\J

Refereed

ASSESSMENT OF SELENIUM STATUS IN MARES AND FOALS UNDER PRACTICAL MANAGEMENT CONDITIONS

J. Lee, BS; 1 E.S. McAllister, VMD; a R.W. Scholz, PhD 1.

S U M M A R Y

Thirty gravid mares of different breeds and in steady- state selenium (Se) nutrition were used to demonstrate a significant positive correlation between blood Se concen- tration and glutathione peroxidase (GSH-Px) activity. The relationship between blood Se and GSH-Px activity was higher in mares than in foals at parturition. The Se status of foals was also significantly correlated with that of the respective mares at parturition under practical manage- ment conditions where wide variations in dietary Se during gestation were present. Selenium concentration of milk was less than one-fourth of that in colostrum within one day postpartum and was considered a minor source of Se for the foals regardless of Se status of the mares during gestation. There were no significant differences in blood Se concen- tration between mares and foals of low Se status at partu- rition or during a 4-week postpartum interval. For mare/ foal pairs of adequate Se status, however, the differences were significant throughout the experimental period. Foals in this group had higher blood Se concentration than either foals or mares of low Se status. Plasma vitamin E concen- tration was elevated 3-4 fold in all foals by 1 day postpar- tum, following colostrum ingestion, and was independent of the Se status of their dams. We did not observe clinical signs of white muscle disease during a 4-week postpartum

Authors' addresses: 1Department of Veterinary Science, Pennsylvania State University, University Park, PA 16802, 2Centre Equine Practice, Centre Hall, PA 16828. *Address correspondence to R.W. Scholz. Acknowledgements:The authors thank G.C. Loop and L.J. Muchinsky for their assistance. We are also indebted to each of the stable owners, for without their cooperation and patience, this study would not have been possible.

period or differences in serum enzymes indicative of muscle damage in any of the foals regardless of their Se status.

I N T R O D U C T I O N

The essentiality of Se in trace amounts for the growth, reproduction and health of domestic animals is well estab- lished. In the equine species, muscle dystrophy or white muscle disease (WMD) has been associated with Se and/or vitamin E deficiency. TM Symptoms of muscle degenera- tion in foals with WMD generally appear during the first weeks of life. 1 This is important in the context that early neonatal Se status in foals is largely dependent upon the Se status of the mare during gestation, s Foals generally are born with a lower blood Se concentration than their dams, although this disparity diminishes when Se status of the dam during gestation is comparatively low. s'6 Selenium concentration is higher in colostrum than in mares' milk; s,7 however, the relatively low amount of Se in milk casts doubt on it being a major source of Se in suckling foals.

Forages and grains grown in Pennsylvania, as in the Northeastern United States in general, are relatively low in Se content. 8 Diseases associated with Se deficiency, par- ticularly in grazing animals, frequently become apparent unless some form of supplementation is undertaken. Supple- mentation of Se to mares during gestation may reduce the incidence of Se deficiency in their foals but the amount transferred via the placenta and milk may be insufficient. The purpose in the present study was to assess the Se status of mares and their foals under practical management con- ditions in central Pennsylvania by measuring Se concentra- tion and glutathione peroxidase activity of blood. We were able to locate stables where Se supplementation to the feed

240 JOURNAL OF EQUINE VETERINARY SCIENCE

of mares during gestation was undertaken and other stables where no supplemental Se was provided. This feature enabled us to evaluate the Se status of mares and foals over a wide range of dietary Se exposure.

M A T E R I A L A N D M E T H O D S

Thirty gravid mares of various breeds from 9 different stables were used in an initial experiment to determine the relationship in our laboratory between Se and glutathione peroxidase (GSH-Px, EC 1.11.1.9) activity in whole blood. The mares selected for this portion of the study represented Standardbred, Appaloosa, Quarter Horse, Thoroughbred and Percheron breeds and were sampled via jugular veni- puncture an average of 23 days prior to parturition. For the remaining experiments on temporal relationships among variables, 15 mare/foal pairs were selected representing each of the breeds indicated. The age of the mares at parturition ranged from 6--11 years. These mares and their foals were classified into 2 groups, low and adequate Se status, based upon the Se content of the total ration. Management practices at each stable differed; however, an estimate of the amount of Se consumed on a dry matter basis by the mares at the time of parturition was determined by sampling all food components individually. The NRC has established a Se requirement of 0.1 mg/kg diet for horses. 9 In two stables Se was not provided as a feed supplement and the Se intake for these mares was esti- mated to be 0.051 (n = 2) and 0.055 (n = 3) mg/kg diet, respectively. This concentration of Se is typical for grains and forages grown on Se-poor soils in Pennsylvania. 1~ These 5 mares and their foals constituted the low Se group. For the remaining stables in which Se supplementation was provided in the feed, the amount of Se consumed by the mares was estimated to be 0.102 (n = 3), 0.136 (n = 2), 0.151 (n = 2) and 0.160 (n = 3) mg/kg diet, respectively. These 10 mares and their foals constituted the adequate Se group. In each stable mares had ad libitum access to water and mixed timothy-alfalfa hay and were fed fixed amounts of locally grown oats. Some stables provided their mares with access to mixed grass-legume pasture as well as various purchased forms of mineral and/or protein supple- ments.

Blood was collected from mares and foals at parturi- tion (prior to suckling), and at postpartum intervals of 1,3,7,14 and 28 days, by jugular venipuncture into vacuum tubes containing EDTA as anticoagulant or into clot tubes without anticoagulant to provide serum. Colostrum and milk were collected into plastic bottles specifically cleaned for that purpose. All samples were kept on ice following collection and during transport to the laboratory. Samples for Se determination were stored frozen at -70~ until the time of analysis. Enzyme analysis was undertaken the same day blood was received at the laboratory. Selenium

40 �9

..j. 30 �9

xE 20 �9

, J o "13 o 1 0 _o II1

0 , L i I I I , I 0.0 0.1 0.2 0.3 0.4

Blood Se (lag/ml)

Figure 1. Blood glutathione peroxidase (GSH-Px) activity as a function of whole blood selenium in mares prior to parturition. One munit represents the oxidation of I nmole NADPH/min at 25~

concentration in blood, colostrum, milk and individual dietary components was determined by the fluorometric method of Whetter and Ullrey. 1~ Glutathione peroxidase activity in whole blood 1~ and serum activities of aspartate aminotransferase la (AST, EC 2.6.1.1) and creatine phos- phokinase 13 (CPK, EC 2.7.3.2) were determined spectro- photometrically. Plasma vitamin E was quantitated by the method of Hansen and Warwick. TM

Data were treated statistically by regression analysis or repeated measures analysis of variance, were appropri- ate. is Significant differences (P < 0.05) among treatment means were determined using Tukey's multiple compari- sons procedure.

R E S U L T S

The management practices involving Se supplemen- tation tO the mares during gestation differed considerably among the stables used in these studies. However, the relationship between blood Se and Se intake of the mares was ascertained by assuming an average expected feed consumption (% body wt) during late gestation of 1.25% for forage and 0.75% for concentrate, respectively. 9 For mares of low Se status, average blood Se concentration from the two stables was 0.047 and 0.064 gg/ml, respec- tively. For mares of adequate Se status, these values ranged from 0.149-0.180 ktg/ml, respectively.

The relationship between whole blood Se and GSH-Px activity determined for 30 mares prior to parturition is illustrated in Figure 1. These data were best described statistically by the linear regression equation Y = 8.69 + 77.57X and were significantly correlated (r = 0.89, P < 0.01). Blood Se was also significantly correlated with GSH-Px activity of foals at parturition (Figure 2) but to a

Volume 15, Number 5, 1995 241

2 5 -

~ 2 0

t 15

E x lO

~ 5 O _o ID

S 0 - I I I I 0.05 0.05 0.10 0.15 0.20

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Figure 2, Blood glutathione peroxidase (GSH-Px) activity as a function of whole blood selenium in foals at parturition. One munit represents the oxidation of 1 nmole NADPH/min at 25 ~

0.20

m r o o.15

~ O.lO Q if) "O O _o m 0 .05

o.oo I f I ] I I 0.00 0.05 0.10 0.15 0.20 0.25 0.30

B l o o d S e (~:J/ml) , M a r e

Figure 3, The re la t ionship b e t w e e n blood selenium concentration in mares and foals at parturition.

lesser extent than that determined for the mares. A qua- dratic regression equation best described these data and was determined to be Y = 6.92 + 107X - 96X 2 (r = 0.64, P < 0.05).

Blood Se concentration of foals at parturition was also highly correlated with maternal blood Se concentration as shown in Figure 3. This relationship for the 15 mare/foal pairs was best described statistically by the quadratic regression equation Y = 0.079 - 0.36X + 2.00X 2 (r = 0.76, p < 0.01).

Selenium concentration was determined in colostrum prior to suckling and in milk at postpartum intervals for mares of low and adequate Se status (Figure 4). At partu- rition, Se concentration of colostrum was significantly higher (P < 0.05) for mares of adequate Se status (0.085 gg/ ml) compared with mares of low Se status (0.045 lag/ml).

E

o~

0.10

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I I

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Days P o s t p a r t u m

Figure 4, Selenium concentration in colostrum and milk at postpartum intervals. Data points are means of 5 (low Se group) and 10 (adequate Se group) indiv idual observations, respectively, and those sharing a common letter are not significantly different (P>O.05).

Within one day postpartum, Se concentration in the milk was reduced significantly (P < 0.05) compared to the colostrum at parturition and showed an overall gradual decline thereafter in both groups during the postpartum period studied. Although milk Se concentration was al- ways higher in mares of adequate Se status, compared with mares of low Se status, differences between the two groups were not significant (P > 0.05) after the first sampling at parturition.

Figure 5 illustrates the relationship between blood Se in mares and their foals over a 4-week interval following parturition. Data in the figure were obtained from 5 mare/ foal pairs each representing adequate (panel A) and low (panel B) Se status, respectively. Daily Se intake by the mares prior to parturition for the two groups was estimated to be 0.149 and 0.053 mg/kg feed dry matter, respectively. Maternal Se status prior to parturition clearly was shown to influence blood Se concentration of the foals at parturition and at the intervals indicated postpartum. Mares of ad- equate Se status had blood Se concentration that was significantly higher (P < 0.05) than that of their foals at each sampling interval. Blood Se concentration in mares and foals of low Se status did not differ (P > 0.05) at parturition or at any postpartum interval, although it was consistently lower on average in foals compared to mares. It was also apparent that foals born to mares of adequate Se status had higher blood Se concentration than either mares or foals of low Se status.

The activities of serum AST and CPK enzymes at parturition and at postpartum intervals in foals born to mares of low and adequate Se status are presented in Table 1. The serum enzyme profile shown for AST and CPK was similar in both groups of foals. Serum AST activity was increased at day 1 postpartum compared to day 0 or at the time of birth. This elevated level, which is not considered

242 J O U R N A L OF E Q U I N E V E T E R I N A R Y S C I E N C E

E O) E

(n "O O _o rn

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0.2

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I

i l l i i i

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o.1 - [ ] Low Se Status

e �9 ' " ; ~ v

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0

m

I , I . ~ I I 0 01 3 7 14 2 8

Days Postpartum

Figure 5 , Blood selenium concentration at parturition and at postpartum intervals in mares and foals of adequate (panel A) and low (panel B) selenium status. Data points are means of 5 individual observations and those sharing a common letter within a panel are not significantly diffe rent (P > 0.05).

abnormal, did not change significantly (P > 0.05) through- out the postpartum period in which blood samples were taken. Serum CPK activity showed considerable variation among foals with the elevated activity at day 1 postpartum in foals of low Se status being significantly higher (P < 0.05) than its activity at subsequent intervals postpartum.

Plasma vitamin E concentration in foals of low and adequate Se status at postpartum intervals is shown in Figure 6. There were no significant differences (P > 0.05) between the two groups at any interval. However, a marked increase (P < 0.05) in plasma vitamin E in both groups was

sO0 "~ I se statu" I

X -O-Adequate

~, "...

.-:w 400 ~ ' ~

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o I I I I I I 01 3 7 14 28

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Figure 6 , Plasma vitamin E concentration at parturition and at postpartum intervals in foals of adequate and low selenium status. Data points are means of 5 individual observations and those sharing a common letter are not significantly different (P>0.05).

observed from birth to postpartum day 1. The magnitude of this increase was 3-4 fold. Plasma vitamin E concentration decreased in both groups of foals following postpartum day 1 but remained significantly higher (P < 0.05) than its concentration at birth through postpartum day 14 of the study.

D I S C U S S I O N

There was a high correlation between whole blood Se and GSH-Px activity in mares which agrees favorably with results of previously reported studies.6,1 ~1 o However, this relationship in blood was not as good in foals at birth and may represent age differences in the amount of Se that is incorporated into the GSH-Px enzyme during erythropoi- esis.J9 Because of its simplicity in a clinical setting, it may be assumed that GSH-Px activity determination would be the preferred procedure to assess Se status in horses under most conditions. However, several limitations in its use as a sole indicator of Se status should be considered, such as

Table 1 . Serum Enzyme Activity of Foals of Low and Adequate Selenium Status at Parturition and at Postpartum Intervals

Days Postpartum Enzyme 0 1 3 7 14 28 AST (U/L) A Low Se foals 36+6 ac 71 +-6 a'b 8 0 + - 5 a'b 101 +5 b 102+8 b 97+-11 b Adequate Se foals 41 +9 a 70+4 a'b 81 +14 a'b 109+9 b 117+4 b 113+5 b

CPK (U/L) B LOW Se foals 104_+13 a,b 198+_31 b 70+--13 ~ 76+-10 a 59+6 a 51+-12 a Adequate Se foals 84_+13 a 84+_12 a 59_+7 a 122+21 a,b 58+4 a 63+4 a

AAspartate aminotransferase 8Creatine phosphokinase CValues represent means _+ SEM, n=5 (low Se foals) or 10 (adequate Se foals). For each enzyme, means having a common letter in their superscripts are not significantly different (P>0.05),

Volume15, Number 5, 1995 243

stability during transport and storage, steady-state Se sta- tus of the animals, interlaboratory differences in assay procedures, among others. Whole blood GSH-Px activity determinations would be of questionable value, for ex- ample, if used to assess Se status after large changes in Se supplementation had been recently undertaken. In this event, actual blood Se analysis would be advisable in addition to GSH-Px activity determination. Nevertheless, an obvious advantage of blood GSH-Px activity determi- nation is that longer-term Se status is estimated regardless of recent changes in parenteral or dietary Se supplementa- tion. Other dietary factors can also influence Se metabo- lism and homeostasis, 2~ so care should be exercised in these interpretations as in assessments of most other deter- minations in clinical chemistry. In the absence of con- founding factors, increases in dietary Se over a rather wide range results in elevated Se concentration and GSH-Px activity in various tissues of many livestock species. 21 Even this has its limitations, however, because at high Se intakes GSH-Px activity and Se concentration in blood may not be highly correlated. 21 It has been shown previ- ously in foals and mature geldings that serum Se concen- tration does not correlate well with high levels of dietary Se supplementation. 22,23

Selenium status of the mares during gestation was reflected by the Se status of the foals at parturition and during the 4-week period postpartum in our studies. These results complement previous studies where it was shown that blood Se concentration in foals was lower than in dams in all cases studied except in one case where a mare had a very low Se status, s's Additionally, parenteral supplemen- tation of Se to mares during gestation has been shown to increase blood Se concentration of foals at birth. 8 It has also been reported that the concentration of Se, although quite variable, is far greater in mares' colostrum than in milk. 7 This indicates that after the conversion of colostrum to milk relatively small amounts of Se are provided to nursing foals regardless of the Se status of the lactating mare. This may also contribute to the occurrence of WMD in foals which is generally observed during the early neonatal period. 1 It is also emphasized that factors other than uncomplicated Se deficiency may predispose to WMD in foals. 17 None of the foals of low Se status in our study developed clinical or subclinical signs of WMD at any time during the study or after the 4-week postpartum period. These foals may be considered Se-deficient or inadequate, however, based on comparison with literature values for blood Se and GSH-Px activity. For example, Caple et al. ~6 found an average blood GSH-Px activity (using an assay procedure similar to ours) of 11.8 munits/mg Hb and whole blood Se concentration of 0.019 ktg/ml in mares who had given birth to foals that were affected with WMD. In related studies, Maylin et al. s found an average blood GSH- Px activity of 14.9 munits/mg Hb and blood Se concentra- tion of 0.042 ktg/ml in 6 mares from a farm where a foal had

died of WMD. Foals affected with WMD in another study had an average serum Se concentration of 0.032 ktg/ml. 4 Selenium concentration in whole blood would likely be similar because it has been shown that the Se content of blood plasma and whole blood is approximately the same in horses, s Others have reported GSH-Px activity in whole blood of 5 foals with WMD to range between 5-12 munits/ mg Hb. 24 These investigators have suggested further that blood GSH-Px activity < 15 munits/mg Hb and blood Se concentration <0.06 t.tg/ml are indicative of deficiency in foals.

We did not observe differences between foals of adequate and low Se status in elevations of serum enzymes indicative of muscle damage. Similar observations have been reported for Thoroughbred racehorses with wide differences in Se status. TM This suggests that either the Se status of the foals was adequate by itself to prevent muscle degeneration or that other factors were also instrumental. One of these factors could be the vitamin E status of the foals. We also determined the serum vitamin E concentra- tion of all foals at parturition and at postpartum intervals and found it to rise sharply following the ingestion of colostrum, which was expected. The enhanced vitamin E status of all foals after colostrum ingestion likely contrib- uted to their antioxidant defense, particularly those of low or marginal Se status in these studies. This supports previ- ously reported research where it was demonstrated that foals born in Se-deficient areas do not necessarily develop WMD.16,17,24 Others have claimed, however, that WMD in foals is caused by combined vitamin E and Se deficiency in mares during late gestation. 4

It is concluded that the Se status of mares during gestation has a significant effect on the Se status of their foals at parturition. This is of practical significance be- cause only modest amounts of Se are available to suckling foals through milk regardless of the Se status of the mares. Foals born to mares of low Se status did not develop clinical signs of WMD over a 4-week period postpartum or show abnormally high elevations in serum enzymes in- dicative of muscle damage. Factors other than an uncom- plicated Se inadequacy should be considered in the etiol- ogy of WMD in foals.

R E F E R E N C E S

1. Dodd DC, BlakelyAA, Thornbury RS, Dewes HF: Muscle degeneration and yellow fat disease in foals. NZ VetJ 1960;8:45- 50.

2. Gabbedy B J, Richards RB: White muscle disease in a foal. Australian Vet J 1970;46:111 - 112.

3. Wilson TM, Morrison HA, Palmer NC, Finley GG, vanDreumel AA: Myodegeneration and suspected selenium/ vitamin E deficiency in horses. J Amer Vet Med Assoc 1976;169:213-217.

4. Higuchi T, Ichijo S, Osame S, Ohishi H: Studies on serum selenium and tocopherol in white muscle disease of foal. Japanese J Vet Sci 1989;51:52-59.

244 JOURNAL OF EQUINE VETERINARY SCIENCE

5. Bergsten G, Holmback R, Lindberg P: Blood selenium in naturally fed horses and the effect of selenium administration. Acta Vet Scand 1970; 11:571-576.

6. Maylin GA, Rubin DS, Lein DH: Selenium and vitamin E in horses. Comell Vet 1980;70:272-289.

7. Breedveld L, Jackson SG, Baker JP: The determination of a relationship between the copper, zinc and selenium levels in mares and those in their foals. J Equine Vet Sci 1988;8:378-382.

8. Kubota J, Allaway WH, Carter DL, Cary EE, Lazar VA: Selenium in crops in the United States in relation to selenium- responsive diseases of animals. J Agr Food Chem 1967; 15:448- 453.

9. NRC: Nutrient Requirements of Horses. National Research Council, Washington, DC, 1989.

10. Scholz RW, Hutchinson L J: Distribution of glutathione peroxidase activity and selenium in the blood of dairy cows. Amer J Vet Res 1979;40:245-249.

11. Whetter PA, UIIrey DE: Improved fluorometric method for determining selenium. J Assoc Official Anal Chem 1978;61:927- 930.

12. Anonymous: Aspartate aminotransferase (AST/GOT). Quantitative, kinetic determination of AST activity in serum or plasma at 340 nm (Procedure No. 58-UV ). St. Louis, MO: Sigma Diagnostics, 1991.

13. Anonymous: Creatine phosphokinase (CPK). Quantitative, kinetic determination in serum or plasma at 340 nm. (Procedure No. 45-UV). St. Louis, MO: Sigma Diagnostics, 1985.

14. Hansen LG, Warwick W J: Afluorometric micromethod for serum vitamins A and E. Amer J Clin Pathol 1969;51:538-546.

15. Minitab| Release 10 for Windows, State College, PA, 1994.

16. Caple lW, Edwards JA, Forsyth WM, Whiteley P, Sefth RH, Fulton L J: Blood glutathione peroxidase activity in horses in relation to muscular dystrophy and selenium nutrition. Australian Vet J 1978;54:57-60.

17. Roneus B: Glutathione peroxidase and selenium in the blood of healthy horses and foals affected by muscular dystrophy. Nord Vet-Med 1982;34:350-353.

18. Blackmore D J, Campbell C, Dant C, Holden JE, KentJE: Selenium status of Thoroughbreds in the United Kingdom. Equine Vet J 1982;14:139-143.

19. Hoekstra WG: Glutathione peroxidase activity of animal tissues as an index of selenium status. In: (Hemphill DD, ed.),Trace Substances in Environmental Health, Proceedings, University of Missouri, 9th Annual Conference on Trace Substances in Environmental Health, 1976;pp.331-337.

20. Ganther HE, Haferman DG, Lawrence RA, Serfass RE, Hoekstra WG: Selenium and glutathione peroxidase in health and disease - a review. In: (Prasad AS, ed.). Trace Elements in Human Health and Disease Vol II, New York:Academic Press 1976;pp. 165-234.

21. UIIrey DE: Biochemical and physiological indicators of selenium in animals. JAn Sci 1987;65:1712-1726.

22. Stowe HD: Serum selenium and related parameters of naturally and experimentally fed horses. J Nutr 1967;93:60-64.

23. Shellow JS, Jackson SG, Baker JP, Cantor AH: The influence of dietary selenium levels on blood levels of selenium and glutathione peroxidase activity in the horse. J An Sci 1985;61:590-594.

24. Dill SG, Rebhun WC: White muscle disease in foals. Compend Cont Educ 1985;7:$627-$635.

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