1
2
Tannins as feed additives to modulate ruminal biohydrogenation: effects on
3
animal performance, milk fatty acid composition and ruminal fermentation
4
in dairy ewes fed a diet containing sunflower oil
5
6
Pablo G. Toral, Gonzalo Hervás, Elena Bichi, Álvaro Belenguer, Pilar Frutos*
7
8
9
Instituto de Ganadería de Montaña (CSIC-Universidad de León), Finca Marzanas s/n.
10
24346 Grulleros, León, Spain
11
12
13
*
14
p.frutos@eae.csic.es
Corresponding author: Tel: + 34 987 317 064; Fax: + 34 987 317 156; E-mail address:
15
16
17
18
1
19
Abstract
20
In vitro studies have suggested that feeding tannins to ruminants can favourably
21
alter ruminal biohydrogenation of dietary linoleic acid, enhancing accumulation of
22
trans-11 18:1 (VA, vaccenic acid) in the rumen and thereby the content of some human
23
health promoting fatty acids, such as VA and cis-9 trans-11 18:2 (rumenic acid, RA), in
24
dairy or meat products. However, reports on impacts of these phenolic compounds on
25
milk fatty acid (FA) profile are very limited and inconsistent. Therefore, fourteen Assaf
26
ewes in mid lactation were used to examine effects of addition of a mixture (1:1, w/w)
27
of two commercial oenological extracts of quebracho condensed tannins (CT) and
28
chestnut hydrolysable tannins (HT) to a diet containing sunflower oil (SO) on animal
29
performance, milk yield and composition, and ruminal fermentation. All sheep received
30
a total mixed ration based on alfalfa hay and a concentrate (400:600), supplemented
31
with 20 g of SO/kg dry matter (DM) plus 0 (Control; n=7) or 10 (TAN; n=7) g of
32
tannins/kg DM. Milk production and composition was analysed on days 0, 3, 6, 9, 12,
33
15, 18, 21, 24 and 27 on treatments, and milk FA profile on days 0, 3, 6, 12, 18 and 27.
34
Neither DM intake nor milk, or its component, yield was affected by TAN treatment.
35
Similarly, addition of the extract of tannins to a SO containing ration did not alter
36
concentrations of the major FA classes in milk (i.e., saturates, monounsaturates,
37
polyunsaturates), had very limited effects on the proportion of particular FA, and was
38
not able to enhance milk VA and RA enrichment above that achieved with SO
39
supplementation. Temporal changes in milk FA composition were characterized by an
40
increase in unsaturated FA with 18 carbons, mainly cis and trans 18:1, and a
41
concomitant reduction in most short and medium chain saturates (6:0 to 12:0 and 16:0;
42
P<0.05) attributable to the presence of SO in the diet. Addition of tannins did not affect
43
ruminal fermentation parameters (i.e., pH, lactate, ammonia, total volatile fatty acid
2
44
concentrations) measured after 28 days. Reasons for the lack of effects of either type
45
(quebracho CT and chestnut HT) or amount of tannins in the diet is discussed.
46
Keywords: chestnut tannin; conjugated linoleic acid; quebracho; sheep; vaccenic acid
47
Abbreviations: BH, biohydrogenation; BW, body weight; CLA, conjugated linoleic
48
acid; CT, condensed tannins; DM, dry matter; FA, fatty acid; FAME, fatty acid methyl
49
esters; HT, hydrolysable tannins; PEG, polyethyleneglycol; RA, rumenic acid; SO,
50
sunflower oil; T, time; TMR, total mixed ration; VA, vaccenic acid; VFA, volatile fatty
51
acid.
52
53
1. Introduction
54
Cis-9 trans-11 18:2, rumenic acid (RA), is the major isomer of conjugated linoleic
55
acid (CLA) in ruminant derived food products and is considered to be the principal
56
component of potential human health promoting effects which have been attributed to
57
CLA (Pariza et al., 2001). The main source of RA is endogenous synthesis, via
58
desaturation of trans-11 18:1, vaccenic acid (VA), in the mammary gland or other body
59
tissues (Palmquist et al., 2005). In ruminants, dietary linoleic acid (cis-9 cis-12 18:2) is
60
biohydrogenated in the rumen by microorganisms, resulting in accumulation of a wide
61
range of intermediates, such as RA, which is then reduced to VA and finally to 18:0,
62
stearic acid (Palmquist et al., 2005). A great deal of effort has been directed towards
63
promoting accumulation of VA in the rumen as a means of increasing the content of
64
CLA in meat or milk. Supplementation of ruminant diets with linoleic acid-rich oils,
65
such as sunflower oil (SO), is known to enhance milk CLA content, presumably through
66
increased ruminal formation of VA (Palmquist et al., 2005; Bernard et al., 2010).
67
Another way to cause accumulation of VA is by inhibiting the last step of rumen
68
biohydrogenation (BH) using marine lipids (e.g., fish oils and algae, Shingfield et al.,
3
69
2006; Toral et al., 2010a, 2010b) or, according to recent studies (Khiaosa-Ard et al.,
70
2009; Vasta et al., 2009a, 2009b), tannins.
71
However, reports on effects of tannins on ruminal BH and meat or milk fat
72
composition are few and contradictory. While in vitro experiments (Khiaosa-Ard et al.,
73
2009; Vasta et al., 2009a) show positive effects on rumen VA accumulation, in vivo
74
studies seem to suggest no significant or even negative effects (Benchaar and
75
Chouinard, 2009; Cabiddu et al., 2009; Vasta et al., 2009b).
76
Despite the long-lasting generalization that tannins were toxic to ruminants or
77
impaired animal performance when eaten, it is now recognized that these phenolic
78
compounds can be detrimental, innocuous or beneficial depending on type and chemical
79
structure, amount ingested and animal species (Makkar, 2003; Mueller-Harvey, 2006),
80
which is probably related to their known effects on the ruminal microbiota (McSweeney
81
et al., 2001). Variations in the susceptibility of microbial populations from different
82
ruminants to effects of dietary tannins (Pell et al., 2000; Frutos et al., 2004) may also be
83
applicable to the populations of bacteria responsible for the different steps of BH of
84
unsaturated fatty acids (FA) in the rumen. Furthermore, there appears to be many
85
similarities, but also substantial divergences, among ruminant species in metabolism of
86
dietary lipids, although most research has been undertaken with dairy cows and goats,
87
and much less is known about the BH process in dairy ewes (Chilliard et al., 2003;
88
Shingfield et al., 2010).
89
It was against this background that our study was completed to examine effects of
90
addition of a mixture of commercially available tannins to a diet containing SO, as a
91
source for rumen VA production, on animal performance, milk yield and composition,
92
including the FA profile, and ruminal fermentation characteristics in sheep
93
4
94
2. Materials and methods
95
2.1. Animals, experimental diets and management
96
Fourteen multiparous Assaf ewes (body weight, BW = 92.5 ± 2.86 kg) at week 13
97
of lactation at the beginning of the experiment were used. Sheep were housed in
98
individual tie stalls and randomly divided into 2 groups, balanced for milk production,
99
BW, days postpartum, number of lactations, and voluntary feed intake, and each was
100
assigned to one of two experimental treatments being: Control or supplemented with
101
tannins (TAN).
102
Diets were prepared weekly and consisted of a total mixed ration (TMR) based on
103
alfalfa hay (particle size >4 cm) and a concentrate (forage:concentrate ratio 400:600),
104
supplemented with 20 g of SO/kg of dry matter (DM) plus 0 (Control diet) or 10 (TAN
105
diet) g of tannins/kg DM. The tannin supplement was a 1:1 (w/w) mixture of two
106
commercial oenological extracts (TanicolMox and Vinitanon; Agrovin S.A., Alcázar de
107
San Juan, Spain) containing both condensed and hydrolysable tannins (Table 1). The
108
ingredients and chemical composition of the diets, which included liquid molasses to
109
avoid selection of dietary components, are also in Table 1. Prior to commencing the
110
experiment, all ewes were fed the TMR without SO for a 2 week adaptation. Clean
111
water was always available and fresh diets were fed daily ad libitum at 09:00 and 19:00
112
h.
113
Ewes were milked daily at approximately 08:30 and 18:30 h in a 1 × 10 stall
114
milking parlour (DeLaval, Madrid, Spain). The experimental conditions were the same
115
for all the sheep. The experiment lasted for 4 weeks, and was completed in accordance
116
with Spanish Royal Decree 1201/2005 for the protection of animals used for
117
experimental purposes.
118
2.2. Measurements and sampling procedures
5
119
2.2.1. Diets
120
Intake of DM was recorded daily by weighing the amount of TMR offered and
121
refused by ewe. Samples of the diets and orts were collected daily, stored at –30ºC and
122
then freeze dried.
123
2.2.2. Milk
124
Individual daily milk yield was recorded on days 0, 3, 6, 9, 12, 15, 18, 21, 24 and
125
27. Milk samples were collected at the same time from each ewe and stored at 4ºC with
126
natamycin until analyzed for fat, crude protein and total solids. Milk FA composition
127
was determined in unpreserved samples collected on days 0, 3, 6, 12, 18 and 27 of the
128
experiment that were stored at –30ºC until analysis.
129
2.2.3. Ruminal fluid
130
After 28 days on treatments, ewes were milked at 08:30 h and given free access to
131
the diets as on other days. After 1 h the TMR was removed and 3 h later samples of
132
ruminal fluid were collected from each ewe using a stomach tube, and checked to
133
ensure that they did not contain saliva. Rumen fluid was strained through two layers of
134
muslin cloth, pH was measured and 4 ml were acidified with 4 ml of 0.2 M HCl for
135
determination of ammonia. Further aliquots of 4 and 0.8 ml of ruminal fluid were
136
collected for lactic acid and volatile fatty acid (VFA; deproteinized with 0.5 ml of 20 g/l
137
metaphosphoric and 4 g/l crotonic acids in 0.5 M HCl) determinations. All samples
138
were stored at –30ºC until analysis.
139
2.2.4. Body weight
140
Ewes were weighed at the beginning and end of the experiment.
141
2.3. Chemical analysis
142
2.3.1. Diets
6
143
TMR samples were analysed for DM (ISO 6496:1999), ash (ISO 5984:2002) and
144
crude protein (ISO 5983-2:2009). Neutral and acid detergent fibre were determined by
145
methods described by Mertens (2002) and the AOAC (2006; Official Method 973.18),
146
respectively, using an Ankom2000 fiber analyzer (Ankom Technology Corp., Macedon,
147
NY, USA). Neutral detergent fibre was assayed with sodium sulphite and α-amylase
148
and expressed with residual ash (the latter also for acid detergent fibre). The content of
149
ether extract in the diets was determined by the Ankom filter bag technology (AOCS,
150
2008; Procedure Am 5-04).
151
2.3.2. Milk
152
Crude protein, fat and total solid concentrations were determined by infrared
153
spectrophotometry (ISO 9622:1999), using a Milko-Scan 255 A/S N (Foss Electric,
154
Hillerod, Denmark).
155
For milk FA composition analysis, lipids in 1 ml of milk were extracted using
156
diethyl ether and hexane (5:4, v/v) and transesterified to fatty acid methyl esters
157
(FAME) using freshly prepared methanolic sodium methoxide, as outlined by
158
Shingfield et al. (2003), with tridecanoic acid (Nu-Chek Prep, Elysian, MN, USA) as an
159
internal standard. Methyl esters were separated and quantified using a gas
160
chromatograph (Agilent 7890A GC System, Santa Clara, CA, USA) equipped with a
161
flame-ionization detector and a 100 m fused silica capillary column (0.25 mm i.d., 0.2-
162
μm film thickness; CP-SIL 88, Chrompack 7489, Varian Iberica S.A., Madrid, Spain)
163
and He as the carrier gas. Total FAME profile in a 2 μl sample volume at a split ratio of
164
1:50 was determined using a temperature gradient programme (Shingfield et al., 2003).
165
Isomers of 18:1 were further resolved in a separate analysis under isothermal conditions
166
at 170ºC (Shingfield et al., 2003). Peaks were identified based on retention time
167
comparisons with authentic standards (GLC 463 and U-45-M, Nu-Chek Prep., Elysian,
7
168
MN, USA; 18919-1AMP, L6031, L8404 and O5632, Sigma Aldrich, Madrid, Spain;
169
21-1614-7, Larodan Fine Chemicals AB, Malmö, Sweden). Identification of 18:1
170
isomers was verified based on FAME standard mixtures when available,
171
chromatograms reported in the literature (e.g., Shingfield et al., 2003; Kramer et al.,
172
2008) and by comparison with samples for which reference measures had been made
173
(Toral et al., 2010c).
174
2.3.3. Rumen fluid
175
Ammonia and lactic acid concentrations were determined by colorimetric
176
methods (Weatherburn, 1967, and Taylor, 1996; respectively) and VFA by gas
177
chromatography using crotonic acid as an internal standard (Ottenstein and Bartley,
178
1971).
179
2.4. Statistical analysis
180
All analyses used the Statistical Analysis System (2003). Data on DM intake, milk
181
yield and composition, as well as milk FA composition, were analyzed by repeated
182
measures analysis, using the MIXED procedure, and assuming a covariance structure
183
fitted on the basis of Schwarz’s Bayesian information model fit criterion. The statistical
184
model included fixed effects of diet (D), time (T), their interaction and the initial record
185
measured at week 0 (covariate). One-way analysis of variance, using the MIXED
186
procedure of the SAS (2003), was applied to data on rumen fermentation (i.e., pH,
187
ammonia, lactate and VFA) and BW changes. In both analyses (i.e., repeated
188
measurement and one-way analysis of variance), animal was nested within diet and used
189
as the error term to contrast effects of tannin supplementation.
190
Differences were declared significant if P<0.05 and values of 0.05<P<0.10 were
191
interpreted as tendencies towards significance. Least square means adjusted for the
192
covariance are reported.
8
193
194
3. Results
195
Neither DM intake nor milk, or its component, yield was affected by diet
196
supplementation with 10 g/kg DM of the mixture of tannin extracts (Table 2). The same
197
lack of differences ocurred for milk fat, CP and total solid contents, as well as for BW
198
change.
199
Tannin supplementation did not impact the concentration of the major classes of
200
FA in milk (i.e., saturated, monounsaturated, and polyunsaturated FA) and had very
201
limited effects on the proportion of some minor FA (Table 3). None of the even chain
202
saturated FA in milk were affected by tannin inclusion, and only the minor odd chain
203
saturate 17:0 was decreased (P<0.05), although the magnitude of this change was small.
204
For both treatments, most short and medium even chain saturated FA (6:0 to 12:0 and
205
16:0) were reduced during the first days on the experiment (P<0.05).
206
Oleic acid was the most abundant milk monounsaturated FA, at 15.6 g/100 g FA,
207
and increased with time on diet (P<0.001). In contrast, cis-9 14:1 (P<0.05) and cis-9
208
16:1 (P=0.072) were reduced in response to tannin supplementation, although these
209
changes were not quantitatively important. With regards to trans monounsaturated FA,
210
there was a 2.7 fold increase in VA on day 3, but no further improvement occurred.
211
Tannin had no effect on milk polyunsaturated FA. The CLA content was
212
relatively high, at 2.24 g/100 g FA, and the peak containing RA was more than 0.95 of
213
total CLA. Under the chromatographic conditions applied, it was not possible to
214
separate the RA from trans-7 cis-9 and trans-8 cis-10 CLA. However, based on
215
previous determinations (e.g., Hervás et al., 2008; Toral et al., 2010a, 2010b), the RA
216
would be expected to account for ≥92% of peak area. RA content increased from 0 to 3
217
days on both diets, with a trend towards a D × T interaction (P=0.089) and, as a
9
218
consequence, for total CLA (P=0.090), because the increase was slightly more in the
219
animals supplemented with tannins.
220
221
Addition of tannin extracts had no effect on rumen pH, lactate, total and molar
proportions of VFA, or ammonia concentrations (Table 4).
222
223
4. Discussion
224
This study was conducted to test the hypothesis that feeding tannins to dairy ewes
225
would favourably modify ruminal BH of dietary linoleic acid and thereby enhance milk
226
fat CLA without inducing negative effects on performance. A number of experiments
227
conducted by our group have examined effects of SO supplementation on performance
228
and milk composition, including milk FA profile, in lactating ewes (Hervás et al., 2008;
229
Toral et al., 2010a, 2010b) and, therefore, differences attributable to the presence of SO
230
will only be mentioned in passing.
231
4.1. Animal performance and milk composition
232
The lack of effects on DM intake, BW, and milk production and composition due
233
to addition of tannins to the diet is consistent with results reported by Benchaar et al.
234
(2008) who observed that inclusion of a quebracho condensed tannin extract in the diet
235
of dairy cows altered neither DM intake nor milk production or composition. In grazing
236
dairy ewes, Molle et al. (2009) found no effect of the condensed tannins of Hedysarum
237
coronarium on intake, but milk yield tended to be reduced and milk fat content was
238
lower. In contrast, Wang et al. (1996) reported an increase of 21% in milk yield of ewes
239
fed Lotus corniculatus versus ewes under the same feeding regime but dosed with
240
polyethyleneglycol (PEG, a non-ionic detergent used to complex and inhibit effects of
241
tannins). Changes in milk yield were accompanied by an increase in the lactose
242
concentration and, after 9 weeks on treatment, a decrease in fat content (Wang et al.,
10
243
1996). However, in a 14 day experiment with dairy cows fed Lotus corniculatus (Turner
244
et al., 2005), an increase of 13% in milk yield was not accompanied with changes in
245
milk fat or lactose concentrations.
246
These apparently inconsistent findings are probably related to ruminant species,
247
time on diets, type of tannins and dose. Tannins have been tentatively classified into
248
two major groups being: hydrolysable (HT) and condensed (CT), although this
249
classification has not been very helpful in predicting animal responses (Mueller-Harvey,
250
2006). Structural and chemical dissimilarities between HT and CT may offer an
251
explanation for differences in their biological effects and, therefore, results obtained
252
using a particular type of tannins cannot be applied to others. In our study, the reason to
253
use a combination of both CT and HT, instead of only CT, was to examine the
254
possibility that some phenolic metabolites deriving from HT degradation in the rumen,
255
may appear in the milk, giving it added value.
256
A key issue when using plant extracts as feed additives is dosage. The amount of
257
the tannins used in our study was chosen with the aim of favourably modulating ruminal
258
BH of dietary linoleic acid without impairing animal performance, and of being
259
practical. Most doses of tannins evaluated in the studies quoted above ranged from 4.5
260
g/kg DM (Benchaar et al., 2008; Benchaar and Chouinard, 2009) to 44.5 g/kg DM
261
(Wang et al., 1996). Furthermore, the lack of standardisation of analysis of this group of
262
phenolic compounds, and the use of different standards to express tannin
263
concentrations, means that among experiment comparisons can seldom be made with
264
confidence (Makkar, 2003; Álvarez del Pino et al., 2005).
265
4.2. Milk fatty acid composition
266
Available reports on impacts of tannin consumption on milk FA profile are
267
inconsistent. In cows or sheep grazing tannin containing legumes, changes in the milk
11
268
content of 18 carbon FA occurred when PEG was supplemented, indicating that tannins
269
could be responsible for a decrease (Turner et al., 2005) or an increase (Cabiddu et al.,
270
2009) in the extent of ruminal BH of dietary PUFA to 18:0. In contrast, diet
271
supplementation with 6.7 g/kg DM of quebracho CT extract resulted in no change in the
272
FA composition of bovine milk (Benchaar and Chouinard, 2009), in agreement with our
273
results. This may suggest the ineffectiveness of the type of tannins used, the low dosage
274
rate, or both, at exerting major effects on rumen BH, while most temporal changes in
275
milk FA profile were likely explained by the presence of SO in the diet. These temporal
276
changes were characterized by an increase in unsaturated FA with 18 C (mainly cis and
277
trans 18:1, including VA) and a concomitant reduction in most short and medium chain
278
saturates (6:0 to 12:0 and 16:0), which is consistent with previous reports in ewes fed
279
SO (Hervás et al., 2008; Toral et al., 2010a, 2010b).
280
Inclusion of tannins in the diet augmented neither milk VA and RA contents
281
above that achieved with SO, nor the low levels of trans-10 18:1. The lack of shift in
282
the distribution of trans 18:1 isomers probably reflected the proportion of forage being
283
sufficiently high to maintain normal rumen function and prevent an increase in trans-10
284
18:1 at the expense of trans-11 18:1. The composition of the ration might also have had
285
an influence in the response to tannins, since Vasta et al. (2009b) showed that addition
286
of 104 g/kg DM of a quebracho CT extract increased the content of both trans-11 and
287
trans-10 18:1 in the rumen of lambs fed high concentrate diets, but not in the rumen of
288
those offered only fresh herbage, despite the very high dose of tannin extract fed.
289
4.3. Rumen fermentation characteristics
290
A number of studies have demonstrated that effects of tannins on ruminal
291
fermentation is dose dependent, and a negative effect only occurs when they are fed at
292
high concentrations (Hervás et al., 2003; Mueller-Harvey, 2006). Moreover, because
12
293
HT can be readily degraded by rumen microbiota (McSweeney et al., 2001), it has been
294
suggested that these compounds may not exert effects on nutrient digestion in the
295
gastrointestinal tract (Foley et al., 1999). However, that assertion was based on
296
observations of the dose dependent action of tannic acid, and it is now widely accepted
297
that both CT and HT can affect ruminal fermentation (Makkar, 2003; Frutos et al.,
298
2004). Inclusion of SO in ewe diets has been previously shown to have no detrimental
299
effect on ruminal nutrient digestion (Hervás et al., 2008; Toral et al., 2010c).
300
301
5. Conclusions
302
To our knowledge, this is the first study using tannins as feed additives in which
303
effects of their addition to a linoleic acid rich diet has been examined in high producing
304
dairy ewes. Addition of a commercial mixture of condensed and hydrolysable tannin
305
extracts to a diet containing sunflower oil had no effect on ruminal fermentation and
306
animal performance, or an important impact on milk FA profile in lactating ewes. The
307
low dose (i.e., 10 g/kg DM), the type of tannins (i.e., 1:1 quebracho CT and chestnut HT
308
extracts), or both, may have been responsible for the lack of change in milk VA and RA
309
content.
310
Further research investigating possible effects of these phenolic compounds, at
311
higher inclusion rates, is advisable to establish the potential of tannins to modify
312
ruminal BH and milk fatty acid composition.
313
314
Acknowledgements
315
The authors thank the research farm staff for their help in the field work and C.
316
Delavaud (INRA, Clermont-Ferrand-Theix, France) for useful discussion during the
317
identification of milk fatty acids. They also thank Prof. K.J. Shingfield (MTT Agrifood
13
318
Research Finland) for helpful comments and revision of the manuscript. P.G. Toral and
319
E. Bichi were granted fellowships from the CSIC (I3P and JAE Programmes,
320
respectively).
321
322
References
323
Álvarez del Pino, M.C., Hervás, G., Mantecón, A.R., Giráldez F.J., Frutos, P., 2005.
324
Comparison of biological and chemical methods, and internal and external
325
standards, for assaying tannins in shrub species. J. Sci. Food Agric. 85, 583-590.
326
AOAC, 2006. Official Methods of Analysis of the Association of Analytical
327
Communities, 18th ed. (1st revision). AOAC International, Gaithersburg, MD,
328
USA.
329
330
AOCS, 2008. Official Methods and Recommended Practices of the American Oil
Chemist's Society, 5th ed. (2nd printing). AOCS, Urbana, IL, USA.
331
Benchaar, C., Chouinard, P.Y., 2009. Assessment of the potential of cinnamaldehyde,
332
condensed tannins, and saponins to modify milk fatty acid composition of dairy
333
cows. J. Dairy Sci. 92, 3392-3396.
334
Benchaar, C., McAllister, T.A., Chouinard, P.Y., 2008. Digestion, ruminal
335
fermentation, ciliate protozoal populations, and milk production from dairy cows
336
fed cinnamaldehyde, quebracho condensed tannin, or Yucca schidigera saponin
337
extracts. J. Dairy Sci. 91, 4765-4777.
338
Bernard, L., Mouriot, J., Rouel, J., Glasser, F., Capitan, P., Pujos-Guillot, E.,
339
Chardigny, J.M., Chilliard, Y., 2010. Effects of fish oil and starch added to a diet
340
containing sunflower-seed oil on dairy goat performance, milk fatty acid
341
composition and in vivo delta-9 desaturation of [13C] vaccenic acid. Brit. J. Nutr.
342
104, 346-354.
14
343
Cabiddu, A., Molle, G., Decandia, M., Spada, S., Fiori, M., Piredda, G., Addis, M.,
344
2009. Responses to condensed tannins of flowering sulla (Hedysarum coronarium
345
L.) grazed by dairy sheep. Part 2: Effects on milk fatty acid profile. Livest. Sci.
346
123, 230-240.
347
Chilliard, Y., Ferlay, A., Rouel, J., Lamberett, G., 2003. A review of nutritional and
348
physiological factors affecting goat milk lipid synthesis and lipolysis. J. Dairy Sci.
349
86, 1751-1770.
350
Foley, W.J., Iason, G.R., McArthur, C., 1999. Role of secondary metabolites in the
351
nutritional ecology of mammalian herbivores: how far have we come in 25 years?,
352
in: Jung, H-J.G., Fahey Jr., G.C. (Eds.), Nutritional Ecology of Herbivores.
353
American Society of Animal Science, IL, USA, pp. 130-209.
354
Frutos, P., Hervás, G., Giráldez, F.J., Mantecón, A.R., 2004. An in vitro study on the
355
ability of polyethylene glycol to inhibit the effect of quebracho tannins and tannic
356
acid on rumen fermentation in sheep, goats, cows and deer. Aust. J. Agric. Res.
357
55, 1125-1132.
358
Hervás, G., Frutos, P., Giráldez, F.J., Mantecón, A.R., Álvarez del Pino, M.C., 2003.
359
Effect of different doses of quebracho tannins extract on rumen fermentation in
360
ewes. Anim. Feed Sci. Technol. 109, 65-78.
361
Hervás, G., Luna, P., Mantecón, A.R., Castañares, N., de la Fuente, M.A., Juárez, M.,
362
Frutos, P., 2008. Effect of diet supplementation with sunflower oil on milk
363
production, fatty acid profile and ruminal fermentation in lactating dairy ewes. J.
364
Dairy Res. 75, 399-405.
365
Khiaosa-Ard, R., Bryner, S.F., Scheeder, M.R.L., Wettstein, H.R., Leiber, F., Kreuzer,
366
M., Soliva, C.R., 2009. Evidence for the inhibition of the terminal step of ruminal
367
alpha-linolenic acid biohydrogenation by condensed tannins. J. Dairy Sci. 92,
15
368
177-188.
369
Kramer, J.K.G., Hernandez, M., Cruz-Hernandez, C., Kraft, J., Dugan, M.E.R., 2008.
370
Combining results of two GC separations partly achieves determination of all cis
371
and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as
372
demonstrated using Ag-ion SPE fractionation. Lipids 43, 259-273.
373
Makkar, H.P.S., 2003. Effects and fate of tannins in ruminant animals, adaptation to
374
tannins, and strategies to overcome detrimental effects of feeding tannin-rich
375
feeds. Small Rumin. Res. 49, 241-256.
376
McSweeney, C.S., Palmer, B., McNeill, D.M., Krause, D.O., 2001. Microbial
377
interactions with tannins: nutritional consequences for ruminants. Anim. Feed
378
Sci. Technol. 91, 83-93.
379
Mertens, D.R., 2002. Gravimetric determination of amylase-treated neutral detergent
380
fiber in feeds with refluxing in beakers or crucibles: Collaborative study. J.
381
AOAC Int. 85, 1217-1240.
382
Molle, G., Decandia, M., Giovanetti, V., Cabiddu, A., Fois, N., Sitzia, M., 2009.
383
Responses to condensed tannins of flowering sulla (Hedysarum coronarium L.)
384
grazed by dairy sheep. Part 1: Effects on feeding behaviour, intake, diet
385
digestibility and performance. Livest. Sci. 123, 138-146.
386
387
388
389
Mueller-Harvey, I., 2006. Unravelling the conundrum of tannins in animal nutrition and
health. J. Sci. Food Agric. 86, 2010-2037.
Ottenstein, D.M., Bartley, D.A., 1971. Improved gas chromatography separation of free
acids C2–C5 in dilute solution. Anal. Chem. 43, 952–955.
390
Palmquist, D.L., Lock, A.L., Shingfield, K.J., Bauman, D.E., 2005. Biosynthesis of
391
conjugated linoleic acid in ruminants and humans, in: Taylor, S.L. (Ed.),
16
392
Advances in Food and Nutrition Research. Vol. 50. Elsevier Academic Press, San
393
Diego, CA, USA, pp. 179-217.
394
395
Pariza, M.W., Park, Y., Cook, M.E., 2001. The biologically active isomers of
conjugated linoleic acid. Prog. Lipid Res. 40, 283-298.
396
Pell, A.N., Woolston, T.K., Nelson, K.E., Schofield, P., 2000. Tannins: Biological
397
activity and bacterial tolerante, in: Brooker, J.D. (Ed.), Tannins in Livestock and
398
Human Nutrition. Aust. Ctr. Int. Agric. Res. ACIAR Proceedings No. 92,
399
Adelaida, Australia, pp. 111-117.
400
SAS, 2003. SAS User’s Guide: Statistics, Version 9.1. SAS Inst. Inc., Cary, NC, USA.
401
Shingfield, K.J., Ahvenjärvi, S., Toivonen, V., Äröla, A., Nurmela, K.V.V., Huhtanen,
402
P., Griinari, J.M., 2003. Effect of dietary fish oil on biohydrogenation of fatty
403
acids and milk fatty acid content in cows. Anim. Sci. 77, 165-179.
404
Shingfield, K.J., Reynolds, C.K., Hervás, G., Griinari, J.M., Grandison, A.S., Beever,
405
D.E., 2006. Examination of the persistency of milk fatty acid composition
406
responses to fish oil and sunflower oil in the diet of dairy cows. J. Dairy Sci. 89,
407
714-732.
408
409
Taylor, K.A.C.C., 1996. A simple colorimetric assay for muramic acid and lactic acid.
Appl. Biochem. Biotechnol. 56, 49-58.
410
Toral, P.G., Frutos, P., Hervás, G., Gómez-Cortés, P., Juárez, M., de la Fuente, M.A.,
411
2010a. Changes in milk fatty acid profile and animal performance in response to
412
fish oil supplementation, alone or in combination with sunflower oil, in dairy
413
ewes. J. Dairy Sci. 93, 1604-1615.
414
Toral, P.G., Hervás, G., Gómez-Cortés, P., Frutos, P., Juárez, M., de la Fuente, M.A.,
415
2010b. Milk fatty acid profile and dairy sheep performance in response to diet
17
416
supplementation with sunflower oil plus incremental levels of marine algae. J.
417
Dairy Sci. 93, 1655-1667.
418
Toral, P.G., Shingfield, K.J., Hervás, G., Toivonen, V., Frutos, P., 2010c. Effect of fish
419
oil and sunflower oil on rumen fermentation characteristics and fatty acid
420
composition of digesta in ewes fed a high concentrate diet. J. Dairy Sci. 93, 4804-
421
4817.
422
Turner, S.-A., Waghorn, G.C., Woodhard, S.L., Thomson, N.A., 2005. Condensed
423
tannins in birdsfoot trefoil (Lotus corniculatus) affect the detailed composition of
424
milk from dairy cows. Proc. N. Z. Soc. Anim. Prod. 65, 283-289.
425
426
Vasta, V., Makkar, H.P.S., Mele, M., Priolo, A., 2009a. Ruminal biohydrogenation as
affected by tannins in vitro. Brit. J. Nutr. 102, 82-92.
427
Vasta, V., Mele, M., Serra, A., Scerra, M., Luciano, G., Lanza, M., Priolo, A., 2009b.
428
Metabolic fate of fatty acids involved in ruminal biohydrogenation in sheep fed
429
concentrate or herbage with or without tannins. J. Anim. Sci. 87, 2674-2684.
430
Wang, Y., Waghorn, G.C., McNabb, W.C., Barry, T.N., Hedley, M.J., Shelton, I.D.,
431
1996. Effect of condensed tannins in Lotus corniculatus upon the digestion of
432
methionine and cysteine in the small intestine of sheep. J. Agr. Sci. 127, 413-
433
421.
434
435
Weatherburn, M.W., 1967. Phenol-hypochlorite reaction for determination of ammonia.
Anal. Chem. 39, 971–974.
436
18
437
Table 1
438
Ingredients and chemical composition of the experimental dietsa.
Diet
Control
TAN
Ingredients (g/kg of fresh matter)
Dehydrated alfalfa hay
Whole maize grain
Whole wheat grain
Soybean meal solvent 44% CP
Beet pulp, pellets
Molasses, liquid
Feed supplement c
Sunflower oil d
Tannin extract e
Chemical composition (g/kg DM)
Organic matter
Crude protein
Neutral detergent fibre
Acid detergent fibre
Ether extract
SEDb
P
393
185
120
147
66
48.0
23.6
17.4
0
390
183
119
146
65
47.5
23.4
17.4
8.7
-
-
892
173
256
149
46
888
170
258
148
44
5.0
2.1
6.8
3.9
3.4
0.45
0.38
0.83
0.72
0.53
439
a
For each experimental diet, n=4
440
b
SED = standard error of the difference.
441
c
Containing (g/kg): salts [NaHCO3 (458.3), CaCO3 (250.0), NaCl (125.0)], minerals
442
and vitamins (104.2), and wheat bran (62.5).
443
d
444
(36.4), and cis-9 cis-12 18:2 (50.3).
445
e
446
commercialized by Agrovin S.A. (Alcázar de San Juan, Spain): TanicolMox is reported
447
to be condensed tannins extracted from quebracho (Schinopsis lorentzii; 900 g/kg
448
extract), and Vinitanon are hydrolysable tannins extracted from chestnut (Castanea
449
sativa; 650 g/kg extract).
Containing (g/100 g total fatty acid methyl esters): 16:0 (5.5), 18:0 (4.4), cis-9 18:1
The tannin extract was a mixture 1:1 (w/w) of two oenological extracts
450
19
451
Table 2
452
Initial body weight and BW change throughout the experiment, dry matter intake, and
453
milk yield and composition, in ewes fed a total mixed ration containing 20 g of
454
sunflower oil/kg DM, without tannins (Control) or supplemented with 10 g of tannin
455
extract/kg DM (TAN).
Control
90.8
0.8
2481
TAN
94.3
1.6
2621
SEDa
5.86
1.65
108.7
D
0.57
0.64
0.23
Pb
T
0.04
1802
103.3
86.7
291.5
1878
105.5
90.4
302.2
64.8
6.71
3.53
11.09
0.26
0.74
0.31
0.34
<0.01
<0.001
<0.001
<0.001
0.15
0.29
0.23
0.13
5.61
4.82
16.13
5.72
4.85
16.08
0.246
0.090
0.271
0.66
0.73
0.88
<0.001
<0.001
<0.001
0.44
0.48
0.30
Diet
Initial BW (kg)
BW change (kg)
DM intake (g/d)
Yield (g/d)
Milk
Fat
Crude protein
Total solids
Composition (g/100 g)
Fat
Crude protein
Total solids
D×T
0.92
456
a
SED = standard error of the difference for D effects.
457
b
Probability of significant effects due to experimental diet (D), time on diet (T), and
458
interaction (D × T).
459
20
460
Table 3
461
Milk fatty acid profile in ewes fed a total mixed ration containing 20 g of sunflower
462
oil/kg DM, without tannins (Control) or supplemented with 10 g of tannin extract/kg
463
DM (TAN).
Pb
Diet
Fatty acid (g/100 g total
FA)
Saturates
4:0
5:0
6:0
7:0
8:0
9:0
10:0
11:0
12:0
14:0
15:0
16:0
17:0
18:0
19:0
20:0
21:0c
22:0
23:0
Monounsaturates
cis-9 14:1
cis-9 16:1d
trans-9 16:1
cis-9 18:1e
cis-11 18:1
cis-12 18:1
cis-13 18:1
cis-15 18:1
cis-16 18:1
trans-4 18:1
trans-5 18:1
trans-6, -7, -8 18:1
trans-9 18:1
trans-10 18:1
trans-11 18:1
trans-12 18:1
trans-16 18:1f
Control
TAN
SEDa
D
5.45
0.02
3.49
0.03
3.00
0.05
8.60
0.06
4.05
10.52
0.73
22.01
0.43
7.62
0.08
0.18
0.05
0.11
0.04
5.66
0.02
3.59
0.02
3.00
0.04
8.31
0.05
4.14
10.17
0.67
21.13
0.38
7.92
0.09
0.18
0.05
0.11
0.04
0.118
0.002
0.223
0.005
0.263
0.009
0.702
0.006
0.251
0.181
0.050
0.626
0.022
0.516
0.004
0.009
0.001
0.004
0.002
0.10
0.85
0.67
0.28
0.98
0.37
0.69
0.32
0.71
0.08
0.25
0.19
0.02
0.58
0.17
0.70
0.34
0.45
0.36
<0.001
0.01
0.00
0.02
<0.001
0.03
<0.001
0.29
0.04
0.17
0.07
0.04
<0.001
0.19
<0.001
<0.001
<0.001
<0.001
<0.001
0.15
0.64
0.15
0.78
0.35
0.78
0.47
0.30
0.62
0.26
0.10
0.17
0.55
0.17
0.43
0.24
0.98
0.51
0.33
0.22
1.13
0.37
15.88
0.52
0.47
0.08
0.09
0.07
0.02
0.01
0.35
0.43
0.72
4.01
0.67
0.40
0.19
1.06
0.31
15.27
0.50
0.50
0.08
0.09
0.07
0.02
0.01
0.35
0.43
0.68
4.09
0.70
0.41
0.010
0.037
0.080
0.649
0.029
0.034
0.004
0.003
0.005
0.001
0.001
0.023
0.018
0.092
0.352
0.032
0.026
0.01
0.01
0.07
0.19
0.43
0.08
0.36 <0.001
0.46
0.23
0.44
0.21
0.60 <0.01
0.48 <0.01
0.51 <0.001
0.95
0.99
0.68 <0.01
0.97
0.06
0.91
0.12
0.66
0.33
0.81
0.01
0.27
0.07
0.47 <0.001
0.07
0.08
0.29
0.33
0.35
0.29
0.62
0.89
0.09
0.68
0.88
0.18
0.01
0.78
0.12
0.46
0.04
T
D×T
21
cis-11 20:1
cis-13 22:1
cis-15 24:1
Polyunsaturates
cis-9 cis-12 18:2
cis-9 trans-12 18:2
trans-9 cis-12 18:2
trans-9 trans-12 18:2
cis-9 trans-11 CLAg
trans-11 trans-13 CLA
trans-12 trans-14 CLA
other trans-trans CLAh
Σ CLAi
cis-6 cis-9 cis-12 18:3
cis-9 cis-12 cis-15 18:3
cis-11 cis-14 20:2
cis-8 cis-11 cis-14 20:3
cis-11 cis-14 cis-17 20:3
cis-5 cis-8 cis-11 cis-14
20:4
cis-5 cis-8 cis-11 cis-14
cis-17 20:5j
cis-7 cis-10 cis-13 cis-16
22:4
cis-7 cis-10 cis-13 cis-16
cis-19 22:5
cis-4 cis-7 cis-10 cis-13
cis-16 cis-19 22:6
0.02
<0.01
<0.01
0.02
<0.01
<0.01
0.002
0.001
0.001
0.79 <0.001
0.36 <0.001
0.80 <0.001
0.21
0.96
0.05
1.92
0.10
0.04
0.01
2.15
0.04
<0.01
<0.01
2.26
0.04
0.31
0.03
0.02
0.01
2.00
0.09
0.04
0.01
2.07
0.04
<0.01
0.01
2.16
0.04
0.33
0.02
0.02
0.01
0.081
0.006
0.002
0.001
0.216
0.003
0.001
0.001
0.261
0.004
0.015
0.003
0.002
0.001
0.37
0.50
0.73
0.73
0.71
0.86
0.33
0.08
0.68
0.93
0.12
0.84
0.77
0.90
<0.001
<0.001
0.15
0.01
0.01
<0.001
0.35
<0.001
<0.01
<0.001
<0.001
<0.001
<0.01
<0.001
0.74
0.83
0.64
0.03
0.09
0.25
0.77
0.47
0.09
0.46
0.75
0.18
0.06
0.53
0.16
0.15
0.006
0.09 <0.001
0.66
0.03
0.04
0.001
0.21 <0.001
0.51
0.02
0.02
0.002
0.64 <0.001
0.55
0.08
0.08
0.002
0.89 <0.001
0.99
0.02
0.02
0.001
0.36 <0.001
0.91
464
a
SED = standard error of the difference for D effects.
465
b
Probability of significant effects due to experimental diet (D), time on diet (T), and
466
their interaction (D × T).
467
c
Contains trans-10 cis-12 CLA as a minor component.
468
d
Coelutes with 17:0 anteiso.
469
e
Contains trans-13, -14, -15 18:1 and cis-10 18:1 as minor components.
470
f
Coelutes with cis-14 18:1.
471
g
Contains trans-7 cis-9 CLA and trans-8 cis-10 CLA as minor components.
472
h
Sum of trans-7 trans-9 CLA + trans-8 trans-10 CLA + trans-9 trans-11 CLA + trans-
473
10 trans-12 CLA.
474
i
Total conjugated linoleic acid.
22
475
j
Coelutes with 24:0.
476
23
477
Table 4
478
Ruminal fermentation characteristics in ewes fed a total mixed ration containing 20 g of
479
sunflower oil/kg DM, without tannins (Control) or supplemented with 10 g of tannin
480
extract/kg DM (TAN).
Diet
Control
pH
6.76
Ammonia (mg/l)
185
Lactate (mmol/l)
1.03
Total VFAc (mmol/l)
110.7
Molar proportions (mol/mol)
Acetate
0.60
Propionate
0.19
Butyrate
0.18
d
Others
0.03
Acetate:propionate ratio
3.41
TAN
6.76
169
1.12
105.4
SEDa
0.089
21.6
0.092
6.81
Pb
0.95
0.49
0.35
0.46
0.63
0.17
0.17
0.03
3.79
0.013
0.021
0.016
0.003
0.393
0.07
0.42
0.76
0.43
0.35
481
a
SED = standard error of the difference.
482
b
Probability of significant effects due to experimental diet.
483
c
VFA = volatile fatty acids.
484
d
Calculated as the sum of isobutyrate, isovalerate, valerate and caproate.
24