Effects of long-term supplementation of dairy cow diets with rumen-protected conjugated linoleic acids (CLA) on performance, metabolic parameters and fatty acid profile in milk fat.

Abstract

The supplementation of conjugated linoleic acids (CLA) to the rations of dairy cows represents an opportunity to reduce the content of milk fat. Therefore, CLA have the potential beneficial effect of reducing energy requirements of the early lactating cow. The present study aimed at the examination of long-term and posttreatment effects of dietary CLA intake on performance, variables of energy metabolism-like plasma levels of non esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB), and fatty acid profile in milk fat. Forty-six pregnant German Holstein cows were assigned to one of three dietary treatments: (1) 100 g/ d of control fat supplement (CON), (2) 50 g/d of control fat supplement and 50 g/ d of CLA supplement (CLA-1) and (3) 100 g/d of CLA supplement (CLA-2). The lipid-encapsulated CLA supplement consisted of approximately 10% of trans-10, cis-12 CLA and cis-9, trans-11 CLA each. The experiment started 1 d after calving and continued for about 38 weeks, divided into a supplementation (26 weeks) and a depletion period (12 weeks). Over the first 7 weeks of treatment, 11 and 16% reductions in dry matter intake compared to control were observed for the cows fed CLA-1 and CLA-2 supplements respectively. Consequently, the calculated energy balance for these two CLA groups was lower compared to the control. Plasma levels of NEFA and BHB remained unaffected. Later in lactation the highest CLA supplementation resulted in a reduction of milk fat content of 0.7%. However, no reduction in milk fat yield, and accordingly no milk fat depression (MFD), could be shown. The trans-10, cis-12 CLA in milk fat increased with increasing dietary CLA supplementation in a dose-dependent manner. The proportion of C16 in milk fat was decreased by the highest CLA supplementation. With the exception of an increase in plasma glucose level in the CLA-2 group, no post-treatment effects were observed. Overall, under the conditions of the present study no improvement in the calculated energy balance by CLA supplementation could be shown for the entire evaluation period.

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Effects of long-term supplementation of dairy cow diets with rumen-Effects of long-term supplementation of dairy cow diets with rumen-
protected conjugated linoleic acids (CLA) on performance, metabolic protected conjugated linoleic acids (CLA) on performance, metabolic
parameters and fatty acid profile in milk fat parameters and fatty acid profile in milk fat
Julia Pappritza; Ulrich Meyera; Ronny Kramerb; Eva-Maria Weberc; Gerhard Jahreisb; Jürgen Rehagec;
Gerhard Flachowskya; Sven Dänickea
a Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal
Health, Braunschweig, Germany b Institute of Nutrition, Friedrich-Schiller-University Jena, Germany c
Clinic for Cattle, University of Veterinary Medicine Hannover, Germany
First published on: 04 March 2011
To cite this Article To cite this Article Pappritz, Julia , Meyer, Ulrich , Kramer, Ronny , Weber, Eva-Maria , Jahreis, Gerhard , Rehage, Jürgen ,
Flachowsky, Gerhard and Dänicke, Sven(2011) 'Effects of long-term supplementation of dairy cow diets with rumen-
protected conjugated linoleic acids (CLA) on performance, metabolic parameters and fatty acid profile in milk fat',
Archives of Animal Nutrition, 65: 2, 89 — 107, First published on: 04 March 2011 (iFirst)
To link to this Article: DOI: To link to this Article: DOI: 10.1080/1745039X.2011.552275
URL: URL: http://dx.doi.org/10.1080/1745039X.2011.552275
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Page 2

Effects of long-term supplementation of dairy cow diets with rumen-
protected conjugated linoleic acids (CLA) on performance, metabolic
parameters and fatty acid profile in milk fat
Julia Pappritza, Ulrich Meyera, Ronny Kramerb, Eva-Maria Weberc,
Gerhard Jahreisb, Ju ¨ rgen Rehagec, Gerhard Flachowskyaand Sven Da ¨ nickea*
aInstitute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for
Animal Health, Braunschweig, Germany;bInstitute of Nutrition, Friedrich-Schiller-University
Jena, Germany;cClinic for Cattle, University of Veterinary Medicine Hannover, Germany
(Received 15 July 2010; accepted 16 November 2010)
The supplementation of conjugated linoleic acids (CLA) to the rations of dairy
cows represents an opportunity to reduce the content of milk fat. Therefore, CLA
have the potential beneficial effect of reducing energy requirements of the early
lactating cow. The present study aimed at the examination of long-term and post-
treatment effects of dietary CLA intake on performance, variables of energy
metabolism-like plasma levels of non esterified fatty acids (NEFA) and ß-
hydroxybutyrate (BHB), and fatty acid profile in milk fat. Forty-six pregnant
German Holstein cows were assigned to one of three dietary treatments: (1) 100 g/
d of control fat supplement (CON), (2) 50 g/d of control fat supplement and 50 g/
d of CLA supplement (CLA-1) and (3) 100 g/d of CLA supplement (CLA-2). The
lipid-encapsulated CLA supplement consisted of approximately 10% of trans-10,
cis-12 CLA and cis-9, trans-11 CLA each. The experiment started 1 d after
calving and continued for about 38 weeks, divided into a supplementation (26
weeks) and a depletion period (12 weeks). Over the first 7 weeks of treatment, 11
and 16% reductions in dry matter intake compared to control were observed for
the cows fed CLA-1 and CLA-2 supplements respectively. Consequently, the
calculated energy balance for these two CLA groups was lower compared to the
control. Plasma levels of NEFA and BHB remained unaffected. Later in lactation
the highest CLA supplementation resulted in a reduction of milk fat content of
0.7%. However, no reduction in milk fat yield, and accordingly no milk fat
depression (MFD), could be shown. The trans-10, cis-12 CLA in milk fat
increased with increasing dietary CLA supplementation in a dose-dependent
manner. The proportion of C16in milk fat was decreased by the highest CLA
supplementation. With the exception of an increase in plasma glucose level in the
CLA-2 group, no post-treatment effects were observed. Overall, under the
conditions of the present study no improvement in the calculated energy balance
by CLA supplementation could be shown for the entire evaluation period.
Keywords: conjugated linoleic acid; dairy cows; feed intake; energy balance; fatty
acids; milk fat percentage
*Corresponding author. Email: sven.daenicke@fli.bund.de
Archives of Animal Nutrition
Vol. 65, No. 2, April 2011, 89–107
ISSN 1745-039X print/ISSN 1477-2817 online
? 2011 Taylor & Francis
DOI: 10.1080/1745039X.2011.552275
http://www.informaworld.com
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1.Introduction
During the transition period, the energy intake of dairy cows is often inadequate to
meet the requirements for maintenance and milk synthesis and the animals
compensate for this gap through the mobilisation of adipose depots. As a
consequence, they experience a negative energy balance (Drackley 1999). In order
to prevent potential negative consequences on health and reproduction, the objective
of prepartal feeding is to achieve an optimal body condition of the cow at time of
parturition. Furthermore, the energy savings at the onset of lactation can cause an
improvement in the energy balance. Milk fat represents the major energy cost in the
production of milk components and is the most variable component in the milk of
ruminants. In addition to diet-induced reduction of milk fat production (milk fat
depression, [MFD]) by low fibre diets or marine oil diets (Bauman and Griinari
2003), the use of dietary conjugated linoleic acid (CLA) supplements is known to
reduce the milk fat yield in dairy cows (Bernal-Santos et al. 2003; Selberg et al. 2004;
Castaneda-Gutierrez et al. 2005). After abomasal infusion of pure CLA isomers,
trans-10, cis-12 CLA was identified as the main isomer responsible for MFD
(Baumgard et al. 2000a). The involved mechanisms are based on the inhibition of the
mammary expression of enzymes necessary for the synthesis of milk fat (Baumgard
et al. 2002b). Due to a CLA-induced reduction of milk fat yield the fatty acid pattern
of milk changed. The trans-10, cis-12 CLA supplementation resulted in a more
dramatic decrease in fatty acids originating from de novo synthesis (5C16) compared
with preformed fatty acids (4C16) (Chouinard et al. 1999; Baumgard et al. 2000a;
Kraft et al. 2001; Shingfield et al. 2009). In a study by Sippel et al. (2009) who fed up
to 51 g trans-10, cis-12 CLA/d for 4 weeks in mid-lactation the milk fat content
decreased dose-dependently by up to 39% while milk yield and other milk
components were unaltered. Consequently, the calculated energy balance was
improved for the treatment groups. Moore et al. (2004) and Odens et al. (2007)
observed a decrease in milk fat content as well, but the concomitant numeric increase
in milk yield resulted in an unaltered calculated energy balance, after feeding 37 g
and 30 g trans-10, cis-12 CLA for 3 and 6 weeks post partum (p.p.), respectively.
Post-supplementation effects were only determined after short-term supplementation
(9 weeks p.p.) of CLA from Castaneda-Gutierrez et al. (2005) and they observed no
influence of CLA on the performance parameters after the end of supplementation.
The milk fat depressing properties of CLA, especially trans-10, cis-12 CLA, and
their effects on the fatty acid pattern of milk fat of dairy cows are well documented.
However, the long-term and post-treatment effects after long-term supplementation
have not been investigated yet, and the results regarding energy metabolism were
inconsistent. Therefore, the present study aimed at the examination of these effects
on performance, fatty acid profile in milk fat, calculated energy balance and plasma
levels of non esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB) as
variables meaningful for energy metabolism.
2.Materials and methods
2.1.Animals, treatments and experimental design
The study was carried out at the experimental station of the Friedrich-Loeffler-
Institute (FLI) in Braunschweig. The experiment was conducted according to the
European Community regulations concerning the protection of experimental
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animals and the guidelines of the LAVES (Lower Saxony State Office for Consumer
Protection and Food Safety, Germany, File Number 33.14.42502-04-071/07). Forty-
six pregnant German Holstein cows were assigned to one of three dietary treatments
according to the mean live weight (627 + 9 kg, only cows), mean number of
lactation (1.9 + 0.1) and milk yield of previous lactation (5797 + 122 kg, 200 d
milk yield): (1) 100 g/d of control fat preparation (CON, n ¼ 15); (2) 50 g/d of CLA
preparation and 50 g/d of the control fat preparation (CLA-1, n ¼ 15); (3) 100 g/d of
CLA preparation (CLA-2, n ¼ 16). Control fat and CLA preparation were added to
the diet in a rumen-protected form. CLA supplementation was started one day after
calving and continued for 26 weeks (supplementation period). The experimental
period started 3 weeks ante partum (a.p.) and lasted until 12 weeks after end of
supplementation (depletion period).
During the treatment period the cows were fed a partial mixed ration (PMR)
for ad libitum consumption consisting of 37% concentrate and 63% silage (60%
maize silage, 40% grass silage based on dry matter [DM] content). During the
supplementation period, each cow received additionally 4 kg concentrate from
the concentrate station (TYPE RIC, Insentec, B.V., Marknesse, The Netherlands).
The composition of the concentrate is presented in Table 1. The diets were formulated
to meet the nutritional requirements of the cows stated by the German Society of
Nutrition Physiology (GfE 2001). The concentrate, which was included in the PMR,
consisted of the same components as the concentrate provided by the concentrate
Table 1.
(PMR).
Components and chemical composition of concentrates and partial mixed ration
Concentrate
PMR
(n ¼ 12) CON (n ¼ 5) CLA (n ¼ 5)
Components [%]
Wheat
Dried sugar beet pulp
Rapeseed meal
Soybean meal
Soybean oil
Calcium carbonate
Mineral feed*
CLA supplement
Control fatty acid supplement
Dry matter [g/kg]
Nutrients [g/kg DM]
Total ash
Crude protein
Ether extract
Crude fibre
Acid detergent fibre
Neutral detergent fibre
Energy?[MJ NEL/kg DM]
Trans-10, cis-12 CLAþ[g/kg DM]
38.50
29.00
20.00
6.50
1.00
0.50
2.00
–
2.50
889 + 10
38.50
29.00
20.00
6.50
1.00
0.50
2.00
2.50
–
887 + 11426 + 20
71 + 5
187 + 1
59 + 3
88 + 6
123 + 12
258 + 10
8.8
0.02
74 + 3
187 + 5
53 + 4
89 + 4
124 + 10
256 + 5
8.8
2.25
69 + 4
118 + 10
32 + 3
193 + 11
225 + 15
425 + 17
6.8
0.01
Notes: *Per kg mineral feed: 140 g Ca, 120 g Na, 70 g P, 40 g Mg, 6 g Zn, 5. 4 g Mn, 1 g Cu, 100 mg I, 40
mg Se, 5 mg Co, 1,000,000 IU vitamin A, 100,000 IU vitamin D3, 1500 mg vitamin E;?Calculation based
on nutrient digestibilities measured with wethers (GfE 1991);
concentrations in concentrates and silage; Means + SD.
þCalculation based on analysed
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station (concentrate CON and CLA), but without the fatty acid supplements
(Table 1). Supplemental CLA was included in the diet as a rumen-protected
commercial CLA preparation (Lutrell1pure, BASF SE, Ludwigshafen, Germany)
and added to the concentrate provided by the concentrate station. The two
predominant CLA isomers were trans-10, cis-12 CLA and cis-9, trans-11 CLA
(analysed proportion of both isomers: 12% of total fatty acid methyl esters [FAME]).
The concentrate fed to the control group contained a rumen-protected fat
preparation (Silafat1, BASF SE, Ludwigshafen, Germany) in which the conjugated
linoleic acids were substituted by a corresponding amount of stearic acid (Table 2).
During the depletion period the cows received only the PMR, without extra
concentrate. The cows were housed in group pens according to their feeding group.
The PMR was provided in 15 self-feeding stations (TYPE RIC, Insentec, B.V.,
Marknesse, The Netherlands) per group. The cows had free access to water.
2.2.Sample collection
Each cow was equipped with an ear transponder recording continuously the daily
individual water intake and feed intake. Representative concentrate samples were
taken once, grass and maize silage samples were taken twice a week, while PMR
samples were collected daily and pooled over approximately 4 weeks.
To ascertain the net energy lactation (NEL), balance studies with four wethers
each were carrried out for the three applied concentrates (CON, CLA and PMR)
and for maize and grass silage, following the standard procedure described by the
GfE (1991).
Cows were milked at 05:30 and 15:30 and the individual milk yield was recorded
by the milking system. Milk samples for the analysis of milk composition were taken
twice a week in the morning and again in the afternoon of the same day. The milk
samples were conserved with bronopol and stored at 88C until they were analysed.
For the analysis of the fatty acid profile in milk fat, milk samples of 100 ml were
collected twice a day at weeks 1, 4 and 12 p.p., at the end of CLA supplementation,
and at weeks 2 and 10 after terminating the CLA supplementation. The samples were
stored at –208C until they were freeze dried. The live weight was recorded
automatically daily.
The blood samples were obtained from the Vena jugularis externa. The date of
sampling depended on the time relative to calving (3, 2 and 1 weeks a.p.; 1 day and 1,
Table 2.Fatty acid profile of fat supplements.*
Fatty acid [% of total FAME?] CON CLA
C16:0
C18:0
C18:1cis-9
Conjugated linoleic acid
C18:2cis-9, trans-11
C18:2trans-10, cis-12
Other CLA
Other FA
10.89
87.30
50.01
10.89
50.31
10.66
0.06
0.02
0.15
1.58
11.99
11.88
0.95
3.32
Notes:*SupplementalCLAwasincludedintheconcentrateportionasarumen-protectedCLApreparation,
for the control group CLA was substituted by stearic acid (C18:0);?FAME, Fatty acid methyl ester.
92 J. Pappritz et al.
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2, 3, 5, 7, 10, 15, and 20 weeks p.p.) and the end of CLA supplementation (26 weeks
p.p., 1, 2, 4, 6, 8 and 10 weeks after the end of supplementation). Body condition
score (BCS) of each animal was recorded at each blood sampling time using a five-
point system (Edmonson et al. 1989) by two persons, and the average of these two
scores was the assigned value. Back fat thickness (BFT) of each cow was measured at
the sacral region by using ultrasound at 3 weeks a.p., 1 day, 3, 10, 15 weeks p.p., at
the end of supplementation and 2, 6 and 10 weeks after end of supplementation
(Staufenbiel 1997). The measuring point was on a line between the upper range of
tuber coxae and tuber ischiadicum.
As the investigations presented in this paper are part of a comprehensive project,
additional samples were taken for further analysis. The corresponding results are not
presented here, but a systematic effect on the recorded performance parameters
cannot be ruled out.
2.3.
The silage and PMR samples were dried at 608C for 72 h. All feed samples were
ground to pass through a sieve with 1 mm pore size for analysing the contents of
nutrients according to the methods of the VDLUFA (Bassler 1976). Milk samples
were analysed for fat, protein, lactose and the somatic cell count (SSC) using an
infrared milk analyser (Milkoscan FT 6000 combined with a Fossomatic 500, Foss
Electric, Hillerød, Denmark). Before analyzing the fatty acid profile in milk fat, the
milk samples were heated up to 408C and homogenised by Ultra Turrax (T25, Janke
& Kunkel, IKA1-Labortechnik, Germany) treatment. Afterwards, morning and
evening milk were mixed according to their milk yields and freeze dried. The fat-
extraction of freeze dried milk was accomplished according to Soxhlet listed in
VDLUFA (Bassler 1976). Total milk fat was converted into FAME by the use of
sodium methoxide as catalyst.
The lipid content of the feed samples was extracted according to Folch et al.
(1957). After this a trans-esterification with Boron trifluoride (BF3) followed to
produce FAME. The emerged extracts were purified by thin-layer chromatography
(SIL G-25 UV254, Machery-Nagel, Germany).
All sample FAME extracts were analysed via gas chromatography ([GC]; GC-
17A Version 3, Schimadzu, Japan) equipped with an auto sampler and flame
ionisation detector. Two different GC procedures were necessary to analyse the
FAME profile of these samples. The first GC method determined the identity and
general FA profile from 4–25 carbon length FA using a medium polarity column
(DB-225ms, 60 m x 0.25 mm inner diameter [i.d.]; 0.25 mm; J&W Scientific,
Germany). The second GC method separated the cis and trans isomers of C18:1using
a high polarity column (SelectTMFAME, 200 m x 0.25 mm, i.d.; 0.25 mm; Varian
Inc., Germany). Various reference standards were used as FAME mix to identify FA
peaks: No. 463, 674, (Nu-Chek Prep, Inc., Elysian, US), BR2, BR4, ME 93
(Larodan; Malmo ¨ , Sweden), Supelco137 Component FAME Mix, PUFA No. 3,
conjugated linoleic acid, linoleic-, linolenic- and octadecenoic acid methyl ester mix
(Supelco; Bellefonte, US). The results were expressed as percentage values of total
FAME.
The centrifugation of plasma (heparin and EDTA) and serum was performed
after sampling and the samples were stored at –808C for further analysis. Plasma
NEFA and glucose levels were determined by enzymatic analysis using commercial
Analysis
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kits (NEFA HR[2] R1þR2 Set, Wako Chemicals GmbH, Neuss, Germany;
Hexokinase Fluid 5þ1, MTI Diagnostics GmbH, Idstein, Germany). Plasma
concentrations of BHB were quantified using a commercial kit (Ranbut, RB 1008;
Randox Laboratories GmbH, Wu ¨ lfrath, Germany). These procedures were
performed using the Cobas Mira Plus Chemistry Analyser (F. Hoffmann-La Roche
Ltd, Basel, Switzerland).
2.4.Calculations
Net energy lactation (NEL) of the used feedstuffs was calculated by using the
nutrient digestibilities from the studies with the wethers according to GfE (1991).
Fat-corrected milk (FCM) was estimated according to Gaines (1928):
FCM [kg/d] = ((milk fat [%] ?0:15Þ þ 0.4) ? milk yield [kg/d]
ð1Þ
The energy balance was calculated by subtracting the daily requirement for
maintenance (GfE 1991) and the daily requirement for milk production (Tyrrell and
Reid 1965) from the daily energy intake.
Maintenance requirement [MJ NEL/d] = 0.293 ? Metabolic live weight [kg0:75?
ð2Þ
Energy content of milk ½MJ NEL=kg?
¼ 0:95 þ 0.38 ?Milk fat [%] þ 0.21 ? Milk protein [%] + 0:07
ð3Þ
Requirement for milk production [MJ NEL/d]
¼ Energy content of milk [MJ NEL/kg] ? Milk yield [kg/d]
ð4Þ
Daily feed intake, live weight and milk yield values were condensed to weekly
means before data analysis. For the calculation of energy balance and milk yields,
the weekly means were used. The proportions of fatty acids in milk fat, which were
lower than the detection limits, were considered as zero in evaluating the data.
2.5.Statistical analysis
The results are presented as least square means (LS means) and standard error (SE)
of the mean. All data were analysed by a one-way ANOVA followed by the Tukey
test. Differences were considered to be significant at p 50.05, trends were declared at
p 50.1. All analyses were performed with SAS (Software package, Version 9.1, SAS
Institute, Cary, NC, USA).
Owing to technical conditions immediately after calving, the evaluation of
performance parameters and milk components was not possible for the first two
weeks p.p. To examine the distinct physiological stages during lactation in more
detail, the supplementation period was separated for analysis into two periods:
Period 1, weeks 2–7 and Period 2, weeks 8–26.
Milk yield, milk components, dry matter intake (DMI), net energy intake,
calculated energy balance and the change in live weight were analysed by using the
PROC MIXED procedure with a compound symmetry covariance structure. The
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model contained the supplementation and week of lactation as fixed factors and
the interaction between both factors. The individual cow effects resulting from the
frequent measurements in the course of the experiment were considered by the
repeated procedure.
Change of BCS and BFT, plasma levels of NEFA, BHB and glucose as well as
milk fatty acid profiles were analysed using the PROC MIXED procedure.
Supplemental CLA was considered as a fixed effect. The influence of the individual
cow on the data was considered in the model as random effect.
3. Results
Out of 46 cows, 43 completed the full 38 weeks of treatment. Two cows were
removed from the trial due to sickness (distorsion [week 8, CON], displacement of
abomasum [week 26, CLA-1]), and one cow had to be taken out of the experiment
due to serious mastitis problems (week 26, CLA-1).
Only slight differences in the contents of nutrients were detected between the two
different concentrate feeds (Table 1). The concentration of the trans-10, cis-12 CLA
isomer in concentrates and silages was calculated based on the analysed
concentrations in these feed components. In the CLA concentrate the concentration
of trans-10, cis-12 CLA amounted to 2.25 g/kg DM. Thus the CLA-1 and CLA-2
groups had intakes of 4 and 8 g trans-10, cis-12 CLA/d respectively. The analysed
proportion of trans-10, cis-12 CLA in the applied rumen-protected CLA preparation
amounted to 12% of total FAME (Table 2).
Performance data for the three periods are presented in Table 3. As expected for
a cow trial including nearly the complete lactation period, most measured
parameters changed markedly over the lactation period. The development of DMI
over the course of the trial is shown in Figure 1. The recorded DMI in the 2nd and
3rd week a.p. was similar in the three groups (CON, 13.98 kg/d; CLA-1, 14.50 kg/d;
and CLA-2, 13.95 kg/d). During the whole treatment period each cow consumed
daily 4 kg of concentrate feed containing the respective fat supplements.
In Period 1 of supplementation the control group consumed significantly more
total DM (11–16%) than the two CLA groups. There was a significant interaction
between supplementation and week of lactation during the whole supplementation
period because cows fed CLA had a more pronounced increase of DM intake in the
second week of lactation. No differences in the DMI were observed between the
treatment groups in Period 2 of supplementation and depletion period.
At the beginning of recording the milk yield there were no significant treatment
differences (Figure 2). After the third week of lactation the cows fed CLA had a
steeper increase in milk yield compared to the control group, resulting in a significant
interaction between supplementation and week of lactation. Overall, the contents of
fat and protein, content and yield of lactose and somatic cell count (SCC) were not
affected by treatment.
Over Period 2 of the supplementation, CLA supplementation caused a 7 and
12% dose-dependent significant reduction in milk fat content of the CLA-1 and
CLA-2 groups respectively (Figure 3). After finishing the supplementation, the milk
fat content of the CLA groups reached the same level like the control group. There
was a significant interaction between supplementation and week of lactation due to
the reduction of milk fat content in the first weeks of lactation and the increase after
end of supplementation. A tendency in reduction of the milk protein content (5%,
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Table 3.
Milk yield and milk composition and energetic variables in Period 1 (early lactation, weeks 2–7 of lactation), Period 2 (until end of CLA
supplementation, week 8–26 of lactation) and Period 3 (depletion period, 12 weeks).
Supplementation groups
p-value
Parameter
CON*
CLA-1{
CLA-2{
SUP?
Week
SUP x Week
Period 1
(n ¼ 15)
(n ¼ 15)
(n ¼ 16)
DMIx[kg/d]
21.1a+0.7
18.5b
+0.7
17.8b+0.7
0.003
50.001
50.001
Milk yield [kg/d]
30.4
+2.1
32.4
+2.1
30.9
+2.0
0.768
50.001
0.035
Milk fat [%]
4.43 +0.15
4.48
+0.15
4.24 +0.14
0.492
50.001
0.024
Milk fat [kg/d]
1.37 +0.12
1.48
+0.12
1.32 +0.11
0.630
0.306
0.380
FCMk[kg/d]
33.6
+2.6
36.0
+2.6
32.7
+2.6
0.653
0.015
0.802
Milk protein [%]
3.12 +0.05
3.11
+0.05
3.14 +0.04
0.897
50.001
0.849
Milk protein [kg/d]
0.94 +0.06
1.00
+0.06
0.96 +0.06
0.735
0.001
0.260
Milk lactose [%]
4.81 +0.04
4.77
+0.04
4.81 +0.04
0.790
50.001
0.216
Milk lactose [kg/d]
1.46 +0.10
1.56
+0.10
1.49 +0.09
0.719
50.001
0.076
SCC{[log10/ml]
4.85 +0.13
5.06
+0.13
4.93 +0.12
0.511
0.011
0.876
Net energy intake [MJ/d]
142.8a+4.3
126.3b
+4.3
121.9b+4.2
0.003
50.001
50.001
Live weight [kg]
526
+16
549
+16
531
+15
0.587
50.001
0.500
Energy balance [MJ/d]
14.9a+5.4
712.3b
+5.4
78.3b+5.2
0.002
50.001
0.039
Period 2
(n ¼ 14)
(n ¼ 15)
(n ¼ 16)
DMI [kg/d]
21.6
+0.6
22.4
+0.6
21.2
+0.6
0.404
0.033
0.050
Milk yield [kg/d]
30.2
+1.6
34.2
+1.6
33.0
+1.6
0.223
50.001
0.672
Milk fat [%]
4.11a+0.13
3.81ab+0.13
3.60b+0.12
0.021
50.001
0.562
Milk fat [kg/d]
1.23 +0.07
1.30
+0.07
1.19 +0.07
0.515
50.001
0.239
FCM [kg/d]
30.9
+1.7
33.7
+1.7
31.4
+1.7
0.484
50.001
0.314
Milk protein [%]
3.24 +0.05
3.14
+0.05
3.09 +0.05
0.088
50.001
0.044
Milk protein [kg/d]
0.97 +0.05
1.07
+0.05
1.01 +0.05
0.352
50.001
0.850
Milk lactose [%]
4.81 +0.03
4.79
+0.03
4.83 +0.03
0.683
50.001
0.111
Milk lactose [kg/d]
1.45 +0.07
1.64
+0.07
1.59 +0.07
0.193
50.001
0.825
SCC [log10/ml]
4.85 +0.12
5.08
+0.12
4.97 +0.11
0.412
0.187
0.796
Net energy intake [MJ/d]
144.3
+3.8
148.8
+3.8
141.7
+3.7
0.410
0.003
0.018
Live weight [kg]
567
+15
591
+15
576
+15
0.521
50.001
0.987
Energy balance [MJ/d]
15.3
+2.7
10.4
+2.6
10.8
+2.5
0.359
50.001
0.031
(continued)
96 J. Pappritz et al.
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Table 3.
(Continued).
Supplementation groups
p-value
Parameter
CON*
CLA-1{
CLA-2{
SUP?
Week
SUP x Week
Period 3
(n ¼ 13)
(n ¼ 13)
(n ¼ 16)
DMI [kg/d]
19.2
+0.5
19.6
+0.5
18.9
+0.5
0.621
50.001
0.136
Milk yield [g/d]
24.0
+1.1
25.3
+1.1
24.5
+1.0
0.665
50.001
0.999
Milk fat [%]
4.16 +0.12
4.21 +0.12
4.26 +0.11
0.849
50.001
50.001
Milk fat [kg/d]
0.99 +0.05
1.07 +0.05
1.04 +0.05
0.568
50.001
0.011
FCM [kg/d]
24.7
+1.2
26.5
+1.2
25.8
+1.1
0.518
50.001
0.432
Milk protein [%]
3.38 +0.05
3.34 +0.05
3.34 +0.05
0.803
50.001
0.153
Milk protein [kg/d]
0.80 +0.03
0.84 +0.03
0.81 +0.03
0.680
50.001
0.923
Milk lactose [%]
4.73 +0.03
4.74 +0.03
4.73 +0.03
0.963
50.001
0.273
Milk lactose [kg/d]
1.14 +0.05
1.2
+0.05
1.16 +0.04
0.612
50.001
0.999
SCC [log10/ml]
4.97 +0.12
5.10 +0.12
5.11 +0.11
0.637
0.003
0.759
Net energy intake [MJ/d]
124.5
+3.4
127.0
+3.4
122.3
+3.2
0.610
0.391
0.164
Live weight [kg]
609
+16
627
+16
613
+15
0.688
50.001
0.980
Energy balance [MJ/d]
12.1
+2.1
8.9
+2.1
7.4
+1.9
0.251
50.001
0.586
Notes: *CON, Cows fed the fat supplement without CLA;{CLA-1, Cows fed 50 g CLA supplement/d;{CLA-2, Cows fed 100 g CLA supplement/d;?SUP, Supplementation;
xDMI, Dry matter intake;kFCM, 4% fat corrected milk;{SCC, Somatic cell count;abValues with different superscripts within one period and within one are significantly
different (p 50.05); LS means + SE.
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p ¼ 0.088) was observed for the highest supplementation group (CLA-2). There was
a significant interaction between supplementation and week of lactation in Period 2
of supplementation because of the increase of milk protein content in CLA-2 group.
The control group had a significantly higher NEL intake in Period 1 of
supplementation in comparison to the two CLA groups. Live weight (LW) remained
unaffected by the CLA supplementation and all groups showed an almost linear
increase in the live weight with similar weekly LW gains. The calculated energy
balance declined significantly (CLA-1, –27 MJ/d and CLA-2, –23 MJ/d) by CLA
Figure 1.
periods for the feeding groups. —?—, Control group (n ¼ 15/14); ---¤---, CLA-1 group
(n ¼ 15/13); ---4---, CLA-2 group (n ¼ 16).
Development of dry matter intake (means) in supplementation and depletion
Figure 2.
for the feeding groups. —?—, Control group (n ¼ 15/14); ---¤---, CLA-1 group
(n ¼ 15/13); ---4---, CLA-2 group (n ¼ 16).
Development of milk yield (means) in supplementation and depletion periods
98 J. Pappritz et al.
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feeding compared with controls in the first weeks of lactation (Period 1) (Figure 4).
After 7 weeks of lactation, the animals of the CLA groups reached a steady energy
balance, whereas the control group had a positive calculated energy balance during
the entire evaluation period.
Table 4 shows the effects of supplementing rumen-protected CLA on indicators
of energy metabolism. BCS and BFT remained unaffected by CLA supplementation.
Plasma concentrations of glucose, BHB and NEFA did not differ between
treatments over the 26-week supplementation period, although there was a trend
Figure 3.
periods for the feeding groups. —?—, Control group (n ¼ 15/14); ---¤---, CLA-1 group
(n ¼ 15/13), ---4---, CLA-2 group (n ¼ 16).
Development of milk fat content (means) in supplementation and depletion
Figure 4.
periods for the feeding groups. —?—, Control group (n ¼ 15/14); ---¤---, CLA-1 group
(n ¼ 15/13); ---4---, CLA-2 group (n ¼ 16).
Development of net energy balance (means) in supplementation and depletion
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(p ¼ 0.057) for lower plasma NEFA concentrations in the CLA-2 group over Period
2 of supplementation. In the depletion period, plasma NEFA and BHB
concentrations remained unaffected by treatment whereas the plasma glucose
concentration was significantly higher in the CLA-2 group.
For the analysis of the fatty acid profile of milk fat (Table 5) the trial was
separated in to supplementation and depletion periods. The addition of the CLA
supplement did not alter the proportion of de novo synthesised (5C16) and
preformed (4C16) milk fatty acids, but reduced significantly the proportion of C16:0
and C16:1in total (originating from both sources) in the CLA-2 group during the
supplementation period. The proportion of trans-10, cis-12 CLA in milk fat
increased significantly and dose-dependently in the CLA groups, but the actual
increase was minimal in level. The proportion of cis-9, trans-11 CLA remained
unaffected by CLA supplementation. In the depletion period, no differences in the
fatty acid profile in milk fat were observed between the three treatment groups.
4.
In short term studies (510 weeks) the effects of dietary supplemented CLA,
especially of the trans-10, cis-12 isomer, on performance and metabolic parameters
Discussion
Table 4.
Period 1 (early lactation, weeks 2–7 of lactation), Period 2 (until end of CLA supplementation,
weeks 8–26 of lactation) and Period 3 (depletion period, 12 weeks).
Body condition score (BCS), back fat thickness (BFT) and plasma metabolites in
Supplementation groups
p-value
SUP?
CON*CLA-1{
CLA-2{
Period 1
BCS$
BFT [cm]
Plasma metabolites
Glucose [mmol/l]
NEFAj[mEq/l]
BHB{[mmol/l]
(n ¼ 15)
3.08 +0.04
2.55 +0.07
(n ¼ 15)
3.10 +0.04
2.71 +0.07
(n ¼ 16)
3.11 +0.04
2.62 +0.07
0.899
0.262
3.5
700 +62
0.6
+0.13.6
659 +60
0.7
+0.1 3.7
693 +58
0.7
+0.10.282
0.877
0.862
+0.1
+0.1
+0.1
Period 2
BCS*
BFT [cm]
Plasma metabolites
Glucose [mmol/l]
NEFA [mEq/l]
BHB [mmol/l]
(n ¼ 14)
2.99 +0.06
2.32 +0.07
(n ¼ 15)
2.99 +0.06
2.39 +0.07
(n ¼ 16)
3.08 +0.06
2.44 +0.06
0.442
0.455
3.7
289 +21
0.5
+0.13.6
250 +20
0.5
+0.13.8
217 +20
0.5
+0.10.273
0.057
0.340
+0.03
+0.03
+0.03
Period 3
BCS*
BFT [cm]
Plasma metabolites
Glucose [mmol/l]
NEFA [mEq/l]
BHB [mmol/l]
(n ¼ 14)
3.04 +0.06
2.24 +0.08
(n ¼ 13)
3.03 +0.06
2.35 +0.08
(n ¼ 16)
3.05 +0.06
2.26 +0.08
0.975
0.557
3.7b+0.1
245 +27
0.4
+0.02
3.9ab+0.1
319 +26
0.4
3.9a+0.1
306 +25
0.4
+0.02
0.027
0.121
0.996
+0.02
Notes:$Use of a BCS chart with a scale from one (under conditioned) to five (over conditioned); *CON,
Cows fed the fat supplement without CLA;{CLA-1, Cows fed 50 g CLA supplement/d;{CLA-2, Cows fed
100 g CLA-supplement/d;
hydroxybutyrate;
significantly different (p50.05); LS means+SE.
?SUP, Supplementation;
abValues with different superscripts within one period and within one row are
jNEFA, non esterified fatty acids;
{BHB, b-
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Table 5.
Fatty acid profile in milk fat in supplementation period (4 dates) and depletion period (2 dates).
Fatty acid
[% of
total
FAMEj]
Supplementation period
p-value
SUP?
Depletion period
p-value
SUP
CON*
(n ¼ 15/14)
CLA-1{
(n ¼ 15)
CLA-2{
(n ¼ 16)
CON
(n ¼ 14)
CLA-1
(n ¼ 13)
CLA-2
(n ¼ 16)
C4:0
3.50
+0.08
3.56
+0.08
3.61
+0.07
0.598
3.30
+0.08
3.25
+0.08
3.26
+0.07
0.889
C6:0
2.37
+0.06
2.42
+0.06
2.43
+0.06
0.727
2.69
+0.07
2.64
+0.07
2.75
+0.07
0.502
C8:0
1.31
+0.05
1.34
+0.05
1.37
+0.04
0.709
1.45
+0.05
1.47
+0.05
1.55
+0.04
0.255
C10:0
2.91
+0.13
3.00
+0.13
3.08
+0.13
0.685
3.20
+0.13
3.36
+0.13
3.52
+0.12
0.199
C12:0
3.12
+0.14
3.16
+0.14
3.28
+0.14
0.692
3.48
+0.14
3.65
+0.14
3.82
+0.13
0.223
C14:0
10.02
+0.29
10.27
+0.29
10.77
+0.28
0.168
11.30
+0.23
11.45
+0.23
11.83
+0.21
0.213
C14:1
0.92
+0.05
0.87
+0.05
0.89
+0.05
0.841
1.22
+0.07
1.24
+0.07
1.14
+0.06
0.523
C15:0
1.78
+0.08
1.74
+0.08
1.76
+0.08
0.944
1.88
+0.06
1.78
+0.06
1.86
+0.06
0.453
C16:0
30.41a
+0.49
29.39ab+0.49
28.62b
+0.47
0.039
34.76
+0.72
34.01
+0.72
33.03
+0.67
0.224
C16:1
2.06
+0.11
2.00
+0.11
1.90
+0.10
0.573
2.27
+0.12
2.27
+0.12
2.10
+0.11
0.494
C17:0
1.62
+0.05
1.60
+0.05
1.60
+0.05
0.960
1.34
+0.04
1.29
+0.04
1.33
+0.04
0.696
C18:0
10.22
+0.34
10.91
+0.34
10.96
+0.33
0.219
8.56
+0.37
8.78
+0.37
9.21
+0.35
0.438
C18:1trans
2.15
+0.15
2.27
+0.15
2.27
+0.14
0.811
1.92
+0.10
1.79
+0.10
1.77
+0.10
0.549
C18:1cis-9
20.92
+0.63
20.60
+0.63
20.57
+0.61
0.910
17.16
+0.61
17.66
+0.61
17.51
+0.57
0.835
C18:2trans-9, trans-12
0.30
+0.02
0.32
+0.02
0.33
+0.02
0.700
0.07
+0.01
0.08
+0.01
0.07
+0.01
0.424
C18:2cis-9, cis-12
1.90
+0.06
1.99
+0.06
1.97
+0.06
0.574
1.30
+0.06
1.34
+0.06
1.27
+0.05
0.702
Conjugated linolic acid
C18:2cis-9, trans-11
0.57
+0.04
0.60
+0.04
0.58
+0.04
0.868
0.59
+0.03
0.54
+0.03
0.51
+0.03
0.166
C18:2trans-10, cis-12
0.004c+0.001
0.02b
+0.001
0.03a
+0.001
50.001
0.00
0.00
0.00
Other CLA
0.17
+0.01
0.16
+0.01
0.16
+0.01
0.628
0.09
+0.004
0.08
+0.004
0.08
+0.003
0.219
C18:3
0.41
+0.02
0.41
+0.02
0.42
+0.02
0.788
0.27
+0.01
0.29
+0.01
0.28
+0.01
0.542
C20:0
0.13
+0.005
0.13
+0.005
0.14
+0.005
0.449
0.13
+0.01
0.14
+0.01
0.14
+0.01
0.260
Other
3.21
+0.10
3.24
+0.10
3.26
+0.09
0.979
3.02
+0.06
2.89
+0.06
2.97
+0.06
0.475
Summation
5C16
26.55
+0.66
26.98
+0.66
27.74
+0.64
0.419
29.35
+0.59
29.64
+0.59
30.55
+0.55
0.301
C16
32.46a
+0.49
31.37ab+0.49
30.53b
+0.48
0.025
37.03
+0.76
36.27
+0.76
35.13
+0.71
0.191
4C16
40.97
+0.97
41.66
+0.97
41.73
+0.94
0.826
33.62
+0.97
34.08
+0.97
34.32
+0.91
0.870
Notes: *CON, Cows fed the fat supplement without CLA;{CLA-1, Cows fed 50 g CLA-supplement/d;{CLA-2, Cows fed 100 g CLA-supplement/d;?SUP, Supplementation;
jFAME, Fatty acid methyl ester;abcValues with different superscripts within one period in the rows are significantly different (p50.05); LS means+SE.
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of dairy cows have been well investigated. However, data regarding the long-term
effects of CLA supplementation on dairy cows is still rare as most examinations were
conducted either during the transition period and early lactation (Moore et al. 2004;
Selberg et al. 2004; Castaneda-Gutierrez et al. 2005; Odens et al. 2007) or during a
few weeks in mid lactation (Giesy et al. 2002; de Veth et al. 2005; Suksombat and
Chullanandana 2008; Piamphon et al. 2009; Sippel et al. 2009; Moallem et al.
2010). Only Perfield et al. (2002) and Bernal-Santos et al. (2003) performed studies
over a 20-week period of lactation treatment, but their CLA supplement was a
mixture of four isomers, including trans-8, cis-10 CLA; cis-9, trans-11 CLA; trans-
10, cis-12 CLA; and cis-11, trans-13 CLA. The aim of the present study was to
investigate the long-term effects of trans-10, cis-12 CLA on dairy cows. Each
animal of the CLA groups received 5 or 10 g of trans-10, cis-12 CLA per day from
1 d p.p. to the 26th week of lactation. The cis-9, trans-11 isomer, which did neither
affect the rates of milk fat synthesis in a study by Baumgard et al. (2002a) nor the
lipid metabolism of lactating cows, was present at almost the same proportion due
to the manufacturing process of the CLA preparation. To determine possible post-
supplementation effects, the animals were observed for further 12 weeks after the
supplementation period of the study.
We aimed at an individual dietary intake of 5 and 10 g trans-10, cis-12 CLA/d
in groups CLA-1 and CLA-2 respectively. However, in both groups the calculated
intakes, basedon the analysedconcentrations
approximately 20% lower than expected (4 and 8 g trans-10, cis-12 CLA/d
respectively).
In the present study, the DMI of the two CLA groups was reduced significantly
by 12 and 16% in early lactation. The similar DMI in the three groups in the 2nd and
3rd week a.p. showed that the differences in the first weeks are not ascribable to
differences before the start of the trial. Furthermore, we measured that each animal
consumed the 4 kg of concentrate feed, which included the fat supplement.
Therefore, the sensory properties can not count for the decrease in DMI. The lower
DMI concerns the PMR, which consisted of the same components for all three
groups. Data concerning the effects of dietary supplemented CLA on the feed intake
of dairy cows during the first weeks of lactation are mostly consistent and in contrast
to our findings. Moore et al. (2004) and Castaneda-Gutierrez et al. (2005) worked
with supplements consisting of various CLA isomers and different trans-10, cis-12
CLA concentrations (9, 12, 25 and 37 g/d). Even with such high concentrations, no
influence on DMI in early lactation was observed. Piamphon et al. (2009) reported a
slightly increased DMI when 8 g trans-10, cis-12 CLA was added to diet during mid-
lactation. However after the abomasal infusion of 10 g trans-10, cis-12 CLA/d,
Baumgard et al. (2000a) found a tendency towards a reduced DMI (p ¼ 0.07), while
abomasal infused cis-9, trans-11 CLA did not affect the DMI. Thus a CLA-related
transitory depression in DMI seems to be possible. The mechanisms behind this
effect are not yet understood, but seem to be regulated at the metabolic level since the
CLA-containing supplements were consumed completely by all cows. In the trial of
Moallem et al. (2010), DMI was reduced significantly by 2.5% after the dietary
supplementation of 5 g trans-10, cis-12 CLA and the authors supposed a relation
with the saturation of the fatty acids in the diet. With the energy content of feed
being nearly similar, the significantly lower net energy intake of the CLA-groups
during the first weeks of lactation can be explained by the lower DMI of these
animals in this time.
inthe concentrates, were
102J. Pappritz et al.
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