Serum Retinol and Beta-Carotene Concentrations in US Dairy Cows

Serum Retinol and Beta-Carotene Concentrations in US Dairy Cows

T.H. Herdt†, W. Seymour‡

†Department of Large Animal Clinical Science and Diagnostic Center for Population and
Animal Health, Michigan State University

‡Roche Vitamins, Inc.

Beta-carotene is a carotinoid present naturally in plant tissue. In un-supplemented
herbivore diets it serves as the primary source of vitamin A activity. In most modern
livestock diets, however, vitamin A is added as a supplement, usually in the form of
retinyl acetate. This raises the question of the role of beta-carotene in dairy cow diets in
which adequate vitamin A has been added as a supplement.

There is some evidence that beta-carotene may have nutritional roles in addition to being
a precursor for vitamin A. Specifically, dietary beta-carotene may benefit animal health
and performance by serving as an antioxidant, enhancing immune function, and by
promoting steroidogenesis, the latter especially within the corpus luteum. The strength of
evidence supporting the importance of these functions in cattle is variable (Weiss, 1998).
Studies with dairy cattle have revealed either positive or no effects of supplemental beta-
carotene on reproduction and mammary gland health (Weiss, 1998). From a recent study
(Arechiga et al., 1998) designed to test effects of beta-carotene in cattle under heat stress,
a significant increase in milk production of cows supplemented with 400 mg/day beta-
carotene was reported in each of three experiments, regardless of ambient temperature.
Other recent information from in vitro studies suggests that beta-carotene may stimulate
the production of progesterone from the corpus luteum (Arikan and Rodway, 2000),
although the results appear variable (O’Shaughnessy and Wathes, 1988).

The variability in both in vivo and in vitro responses to supplemental beta-carotene in
cattle may be associated with variation in the beta-carotene status of the animals, prior to
supplementation or tissue collection. In order to better evaluate the potential benefit of
beta-carotene supplements, more data is needed concerning the beta-carotene status of
US dairy cattle. With that as a goal, the objective of this study was to determine
descriptive statistics for serum beta-carotene concentrations in a random sampling of
healthy US dairy cattle. In addition, the sampling design allowed us to examine for
regional differences, within a specific time period. Furthermore, the relationship between
serum beta-carotene and retinol concentrations was evaluated.

Materials and Methods

Samples analyzed were from the 1996 National Animal Health Monitoring System study
of US dairy herds. For this study, herds were selected from four regions across the
country, Northeast (NY, PA, VT), Midwest (MN, WI, MI, IA, IN, IL, OH, MO), West
(CA, OR, WA, ID, NM, TX), and Southeast (FL, KY, TN). The selection of herds was
designed to reflect the size of the dairy cow population in each area such that total
sampling was representative of the US dairy cow population (NAHMS, 1996). On each

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farm, cows from the entire population of healthy adults were randomly selected for blood
sampling. Cows designated as sick, or that were scheduled for culling were not included
in the samples analyzed in this study. The blood samples used in this analysis were
collected in April or May of 1995. The samples were frozen at –80°C during storage.
Herds were designated as being “pastured” if at sometime during the year dry or lactating
cows received and estimated 90% or more of their forage from pasture. No record was
made of whether or not the cows from which blood samples were taken were currently at
pasture. Herds were further classified by size; 1 – 1 to 49, 2 – 50 to 99, 3 – 100 to 299,
and 4 – 300+.

The data analyzed for this report were not part of the original study design, so we were
using the sample amount that remained after the designed analyses were completed.
Therefore, in some cases there was not sufficient sample volume for beta-carotene and
retinol analysis.

Beta-carotene and retinol were extracted from the samples with an equal volume of
ethanol and twice the sample volume of hexane. Quantification was by a single,
modified-reverse-phase HPLC procedure employing a C18 column and a mobile phase of
acetonitrile, methylene chloride, and methanol (70:20:10). Detection was by absorbance
at 225 nm (retinol) and 450 nm (beta-carotene). A commercial kit (Sigma) was used to
determine cholesterol concentrations in serum.

Statistical evaluation was by a step-down procedure using a mixed-model ANOVA. (Proc
Mixed, SAS). Initial fixed independent variables were region, pasture, and herd size.
Herd was analyzed as a random variable. Serum cholesterol was included in the model as
an independent covariate. A single interaction effect, pasture by region, was also
included in the initial model. Variables were eliminated from the model if the
significance (P) value for their effect in the aggregate model was greater than 0.2.

Results

Descriptive statistics
Samples with suitable volume for analysis were obtained from 358 animals distributed
among 35 herds. There were 11 herds in the Midwest, 10 herds in the Northeast, 7 herds
in the Southeast, and 7 herds in the West. The modal number of animals tested per herd
was 10, with a range of 7 to 18. The herds were distributed approximately equally
between pastured (17) and non-pastured (18). Herds were also distributed approximately
equally among size classifications: 1 – 8, 2 – 10, 3 – 9, and 4 – 8. However, there was
not even distribution of pastured herds among herd size classifications, as illustrated in
figure 1. Thus, herd-size and pasture effects were confounded.

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Figure 1. Bars are stacked by herd size, as indicated by shade of gray. The
grouping of regions is by pasture (Y – yes, N – no). Note that all but one of the
large herds were not pastured, while the large majority of the small herds were
pastured, confounding the effects of herd size and pasture.

The distribution of serum beta-carotene concentrations is in figure 2. Note that the
distribution is strongly skewed to the right, and is not normal. This distribution is
characteristic of many biological variables in which there is a lower limit (zero) but no
rigidly controlled upper limit. Log transformation results in an approximate
normalization of the distribution, as illustrated in figure 3.
45
40
35
30
25
20
Frequency 15
10
5
0
0
6
1.2 2.4 3.6 4.8
7.2 8.4 9.6
12
10.8
More
Beta-carotene (ug/ml)

Figure 2. Frequency distribution of untransformed beta-carotene values.

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35
30
25
20
15
Frequency
10
5
0
-1
1
-1.8 -1.4
-0.6 -0.2 0.2 0.6
1.4 1.8 2.2 More
Log Serum Beta-carotene (ug/ml)
Figure 3. Distribution of log-transformed serum beta-carotene
concentrations. This distribution deviates significantly (p<0.05) from
normal, but is far closer to normal than the untransformed data.

The distribution of serum retinol concentrations is in figure 4. This distribution is
normal, which is characteristic of many biological variables that are under homeostatic
control.
50
45
40
35
30
25
20
Frequency 15
10
5
0
20
80 140 200 260 320 380 440 500 560 More
Serum Retinol (ng/ml)
Figure 4. Distribution of serum retinol concentration. This distribution
approximates normal.

Serum beta-carotene concentrations were correlated significantly (p<0.02) with serum
retinol and cholesterol, but the R2 values were low, 0.06 and .025 respectively.

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Scattergams illustrating the association between serum beta-carotene, serum cholesterol,
and serum retinol are in figures 5 and 6.

700
500
450
600
400
500
350
300
400
250
300
200
200
Serum retinol (ng/ml)
150
Serum cholesterol (mg/dl) 100
100
50
0
0
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
Serum beta-carotene (ug/ml)
Serum beta-carotene (ug/ml)
Figures 5 and 6. The association between serum beta-carotene and serum retinol and
cholesterol concentrations. The associations between these variables are not strong,
but note that while low values of beta-carotene are associated with both high and low
values of retinol or cholesterol, no low values of these two variables are associated
with higher values of beta-carotene.

Unadjusted beta-carotene means by region and pasture are in tables 1 and 2.

Midwest
Northeast
Southeast
West
Mean
(µg/ml)
1.24 2.14 2.15 2.67
Standard
0.99 1.35 2.31 2.27
deviation
Table 1. Means and standard deviations of serum beta-carotene concentrations
related to region.

Pastured
Not
pastured
Mean (µg/ml)
2.41
1.68
Standard deviation
2.0
1.64
Tables 2. Means and standard deviations of serum beta-carotene concentrations
related to use of pasture in the herd. It is unknown whether or not the animals from
which the measures were made were receiving pasture at the time of sampling.

In univariate analysis of log-transformed values, the Midwest mean is lower (P<0.05)
than each of the other three regions. None of the other regions are significantly different
from one another. In univariate analysis of log-transformed values, pastured herds have
higher (P<0.05) mean serum beta-carotene than non-pastured herds.

Model building
Normal distribution is an important assumption for analysis of variance. Therefore, the
log transformed beta-carotene values were used for model building. Independent
variables remaining in the final model were herd (P<0.001), cholesterol (P<.001), and

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pasture (P=0.08), although pasture was marginally significant. Herd accounted for the
major portion (68%) of the variation. Variables dropped from the model included region,
herd size, and region by pasture interaction.

Discussion

Beta-carotene is the dietary source of vitamin A in the natural diets of ruminants. In this
study, however, the correlation between serum retinol and serum beta-carotene was weak.
Animals with low serum beta-carotene had a broad range of serum retinol concentrations.
The lack of association between retinol and beta-carotene at the low end of the beta-
carotene distribution probably reflects the addition of retinyl acetate to the diets of many
of the animals sampled, thus making beta-carotene unnecessary as a dietary source of
vitamin A. At the higher end of the serum beta-carotene concentration spectrum, there
were no animals with low serum retinol. This perhaps indicates that when beta-carotene
is available the animals will have adequate vitamin A status.

Beta-carotene, like alpha-tocopherol is carried nearly exclusively in the lipoprotein
fraction of blood serum (Chew et al., 1993). Therefore, the concentration of serum
lipoproteins could be expected to influence serum beta-carotene concentration. Serum
cholesterol also is exclusively a component of the lipoprotein fraction. Therefore,
cholesterol can be used as an estimator of serum lipoprotein concentration. Figure 6
illustrates the relationship between serum cholesterol and beta-carotene. As would be
expected, animals with low serum cholesterol did not have high beta-carotene
concentrations, because there was little lipid fraction into which it could distribute. In
contrast, animals with high serum lipoprotein concentrations, as indicated by high serum
cholesterol, had a broad range of serum beta-carotene concentrations, probably related to
dietary beta-carotene intake.

Beta-carotene is present in large concentration in most fresh forages, but diminishes with
storage. Therefore, it might be expected that animals at pasture would have higher serum
beta-carotene concentrations than those receiving stored forage. However, in this study it
was not known whether or not the animals sampled were receiving pasture at the time of
blood collection, only that some animals on the farm received pasture as their major
forage source at some time of the year. The samples were collected in April and May,
making it unlikely that pasture had been fed for very long in the Midwest or Northeast.
The effect of pasture may have been both from current consumption of pasture, and from
body stores of beta-carotene that had accumulated from previous pasture feeding.

Perhaps the most striking observation in this study was the large variance component due
to herd. This means that environmental factors, likely related to nutrition, have a large
influence on serum beta-carotene concentration. This implies that serum beta-carotene is
readily amenable to manipulation by dietary management. In addition, the general
distribution of serum beta-carotene values was well below the 3 µg/ml concentration
suggested as minimal by some authors (Frye et al., 1991). Therefore, there may be an
opportunity to manage serum beta-carotene in dairy cattle by dietary supplements.
Further epidemiological research is necessary to better elucidate the relationship between
serum beta-carotene concentrations and health and performance variables.

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