated. Therefore, "low" levels of a given mineral in horse hair may not reflect a dietary deficiency of that mineral but instead could indicate excess of antagonistic minerals or other interferring agents 17~ in the diet. Basic to an understanding of hair mineral analysis is the fact that the minerals must first pass through the complex systems of ingestion, absorption and circulation before being deposited in the hair. Therefore, hair minerals may also reflect metabolic activities in addition to mineral status of the individual animal. ~8'I9~Though none of the horses sampled had overt clinical signs of imbalances or disease, it is possible that subclinicalproblems existed. Blood samples unfortunately were notdrawn forevaluation of either mineral content or metabolic parameters, due to constraints beyond the control of the authors. The results are therefore reflective of a population of horses with no obvious clinical disease or diet imbalance but which may have had subclinical problems, and reflects the average horse population in Southern Arizona. The inter-element relationships of feed mineral content compared to hair minerals indicate that change in the

415

Table 7. Hair-feed mineral correlations (p<.05, two-tailed test) on log transformed data (from Arizona horses). Feed Mineral Hair mineral(s) affected Ca

Ca (-. 128", 1.6"*) Na (-.171,2.9), K (-.306,9.4) P (-.144, 2.1), Pb (-.120,1.4)

P

P (-.274, 7.5) Ca (-.301,9.1), Na (-.181,3.3), K (-.133, 1.8), Fe (-.162, 2.6) Ni (-.128, 1.6)

Mg

Ca (-.267, 7.1), Na (-.268, 7.2), K (-.214, 4.6) Zn (-.099, 1.0), P (-.241,5.8) Pb (-.118, 1.4) Ni (-.105, 1.1)

Na

Na (-1.0, 1.0), K (-.237, 5.6), P (-.128, 1.6) Pb (-.103, 1.1), Cd (-.123, 1.5), Ni (-.115, 1.3)

K

K (-.179, 3.2), Mn (-.106, 1.1), Cd (-.124, 1.5) Ni (-.137, 1.9)

Fe

Ca (-.162, 2.6), Na (-.279, 7.8), K (-.307, 9.4) Zn (-.121, 1.5), P (-.136, 1.8), Pb (-.161, 2.5) Cd (-.137, 1.9), Ni (-.120, 1.4)

Cu

Ca (-.265, 7.0), Na (-.266, 7.1 ), K (-.228, 5.2) P (-.259, 6.7), Pb (-.129, 1.7), Ni (-.123, 1.5)

Zn

Ca (-.229, 5.2), Na (-.310, 9.6), K (-.222, 4.9) P (-.208, 4.3), Pb (-.125, 1.6), Ni (-.111, 1.2)

Mn

Ca (-.221,4.9), Na (-.340, 11.6), K (-.250, 6.2) Zn (-.141,2.0), P (-.173, 3.0), Pb (-.121, 1.5) Mn (-.107, 1.1)

Pb

Ca (-.193, 3.7), Na (-.126, 1.6), K (-.192, 3.7), Zn (-.106, 1.1), P (-.166, 2.7), AI (-.107, 1.1) Ni (-.126, 1.6)

Hg

Na (-.161,2.6), K (-.201,4.0), Cr (-.239, 5.7) P (-.177, 3.1)

Cd

Na (-.279, 7.8) K (-.272, 7.4), Cr (-.120, 1.4) Pb (-.147, 2.2), Cd (-.I 12, 1.2)

AI

Ca (-.159, 2.5), Na (-.242, 5.8), K (-.282, 7.9) P (-.171,2.9), Pb (-.165, 2.7), Cd (-.139, 1.9) Ni (-. 123, 1.5)

Ni

Ca (-.260, 6.8), Na (-.217, 4.7), K (-.173, 3.0) P (-.218, 4.7), Pb (-.104, 1.1), Hg (-.104, 1.1) Cd (-.115, 1.3), Ni (-.137, 1.9)

*r = Pearson'scorrelation coefficientindicating + or - relationship **% = % of variation explainedby feed variation (r2 x 100)

concentration of a mineral in a feed ration (independent variable) will illicit a change in hair mineral(s) content (dependent variable). However, changes will rarely be on a one-to-one basis and more than one mineral will be affected. Therefore, it appears quite possible that a dependent mineral, which is either low or high in horse hair, can be manipulated by choosing an independent mineral which will affect the dependent mineral in the desired direction (synergistic/antagonistic). 2° The choice of minerals to be supplemented will often represent a compromise, as due regard must be given to the other dependent minerals. Further studies are already underway to confirm the validity of this approach. 416

In conclusion, due to the complex inter-relationships, testing for only a few individual minerals in horse hair is not adequate for determining the mineral status of an animal. Indiscriminate supplementation or megadoses of minerals should be discouraged on the basis that the status of other minerals in the animal may be adversely affected. Intervention studies are in progress to determine the effect of mineral supplementation on the mineral status in horses.

REFERENCES 1. Jones, D: Fortune tellingwith horse hair, Horsemen Magazine, pp. 56-62,1977. 2. McLean, L: Hair analysis in the field, Equine Vet Data, 4:161-164,169,1983a. 3. Sippel, WL. et ah Nutritional consultation in horses by art of feed, blood and hair analysis, Proc. AAEP 10:139-152,1964. 4. Combs, DK, Goodrich, RD, Meiske, JL. Mineral concentrations in hair as indicators of mineral status: A review, J Anita Sci, 54(2),1982. 5. Cope L, Hintz HF: Influence of month, color, age, corticosteroids and dietary molybdenum on mineral concentration of equine hair, Am J Vet Res 43(7):1981. 6. Fisher DD et al: Switch hair as an indicator of magnesium and copper status of beef cows, AmerJ VetRes,46:2235-2240,1985. 7. Wysocki AA, Klett RH: Hair as an indicator of the calcium and phosphorus status of ponies, J Anim Sci32:74-78,1971. 8. LeRoy RF: Effects of washing on trace mineral content of human hair, J Orththo Med 1:120-125,1986. 9. Taras MJ Ed: Standard methods for the examination of water and wastewater. Methods 223D. 13th Edition, Published by by the American Public Health Association, Washington, DC, 1971. 10. Von Loon J C: Se/ectedmethods of trace meta/ analysis in biological and environmental samples, John Wiley and Sons, inc., New York, N.Y., 1985, 357 pp. 11. Hinrichs DD: Multivariab/e regression analysis, A computarprogram, Washougal, Wash., 1985. 12. Spiegel M R: Probabilityand Statistics, McGraw-Hill Book Co., New York, N.Y., 1975. 13. Boarks G, McGilvray J: Interpretationand uses of medical statistics, Blackwell Scientific Publishing, Oxford, England, 1975. 14. Arkin H, Colton R: Statistical Methods, Harper & Row Publishers, Inc., New York, N.Y., 1975. 15. U.S. Dept. of Commerce: Toxic trace metals in mammalian hair and hails, PB 80-103997, National Tech. information Service, Springfield, VA, 1979, 16. Hambridge KM: Hair Analysis: Worthless for vitamins, limited for minerals, AmerJ C/in Nutr, 36:943-949,1982. 17. Hintz HF: Horse Nutrition, ARCO Publishing, Inc., New York, N.Y., 1983. 18. Eck P: Energy, Hea/thview Publications, Charlottesville, VA, pp 210,1985. 19. Hopps HC: The biological bases for using hair and nail for analysis of Trace Minerals, D Hemphill, Ed. Trace Substances in Environment Health. VIII, University of Missouri, Columbia, Missouri, 1974. 20. Passwater RA, Cranton EM: Trace elements, hair analysis andnutrition, Keats Publishing, New Canaan, Conn., 1983, 384 pp. 21. National Research Council Nutrient Requirements of the horse, 5th Ed., National Academy Press, Washington, DC, 1989.

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