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Effect of Ca and P supplementation on the haematological parameters and content of selected minerals in the blood of young farmed fallow deer males (Dama dama)

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The aim of the study was to assess the effect of supplementation of feed rations with increased calcium and phosphorus doses on the haematological parameters and plasma zinc (Zn), phosphorus (P), magnesium (Mg), copper (Cu), calcium (Ca), and iron (Fe) content as well as the body weight and the growth and development of the first antler in farmed fallow deer (Dama dama Linnaeus, 1758). The mean level of erythrocytes (RBC), haemoglobin (HGB), and haematocrit (HCT) was increased in the Ca- and P-supplemented group after the treatment period. The change was statistically significant (p < 0.05) in the case of RBC and HCT. The other haematological parameters (mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and platelet count (PLT)) were reduced. An increase in the Zn content was observed in the plasma of slaughtered animals. The concentration of other minerals (P, Mg, and Cu only in group II receiving a higher level of Ca and P in the feeding dose; Ca and Fe only in group I supplemented with a lower content of Ca and P in a nutritional dose) in blood plasma decreased slightly after the supplementation period and declined further after the slaughter. Noteworthy, there was a significant increase in the plasma Cu and Fe levels in group I in group II, respectively, in the post-supplementation period. No significant differences were observed in the body weight between the groups, but there was a beneficial effect of the higher Ca and P dose in the feed ration for the farmed fallow deer on the length of the first antler (p < 0.05). The antlers of animals in group II were on average 2.3 cm longer than in group I.
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ORIGINAL ARTICLE
Effect of Ca and P supplementation on the haematological
parameters and content of selected minerals in the blood
of young farmed fallow deer males (Dama dama)
Katarzyna Tajchman
1
&Żaneta Steiner-Bogdaszewska
2
&Edyta Kowalczuk-Vasilev
3
&Roman Dąbrowski
4
Received: 21 February 2019 /Accepted: 10 July 2019
#The Author(s) 2019
Abstract
The aim of the study was to assess the effect of supplementation of feed rations with increased calcium and phosphorus doses on
the haematological parameters and plasma zinc (Zn), phosphorus (P), magnesium (Mg), copper (Cu), calcium (Ca), and iron (Fe)
content as well as the body weight and the growth and development of the first antler in farmed fallow deer (Dama dama
Linnaeus, 1758). The mean level of erythrocytes (RBC), haemoglobin (HGB), and haematocrit (HCT) was increased in the Ca-
and P-supplemented group after the treatment period. The change was statistically significant (p< 0.05) in the case of RBC and
HCT. The other haematological parameters (mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean
corpuscular haemoglobin concentration (MCHC), and platelet count (PLT)) were reduced. An increase in the Zn content was
observed in the plasma of slaughtered animals. The concentration of other minerals (P, Mg, and Cu only in group II receiving a
higher level of Ca and P in the feeding dose; Ca and Fe only in group I supplemented with a lower content of Ca and P in a
nutritional dose) in blood plasma decreased slightly after the supplementation period and declined further after the slaughter.
Noteworthy, there was a significant increase in the plasma Cu and Fe levels in group I in group II, respectively, in the post-
supplementation period. No significant differences were observed in the body weight between the groups, but there
was a beneficial effect of the higher Ca and P dose in the feed ration for the farmed fallow deer on the length of
the first antler (p< 0.05). The antlers of animals in group II were on average 2.3 cm longer than in group I.
Keywords Dama dama .Supplementation .Haematology .Minerals .Farming
Introduction
Cervids undergo spectacular physiological changes during the
year. The antler cycle begins in spring with the cast of the old
set. Afterwards, a new antler grows and is cleaned from velvet
at the end of summer (Suttie et al. 1991). However, it has been
evidenced that the first stage of cervid nutrition can exert an
effect on antler size and weight. The correlation between the
skeleton size and the antler weight is one of the longest known
allometric dependencies occurring in animals (Zannèse et al.
2006). The growth rate and size of the first antlers developed
by young males in the second year of life depend on the
quality of food provided by their mothers and by the habitat
and ecological conditions existing during the winter period
(García et al. 1999; Gómez et al. 2008; Janiszewski et al.
2008). Bartoš(1980)aswellasBartošand Bubenik (2011)
have shown that behavioural aspects, e.g. dominance in group
and social structures or other stress factors, can also be a cause
of formation of larger antlers in deer.
Electronic supplementary material The online version of this article
(https://doi.org/10.2478/s11756-019-00310-2) contains supplementary
material, which is available to authorized users.
*Katarzyna Tajchman
katarzyna.tajchman@up.lublin.pl
1
Department of Ethology and Animal Welfare, Faculty of Biology,
Animal Sciences and Bioeconomy, University of Life Sciences in
Lublin, Akademicka 13, 20-950 Lublin, Poland
2
The Witold Stefański Institute of Parasitology, Polish Academy of
Sciences, Research Station in Kosewo Górne,
11-700 Mrągowo, Poland
3
Institute of Animal Nutrition and Bromatology, Faculty of Biology,
Animal Sciences and Bioeconomy, University of Life Sciences in
Lublin, Akademicka 13, 20-950 Lublin, Poland
4
Department and Clinic of Animal Reproduction, Faculty of
Veterinary Medicine, University of Life Sciences in Lublin, Głęboka
30, 20-612 Lublin, Poland
Biologia
https://doi.org/10.2478/s11756-019-00310-2
The length of the first antler in deer is an index trait of the
ontogenic quality and a phenotypic trait that reflects the qual-
ity of nutrition (Gaspar-López et al. 2008). The first antler
grows with an average rate of 1.95 ± 0.05 cm per week, which
is the highest at week 14 (Gaspar-López et al. 2008). The
conditions provided to a young male in the early months of
life exert important effects on the final size and fitness of the
adult male. Intensive antler growth (antlerogenesis) requires a
supply of a large amount of calcium and phosphorus in a very
short time. This demand for the red deer is ca. 100 g/day, while
only ca. 34 g/day is sufficient for efficient growth of skeletal
bones (Cervus elaphus Linnaeus, 1758). These animals are
not able to acquire such an amount of mineral ingredients with
natural nutrition (Chen et al. 2008).
The average percent mineral content in the first antler ofthe
Iberian deer (Cervus elaphus hispanicus Hilzheimer, 1909)
was shown to be 34% Ca, 31% P, and 44% K. Even the best
post-weaning nutrition cannot compensate for the nutrition
obtained during lactation (Landete-Castillejos et al. 2007a).
It has been found that a 3-kg antler contains 536 g of calcium,
348 g of which is absorbed in the final period of antler
mineralisation (Gómez et al. 2012). Landete-Castillejos et al.
(2007b) proved that total antler formation requires displace-
ment of approx. 400 g of Ca and 200 g of P from the skeleton,
which means transport of even 8.4 g of Ca and 2.4 g of P on
each day of the most intensive growth. This demonstrates the
scale of the demand for these minerals (Landete-Castillejos
et al. 2007b;Kuba2014). Calcium is the basic component
of bones and is present in all animal tissues and body fluids.
In terms of the content, phosphorus is the second bone com-
ponent after calcium; its deficiency causes inhibition of
growth and reduction of animal productivity and fertility.
These two components are highly important in Cervidae,
since these animals produce new antlers every year. The peri-
od of antler growth is a time of a rich blood supply, which is a
carrier of nutrients, e.g. minerals required for development of
antlers (Landete-Castillejos et al. 2007b)
In winter, farm fawns are usually fed with farm feed, which
can be supplemented with mineral additives. In turn, they
spend summer in pastures. As in domesticated ruminants, this
change in feeding can be related with a period of adaptation of
rumen microflora and microfauna. This period is frequently
associated with diarrhoea, which may cause changes in the
haematological profile (Kováčet al. 1997). Moreover blood
is a very sensitive indicator of metabolic changes in both the
physiological and pathological status of animals (Weiss and
Ward ro p 2010). In such situations, the haematological indices
facilitate immediate detection of emerging potential anomalies
or changes in the organism (Neumefister et al. 2001). The
influence of many intra- and extrasystemic factors may lead
to disruption of the homeostatic balance in the organism,
which can be reflected in distinct changes in the values of
individual blood components. The aim of the study was to
determine the effect of supplementation with increased doses
of Ca (calcium) and P (phosphorus) in the diet on the haema-
tological parameters and plasma content of Zn (zinc), P (phos-
phorus), Mg (magnesium), Cu (copper), Ca (calcium), and Fe
(iron). The impact of the supplementation on the body weight
as well as growth and development of the first antler in farmed
fallow deer was examined in the study.
Materials and methods
Experimental design
The research was carried out at the Research Station of the
Institute of Parasitology, Polish Academy of Sciences,
Kosewo Górne (Region of Warmia and Mazury; Poland;
53
o
48N; 21
o
23E) in 20162017 (DecemberAugust).
The study involved fawns born in the same year (the same
age). In order to provide dose adjustment for males, the ani-
mals had to be placed in different enclosures, thus modifying
the social environment. Both research groups had the same
social structures and were influenced by the same stress fac-
tors. It has been demonstrated that fallow deer live in unisex-
ual groups for most of the year (Braza et al. 1990). All animals
were adapted to routine management and maintained in good
health and body condition during the experiment. Handling
procedures and sampling frequency were designed to reduce
stress and health risk for the animals. During the experiment,
no negative behaviour and anomalies were observed. No re-
cord of individual intake of feed was attempted at any stage.
The 24 male fawns in the first year of life were divided into
two equal groups (n= 12) receiving different nutrition for
5months(DecemberApril):
group I - standard nutrition supplemented with a comple-
mentary mineral feed mixture for fawns Cielak B
Plus©from LNB (Cargill, Poland). The supplement
constituted 2.5% of the standard farm nutrition - 6.5 g
(Table 1),
group II - standard nutrition supplemented with a com-
plementary mineral feed mixture for fawns Cielak B
Plus©from LNB (Cargill, Poland). The supplement
constituted 2.5% of the standard farm nutrition - 6.5 g;
it was additionally enriched with monocalcium phosphate
(0.9 g) to increase the Ca and P level up to 25% and 13%,
respectively (Table 1).
The demand for Ca and P in cervids (animals exceeding
60 kg of body weight) is 2.7 g per day and 2.2 g per day,
respectively (NRC 2007). These recommendations are indis-
pensable for animal survival. The antler mineralisation pro-
cess is such a big effort for the organism (Landete-Castillejos
et al. 2012) that the typical diet may be insufficient to cover
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the large demand for Ca and P at this time. At the beginning of
the experiment, the animals from both groups were at the age of
6 months and weighed on average by half less (29.3 kg); there-
fore, the supplementation was adjusted. The first experimental
group (I) received 1.2 g of Ca per day and 0.47 g of P per day,
while the second experimental group (II) received 1.8 g of Ca
per day and 0.9 g of P per day as well as standard farm nutrition.
To increase the concentration of Ca and P in the nutritional
dose, the animals were administered Cielak B Plusfrom
LNB (Cargill, Poland) and monocalcium phosphate.
Measurements
The animals spent the winter (DecemberApril) in a shelter
situated in the wintering ground divided into two identical
rooms, where the amount of feed taken was controlled. The
walls of the shelter were made of horizontal wooden boards
and provided protection against the adverse impact of weather
conditions. In the shelter, there was a feeding rackfor haylage,
water containers, and feeding troughs for concentrated feed.
The concentrate was provided once daily. The animals had
free access to water over the entire wintering period. After
the winter period, the animals stayed on the available pasture
without supplementation (MayAugust). In European coun-
tries (e.g. the Czech Republic, Denmark, France, and Great
Britain), it is common practice that weaned red or fallow deer
calves are wintered indoors from September to the following
spring in order to protect them from aversive weather condi-
tions, reduce expenses for winter feeding, and encourage tam-
ing of the animals (Bartošand Šiler 1993). The animals were
provided with adequate space during the winter and grazing
periods as recommended in DEFRA (2018), FEDFA (2018),
and Mattiello (2009). Body weight was measured with the use
of a set of MP 800 sensors coupled with a Tru-test DR 3000
weight reader. As declared by the manufacturer, the accuracy
of this set is ± 1% and the minimum resolution is 100 g. To
obtain additional information about the ontogeny of the young
fallow deer males, the growing antlers with the pedicle (at the
skull base) were measured when the development was com-
pleted. The measurements were carried out using a tape mea-
sure at the age of 14 months (17 August 2017) and the mean
values of the measurement of both beams were noted.
Sampling
Bloodwassampledfromvena jugularis externa always at the
same time (from 1 to 3 h after dawn) to avoid variations associ-
ated with circadian rhythms before the supplementation period
(28 December 2016), after the supplementation period (20 April
2017), and during slaughter (17 September 2017). There was no
need for sedation and it was carried out as in research Langridge
(1992), García et al. (2002), and Gaspar-López et al. (2011). For
haematological analyses, 5-ml volumes of blood were collected
into vacuum tubes containing an anticoagulant agent (EDTA).
The samples were chilled (48 °C) for 15 min after collection and
analysed 2 or 3 h later. The analyses were carried out with the use
of an automatic haematological analyser Mythic 18.The device
was calibrated each time before the analysis of the samples. The
blood was analysed to determine the levels of leukocytes
(WBC), erythrocytes (RBC), haemoglobin (HGB), haematocrit
(HCT), mean corpuscular volume (MCV), mean corpuscular
haemoglobin (MCH), mean corpuscular haemoglobin concentra-
tion (MCHC), and platelets (PLT).
The blood samples (5 ml) were mixed with heparin as an
anticoagulant. Plasma for analysis of the biochemical param-
eters was obtained by centrifugation of whole blood at
3000 rpm for 10 min in a laboratory centrifuge (MPW-
350R, MPW Medical Instruments, Warsaw, Poland) at a tem-
perature of 4 °C. After centrifugation, plasma Zn, P, Mg, Cu,
Ca, Fe levels were determined using reagent kits (BioMaxima,
Lublin, Poland) according to the manufacturers protocols and
a random access biochemical analyser Metrolab 2300 GL
Table 1 Composition of the dietary supplement provided to farmed
fallow deer fawns
Premix composition Components Group I Group II
Content
(in 1 kg)
Macronutrients
(%)
Calcium (Ca) 18.3 25
Phosphorus (P) 7.2 13
Sodium (Na) 8 8
Magnesium (Mg) 4.3 4.3
Micronutrients
(mg)
Manganese (Mn) 2000 2000
Zinc (Zn) 3750 3750
Iron (Fe) 2900 2900
Copper (Cu) 375 375
Cobalt (Co) 12.5 12.5
Iodine (I) 50 50
Selenium (Se) 12.5 12.5
Vitamins A (j.m.) 1000000 1000000
D3 (j.m.) 200000 200000
E (mg) 1500 1500
K3 (mg) 40 40
B1 (mg) 30 30
B2 (mg) 140 140
B6 (mg) 30 30
B12 (mcg) 600 600
Folic acid (mg) 20 20
Pantothenic acid (mg) 300 300
Niacin (mg) 600 600
Biotin (mcg) 600 600
Choline chloride (mg) 2000 2000
Bold was used to emphasize that you was examined impact Ca and P
supplementation depending on the amount of these minerals in the dose
Biologia
(Metrolab SA, Buenos Aires, Argentina). Haematological pa-
rameters were determined in the first two terms (before and
after the supplementation, i.e. at the beginning of antler
growth in April), since slaughter exerts adverse effects and
the results may not be comparable to those obtained in the
other periods (Marco and Lavín 1999; Kocan et al. 1981).
The plasma mineral content was determined in the three terms
(before the supplementation, after the supplementation at the
beginning of antler growth in April, and post slaughter - after
ossification of antlers). The article is part of the research of a
large scientific project consisting of several studies.
Statistical analysis
The results were subjected to statistical analysis. The analysed
values were presented as a mean value and standard deviation
in the case of measurable parameters and as cardinality and
percentage in the case of non-measurable variables.
The normality of the distribution of variables in the
analysed groups was verified with the Shapiro-Wilk test. The
differences between the two groups were assessed with the
Studentsttest or with the Mann-Whitney test when the for-
mer could not be applied. The comparison of the haematolog-
ical results and plasma mineral concentrations before and after
the supplementation as well as before the supplementation and
post slaughter was performed with a Studentsttest for depen-
dent variables or a paired-samples Wilcoxon rank test when
the conditions for application of the former test were not met.
The comparison of the weight results obtained in the subse-
quent measurements was carriedout with a Friedman ANOVA
analysis, and pairwise comparisons were performed with a
paired-samples Wilcoxon rank test with Bonferroni correction.
A significance level of p< 0.05 was assumed, which indicated
the presence of statistically significant differences or correla-
tions. The database was compiled and statistical analyses were
carried out in the Statistica 9.1 software (StatSoft, Polska).
Results
In the study, many interesting relationships were observed
between the haematological parameters and the content of
selected minerals supplemented in the nutritional dose.
There was a significant increase in RBC in the study groups
in the post-supplementation period: from 11.035 × 10
6
μl
1
to
12.954 × 10
6
μl
1
in group I and from 10.355 × 10
6
μl
1
to
12.942 × 10
6
μl
1
in group II receiving the higher Ca and P
dose in the diet. Similarly, the increase in HCT was higher in
group II, i.e. from 37.983% to 47.200%, than in group I (from
40.481% to 48.127%). In turn, the MCHC value was reduced
from 40.509 g dl
1
to 38.718 g dl
1
in group I and from
42.825 g dl
1
to 39.725 g dl
1
in group II. There were signif-
icant differences in the blood haematological parameters, i.e.
in the level of RBC, HCT, and MCHC (p<0.05),withinboth
groups and between the groups before the winter supplemen-
tation. There were no significant differences at the end of this
period (Table 2). In contrast, the content of RBC and HCT
changed significantly (p< 0.05) in the post-supplementation
period (Table 3). Furthermore, significant differences in the
level of RBC, HGB, HCT, and MCHC were noted between
both study groups before and after the supplementation.
Additionally, there was a significant decline in PLT after the
supplementation in group I (from 457.182 × 10
3
μl
1
to
378.545 × 10
3
μl
1
,p= 0.033). In group II, there was a signif-
icant change in the WBC value (from 4.808 × 10
3
μl
1
to
4.214 × 10
3
μl
1
,p< 0.001) and MCH (from 15.875 pg to
14.458 pg, p=0.014)(Table4).
The plasma mineral content differed significantly between
the study groups in the pre-supplementation period in the case
of Zn (35.821 μmol L
1
in group I and 45.600 μmol L
1
in
group II, p= 0.001), Mg (0.976 mmol L
1
in group I and
0.825 mmol L
1
in group II, p< 0.001), Cu (14.694 μmol L
1
in group I and 13.964 μmol L
1
in group II, p=0.018),andFe
(41.695 μmol L
1
in group I and 32.005 μmol L
1
in group II,
p= 0.003). After the supplementation period, significant differ-
ences were observed only in the case of Cu (20.780 umol L
1
in
group I and 11.953 umol L
1
in group II, p= 0.035). In turn,
there were differences in the levels of Zn (57.783 μmol L
1
in
group I and 49.650 μmol L
1
in group II, p= 0.001) and P
(1.640 mmol L
1
in group I and 1.838 mmol L
1
in group II,
p= 0.038) in the post-slaughter period following the summer
grazing period. Additionally, the plasma Zn content was in-
creased in the post-slaughter period (by 21.922.1 μmol L
1
ingroupIandbyonly4.0514.5 μmol L
1
in group II). The
contentofotherminerals(P,Mg,andCuonlyingroupII;Ca
and Fe only in group I) in the blood plasma in the post-
supplementation period decreased slightly and was even lower
after the slaughter. Noteworthy, there was a significant increase
in the plasma levels of Cu (from 14.694 μmol L
1
to
20.780 μmol L
1
) in group I and Fe (from 32.005 μmol L
1
to 34.475 μmol L
1
) in group II after the supplementation pe-
riod (Table 5). Both groups showed significant changes in the P
and Cu content in the post-supplementation period and in the
Zn, Mg, and Ca levels after the slaughter (Table 6).
Significant changes in the plasma P and Mg content were
demonstrated in both groups in the pre- and post-
supplementation periods and in the post-slaughter period.
Additionally, there was a significant change in the plasma
Zn and Cu levels in group I between the post-
supplementation and post-slaughter periods. The content of
Ca and Fe in the plasma changed significantly in group I in
the pre- and post-supplementation periods as well as the post-
supplementation and post-slaughter periods. The group re-
ceiving the higher dose of Ca and P in the feed ration (II)
exhibited a significant change in the plasma Zn level after
the supplementation (Table 7).
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The determination of the effects of the supplementation
on blood parameters was accompanied by an analysis of
its impact on the body weight and the length of the first
antler in the farmed fallow deer. There were no significant
differences in the body weight of the animals between the
groups, whereas the increased Ca and P content in the
feed ration was shown to exert a significant positive effect
on the first antler length (p< 0.05). The animals from
group II developed on average 2.3 cm longer antlers than
the individuals in group I (Online Resource: Table A1).
Additionally, there were significant correlations in the
case of the body weight in both groups of the farmed
Table 2 Comparison of haematological parameters in farmed fallow deer before the beginning of the supplementation to the end of the
supplementation
Haematological parameters Group Before supplementation After supplementation
MSDt
a
/U
b
/
Z
c
pMSDt
a
/U
b
/Z
c
p
WBC
(10
3
μl
1
)
I 4.749 0.755 0.077
c
0.938 4.417 0.532 1.165
a
0.250
II 4.808 0.705 4.214 0.639
RBC
(10
6
μl
1
)
I 11.035 0.899 2.234
c
0.025* 12.954 0.535 -0.561
c
0.574
II 10.355 1.377 12.942 0.579
HGB
(g dl
1
)
I 16.363 0.978 1.667
c
0.095 18.609 0.733 -1.356
c
0.174
II 16.133 0.745 18.708 0.569
HCT
(%)
I 40.481 2.345 2.938
c
0.003* 48.127 2.845 -0.869
c
0.384
II 37.983 3.602 47.200 2.979
MCV
(fl)
I 36.790 1.392 0.429
c
0.667 37.100 1.066 1.535
c
0.124
II 36.916 1.942 36.433 1.077
MCH
(pg)
I 14.900 1.418 -1.136
c
0.255 14.354 0.181 -1.561
a
0.126
II 15.875 2.494 14.458 0.258
MCHC
(g dl
1
)
I 40.509 2.439 2.502
c
0.012* 38.718 1.000 1.753
c
0.079
II 42.825 4.499 39.725 1.582
PLT
(10
3
μl
1
)
I 457.181 81.158 0.077
c
0.938 378.545 118.994 52.000
b
0.627
II 445.416 175.665 376.250 156.313
For abbreviations WBC, RBC, HGB, HV-CT, MCV, MCH, MCHC and PLT (see material and methods);
a
the Studentsttest result,
b
,
c
Mann-Whitney
test results, M - mean, SD - standard deviation, *values of correlation coefficients are statistically significant at p<0.05
Table 3 Analysis of changes in
haematological parameters of
farmed fallow deer after the
supplementation period
Haematological parameters Group Change after supplementation
MSDt
a
/U
b
/Z
c
p
WBC
(10
3
μl
1
)
I0.331 0.655 1.309
c
0.190
II 0.594 0.582
RBC
(10
6
μl
1
)
I 1.919 0.941 2.277
c
0.022*
II 2.587 1.032
HGB
(g dl
1
)
I 2.245 0.795 1.487
c
0.136
II 2.575 0.961
HCT
(%)
I 7.645 2.720 -1.837
c
0.066
II 9.216 2.309
MCV
(fl)
I 0.309 2.014 0.738
c
0.460
II 0.483 2.837
MCH
(pg)
I0.545 1.477 1.182
c
0.237
II 1.416 2.381
MCHC
(g dl
1
)
I1.790 2.279 1.795
c
0.072
II 3.100 3.244
PLT
(10
3
μl
1
)
I78.636 140.014 1.328
a
0.199
II 69.166 243.356
For abbreviations WBC, RBC, HGB, HV-CT, MCV, MCH, MCHC and PLT (see material and methods);
a
the
Studentsttest result,
b
,
c
Mann-Whitney test results, M - mean, SD - standard deviation, *values of correlation
coefficients are statistically significant at p<0.05
Biologia
fallow deer in almost all the terms of weighing, except for
term 2 and 3 (Online Resource: Table A2,A3).
Discussion
There are few studies on the effect of supplementation on
haematological parameters and mineral concentration in the
plasma of farmed fallow deer. This type of research is impor-
tant for breeders since this species is the most commonly bred
Cervidae in Poland (FEDFA 2018). Commercial deer breed-
ing in Poland began in 2002. As demonstrated by data pre-
sented in 2014 by the Polish Union of Deer Breeders at the
FEDEFA congress, it can be assumed that there are 6000
9000 red deer and 2500030000 fallow deer in Poland
(Janiszewski et al. 2014).
Table 4 Comparison of haematological parameters in farmed fallow deer between group I and II before the beginning of the supplementation to the end
of the supplementation
Haematological parameters Group I Group II
Before supplementation After
supplementation
Before-after Before
supplementation
After
supplementation
Before-after
MSDMSDZ
c
pMSDMSDZ
c
p
WBC
(10
3
μl
1
)
4.749 0.756 4.417 0.532 1.445 0.149 4.808 0.705 4.214 0.639 3.543 <0.001*
RBC
(10
6
μl
1
)
11.035 0.899 12.954 0.535 4.107 <0.001* 10.355 1.378 12.942 0.579 4.286 <0.001*
HGB
(g dl
1
)
16.363 0.978 18.609 0.733 4.107 <0.001* 16.133 0.745 18.708 0.569 4.286 <0.001*
HCT
(%)
40.482 2.345 48.127 2.846 4.107 <0.001* 37.983 3.602 47.200 2.979 4.286 <0.001*
MCV
(fl)
36.791 1.393 37.100 1.066 2.390 0.017* 36.917 1.942 36.433 1.077 0.286 0.775
MCH
(pg)
14.900 1.419 14.354 0.182 1.415 0.157 15.875 2.495 14.458 0.259 2.451 0.014*
MCHC
(g dl
1
)
40.509 2.439 38.718 1.001 3.230 0.001* 42.825 4.499 39.725 1.582 3.879 <0.001*
PLT
(10
3
μl
1
)
457.182 81.158 378.545 118.994 2.126 0.033* 445.416 175.665 376.250 156.313 1.686 0.092
For abbreviations WBC, RBC, HGB, HV-CT, MCV, MCH, MCHC and PLT (see material and methods);
c
Mann-Whitney test results, M - mean, SD -
standard deviation, *values of correlation coefficients are statistically significant at p<0.05
Table 5 Comparison of the content of Zn, P, Mg, Cu, Ca, and Fe in the plasma of farmed fallow deer before the beginning of the supplementation to the
end of the supplementation and in the post-slaughter period
The content of minerals
in plasma
Group Before supplementation After supplementation Post-slaughter
MSDt
a
/U
b
/Z
c
pMSDt
a
/U
b
/
Z
c
pMSDt
a
/U
b
/Z
c
p
Zn
(μmol L
1
)
I 35.821 8.604 96.000
b
0.001* 35.704 8.727 54.000
b
0.722 57.783 1.814 20.000
b
0.001*
II 45.600 10.892 35.186 10.831 49.650 9.894
P
(mmol L
1
)
I 2.775 0.370 152.000
b
0.107 2.142 0.234 0.621
a
0.542 1.640 0.228 36.000
b
0.038*
II 2.618 0.311 2.146 0.478 1.838 0.227
Mg
(mmol L
1
)
I 0.976 0.152 56.000
b
<0.001* 0.726 0.090 32.000
b
0.069 0.558 0.086 52.000
b
0.265
II 0.825 0.067 0.696 0.124 0.560 0.020
Cu
(umol L
1
)
I 14.694 1.851 124.000
b
0.018* 20.780 14.402 28.000
b
0.035* 4.833 0.566 40.000
b
0.068
II 13.964 6.397 11.953 2.160 4.388 0.570
Ca
(mmol L
1
)
I 3.481 0.690 160.000
b
0.159 2.966 0.278 36.000
b
0.122 2.728 1.075 48.000
b
0.178
II 3.021 0.307 2.776 0.234 2.766 0.538
Fe
(μmol L
1
)
I 41.695 12.468 104.000
b
0.003* 39.404 7.345 1.165
a
0.250 27.070 3.793 60.000
b
0.513
II 32.005 4.027 34.475 7.893 34.660 12.805
For abbreviations Zn, P, Mg, Cu, Ca and Fe (see introduction);
a
the Studentsttest result,
b
,
c
Mann-Whitney test results, M - mean, SD - standard
deviation, *values of correlation coefficients are statistically significant at p<0.05
Biologia
In the present experiments, a decline in the WBC count was
observed in both groups in the spring (after the supplementa-
tion period). In group II supplemented with the increased
amounts of Ca and P, this parameter had higher values, i.e.
0.6 × 10
3
μl
1
, than in group I (0.3 × 10
3
μl
1
). This may
indicate a higher rate of antler growth in the fallow deer, which
is in agreement with the findings reported by Gaspar-López
et al. (2011), who showed that the size of antlers was nega-
tively correlated with the WBC count in cervidsblood. As
shown by their results, larger antler sizes were found in ani-
mals that had lower values of the WBC count, MCHM, and
MPV (Gaspar-López et al. 2011; Weiss and Wardrop 2010).
The WBC and antler score were negatively correlated. This
negative relationship between the leukocyte count and antler
size may be explained by the theory proposed by Coop and
Kyriazakis (1999) that the requirements of the immune system
are only covered after the needs of maintenance of body pro-
tein or reproduction. Another possible explanation is the
weaker condition of the first defence line in animals with
smaller antlers, as suggested by Landete-Castillejos et al.
(2002). This would be associated with a higher expense in
the immune system, reducing the amount of resources avail-
able for antler growth (Landete-Castillejos et al. 2007b).
The mean leukocyte count was slightly higher than that
reported by Mohri et al. (2000) in Persian fallow deer
(Dama mesopotamica Brooke, 1875) and Chapman and
Chapman (1982)infallowdeer(D. dama), but it was the same
as the result presented by English and Lepherd (1981),
Zomborsky et al. (1997), and Kováčet al. (1997). The differ-
ences may have been related to the method applied for immo-
bilisation of the animals during blood collection. The use of
physical methods, as in the case of farm fallow deer, may
result in elevated leukocyte counts, in comparison with
chemical methods. As shown by Tomkins et al. (1991), the
diet may be another cause of reduced WBC and elevated RBC
counts. In the present study, there was an increase in the mean
number of erythrocytes in the post-supplementation period; a
higher increase, i.e. by 2.6 × 10
6
μl
1
, was observed in group
II. Increased blood platelet counts are observed in cervids in
spring (Gaspar-López et al. 2011); however, the value of this
parameter measured in the experimental animals in April was
reduced by 78.6 × 10
3
μl
1
in group I and by 69.2 × 10
3
μl
1
in group II. The increase in the haematological parameters as
well as haemoglobin and haematocrit may support the positive
effect of supplementation with an increased amount of Ca and
P in the diet.
The mean number of erythrocytes in the fallow deer blood
was similar tothe value reported by Mohri et al. (2000)aswell
as Chapman and Chapman (1982). Similarly, the level of
haemoglobin was similar to that shown by Mohri et al.
(2000), BarićRafaj et al. (2011), Chapple et al. (1991), and
Cross et al. (1994) and slightly higher than that reported by
Rosef et al. (2004). The value of haematocrit was higher in the
farmed fallow deer in the post-supplementation period and
similar in the pre-supplementation period to the values report-
ed by Mohri et al. (2000), BarićRafaj et al. (2011), Chapple
et al. (1991), and Cross et al. (1994). The MCV parameter was
slightly lower in the farmed fallow deer than that shown by
Cross et al. (1994)andRosefetal.(2004) and higher than the
value demonstrated by Chapple et al. (1991). The MCH value
was lower than in Persian fallow deer (Mohri et al. 2000),
similartothatreportedbyBarićRafaj et al. (2011)and
Cross et al. (1994), and higher than in investigations conduct-
ed by Chapple et al. (1991). The MCHC parameter exhibited
higher values than those presented by Mohri et al. (2000)or
those determined in other cervid species by Audigé (1992),
Table 6 Analysis of changes in the Zn, P, Mg, Cu, Ca, and Fe content in the plasma of farmed fallow deer in the pre-supplementation and post-
slaughter periods
The content of minerals
in plasma
Group Change after supplementation Change post-slaughter
MSDt
a
/U
b
/Z
c
pMSDt
a
/U
b
/Z
c
p
Zn
(uμmol L
1
)
I1.414 10.718 56.000
b
0.821 20.562 5.514 4.000
b
<0.001*
II 9.862 17.521 1.688 9.843
P
(mmol L
1
)
I0.500 0.548 8.000
b
<0.001* 1.055 0.496 38.000
b
0.051
II 0.506 0.271 0.742 0.418
Mg
(mmol L
1
)
I0.334 0.196 52.000
b
0.627 0.475 0.179 14.000
b
0.000*
II 0.091 0.087 0.222 0.072
Cu
(μmol L
1
)
I 6.608 15.857 4.239
a
0.001* 9.108 1.423 52.000
b
0.265
II 1.375 1.570 10.478 8.099
Ca
(mmol L
1
)
I1.048 0.598 44.000
b
0.313 1.090 0.960 32.000
b
0.021*
II 0.191 0.245 0.365 0.910
Fe
(μmol L
1
)
I 6.864 7.633 1.309
c
0.190 6.995 5.756 52.000
b
0.265
II 3.851 8.520 1.890 16.387
For abbreviations Zn, P, Mg, Cu, Ca and Fe (see introduction);
a
the Studentsttest result,
b
,
c
Mann-Whitney test results, M - mean, SD - standard
deviation, *values of correlation coefficients are statistically significant at p<0.05
Biologia
BarićRafaj et al. (2011), Chapple et al. (1991), and Cross et al.
(1994). The mean number of platelets was higher than the
value reported from Persian fallow deer (Mohri et al. 2000)
or shown by BarićRafaj et al. (2011); yet, it was in the range
reported by Cross et al. (1994). These seasonal changes in the
haematological parameters may have been associated with the
changes in the diet, as demonstrated by DelGuidice et al.
(1992). Nevertheless, it should be borne in mind that haema-
tological parameters usually have higher values in young an-
imals up to 18 months of age (Weiss and Wardrop 2010).
Besides haematological parameters, the content of some
minerals in the blood plasma was analysed. The highest con-
centration of most minerals (P, Mg, Cu only in group II, Ca
and Fe only in group I) was detected in the first period, i.e.
before the supplementation was included in the youngest an-
imals. This can be explained by the high activity of osteoblasts
in calves in bone tissue remodelling (Rosef et al. 2004;Kučer
et al. 2013). Additionally, the slight decrease in the plasma
mineral content in the post-supplementation period may have
been caused by the change in the diet and the onset of the
antler development period. However, in contrast to the
present research, Graham et al. (1962)aswellasMorrisand
Bubenik (1983) showed that the concentration of minerals in
theplasmaremainedstableduringantlergrowth.
The increase in the Zn concentration in the plasma of the
farmed fallow deer observed in the post-slaughter period, i.e.
after cleaning off the velvet and ossification of antlers, may
have been related to the approaching mating season. Zn is
responsible for normal testosterone concentrations and proper
function of the immune system (Weiss and Wardrop 2010;
Bartoskewitz et al. 2007). After antler growth and mat-
ing periods, the immune system of cervids can return to
homeostasis, hence the increased uptake of this mineral
in the blood (Kun et al. 2015). This may also result
from the supplementation, as in the investigations con-
ducted by Suresh et al. (2013).
Scharfe et al. (1998) and Chapple et al. (1991)foundaP
concentration of 2.40 mmol L
1
in neonatal fallow deer. A
similar concentration of this mineral was determined in the
plasma of the farmed fawns in the pre-supplementation peri-
od. The value declined in the subsequent terms with the age of
animals. There were no significant differences between the
study groupsdespite the different P content in the diet. In other
studies, the mean concentration of P at unrestricted access was
3.20 mmol L
1
in a population of young red deer and
1.48 mmol L
1
in adult animals. The decline with age is un-
derstandable, as the P demand is higher during early growth
(Kučer et al. 2013).
It is known that the Mg concentration decreases with age
due to stress susceptibility, defective membrane functions, and
disruption of intracellular calcium metabolism, inflammation,
cardiovascular diseases, including atherosclerosis and ische-
mic injury, diabetes, fibrosis, immune dysfunction, and other
Table 7 Comparison of the content of Zn, P, Mg, Cu, Ca, and Fe in the plasma of farmed fallow deer between group I and II before the beginning of the supplementation to the end of the supplementation
and in the post-slaughter period
The
content
of minerals
in plasma
Group I Group II
Before
supplementation
After
supplementation
Before-
after
Before
supplementation
Post-
slaughter
Before-
post-
slaughter
Before
supplementation
After
supplementation
Before-
after
Before
supplementation
Post-slaughter Before-
post-
slaughter
MSDMSDZ
c
/pMSDMSDZ
c
/pMSDMSDZ
c
/pMSDMSDZ
c
/p
Zn
(μmol L
1
)
37.118 6.121 35.704 8.728 0.153
NS
37.221 5.542 57.783 1.815 3.059
0.002*
45.049 11.590 35.187 10.831 1.961
0.049*
51.338 4.084 49.650 9.895 0.784
NS
P
(mmol L
1
)
2.642 0.418 2.142 0.234 2.497
0.012*
2.695 0.398 1.640 0.228 3.059
0.002*
2.653 0.314 2.147 0.478 3.059
0.002*
2.580 0.241 1.838 0.228 3.059
0.002*
Mg
(mmol L
1
)
1.060 0.162 0.726 0.091 2.803
0.005*
1.033 0.159 0.558 0.086 3.059
0.002*
0.788 0.072 0.697 0.124 2.824
0.005*
0.782 0.065 0.560 0.020 3.059
0.002*
Cu
(μmol L
1
)
14.172 1.644 20.780 14.402 0.968
NS
13.941 1.581 4.834 0.566 3.059
0.002*
10.578 1.249 11.953 2.160 2.197
0.028*
14.866 7.902 4.389 0.570 3.059
0.002*
Ca
(mmol L
1
)
4.014 0.434 2.966 0.278 2.803
0.005*
3.818 0.602 2.728 1.076 2.824
0.004*
2.968 0.103 2.777 0.234 2.275
0.023*
3.132 0.404 2.767 0.538 0.470
NS
Fe
(μmol L
1
)
32.540 2.788 39.404 7.345 2.089
0.037*
34.065 4.364 27.070 3.793 3.059
0.002*
30.623 2.539 34.475 7.894 0.471
NS
32.770 4.817 34.660 12.804 0.470
NS
For abbreviations Zn, P, Mg, Cu, Ca and Fe (see introduction);
c
Mann-Whitney test results, M - mean, SD - standard deviation, *values of correlation coefficients are statistically significant at p<0.05
Biologia
diseases associated with aging (Rayssiguier et al. 1993). The
Mg concentration in the farmed fallow deer was similar to that
reported by Kučer et al. (2013), and a significant decline in
this parameter was noted after the grazing period without
supplementation.
Cu deficiency is common in farmed red deer livestock.
Padillaetal.(2000)andBaoetal.(2010), however, detected
low serum Cu (9.86 μmol L
1
) values in young red deer kept
on pasture. Values lower than 8 μmol L
1
are below the crit-
ical level for Cu deficiency (Mackintosh et al. 1987).
Probably, the distinct decline in the plasma Cu content noted
in the post-slaughter period in the farmed fallow deer was
caused by the preceding pasture period, which is sometimes
characterised by deficiencies of this mineral in pasture-kept
animals. In turn, the study conducted by Rosef et al. (2004)
showed a mean Cu value of 13.0 μmol L
1
. A similar level of
this element was noted in the farmed fallow deer in the pre-
supplementation period before the winter.
The mean values of Ca, Mg, and P in the fallow deer blood
were higher than the concentrations described by Marco and
Lavín (1999) and Rosef et al. (2004). This confirms the pos-
itive effect of the supplementation applied during the winter
period. However, the Ca to Mg ratio is important in the ab-
sorption, utilisation, and excretion of these minerals (Somer
1995). Magnesium and calcium act in conjunction; for in-
stance, they are involved in the regulation of nerve and muscle
tone. A high Ca2+/Mg2+ ratio also predisposes to arterial
spasms and increases catecholamine release (Sheehan and
Seelig 1984). Probably, the changes in the Fe concentration
in the blood of the group I fallow deer were induced by an
unfavourable Ca/Mg ratio in the diet. Evidently, the group II
animals received a better-balanced and more beneficial diet
containing an increased content of Ca and P in the feed ration.
At a proper and constant level of nutrition, the concentration
of Ca and P in the blood does not change despite the period of
antler growth (NRC 2007; Gaspar-López et al. 2011).
Proper nutrition is especially important in Cervidae calves,
whose survival of the first winter is determined by achieve-
ment of normal body weight (Fennessy et al. 1991). A com-
parison of the effect of supplementation of feed for farmed
Iberian red deer (C. elaphus hispanicus) demonstrated a sig-
nificant difference in biometric parameters between the con-
trol group and the group of animals receiving enriched diets
(Olguin et al. 2013). The present study showed no effect of the
supplementation on the body weight in the analysed fallow
deer. In other studies of farmed fallow deer, fawns that spent
winter with does were characterised by an average 5.43%
decline in body weight, in comparison with fawns wintered
under a shelter - 10.24% (Janiszewski et al. 2008).
Appropriate conditions provided to animals during the first
winter are essential, as confirmed by the above-mentioned
experiments and the present study, which demonstrated a pos-
itive effect of the supplementation on the body weight and
antler length. Moreover, the final antler size was negatively
related to the leukocyte count and the body condition had
a more important effect on the final antler size, which was
bigger than that exerted by the body weight, as also con-
firmed by Gaspar-López et al. (2011). However, one can-
not forget that the size of antlers can also be influenced by
animal behaviour and their social structure (Bartoš1980;
Bartošand Bubenik 2011).
The increased Ca and P supply contributed to elevation of
the plasma Zn content and improved some haematological
indices, e.g. RBC, HGB, and HCT. Moreover, the increased
doses of Ca and P in the diet exerted a positive effect on the
concentration of minerals in the blood of cervids. Both animal
groups exhibited a significant increase in the plasma Cu and
Fe levels in group I in the post-supplementation period. Thus,
the supplementation of the fawn nutrition had a positive effect
on animal fitness and antler growth.
Compliance with ethical standards
Ethical statement This study was carried out in strict accordance with
the Polish legislation for the use of animals in research (Act of 15 January
2015 on the protection of animals used for scientific or educational pur-
poses). The protocol was approved by the Local Ethics Committee 0069,
Resolution No. 42/2016 at the University of Warmia and Mazury in
Olsztyn. Animals were restrained using a cushioned crush specifically
designed to restrain deer movements - a handling box (2 m × 2 m ×
0.6 m) only for a few minutes. At that time, the fallowdeer were weighed,
the blood was collected, and the antlers were measured.
Conflict of interest No potential conflicts of interest were reported by
the authors.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made.
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