Effect of Zinc Source and Level on Finishing Cattle
Performance, Carcass Characteristics, and Adipocyte Differentiation
L.J. McBeth, D.R. Stein, A.T.V. Pillai,
M.J. Hersom, C.R. Krehbiel, U. DeSilva, R.D. Geisert,
J.R. Malayer, J.B. Morgan, C. K. Larson,
and R.L. Ball
Story in Brief
The objective of the present experiments was to determine the
effects of zinc source and level on feedlot performance and carcass
characteristics, and to determine the effects of zinc source and level on
growth and differentiation of primary adipocytes in cell culture. For performance response variables, overall
body weight, average daily gain, dry matter intake, and feed efficiency did not
differ among treatments. Although not
statistically significant, when steers were fed 90 ppm Zn, marbling score was
4.0% greater and 12th-rib fat was 21.8% lower for steers fed organic compared
with inorganic Zn. In vitro, results
suggest that soluble Zn methionine decreased expression of peroxisome
proliferator-activated receptor-γ and stearoyl CoA desaturase in cell
cultures of stromal-vascular cells isolated from intramuscular and subcutaneous
fat depots compared with inorganic Zn.
However, more time intervals between 0 and 24 h and more Zn
concentrations between 0 and 5 µM should be evaluated before conclusions are
drawn.
Key Words:
Adipogenesis, Carcass Quality, Cattle, Feedlot Performance, Zinc
Introduction
Due to the potential for increased bioavailability from amino
acid complexed Zn (Engle et al., 1997), addition of organic sources of Zn to
feedlot cattle diets seems favorable.
Greene et al. (1988) reported higher marbling scores and carcass quality
for steers fed Zn methionine (360 mg/kg of Zn) compared with steers fed ZnO
(360 mg/kg of Zn) or steers fed a control diet (82 mg/kg of Zn). The increase in intramuscular fat deposition
with Zn methionine supplementation was mirrored by increased KPH and external
fat (Greene et al., 1988). McBeth et al.
(2002) recently fed 336 crossbred steers to determine the effects of Zn level
and source on feedlot performance and carcass merit. Thirty ppm of added Availa®Zn resulted in
similar or greater performance compared with 30 ppm of added ZINPRO® Zn
methionine, and numerically greater improvements in performance were observed
when 60 ppm of added Availa Zn was added to the basal diet compared with 60 ppm
of added ZnSO4. When 120 ppm of total
dietary Zn was fed, 50:50 ZnSO4:AvailaZn resulted in 1.6% greater daily gain,
2.7% lower DMI, 4.2% improved efficiency, and 28.2% more choice carcasses than
when 120 ppm from ZnSO4 was fed. Adding
30 ppm of Zn methionine to the control diet (60 ppm Zn from ZnSO4) resulted in
similar carcass advantages; however, feedlot performance was numerically lower
compared with feeding 90 ppm total dietary Zn from ZnSO4. These results suggested increases in daily
gain and DMI with increasing supplemental Zn.
In addition, numeric advantages in feedlot performance and carcass
traits were observed when a combination of Availa Zn and ZnSO4 were fed.
Research regarding the effects of Zn on fat metabolism and
adipogenesis in ruminants is limited.
Tanaka et al. (2001) reported increases in the specific activity of
glycerol phosphate dehydrogenase with the addition of 1 µM of Zn in adipocyte
culture with or without the addition of insulin to the medium. Similarly, the same researchers reported that
addition of fattened cattle serum to the growth medium increased the Zn
concentration of the medium from 1 µM to 3 µM suggesting that Zn concentration
in the medium reflects the adipogenic activity in cattle serum. Murine and in vitro models have also indicated
that Zn has the ability to enhance adipogenesis. Shisheva et al. (1992) reported that addition
of Zn to both growth media and differentiation media increased the
incorporation of glucose into lipids in vitro and illustrated this effect both
independent of insulin and additively with insulin. It has been demonstrated in ruminants that
insulin sensitivity is decreased with age and carcass fat accumulation (McCann
et al., 1986; Eisemann et al., 1997).
This relationship between insulin sensitivity and Zn could offer some
explanation into the mechanism of increased fat deposition with Zn
supplementation.
The purpose of the present experiments was to determine the
effects of zinc source and level on feedlot performance and carcass
characteristics, and to determine the effects of zinc source and level on
growth and differentiation of bovine stromal-vascular cells.
Materials and Methods
Experiment 1. One hundred sixty crossbred steers (avg initial
BW = approximately 320 kg) were delivered to the Willard
Sparks Beef
Research Center
near Stillwater, OK on March 14, 2003. On arrival, steers were individually weighed
and ear tagged. On the following day,
steers were horn tipped as needed, implanted with Revalor-S (Hoechst Roussel
Vet, Clinton, NJ), vaccinated with IBR-PI3-BVD-BRSV (Titanium 5, Intervet,
Millsboro, DE), vaccinated with a seven-way clostridial preparation (Vision 7,
Intervet, Millsboro, DE), and treated for control of external and internal
parasites (Ivomec-Plus injectable, Merial, Duluth, GA). Steers were blocked by initial BW into eight
weight blocks. Body weight (unshrunk)
taken on the day of arrival (d 0) was considered initial weight. Within block steers were assigned randomly to
4 pens (5 steers/pen; 8 pens/treatment).
Treatments included:
1) 60 ppm ZnSO4 (control); 2) control plus 30 ppm ZnSO4; 3) control plus
30 ppm ZnÒMethionine;
and 4) control plus 30 ppm AvailaÒZn. The basal
diet included (DM basis) rolled corn (76.5%), ground alfalfa (10.0%), molasses (4.0%),
yellow grease (2.0%), and a pelleted supplement (7.5%), and was formulated to
meet or exceed NRC (1996) nutrient requirements. Monensin (33 mg/kg of diet) and tylosin (11
mg/kg of diet) were fed. Steers were
gradually adapted to the final 90% concentrate diet by offering 65, 75, and 85%
concentrate diets for 7 d each. Feed
refused was weighed at 28-d intervals and as needed (e.g., following inclement
weather). In addition, diet and
ingredient samples were collected, and DM samples were composited by 28-d
periods, allowed to air dry, and ground in a Wiley mill to pass a 1-mm
screen. Diet samples were analyzed for
N, starch, ash (AOAC, 1996), ADF (Goering and Van Soest, 1970) and Zn. Interim unshrunk BW was determined at 28-d
intervals. Steers were slaughtered at a
commercial facility. Hot carcass weight,
external fat, internal fat, longissimus muscle area, marbling score, yield
grade and quality grade were determined.
Data for BW, DMI, ADG, feed efficiency and normally
distributed carcass characteristics were analyzed as a randomized complete
block design using the Proc Mixed procedure of SAS Release 8.02 (SAS Institute
Inc., Cary, NC).
The model included treatment, and block was included as a random
variable. Pen served as the experimental
unit. Pre-planned comparisons were: 1)
Zn level (60 vs 90 ppm); 2) inorganic vs organic Zn; and 3) Zn Methionine vs
Availa Zn.
Experiment 2. Our goal was to determine if Zn would increase
the expression of genes involved with fat metabolism in cultured stromal-vascular
cells harvested from subcutaneous and intramuscular adipocytes. Intramuscular and subcutaneous adipose
tissues were collected from steers harvested at the Food and Agricultural Products
Center and placed in
sterile Hanks balanced salt solution containing 250 ng/mL amphotericin B and
0.2 mg/mL gentamycin for transportation to the laboratory (Ohyama et al.,
1998). In the lab, tissue from each
depot was digested in Hanks balanced salt solution containing 1 mg/mL Type I
collagenase for 1 h at 38ºC with shaking at 170 cycles/min. Subcutaneous adipose tissue was minced into
sections of approximately 1 mm2
with scissors before incubation in the digestion solution. Following digestion, the cell suspensions
were filtered through 250-µm nylon mesh to remove undigested tissue and large
aggregates of cells. The filtrate was
centrifuged at 700 x g for 5 min and decanted to separate floating adipocytes
from the pellet of stromal-vascular cells.
The cell pellet was resuspended and washed three times with DMEM
containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 250 ng/mL
amphotericin B, and 0.2 mg/mL gentamycin (growth medium; Ohyama et al., 1998).
The stromal-vascular cells were seeded on 12-well (22 mm
diameter) tissue culture plates at a density of 1 x 104 cells/cm2. Cells were incubated in the growth medium at
37ºC under a humidified atmosphere of 95% air and 5% CO2. After reaching confluence, the growth medium
was replaced with the differentiation medium composed of 1:1 (vol:vol) mixture of
DMEM/Ham’s F12 media containing 10 µg/mL bovine transferrin, 17 µM biotin, 10
µg/mL bovine insulin, 100 U/mL penicillin, and 100 µg/mL streptomycin with or
without an inorganic or organic Zn concentration of 5 µM. Cells were cultured in these differentiation
media for 0, 12 or 24 h. Total cell
extracts were collected and RNA extracted with 4 mL of TRIzol™ reagent (Gibco, Grand Island, NY).
We used a PCR-based assay to determine mRNA expression of
peroxisome proliferator-activated receptor-γ (PPARγ) and stearoyl CoA
desaturase (SCD-1). Expression of these
genes was evaluated by quantitative RT-PCR utilizing the fluorescent reporter
and 5’ exonuclease assay system (TaqManâ, PE Biosystems, Foster
City, CA). Briefly, reverse transcription of total RNA and
PCR amplification were performed using the TaqManÒ One-Step RT-PCR Master
Mix Reagents Kit, TaqManâ fluorescent probe, and sequence detection primers (PE
Biosystems). TaqManâ
probe specific for target was designed to contain a fluorescent 5’ reporter dye
(TET) and 3’ quencher dye (TAMRA). Each
one-step RT-PCR reaction (25 µL) contained 2X Master Mix without
uracil-N-glycosylase (12.5 µL), 40X MultiScribeÒ and RNAse Inhibitor Mix
(0.625 µl), target forward primer (200 nM), target reverse primer (200 nM), and
the fluorescent labeled target probe (200 nM) designed from the mRNA sequence
for bovine PPARγ and SCD-1. The PCR
amplification was carried out in the ABI PRISMâ 7700 Sequence Detection
System (PE Biosystems). Thermal cycling
conditions were 50oC
for 2 min, 95oC for
10 min followed by 40 repetitive cycles of 95oC for 15 sec and 60oC for 1 min. As a
normalization control for RNA loading, parallel reactions in the same multiwell
plate were performed using 18S ribosomal RNA as target (18S Ribosomal Control
Kit, PE Biosystems).
Data were analyzed as a complete randomized design using the
Mixed procedure of SAS. The model
included terms for fat depot, Zn source, Zn level, time, and the appropriate
interactions.
Results and Discussion
Experiment 1. Effect of Zn source and level on feedlot cattle
performance is shown in Table 1. Overall
(d 0 through 139) body weight, average daily gain, dry matter intake, and feed
efficiency did not differ among treatments.
From d 85 to 112, average daily gain was greater (P=.07) for steers fed
60 ppm Zn compared with 90 ppm Zn. From
day 113 to 139, steers fed Zn methionine had greater (P=.005) average daily
gain than steers fed Availa Zn. Steers
fed Availa Zn were more (P=.01) efficient than steers fed Zn methionine from d
85 to 112, whereas steers fed Zn methionine were more (P=.05) efficient than
steers fed Availa Zn from d 113 to 139.
Overall, steers fed 90 ppm of organic Zn were 2.6% more (P=.16)
efficient than steers fed inorganic Zn.
Effects of Zn source and level on carcass characteristics is
shown in Table 2. Hot carcass weight,
marbling score, 12th-rib fat depth, longissimus muscle area, % kidney, pelvic
and heart fat, and USDA Yield Grade did not differ among treatments. Although not statistically significant, when
steers were fed 90 ppm Zn, marbling score was 4.0% greater and 12th-rib fat was
21.8% lower for steers fed organic compared with inorganic Zn.
Experiment 2. In cultured adipocytes, appearance of PPARγ
is followed by expression of many genes representing the mature adipocyte
phenotype. Because recent research
indicates that Zn may promote adipogenesis (Tanaka et al., 2001), we
hypothesized that zinc might stimulate the expression of PPARγ and subsequently
genes commonly found in mature adipocytes (e.g., SCD-1, which catalyzes the
rate-limiting step in cellular synthesis of monounsaturated fatty acids such as
oleate [C18:1] and palmitoleate [C16:1]).
Based on work in feedlot cattle, we also hypothesized that differences
might occur between inorganic and organic sources of Zn. Figure 1 shows the effect of time, Zn source,
and Zn level on fold expression of PPARγ from subcutaneous and
intramuscular fat cells grown in primary culture (time x Zn source x Zn level
interaction, P<.05). Inorganic Zn did
not affect fold expression of PPARγ at 12 or 24 h. However, 5 µM of organic Zn decreased
expression of PPARγ at both 12 and 24 h.
Expression of PPARγ was decreased at 24 h compared with 12 h of
incubation. At 12 h, expression of SCD-1
was increased fourfold compared with no Zn, whereas organic Zn decreased
expression of SCD-1 approximately twofold (Figure 2). A similar response was observed at 24 h,
although expression of SCD-1 was decreased at 24 h when both sources and levels
of Zn were included in the differentiation media. These results suggest that soluble Zn
methionine decreased expression of PPARγ and SCD-1 in cell cultures of
stromal-vascular cells isolated from intramuscular and subcutaneous fat
depots. However, more time intervals
between 0 and 24 h and more Zn concentrations between 0 and 5 µM should be
evaluated before conclusions are drawn.
Table 1. Effects of
zinc level and source on feedlot cattle performance
|
|
|
|
60 ppm ZnSO4
|
Contrasts
|
|
Item
|
60 ppm ZnSO4
|
30 ppm ZnSO4
|
30 ppm Availa Zn
|
30 ppm Zn Met
|
SEM
|
60 vs
90 ppm
|
Inorganic
vs Organic
|
Availa Zn
vs
Zn Methionine
|
|
BW
|
|
|
|
|
|
|
|
|
|
Initial
|
786
|
783
|
783
|
783
|
14.7
|
--
|
--
|
--
|
|
d 139
|
1312
|
1297
|
1297
|
1317
|
22.5
|
.47
|
.84
|
.19
|
|
Daily gain, lb
|
|
|
|
|
|
|
|
|
|
d 0 – 28
|
4.10
|
3.96
|
4.08
|
3.90
|
.23
|
.61
|
.85
|
.56
|
|
d 29 – 56
|
4.35
|
4.41
|
4.44
|
4.36
|
.22
|
.83
|
.91
|
.78
|
|
d 57 – 84
|
3.98
|
3.91
|
3.74
|
4.08
|
.22
|
.79
|
.88
|
.28
|
|
d 85 – 112
|
3.90
|
3.34
|
3.64
|
3.52
|
.18
|
.07
|
.82
|
.65
|
|
d 113 – 139
|
2.49
|
2.74
|
2.46
|
3.20
|
.17
|
.14
|
.23
|
.005
|
|
d 0 – 139
|
3.79
|
3.70
|
3.70
|
3.84
|
.08
|
.58
|
.74
|
.17
|
|
DM intake, lb/d
|
|
|
|
|
|
|
|
|
|
d 0 – 28
|
22.3
|
21.8
|
21.6
|
21.7
|
.70
|
.30
|
.44
|
.84
|
|
d 29 – 56
|
22.1
|
22.2
|
21.8
|
21.9
|
.74
|
.85
|
.59
|
.90
|
|
d 57 – 84
|
23.9
|
23.0
|
22.8
|
23.2
|
.56
|
.11
|
.39
|
.54
|
|
d 85 – 112
|
24.2
|
23.8
|
22.9
|
23.8
|
.57
|
.29
|
.24
|
.20
|
|
d 113 – 139
|
24.5
|
24.7
|
24.5
|
25.4
|
.52
|
.47
|
.44
|
.22
|
|
d 0 – 139
|
23.4
|
23.1
|
22.7
|
23.2
|
.49
|
.34
|
.41
|
.32
|
|
Feed:Gain, lb/lb
|
|
|
|
|
|
|
|
|
|
d 0 – 28
|
5.53
|
5.63
|
5.31
|
5.63
|
.22
|
.96
|
.60
|
.30
|
|
d 29 – 56
|
5.14
|
5.14
|
4.94
|
5.05
|
.21
|
.70
|
.50
|
.71
|
|
d 57 – 84
|
6.21
|
5.92
|
6.25
|
5.73
|
.32
|
.51
|
.82
|
.25
|
|
d 85 – 112
|
6.24
|
7.18
|
6.44
|
6.82
|
.24
|
.30
|
.25
|
.01
|
|
d 113 – 139
|
10.63
|
9.21
|
10.57
|
7.97
|
.89
|
.19
|
.47
|
.05
|
|
d 0 – 139
|
6.17
|
6.26
|
6.14
|
6.05
|
.09
|
.84
|
.16
|
.40
|
|
Table 2. Effects of
zinc level and source on carcass merit
|
|
|
|
60 ppm ZnSO4
|
Contrasts
|
|
Item
|
60 ppm ZnSO4
|
30 ppm ZnSO4
|
30 ppm Availa
Zn
|
30 ppm Zn Met
|
SEM
|
60 vs.
90 ppm
|
Inorganic
vs
Organic
|
Availa Zn
vs Zn Methionine
|
|
Hot carcass wt., lbs
|
832
|
833
|
837
|
844
|
15.0
|
.54
|
.35
|
.60
|
|
Marbling a
|
395
|
374
|
382
|
396
|
27.0
|
.72
|
.87
|
.71
|
|
12th-rib fat, in.
|
.56
|
.67
|
.54
|
.55
|
.05
|
.64
|
.16
|
.95
|
|
Ribeye area, in2
|
14.03
|
13.59
|
14.29
|
13.96
|
.40
|
.84
|
.39
|
.54
|
|
KPH, %
|
1.93
|
1.97
|
2.00
|
1.94
|
.06
|
.62
|
.75
|
.52
|
|
Yield grade
|
2.98
|
3.40
|
2.87
|
2.98
|
.20
|
.62
|
.20
|
.70
|
|
a300 = slight, 400 = small
|
|
|
|
Figure 1. Effect of
time after dosing (12 or 24 h), Zn source (organic or inorganic) and Zn level
(0 or 5 µM) on fold differences in PPARγ gene expression in subcutaneous
and intramuscular primary cell cultures
|
|
|
|
Figure 2. Effect of
time after dosing (12 or 24 h), Zn source (organic or inorganic) and Zn level
(0 or 5 µM) on fold differences in stearoyl-CoA desaturase gene expression in
subcutaneous and intramuscular primary cell cultures
|
Literature Cited
Association of Analytical Chemists. 1996. 16th ed. Assoc. of
Offic. Anal. Chem., Arlington,
VA.
Eisemann, J.H. et al.
1997. J. Anim. Sci. 75:2084.
Engle, T.E. et al.
1997. J. Anim. Sci. 75:3074.
Greene, L.W. et al.
1988. J. Anim. Sci. 66:1818.
Goering, H.K., and P.J. Van Soest. 1970. Agric. Handbook No. 379, ARS, USDA, Washington, DC.
McBeth, L.J. et al. 2002. Okla. Agr. Exp. Sta.
Res. Rep. P-993:24.
McCann, J.P. et al.
1986. J. Nutr. 116:1287.
NRC. 1996. Nutrient Requirements of Beef Cattle. (7th Ed.). National Academy
Press, Washington
D.C.
Ohyama, M. et al.
1998. J. Anim. Sci. 76:61.
Shisheva, A. et al.
1992. Diabetes 41:982.
Tanaka, S. et al.
2001. J. Anim. Sci. 14:966.
Acknowlegements
Special thanks are conveyed to the Zinpro Corp. for
providing funding and to ContiBeef, Cimarron Feeders for providing
cattle. The authors wish to thank the undergraduate students at the Willard Sparks Beef
Research Center
and the graduate students for help with these experiments.
Copyright 2004 Oklahoma Agricultural Experiment Station
Authors
McBeth, L.J. – Graduate Student
Stein, D.R. – Graduate Student
Pilla, A.V. – Graduate Student
Hersom, M.J. – Assistant Professor, University of Florida
Krehbiel, C.R. – Assistant Professor
DeSilva, U. – Assistant Professor
Geisert, R.D. - Professor
Malayer, J.R. – Associate Professor, Dept. of Physiological
Sciences
Morgan, J.B. – Associate Professor
Larson, C.K. – ZinPro Corporation
Ball, R.L. - Herdsman