Mineral status in beef cattle, live weight changes and immune response as related to selenium and vitamin E supplementation

Two experiments one in 1988 and one in 1989 were conducted to evaluate the mineral status of grazing cattle on two farms in north Florida. Lymphocyte blastogenic response, live weight gain and body condition scores were also evaluated in Experiment 2 as related to the supplemental Se and vitamin E provided in this experiment.

In Experiment 1 serum mineral concentrations of cows presented monthly variations, while in calves mineral concentrations varied as a function of location and/or month of sampling. In Experiment 2 serum mineral concentrations for both cows and calves varied simultaneously as a function of location and sampling month. Variations in serum concentrations were not consistent from year to year or from cows to their calves. With the exception of P for cows in both experiments, and based on the mean concentrations, serum mineral status of both cows and calves was considered adequate in both experiments which may be accounted for by the free-choice consumption of mineral supplement provided to all animals, since soil and forage data showed several minerals to be low to deficient.

Neither Se nor vitamin E had any effect on lymphocyte blastogenic response with any of the mitogens evaluated; however, the health status of cattle was also satisfactory on both farms during the time the experiment was conducted. Cows lost weight from March to October, but BCS in October were slightly higher than in March. Selenium supplemented cows at Williston were the only cows gaining weight from June-October and their calves had higher ADG than controls.

In many areas of the world cattle production depends almost exclusively on forages to provide all required nutrients. Under these circumstances, animal productivity is often seriously limited due to deficiencies or imbalances of essential nutrients. Langlands (1987) indicated that tissues and blood are more reliable means of evaluating mineral status of grazing ruminants than forages due to soil contamination, variability in diet selection or availability of ingested nutrients. Underwood (1981) indicated that mineral concentrations of serum or tissues consistently higher or below the 'normal' concentrations or ranges provide suggestive but not conclusive evidence of dietary deficiency or excess of particular minerals. He also indicated that the choice of tissue or fluid for analysis depends upon the mineral element under investigation. Body condition score (BCS) or changes in body condition is an indicator more reliable in evaluating nutritional status than live weight or changes in live weight and as a result BCS is a useful technique in making management decisions (Kunkle and Sand 1990).

Vitamin E and Se play important roles in the functioning of the immune system. Cell-mediated immunity and humoral immune response were higher in vitamin E supplemented calves (Reddy et al 1986). Selenium supplementation enhanced antibody response in weaned beef calves fed a Se deficient diet (Swecker et al 1989). Supplemental Se along with Vitamin E enhanced serum antibody response to Pasteurella hemolytica in steers new to the feed lot environment (Droke and Loerch 1989). Also, improved disease resistance was reported in dairy cattle with Se and vitamin E supplementation (Smith et al 1985).

Several disease problems were reported in beef cattle grazing native pastures on sandy soils near Bronson, Fla.; however, at a different location on soils with a more clay type of texture (Archer and Williston) the same problems have not been recorded. The disease problems included severe scours and pneumonia, as well as a disease condition resembling shipping fever in calves, and Heinz body anemia in cows. At both locations, soil and forage data indicated mineral deficiencies (Cuesta et al 1992).

The objectives of this study were to evaluate the mineral status of grazing beef cattle, changes in live weight and BCS as well as the immune responsiveness in terms of mitogen-driven blastogenic response as related to Se and vitamin E supplementation.

Two experiments were conducted in north Florida on two farms located near Bronson and Williston, utilizing pregnant crossbred beef cows over 3 years of age. Cows were ¼ to ½ Angus, crossbred with Hereford, Charolais and Brahman. Calving season was from April to June. Animals grazed on bahia grass (Paspalum notatum) pastures throughout the experimental period and received a mineral supplement that contained monensin. Analysis of the free-choice mineral supplement provided to the control animals was: Ca, 15.7%; P, 6.3%; Mg, 2.0%; K, 0.3%; Na, 11.0%; Mn, 2400 ppm; Fe, 7300 ppm; Zn, 2640 ppm; Cu, 448 ppm; Co, 16.4 ppm; Mo, 10.8 ppm; and Se, 0.8 ppm.

In June 1988, 48 pregnant cows were randomly assigned to two treatments at each farm as follows: 1) Control (no supplemental Se) and 2) Supplemental Se. Selenium as sodium selenite was administered as part of the free-choice mineral supplement to provide 23 ppm Se. The average consumption of the mineral supplement was 65 g/h/d and the Se consumption was 1.5 and 0.052 mg/h/d for the Se treated and control animals, respectively.

In March 1989 additional treatments with and without vitamin E were included in combination with each level of Se. Eighty pregnant cows was randomly assigned to the four treatments at each farm as follows: 1) Control (without supplemental Se or vitamin E), 2) Vitamin E, 3) Se and 4) Se + vitamin E.

Vitamin E was administered in boluses of 20 g/cow of a commercial product which provided 10,000 IU of I-tocopherol, administered, in March and June 1989. The Se treated animals at Bronson also received Se as sodium selenate in the drinking water from March to July 10, 1989. The average consumption of the mineral supplement was 65 g/h/d. Combining Se from both the mineral supplement and water, the average Se consumption, other than pasture would be 2.1 and 1.5 mg/h/d for the Se treated groups in Bronson and in Williston, respectively.

For both experiments, blood samples for mineral analysis were taken by jugular puncture in two 15 ml vacutainers. Cows and calves were sampled twice, in June and October of each year, and cows were sampled additionally in March 1989. These samples were centrifuged at 700 g for 20 min and were processed and analyzed according to the procedures described by Fick et al (1979). Serum samples were deproteinated with 10% trichloroacetic acid (TCA) and 1% lanthanum chloride and analyzed for mineral content according to procedures described by Fick et al (1979). Calcium, Mg, Cu, Zn and Fe were analyzed by atomic absorption spectrophotometry with a Perkin-Elmer 5000 (Perkin-Elmer 1980). Phosphorus concentration was determined using a colorimetric procedure described by Harris and Popat (1954).

Blood samples for lymphocyte blastogenic response were obtained from the cows in March, June and October 1989, simultaneously with the blood samples for mineral analysis. These samples were collected by jugular puncture into 60 ml blood collection tubes using acid citrate as anticoagulant. After collection blood samples were placed in a cooler with ice and brought to the Immunology laboratory, where they were processed the following day. Blood samples were centrifuged at 700 g for 20 min and white blood cells (WBC) were then harvested with a Pasteur pipette and mixed with phosphate-buffered saline (PBS) for a total volume of 10 ml. The WBC and the medium were then layered on 5 ml Ficoll/Hypaque and centrifuged at 700 g for 40 min. Lymphocytes sedimented at the interface between ficoll and medium were collected with a Pasteur pipette, washed twice with RPMI-1640 medium and then resuspended in RPMI-1640 supplemented with fetal bovine serum + PBS and antibiotics (penicillin-streptomycin). Cells were enumerated in a hemocytometer and the suspension was adjusted to contain 2,000,000 cells/ml. Fifty Tl of this cell suspension (200,000 cells/ml) was aliquoted, in triplicate, in 96-well tissue culture plates. Fifty TL of the mitogen solution was then added. The volume in each well was adjusted to 200 TL by the addition of 100 TL of medium RPMI plus bovine serum. The concentration of mitogens used were phytohematoglutinin (PHA) 5 Tg/ml, Concanavalin A (Con A) 1 Tg/ml, and recombinant human IL-2 10 U/ml; control cultures without mitogens were used as negative controls. The culture plates were incubated at 370C in a 5% carbon dioxide in air, humidified incubator for 48 hours. Methyl (tritium labeled) thymidine (1 TCi/culture) was added during the last 18 hours of incubation. Cultures were stored at -200C prior to harvesting. Samples were harvested on filter paper using an automated cell harvester, and filters counted using a liquid scintillation counter to determine the incorporation of tritium labeled thymidine into peripheral blood leukocytes.

Lymphocyte stimulation index was calculated as the ratio of counts per minute (CPM) from mitogen stimulated leukocytes over the control (no mitogen) culture.

Live weight of cows and calves as well as BCS of cows was taken at each sampling time. Live weight changes of cows were calculated by difference between sampling dates as follows: March-June, June-October and March-October. Live weight changes of calves were calculated as average daily gain (ADG) for the June to October period. Body condition scores were evaluated based on a 1 to 9 scale (Kunkle and Sand 1990).

Data obtained in these experiments were analyzed statistically using Repeated Measures Analysis of Variance (Freund et al 1986; Gill and Hafs 1971; Milliken and Johnson 1984) with the General Linear Models (GLM) procedure of the SAS System (SAS Institute Inc. 1987). Due to the unequal subclass numbers, least squares means were calculated for all the response variables evaluated.

Mean serum mineral concentrations of cows (Table 1) and calves (Table 2) as related to location and sampling month are shown. With the exception of P and Cu, all serum mineral concentrations in cows presented seasonal variation. Higher (P<0.01) serum concentrations of Mg and Zn were found in October than in June. Serum mineral concentrations were similar for both locations (P>0.05).

Table 1: Serum minerals of cows as related to location and sampling month (Experiment 1)

		June		October
Element	Location	Mean	SE	Mean	SE	CL

P, mg/dl	Bronson	4.2	0.29	3.4	0.24	4.5
	Williston	4.0	0.29	3.9	0.24
	Mean	4.1		3.7
Mg, mg/dl	Bronson	2.2	0.09	2.3	0.05	2
	Williston	2.0	0.09	2.2	0.05
	Mean	2.1**		2.3*
Cu, ppm	Bronson	0.69	0.06	0.71	0.04	0.65
	Williston	0.66	0.06	0.68	0.04
	Mean	0.68		0.70
Zn, ppm	Bronson	0.70	0.04	0.98	0.04	0.6
	Williston	0.73	0.04	0.92	0.04
	Mean	0.71**		0.94*

Least squares means of cows are based on 20 samples.
CL = Critical level (McDowell 1985; McDowell 1987).
*, ** Means having different superscripts in rows differ P<0.05.

Serum mineral concentrations of calves (Table 2) varied with locations and sampling months. Calves at Bronson had higher (P<0.01) serum concentrations of P and higher (P<0.05) Cu and Zn than at Williston. Only in June was serum Mg of calves at Bronson higher than at Williston. Serum P and Cu were higher (P<0.05) in June than in October.

Table 2: Serum minerals of calves as related to location and sampling month (Experiment 1)

		June		October
Element	Location	Mean	SE	Mean	SE	CL

P, mg/dl	Bronson	9.6	0.26	6.5	0.21	4.5
	Williston	8.2	0.28	5.6	0.23
	Mean	8.9*		6.1**
Mg, mg/dl	Bronson	2.1	0.08	2.1	0.04	2.0
	Williston	1.8	0.09	2.1	0.04
	Mean	1.9**		2.1*
Cu, ppm	Bronson	0.82	0.07	0.65	0.04	0.65
	Williston	0.60	0.07	0.54	0.05
	Mean	0.71*		0.60**
Zn, ppm	Bronson	1.18	0.05	1.06	0.04	0.6
	Williston	0.92	0.06	0.96	0.04
	Mean	1.05		1.01

Least squares means of cows are based on 20 samples.
CL = Critical level (McDowell 1985; McDowell 1987).
*, ** Means having different superscripts in rows differ P<0.05.

On the average, only cows were P deficient. Values ranged from 3.43 to 4.22 mg/dl with 74% of the samples below the critical level of 4.5 mg/dl suggested by McDowell (1985). Similar concentrations were reported by Espinoza et al. (1991), with a range of 3.2 to 5.4 mg/dl. On the average, serum Mg concentrations of calves at Williston were low, and 24% of the samples from both cows and calves were below the critical level of 2.0 mg/dl suggested by the NCMN (1973). Similar concentrations were reported by Espinoza with a range of 1.7 to 2.3 mg/dl.

On the average only calves at Williston were Cu deficient, however, 30% of the samples from cows and 43% from calves were below the critical value of 0.65 ppm suggested by McDowell and Conrad (1977). Similar concentrations were reported by Espinoza et al. (1991a) with a range of 0.76 to 1.12 ppm and Merkel et al (1990) with 0.59 ppm in Charolais cattle in Florida. Only 13% of the serum samples from cows were below the critical level of 0.6 to 0.8 ppm of serum Zn suggested by McDowell (1987). Similar concentrations were reported by Espinoza et al (1991) with a range of 0.44 to 0.97 ppm and Merkel et al (1990) with 0.77 ppm.

Mean serum mineral concentrations of cows (Table 3) and calves (Table 4) as related to location and sampling month are shown. Serum concentrations of all minerals in cows varied with locations and sampling month; with most of the variations found in October. At Williston cows had higher (P<0.01) serum Mg than at Bronson in June and October. Only in June serum P at Bronson was higher (P<0.05) than at Williston. Concentrations of serum Zn at Williston were higher than at Bronson in June, but in October they were lower (P<0.01).

Table 3: Mean serum mineral concentrations of cows as related to location and sampling month (Experiment 2)

		March		June		October
Element	Location	Mean	SE	Mean	SE	Mean	SE

P, mg/dl	Bronson	3.2	0.10	3.5	0.12	3.6	0.14
	Williston	3.5	0.10	3.2**	0.12	3.9**	0.14
Mg, mg/dl	Bronson	2.0**	0.04	1.8	0.04	2.0*	0.03
	Williston	2.2*	0.04	1.9	0.04	2.0**	0.03
Cu, ppm	Bronson	0.92	0.03	0.75	0.03	1.02**	0.02
	Williston	0.91	0.03	0.80	0.03	1.19*	0.02
Zn, ppm	Bronson	0.62	0.03	0.83**	0.03	0.58*	0.02
	Williston	0.64	0.03	0.92*	0.03	0.48**	0.02

Means based on 40 samples each location.
CL = Critical levels (McDowell 1985; McDowell 1987) are as follows: Ca, 8 mg/dl; P, 4.5 mg/dl; Mg, 2.0 mg/dl; Cu, 0.65 ppm; Zn, 0.60 ppm; Fe, 1.0 ppm.
*, ** Means in the same month differ P<0.05.

Serum mineral concentrations of calves (Table 4) also varied as a function of the interaction between location and month, except for P and Mg. Higher (P<0.01) serum concentrations of P and Mg were recorded in October than in June, regardless of the location. Serum Cu was lower (P<0.01) in both months at Bronson. Serum Zn at Bronson was higher (P<0.05) only in June. Serum Fe at Williston was higher (P<0.01) than at Bronson in June, but lower (P<0.01) in October.

On the average, only cows were deficient in serum P with 86% of the samples below the critical level of 4.5 mg/dl suggested by McDowell (1985). Mean serum P concentrations ranged from 3.17 to 3.88 mg/dl in cows and 6.31 to 7.31 mg/dl in calves. Similar values for cows were reported by Espinoza et al (1991) with a range of 3.2 to 5.4 mg/dl. On the average, only in June were cows and calves low in serum Mg, and also at Bronson cows were low in October; 38% of the samples from cows and 30% from the calves were below the critical level of 2.0 mg/dl suggested by the NCMN (1973).

Table 4: Mean serum mineral concentrations of calves as related to location and sampling month (Experiment 2)

		June		October
Element	Location	Mean	SE	Mean	SE	CL

Ca, mg/dl	Bronson	11.5*	0.11	14.0*	0.12	8.0
	Williston	7.7**	0.11	12.6**	0.11
P, mg/dl	Bronson	7.1	0.15	7.3	0.18	4.5
	Williston	6.3	0.14	7.0	0.17
Mg, mg/dl	Bronson	2.0*	0.04	2.1*	0.03	2.0
	Williston	1.8**	0.03	2.0**	0.03
Cu, ppm	Bronson	0.31**	0.03	0.95**	0.03	0.65
	Williston	0.72*	0.03	1.05*	0.02
Zn, ppm	Bronson	1.32*	0.05	0.75	0.02	0.6
	Williston	1.18**	0.04	0.75	0.02

Least squares means of calves are based on 30 to 33 samples.
CL = Critical level (McDowell 1985; McDowell 1987).
*, ** Means in the same month differ P<0.05.

On the average, only in June were calves deficient in serum Cu; but 10% of the samples from cows and 30% from calves were below the critical level of 0.65 ppm suggested by McDowell and Conrad (1977). Mean serum Cu ranged from 0.75 to 1.19 ppm in cows and 0.31 to 1.05 ppm in calves. These concentrations are similar to the values reported by Merkel et al (1990) with 0.59 ppm and Espinoza et al (1991) with a range of 0.76 to 1.12 ppm. On the average only cows were low in serum Zn in October; however, 23% of the samples from cows and 7% from calves were below the critical level of 0.6 to 0.8 ppm suggested by McDowell (1987). Mean serum Zn ranged from 0.48 to 0.92 ppm in cows and 0.75 to 1.32 ppm in calves. These concentrations are similar to the values of 0.77 ppm reported by Merkel et al (1990) and a range of 0.44 to 0.97 ppm reported by Espinoza et al (1991). Mean serum mineral concentrations lower than the critical levels were found more often in cows than in calves as follows: P in both experiments, Ca in June for Experiment 1 and Mg in June of Experiment 2. At Williston serum Zn was also lower than the critical levels in October in Experiment 2. Serum Cu was lower in calves in June in Experiment 2. The above information indicates that most of the mineral deficiencies recorded in these experiments were seasonal. With the exception of P, on the average, serum mineral status was relatively satisfactory as a result of providing the free-choice mineral supplement to all the animals, since several minerals were deficient in the forage.

Lymphocyte blastogenic response as related to Se and vitamin E and month of sampling are shown in Table 5. Mitogenic response to PHA and Con A varied with month of sampling and the interaction between vitamin E and location. Vitamin E treated cows at Williston had lower blastogenic response with both PHA (P<0.05) and Con A (P<0.01) than vitamin E untreated cows. At Bronson with vitamin E untreated cows, Con A had a lower response than at Williston. Variations in mitogenic response to IL-2 were due to vitamin E and the interactions between Se and month, and location and month. Vitamin E supplemented cows had lower (P<0.01) response to IL-2 than non control cows (7.76 vs 9.68). Only in March did Se treated cows have higher (P<0.01) response to IL-2 than Se untreated cows. Cows at Williston had lower (P<0.01) mitogenic response to IL-2 in June, but higher (P<0.01) in October than at Bronson. Reddy et al (1987) reported higher lymphocyte blastogenic response to T and B-cell mitogens in dairy calves supplemented with vitamin E (125-500 IU/ animal) than control calves. Parenteral Se supplementation of lambs with barium selenate increased response of blood lymphocytes to PHA and pokeweed mitogens in vitro, while plasma from untreated lambs consistently had lower response (Turner et al 1985).

Table 5: Lymphocyte blastogenic response with PHA, Con A and IL-2 as related to selenium (Se), vitamin E (VE) and month of sampling

		PHA		Con A		IL-2

Month		+ VE	- VE	+ VE	- VE	+ VE	- VE
March	+ Se	39.95	36.73	60.40	68.72	21.29	24.65
	- Se	16.55	26.24	36.13	60.97	8.22	14.66
June	+ Se	35.57	35.83	45.49	42.95	8.15	7.69
	- Se	32.56	29.56	48.65	35.43	8.25	7.68
October	+ Se	43.43	58.14	57.67	98.13	4.51	5.08
	- Se	46.59	83.58	63.92	134.09	4.18	7.70
SE (+ Se)		0.49	0.50	0.43	0.45	0.38	0.40
SE (- Se)		0.49	0.50	0.43	0.43	0.40	0.40
Significance		LVE,	M**	L VE*,	M**	VE, Se M*,	L M**

* P < 0.05.
** P < 0.01.
Lymphocyte stimulation index with mitogens: Concanavalin A (Con A), Phytohematoglutinin (PHA) and human recombinant Interleukin-2 (IL-2).

In this study, it appears that neither vitamin E nor Se had any effect on the lymphocyte blastogenic response with any of the mitogens evaluated. However this experiment was conducted under field conditions and certain factors which might have had some effect on the response were not measured or under direct control such as differences in nutritive value and amount of herbage available, as well as management of the animals. In the case of vitamin E, perhaps closer and more frequent sampling could provide better information on the effect of supplemental boluses or how often the boluses should be administered to the animals under field conditions. On the other hand the report of disease problems at the end of the experiment indicated four cases of scours at Bronson in early April with death in one case.

Cows at Bronson were heavier (P<0.01) than at Williston (404 vs 352 kg) (Table 6). Also, cows treated with Se had higher (P<0.10) initial live weight than the controls (386 vs 370 kg).

Variations in live weight changes were due to the location by Se interaction (P<0.01) in the March-June and June-October periods, but for the whole experiment (March-October) these changes were due to the location-Se-vitamin E interaction (P<0.05). However, the analysis of variance performed by location showed no differences due to the Se-vitamin E interaction.

All cows lost weight in the March-June period, except control cows at Bronson which gained 5.4 kg; however, Se treated cows lost more weight (P<0.05) than the controls (Table 6). Selenium treated cows in June-October at the Williston location gained 14 kg; while the control cows lost weight (P<0.05), and both treated as well as the control cows lost weight at the Bronson location.

Table 6: Mean changes in live weight and condition scores of cows as related to locations, selenium and periods (Experiment 2)

		Bronson		Williston
		Mean	SE	Mean	SE

Period		Weight Changes, kg
March-June	+ Se	-51.5**	4.9	-13.7**	3.3
	- Se	5.4*	4.7	-1.8*	3.1
June-October	+ Se	-12.4	4.4	14.3*	4.1
	- Se	-13.3	4.3	-16.2**	3.8
March-October	+ Se	-64.0**	5.3	-1.4*	4.6
	- Se	-8.2	5.0	-17.9**	4.2
		Condition Score
March	+ Se	4.2**	0.16	4.6**	0.17
	- Se	4.6**	0.15	4.7**	0.16
October	+ Se	5.1**	0.13	4.4***	0.13
	- Se	5.1**	0.12	5.0**	0.12

Least squares means in kg of live weight.
** *** Means within the same period or month having diferent superscript differ P<0.01.

For the whole experiment (March-October) Se treated cows lost more weight at Bronson than the controls; however, at Williston, the reverse was true with less weight loss in the Se treated cows. Higher loss of weight in the March-June period was expected since most of the cows calved during this period. The continuous loss of weight during the June-October period may reflect the effects of lower availability and nutritive value of the forage, since bahiagrass quality is often low in July-September, becoming unpalatable and low in feeding value with increasing maturity (Chambliss and Sollenberger 1991).

Body condition scores of cows at the beginning of the experiment (March) were similar (P>0.05) for both locations and Se levels. They ranged from 4.2 to 4.7. In October Se treated cows at Williston had lower (P<.01) BCS than the controls (4.4 vs 5.0), while both Se treated and control cows at Bronson had 5.1. Despite the high loss of weight in some cows from March-October (Se treated cows at Bronson and controls at Williston), mean BCS of most treatments was better in October than in March, with 5.0 BCS or higher, which is a desirable BCS of productive beef cows under normal grazing management conditions (Kunkle and Sand 1990).

Live weight of calves was similar in June (P>0.05) for both locations (182 and 174 kg at Bronson and Williston, respectively). Selenium treated calves had higher (P<0.05) ADG at Williston than the controls (Table 7), but no differences in ADG were found due to Se levels at Bronson. The higher ADG in the Se treated calves at Williston also coincides with the higher weight gains obtained by their mothers during that period (June-October), as compared to the control cows which lost weight.

Table 7: Average daily gain of calves as related to locations and selenium (Experiment 2)

	Bronson		Williston
	Mean	SE	Mean	SE

+ Se	750	46	849*	36
- Se	812	35	760**	32

Least squares means in grams/d from June to October 1989.
* ** Means within the same column having different superscripts differ P<0.05.

Mineral status and response to supplemental Se and vitamin E were evaluated for grazing beef cattle in north Florida. Serum mineral concentrations for both cows and calves varied simultaneously as a function of location and sampling month. Neither Se nor vitamin E had any effect on lymphocyte blastogenic response with any of the mitogens evaluated. At one of the two locations, Se-supplemented cows gained weight from June-October and their calves had higher average daily gains than controls. With the exception of the P, serum minerals were considered generally adequate, which was likely due to consumption of a free-choice mineral supplement.