Chinese Journal of Animal Nutrition 2007, 19(5)

Effect of Different Levels of Zinc on Blood Physiological and Biochemical

Parameters in Stud Holstein Bulls

XIN Fu-yu1,2 HOU Ming-hai1 LI Wen-li2 LI Yan-qin1 WANG Ling-ling1
SONG Jie1 ZHONG Ji-feng1
(1.Dairy Cow Research Center of Shandong Academy of Agricultural Science, Jinan 250100, China; 2.College of Animal
Science and Technology, Laiyang Agricultural College, Qingdao 266109, China）

Abstract: Thirty breeding Holstein bulls assigned randomly to five groups was conducted to study the effect of different
levels of zinc on blood physiological and biochemical parameters. This experiment involved 5 dietary treatments with
single factorial arrangement, consisting of one control group fed basal diet and four levels of zinc which is 50, 80, 110, 200
mg/kg (by the dry matter of the dietary). The results shows that (1) the effect of different levels of zinc on the activity of
GPT、GOT was extremely significant (P<0.01),but that on AKP, LDH was not significant (P>0.05); (2) the effect of
different levels of zinc on the testosterone contents in serum was extremely significant (P<0.01),but that on zinc contents
in serum was not significant(P>0.05). All of the variety of physiological and biochemical parameters analysed in each
group indicated that the optimal supplementation of zinc was 110 mg/kg by the dry matter in this experiment.
Key words: Zinc; Holstein; Stud Bulls; Physiological and Biochemical Parameters

Since Todd Bertrand had proved zinc was the
essential trace element for animals in 1934, mass of
study had been performed on zinc, it is discovered
that the physiological function of zinc were the most
of all trace element. It has been proved that zinc
participates the composition of 300 kinds of
enzymes and function-proteins of the animal in vivo
now, it participates on the metabolism of proteins,
amino acids, nucleinic acids, fat, carbohydrate and
vitamins as well as the metabolism of other trace
elements, but also relates to the activity of insulin,
glucagons, prostaglandin, gonad stimulating
hormone. It is necessary to the physiological
function such as the development of ossature,
reproduction, immunization, blood clotting,and the
[1-3]
stabilization of biomembrane .Furthermore, zinc
is the composition of many function-proteins such
Received date:2007-01-05
Support project: improved variety project of Shan Dong
Province, No. 2003-3009
Author:Xin Fu-Yu(1982-), male, Postgraduate, major in
ruminant nutrition, E-mail: xinfuyu_2004yjs@126.com
Corresponding author: Hou Ming-Hai(1963-), Professor,
Animal Nutrition expert, E-mail:minghaihou@163.com
as metallothionein, neucleoprotein and some
receptors.
Studies on the requirement of zinc, many
scholars domestic and abroad has focus on the
suitable supplement to diets of swine and cow, but
similar research on breeding bulls is very weak
accordingly[4]. Recent studies have confirmed that
animal feed which is lack of zinc could influence
the growth, development and performance of
breeding bulls acutely. According to practic trophic
analysis, the content of zinc in common diets for
bulls is far lower than NRC standard. This study
was carried out for determining the suitable zinc
supplement for local Holstein herd sire through
detecting some important biochemical indicators in
blood with different levels of zinc supplementation,
aiming to provide a necessary theoretical basis for
scientific raising of herding bulls.
1 Materials and methods
1.1 Experimental design a nd Choice of
experimental bulls
In the experiment,30 healthy pure Holstein
 XIN Fu-yu et al.: Effect of Different Levels of Zinc on Blood Physiological

breeding bulls with basically consistent
physiological condition (815±73 kg in body weight,
2.5~3.5 years of age, normal Semen Collection
twice a week) were assigned at randomly to 5
groups, named group A,B,C,D and E respectively.
Each groups had 6 bulls. Simple factor experiment
design was designed. Group A was set as control
group and the other four groups as experiment
groups. Bulls in control group fed with basic daily
diet while bulls in experiment groups were fed the
diet with different levels of inorganic zinc
supplementation(50, 80, 110, 200 mg/kg,
calculated by dry material).Zinc used in the study
was in form of analytical pure ZnSO4·7H2O.
1.2 Diet composition and feeding management
The rations were determined according to
NRC Nutrient Requirement of Dairy Cow (Edition
[5]
Ⅶ ) .Basic diets is composed of mixed
concentrate, Lucerne and Leymus chinensis. Bulls
were fed fixed quantity (0.5% of body weight)
twice a day. The composition and nutrient of the
mixed concentrate were shown in Table 1.
The livestock house for experiment was
washed and degermed before. Considering that the
time for generation of sperm is about 64~74 days
and the time for sperm migration from
seminiferous rubule to epididymis where the
sperms stored and ripped completely is about
14~18 days[6], The period was divided into two
phases including 3 months of pre-experiment
period and 5 months of experiment period.
Preparing work such as Serial number,
helminthicide and body weight adjuster were done
in pre-experiment period. Feeding management in
the two period was identical.

Table 1 The composition and nutrient levels of the basal diet (air-dry basis,%)
Ingredient Content Nutrient Content
Corn 55 DM 86.3
Soybean meal 24.5 CP 13.69
Wheat bran 16 Ca 1.53
Fish meal 2.0 TP 0.33
NaCl 1.0 Zn(mg/kg) 27
CaHPO4 1.5 NEL (MJ/kg)* 6.97

Total 100.00
Note：The values of nutritient are measured except that NEL is calculated.
1.3 Sample collection and treatment
During experiment period, blood samples were
collected germ freely from vena caudalis in the
middle ten days of each month. The samples were
centrifuged in low temperature (4℃) immediately,
Serum was separated and collected in order to
detect correlated biochemical indicator.
1.4 Detected index and methods
1.4.1 Determination of Zinc content in feed and
serum
Samples of feed and serum were treated by
Wet-digestion method. Determination of Zinc in feed
and serum by Atomic Adsorption Spectrometry.
Operating procedure for serum digestion was:
suction 1ml serum into 25ml conical flask
accurately, add 5 ml High-grade nitric
acid ,digested in constant temperature electric
stove at 200℃. when the mixture become clarify,
add 1 ml high-grade perchloric acid,degested till
the digest turned achromasy and fume white
smoke . Add 10 ml distilled water, degested till the
mixture turned achromasy and fume white smoke
again. Metered volume the digest to 10 ml
accurately. Zinc content was detected with
TAS-990 Atomic Absorption Spectrometer
1.4.2 The determination of testosterone
Special kit was bought from beijing north
institute of biological technology. The operative
procedure was according to its directions.
Chinese Journal of Animal Nutrition www.chinajan.com

Testosterone content in serum was detected with
SN-695 Intelligent γ-radioimmunoanalyzer.
1.4.3 The determination of enzymes activaty
Kits for determinate GPT、GOT、AKP and LDH
activity were bought from Nanjing Jiancheng
Bioengineering Institute，The operative procedure
was according to the directions.。Absorptance was
detected with UV-9200 UV-Vis Spectrophotometer.
1.5 Data processing and statistical analysis
Single factor analysis of variance was
operated by ANOVA process of SPSS 11.5.
Multiple comparison was carried out by LSD
method. Result shown bellow were indicated in
form of “average ± standard deviation”

Table 2 Effect of different levels of Zinc supplement on serum zinc and testosterone
Treatment Serum Zinc (μg/mL) testosterone (pg/mL)
A (Control) 2.32±0.28a 4.88±0.69Aa
B (50 mg/kg) 2.46±0.11a 5.61±0.57Aa
a
C (80 mg/kg) 2.75±0.25 6.40±0.94Aa
D (110 mg/kg) 2.99±0.19a 9.12±1.38Bc
a
E (200 mg/kg) 2.85±0.26 8.51±1.81b
SEM 0.101 0.518
P-value 0.006 0.017
Note：values marked with same small letters in the same column were not significant(P>0.05), with different small letters
were significant(P<0.05), with different capital letters were extremely significant(P<0.01). The same below.

2 Results and analysis there were no distinct differences(P>0.05). With

increasing zinc additive, zinc and testosterone
2.1 Effect of different levels of Zinc supplement
content in serum showed a trend that increased at
on serum zinc and testosterone
the beginning and then decreased.It indicates that
As shown in Table 2, testosterone content in
the optimal zinc supplement for breeding bulls is
experiment groups was higher than control group
between 50 mg/kg and 200mg/kg.
significantly（P<0.01）. Zinc supplementation in
diet elevated the Zinc concentrate in serum, but

Table 3 Effect of different levels of zinc supplement on the activity of GPT、GOT、AKP、LDH

Treatment GPT (Carmen’s unit/ml) GOT (Carmen’s unit/ml) AKP (King's unit/100ml) LDH(U/L)
Aa Aa a
A(Control) 14.17±1.14 22.60±2.40 12.10±1.14 6631.70±705.86a
B(50 mg/kg) 16.04±1.93a 23.56±2.60a 11.58±1.48a 7078.77±714.25a
C(80 mg/kg) 17.80±1.67a 23.74±2.24a 13.49±1.87a 7031.90±735.59a
D(110 mg/kg) 21.99±2.12Bb 33.27±3.14Bb 12.14±1.98a 7133.23±774.44a
a a a
E(200 mg/kg) 16.31±1.83 26.88±2.85 13.65±1.76 6973.76±816.79a
SEM 0.632 0.868 0.364 330.786
P-value 0.155 0.161 0.001 0.000

2.2 Effect of different levels of zinc supplement
on the activity of GPT、GOT、AKP、LDH
Date in table 3 indicates that the activity of
GPT and GOT in experiment groups was higher
than control group significantly(P<0.01), AKP and
LDH activity has no significant change（P>0.05）.
With increasing zinc additive, the activity of serum
LDH rised earlier but decreased later. However,
the activity of serum AKP has no regularity change
among groups.
 XIN Fu-yu et al.: Effect of Different Levels of Zinc on Blood Physiological

3 Discussion there are positive correlation significantly between

serum testosterone and free testosterone, serum
3.1 Effect of different levels of Zinc supplement
testosterone were reported in many articals
on serum zinc
according to concerned research on factors affecting
The content of serum zinc reflect the zinc
the decrease of semen quality so as to reflect the law
nutritional status of bull directly，low level of
of change of seminal plasma testosterone [8-9]. As the
serum zinc could influence the performance of bull
result of the test shows: Zinc supplement enhanced
seriously。the content of trace element is relatively
the synthesis and externalization of testosterone,
stable in various kinds of animal’s blood and
improved productitive performance of bulls.With
changing within physiological range when trace
increasing zinc supplement,serum testosterone show
element is enough both in diet and animal body.
a tendency that increased at the beginning and then
The results showed that, the content of serum zinc
gradually decreased.
is 70-130 ug/100 ml in normal bull. Although
3.3 Effect of different levels of zinc supplement
various kinds of nutritive material intrance and
on the activity of GPT、GOT、AKP、LDH
Exit continuously in blood, the content of serum
In recent years , alkaline phosphatase activities
zinc could recover the balance quickly in healthy
is known as a index for the assessment of zinc
animals. In this trial, the concentration of serum
nutriture. Wright’s research confirmed there were
zinc in test group was higher than that in control
obviously positive correlation between serum zinc
group with zinc supplement, It indicated that zinc
and alkaline phosphatase activities.However,the
supplement in diet could increase the content of
activities of alkaline phosphatase in neutrophil have
serum zinc and improve the zinc nutritional status
nothing to do with that in blood plasma. It is therefore
of bull.Although no significant change was found
considered that simultaneous determination on
in the two groups.(P>0.05),there is a positive
alkaline phosphatase activities both in neutrophil and
correlation between serum zinc and diet zinc in a
in blood plasma can be a reliable determination index
given range. Relative study demonstrated serum
of zinc nutriture
zinc began to descend only when zinc in liver and
GPT and GOT is an important amino acid
bone descend to critical value. 50% of normal
transaminase which exist in chondrosome
serum zinc is the mark of zinc deficiency.
extensively.It plays an important role in protein
3.2 Effect of different levels of Zinc supplement
metabolic process,takes participate in the
on serum testosterone
transform and synthesis of various kinds of
Zinc is an inherent component of cortical
essential amino acids,lies in special important
substance of suprarenal gland.It also participates in
position in amino acid metabolism and reciprocal
regulating the function of appendices
transformation of protein,fat and carbohydrate[11-12].
suprasphenoidalis - sexual gland system. The
The result of this test indicates that zinc
synthesis of testicosteroid depends on the existence
supplement in bull diet can elevate the activities of
of zinc. Zinc plays an important role on testosterone
GPT and GOT significantly, in other words,it can
externalization through affecting the release of
promote the synthesis of protein,decrease the
gonadotropic hormones. Serum testosterone is the
decompose of protein,increase protein deposition
total amount of testosterone in blood circulation, in
in vivo. All result above is identical with the
other words, it’s the sum of free testosterone and
mechanism of growth promotion of zinc.
combined-testosteron. However, what really play
biological action in vivo is the free testosterone 4 Conclusion
which account for only 3% of the total. Most of free ① Different levels of zinc supplement in diet
testosterone exist in seminal plasma. Because of for Holstein breeding bulls elevated the content of
Chinese Journal of Animal Nutrition www.chinajan.com

serum testosterone significantly, enhanced GOT
and GPT significantly too. However, serum
zinc,AKP and LDH have no remarkable chang, but
have the tendency to rise;
②All indexes in the four experiment groups
indicate that suitable dietary zinc supplement can
improve the nutriture of breeding bulls,100mg/kg
addition obtained good results.
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(Editor JI Peng)