It is well established that trans-10, cis-12 conjugated linoleic acid (CLA) is involved in the regulation of lipid metabolism in a number of mammalian species including the pig, cow and human(Reference Bauman, Corl, Peterson, Sébédio, Christie and Adlof1). Numerous studies have characterised the anti-lipogenic effects of trans-10, cis-12 CLA on milk fat synthesis in the lactating cow. In the bovine, reductions in milk fat are known to occur in a predictable and dose-dependent manner in response to post-ruminal infusions(Reference Baumgard, Corl, Dwyer, Saebø and Bauman2–Reference Peterson, Baumgard and Bauman4) or rumen-protected supplements of trans-10, cis-12 CLA(Reference Giesy, McGuire, Shafii and Hanson5–Reference Castañeda-Gutiérrez, Overton, Butler and Bauman7). While the anti-lipogenic effects are well characterised in the lactating cow, the role of trans-10, cis-12 CLA in lipogenesis in other lactating ruminant species is less well defined.
The effects of stage of lactation on milk fat are known to be comparable in the bovine and caprine, but changes in milk fat synthesis to lipid supplements in the diet differ markedly between these species(Reference Chilliard, Ferlay, Rouel and Lamberet8, Reference Chilliard, Glasser, Ferlay, Bernard, Rouel and Doreau9). Typically, milk fat content is increased in response to dietary fat in the goat, but not in the cow, which may reflect inter-species differences in ruminal lipid metabolism and/or the regulation of cellular processes in the mammary gland and the relative importance of key enzymes in the synthesis of milk fatty acids(Reference Chilliard, Ferlay, Rouel and Lamberet8, Reference Chilliard, Glasser, Ferlay, Bernard, Rouel and Doreau9). Recent studies examining short-term intravenous(Reference Schmidely and Morand-Fehr10) or post-ruminal infusions(Reference de Andrade and Schmidely11) of trans-10, cis-12 CLA in goats have reported relatively minor or no effects on mammary lipogenesis. However, rumen-protected supplements of trans-10, cis-12 CLA were reported to decrease milk fat synthesis in goats(Reference Erasmus, Bester, Fourie, Coertze and Hall12, Reference Rovai, Lock, Gipson, Goetsch and Bauman13), but the quantity required to inhibit milk fat synthesis in the goat was much higher than expected based on metabolic live-weight comparisons with the lactating cow. While inconclusive, the available evidence points towards a lower sensitivity to the anti-lipogenic effects of trans-10, cis-12 CLA in the caprine than in the bovine.
Further studies have demonstrated that the anti-lipogenic effects of trans-10, cis-12 CLA on milk fat synthesis in the lactating cow are markedly reduced when diets contain relatively high amounts of rumen-protected unsaturated fatty acids(Reference Gulati, McGrath, Wynn, Thomson and Scott14). Inter-species differences in milk fat responses to lipid supplements(Reference Chilliard, Ferlay, Rouel and Lamberet8, Reference Chilliard, Glasser, Ferlay, Bernard, Rouel and Doreau9) also suggest that the supply of fatty acid precursors available to the mammary gland would have a more critical role on the inhibitory effects of trans-10, cis-12 CLA on milk fat synthesis in the goat compared with the cow.
Eight lactating alpine goats were used in two 4 × 4 Latin-square experiments to examine milk fat responses to incremental inclusion of calcium salts of a mixture of CLA isomers (CaCLA) containing trans-10, cis-12 in the diet. In order to establish the possible role of long-chain fatty acid precursor supply, CaCLA replaced maize grain in concentrate supplements (Experiment 1) or calcium salts of palm oil fatty acids (Experiment 2) in the diet. For both experiments, milk composition responses to the same range in trans-10, cis-12 CLA intakes were evaluated. CaCLA were selected as a source of rumen-protected trans-10, cis-12 CLA since the effects of these lipid supplements on milk fat synthesis have been determined in short-term experiments(Reference Giesy, McGuire, Shafii and Hanson5, Reference de Veth, Gulati, Luchini and Bauman15) and extensively evaluated over an extended period during early(Reference Moore, Hafliger, Mendivil, Sanders, Bauman and Baumgard6, Reference Castañeda-Gutiérrez, Overton, Butler and Bauman7, Reference Bernal-Santos, Perfield, Barbano, Bauman and Overton16) and established lactation in the cow(Reference Perfield, Bernal-Santos, Overton and Bauman17, Reference Piperova, Moallem, Teter, Sampugna, Yurawecz, Morehouse, Luchini and Erdman18), allowing an indirect comparison of the anti-lipogenic effects of trans-10, cis-12 CLA between ruminant species.
Materials and methods
Animal management and experimental design
All experimental procedures were approved by the Institut National de la Recherche Agronomique Animal Care and Use Committee in accordance with the guidelines on the use of animals for experimental purposes implemented in France(19). Eight lactating non-pregnant alpine goats in mid-lactation of mean 250 (se 8·3) d in lactation, 63·9 (se 4·33) kg live weight and producing 2·58 (se 0·121) kg milk/d were used in two experiments each conducted as a 4 × 4 Latin square with 14 d experimental periods. Animals were assigned to Latin squares according to milk yield and milk composition determined over a 4 d period immediately before the start of the experiment. Mean milk yield (kg/d), milk fat, protein and lactose content (g/kg) were 2·59 (se 0·085), 42·0 (se 1·30), 39·6 (se 1·20) and 43·9 (se 1·65), and 2·58 (se 0·235), 39·6 (se 1·35), 36·7 (se 0·40) and 42·9 (se 1·05) for the goats used in Experiments 1 and 2, respectively. Experimental animals were housed in individual stalls and had continuous access to water. Daily rations were fed equal meals at 08.30 and 16.00 hours and goats were milked at 06.00 and 15.30 hours.
Experimental treatments consisted of CaCLA prepared from chemical isomerisation of sunflower-seed oil and containing cis-9, trans-11 CLA and trans-10, cis-12 CLA as major components (Im'prouve ALC, Xeris S.A., Séné, France; Table 1), and included in the diet at a rate of 0, 30, 60 or 90 g/d corresponding to 7·47, 14·9 and 22·4 g of trans-10, cis-12 CLA/d. Supplements of CaCLA were fed for the first 10 d of each experimental period but removed from daily rations during the remaining 4 d in order to minimise treatment carry-over effects. In Experiment 1, CaCLA replaced maize grain in concentrate supplements (treatments C0, C1, C2 and C3, respectively), while in Experiment 2 CaCLA substituted for calcium salts of palm oil fatty acids (CaPO) (treatments CP0, CP1, CP2 and CP3; Ruminer, Aurillac, France; Table 1, respectively). Replacing maize grain with CaCLA supplements also enhanced diet energy content in Experiment 1, whereas treatments CP0, CP1, CP2 and CP3 in Experiment 2 were formulated to be isoenergetic.
Table 1 Fatty acid composition of lipid supplements
CaPO, calcium salts of palm oil fatty acids; CaCLA, calcium salts of conjugated linoleic acid.
Experimental diets
Goats were offered lucerne hay ad libitum supplemented with 0·65 kg DM/d concentrates of variable composition (Table 2). Diets were formulated to supply 110 and 130 % of predicted energy and protein requirements, respectively(Reference Jarrige20). In Experiment 1, CaCLA replaced (on a DM basis) maize grain in concentrate supplements or substituted for calcium salts of palm oil fatty acids in Experiment 2 (Table 2). For both experiments, concentrate supplements were fed at a fixed rate to avoid possible selection of dietary components, maintain the forage:concentrate ratio of the diet and ensure that experimental treatments supplied the targeted amount of trans-10, cis-12 CLA.
Table 2 Ingredient and chemical composition of experimental hay and concentrate supplements
ADF, acid-detergent fibre; CP, crude protein; NDF, neutral-detergent fibre; OM, organic matter; CaPO, calcium salts of palm oil fatty acids.
* A control concentrate supplement fed in experiment 1 (C) or concentrates fed in experiment 2 containing CaPO that were replaced incrementally with 0, 49, 83 or 130 g/kg diet DM of calcium salts of conjugated linoleic acid (CP0, CP1, CP2 and CP3, respectively).
† Declared ingredient and chemical composition do not include supplements of calcium salts of conjugated linoleic acid.
‡ Calcium salts of palm oil fatty acids.
§ Proprietary mineral and vitamin supplement (Centraliment, Aurillac, France) declared as containing (g/kg) sodium (50), calcium (200), phosphorus (45), magnesium (45), zinc (6) and manganese (3·5); (mg/kg) retinyl acetate (206), cholecalciferol (3·0) and dl-tocopheryl acetate (1300).
∥ g/kg DM, unless otherwise stated.
Experimental measurements and sampling
Foods
Individual intakes were recorded daily but only measurements made on days 9 and 10 of each experimental period were used for statistical analysis. During this interval, samples of lucerne hay, concentrate mixtures C, CP, CP1 and CP2, calcium salts of palm oil fatty acids and CaCLA supplements were collected, composited and stored at − 20°C. Feed DM content was determined after drying at 105°C for 24 h. Samples of maize silage, lucerne hay and concentrates were dried at 48°C for 48 h, passed through a 1 mm screen and submitted for the determination of chemical composition according to standard procedures(21). Additional samples of experimental feeds were collected, lyophilised (Thermovac TM-20, Froilabo, Ozoir-la-Ferrière, France) and submitted for fatty acid determinations.
Milk
Milk yields were recorded daily, but only measurements on days 9 and 10 of each experimental period were analysed statistically. Samples of milk were collected from individual goats over four consecutive milkings starting 06.00 hours on day 9 of each experimental period, preserved with potassium dichromate (Merck, Fontenay-Sous-Bois, France) and stored at 4°C until analysed for fat, crude protein and lactose content. Milk fat, crude protein and lactose were determined by near IR spectroscopy(21) calibrated using reference caprine milk samples. Unpreserved milk samples were also collected at 15.30 hours on day 9 and 06.00 hours on day 10, stored at − 20 °C, composited according to yield and submitted for fatty acid analysis.
Live weight
Goats were weighed at the beginning of the experiment and on the last day of each experimental period at 11.00 hours.
Fatty acid analysis
Lipid in lucerne hay, concentrate ingredients and CaCLA supplements was extracted(Reference Folch, Lees, Sloane and Stanley22) and transesterified to fatty acid methyl esters (FAME) by incubation with methanolic hydrochloric acid according to standard procedures(Reference Loor, Ueda, Ferlay, Chilliard and Doreau23) using 23:0 (Sigma, St-Quentin Fallavier, France) as an internal standard. Lipid content and fatty acid composition of CaPO supplements were determined using the same extraction procedure(Reference Folch, Lees, Sloane and Stanley22), with the exception that the filtration step was omitted according to the recommendations of the manufacturer and the organic extract was transesterified to FAME using methanolic hydrochloric acid as a catalyst(Reference Loor, Ueda, Ferlay, Chilliard and Doreau23).
For milk fatty acid determinations, lipid in 1 ml samples was extracted and transesterified to FAME using freshly prepared methanolic sodium methoxide(Reference Christie24, Reference Roy, Ferlay, Shingfield and Chilliard25). Methyl esters were quantified by GLC using a gas chromatograph Trace GC 2000 equipped with a flame ionisation detector (Thermo Finnigan, Les Ullis, France) and a fused silica capillary column (100 m × 0·25 mm internal diameter) coated with 0·2 μm film of cyanopropyl polysiloxane (CP-SIL 88; Chrompack 7489, Middelburg, The Netherlands) using hydrogen as the carrier gas operated at constant pressure (125 kPa). Total FAME profile in a 2 μl sample at a split ratio of 1:40 was determined using a temperature gradient programme(Reference Roy, Ferlay, Shingfield and Chilliard25). Injector and detector temperatures were maintained at 255 and 260°C, respectively. Peaks were routinely identified by comparison of retention times with FAME standards (GLC 463, Nu Chek Prep Inc., Elysian, MN, USA; reference mixture 47 885, Supelco, Bellefonte, PA, USA) and a reference butter oil (CRM 164; Commission of the European Communities, Community Bureau of Reference, Brussels, Belgium) was used to estimate correction factors for short-chain (4:0–10:0) fatty acids(Reference Loor, Ferlay, Ollier, Doreau and Chilliard26). Methyl esters not contained in commercially available standards were identified based on the comparisons with reference milk fat samples of known fatty acid composition based on the GC–MS analysis of 4,4-dimethyloxazoline fatty acid derivatives(Reference Shingfield, Reynolds, Hervás, Griinari, Grandison and Beever27, Reference Shingfield, Chilliard, Toivonen, Kairenius, Givens and Bösze28).
Following GLC analysis, samples of milk fat FAME were evaporated under nitrogen, dissolved in heptane and the distribution of CLA isomers was determined by HPLC using four silver-impregnated silica columns (ChromSpher 5 lipids, 250 × 4·6 mm, 5 μm particle size; Varian Ltd, Walton-on-Thames, UK) coupled in series and 0·1 % (v/v) acetonitrile in heptane as the mobile phase(Reference Shingfield, Ahvenjärvi, Toivonen, Ärölä, Nurmela, Huhtanen and Griinari29). Isomers were identified using an authentic CLA methyl ester standard (O-5632; Sigma-Aldrich, YA-Kemia Limited, Helsinki, Finland) and chemically synthesised trans-9, cis-11 CLA(Reference Shingfield, Reynolds, Lupoli, Toivonen, Yurawecz, Delmonte, Griinari, Grandison and Beever30). Identification was verified by cross-referencing with the elution order reported in the literature(Reference Delmonte, Kataok, Corl, Bauman and Yurawecz31) using cis-9, trans-11 CLA as a landmark isomer.
Statistical analysis
Experimental data were subjected to ANOVA using the general linear model procedure of Statistical Analysis Systems software package version 9.1 (SAS Institute, Inc., Cary, NC, USA) with a model that included the random effects of the goat and fixed effects of period and treatment. Sums of squares for treatment effects were further separated using orthogonal contrasts into single-degree-of-freedom comparisons to test for the significance of linear, quadratic and cubic components of the response to experimental treatments. Least-squares means are reported and treatment effects were declared significant at P < 0·05.
Relationships between CaCLA in the diet, milk fatty acid composition, fat content and milk fat secretion were initially examined by regression analysis using the REG procedure of SAS. In cases where close linear or quadratic associations were identified between experimental variables, the relationship was further explored with an exponential decay model fitted using the Marquardt non-linear algorithm within the NLIN procedure of SAS(Reference deVeth, Griinari, Pfeiffer and Bauman32).
Results
All animals remained in good health during the experiment, but in both experiments, goats did not consume all of the CaCLA supplement offered and therefore the amount of trans-10, cis-12 CLA supplied by experimental treatments was marginally lower than planned.
Experiment 1
Inclusion of CaCLA in the diet had no effect (P>0·05) on DM intake, milk yield, milk protein content, milk lactose concentrations or live weight, but increased linearly (P < 0·01) fatty acid intake and decreased linearly (P < 0·01) milk fat content and yield (Table 3). Compared with the control, treatments C1, C2 and C3 resulted in 19·8, 27·9 and 32·3 % decreases in milk fat yield and 16·2, 22·7 and 29·4 % reductions in milk fat content, respectively. Reductions in milk fat synthesis to CaCLA were also accompanied by changes in milk fatty acid composition characterised by linear (P < 0·05) decreases in 6:0, 8:0, 10:0, 12:0, cis-18:1, 20:2n-6, 20:4n-6, 20:5n-3 and 22:5n-3 and linear (P < 0·05) increases in 18:0, Σ trans-18:1, Σ CLA, 20:0, 22:0 and Σ PUFA concentrations (Table 4). CaCLA supplements also induced linear or quadratic (P < 0·05) decreases in the concentration of fatty acids containing a cis-9 double bond, with the exception of cis-9, trans-13-18:2, and reduced product:substrate ratios for Δ9-desaturase (Table 4). Concentrations of CLA isomers in milk other than trans-11, cis-13 were enhanced linearly (P < 0·05) in response to incremental inclusion of CaCLA in the diet (Table 5).
Table 3 Effect of dietary conjugated linoleic acid (CLA) supplements on nutrient intake, milk production and live weight in lactating goats (Experiment 1)*
CaCLA, calcium salts of conjugated linoleic acid.
* Values represent the mean of days 9 and 10 of treatment.
† Diets based on lucerne hay supplemented with concentrates formulated to supply 0, 30, 60 or 90 g CaCLA/d (C0, C1, C2 and C3, respectively).
‡ Standard error of the mean for sixteen measurements; error df = 6.
§ Significance of linear responses to CaCLA supplements in the diet. Quadratic and cubic responses to CaCLA were NS (P>0·05).
∥ 35 g/kg fat-corrected milk yield (kg) = milk yield (kg) × (313+11·2 × fat content (g/kg))/704(Reference Sauvant51).
Table 4 Effect of dietary conjugated linoleic acid (CLA) supplements on milk fatty acid composition in lactating goats (Experiment 1; g/100 g fatty acids)*
CaCLA, calcium salts of conjugated linoleic acid.
* Milk fatty acid profile on day 10 of treatment.
† Diets based on lucerne hay supplemented with concentrates formulated to supply 0, 30, 60 or 90 g CaCLA/d (C0, C1, C2 and C3, respectively).
‡ Standard error of the mean for sixteen measurements; error df = 6.
§ Significance of linear (L) and quadratic (Q) responses to CaCLA supplements in the diet. Cubic responses to CaCLA were NS (P>0·05).
∥ Containing trans-17-18 : 1 as a minor component.
¶ Coeluting with cis-14-18 : 1 as a minor isomer.
** Fatty acid content of milk fat calculated assuming that lipid in milk is secreted as TAG.
Table 5 Effect of dietary conjugated linoleic acid supplements on milk conjugated linoleic acid isomer concentrations in lactating goats (Experiment 1; mg/100 g fatty acids)*
CaCLA, calcium salts of conjugated linoleic acid.
* Milk fatty acid profile on day 10 of treatment.
† Diets based on lucerne hay supplemented with concentrates formulated to supply 0, 30, 60 or 90 g CaCLA/d (C0, C1, C2 and C3, respectively).
‡ Standard error of the mean for sixteen measurements; error df = 6.
§ Significance of linear responses to CaCLA supplements in the diet. Quadratic and cubic responses to CaCLA were NS (P>0·05).
Incremental inclusion of CaCLA in the diet decreased linearly (P < 0·05) fatty acid secretion in milk due to reductions in both the output of ≤ C16 fatty acids synthesised de novo and C16 fatty acids (Fig. 1 (a)). CaCLA supplements had no effect (P>0·05) on the secretion of >C18 long-chain fatty acids in milk derived from the uptake of circulating plasma lipids (Fig. 1 (a)). Experimental treatments increased linearly (P < 0·001) trans-10, cis-12 CLA output in milk from 14·7 to 163, 296 and 404 mg/d, associated with a mean efficiency of transfer from the diet into milk of 2·37, 2·38 and 2·19 % (se 0·099, P = 0·412) for treatments C1, C2 and C3, respectively.
Fig. 1 Milk fatty acid secretion in goats measured on day 10 of treatment in response to (a) 0, 30, 60 or 90 g calcium salts of conjugated linoleic acid (CLA)/d in the diet (C0 (□), C1 (
), C2 (
) and C3 (■), respectively) or (b) incremental replacement of calcium salts of palm oil fatty acids in the diet with 0, 30, 60 or 90 g calcium salts of CLA/d (CP0 (□), CP1 (), CP2 () and CP3 (■), respectively). Fatty acids in milk categorised according to metabolic origin: ≤ C14 synthesised de novo; ≥ C18 extracted and incorporated into milk fat from circulating plasma lipids; C16 derived from both sources. For both experiments, dietary supplements of calcium salts of CLA decreased linearly (P < 0·01) ≤ C14 and ≥ C16 output but had no effect (P>0·05) on ≥ C18 secretion in milk. Quadratic and cubic responses to experimental treatments were NS (P>0·05). Error bars indicate standard errors of the mean for sixteen measurements.
Experiment 2
Substituting CaCLA for CaPO in the diet had no effect (P>0·05) on DM intake, milk yield, milk protein content, milk lactose concentrations or live weight, but decreased linearly (P < 0·01) milk fat content, milk fat output and fat-corrected milk yield (Table 6). Relative to the control, replacing CaPO with CaCLA in the diet resulted in 17·5, 39·0 and 49·3 % decreases in milk fat yield and 24·0, 33·8 and 35·8 % reductions in milk fat content for treatments CP1, CP2 and CP3, respectively. Supplements CaPO and CaCLA were declared as containing the same amount of fatty acids. Due to measured differences in the supplement lipid content (Table 1), and marginal decreases in forage and concentrate DM intake, incremental replacement of CaPO with CaCLA in the diet resulted in a linear reduction (P < 0·05) in total fatty acid ingestion (Table 6). Replacing CaPO with CaCLA in the diet also altered milk fatty acid composition characterised by linear (P < 0·05) decreases in 6:0, 8:0, 10:0, 12:0, 16:0 and 20:4n-6 and linear or quadratic (P < 0·05) increases in 18:0, Σ trans-18:1, Σ CLA, 18:3n-3, 20:0, 20:2n-6, 22:0 and Σ PUFA concentrations (Table 7). Substituting CaPO with CaCLA in the diet also induced linear or quadratic (P < 0·05) decreases in the concentration of fatty acids containing a cis-9 double bond, other than cis-9, trans-13-18:2 and reduced product:substrate ratios for Δ9-desaturase (Table 7). Incremental inclusion of CaCLA at the expense of CaPO also enhanced in a linear or quadratic manner (P < 0·05) the concentration of all CLA isomers in milk, with the exception of trans-11, cis-13 (Table 8).
Table 6 Effect of replacing calcium salts of palm oil fatty acids in the diet with supplements of conjugated linoleic acid (CLA) on nutrient intake, milk production and live weight in lactating goats (Experiment 2)*
CaCLA, calcium salts of CLA; CaPO, calcium salts of palm oil fatty acids.
* Values represent the mean of days 9 and 10 of treatment.
† Diets based on lucerne hay supplemented with concentrates containing calcium salts of palm oil fatty acids that were replaced incrementally with calcium salts of conjugated linoleic acid to supply 0, 30, 60 or 90 g CaCLA/d (CP0, CP1, CP2 and CP3, respectively).
‡ Standard error of the mean for sixteen measurements; error df = 6.
§ Significance of linear responses to CaCLA supplements in the diet. Quadratic and cubic responses to CaCLA were NS (P>0·05).
∥ 35 g/kg fat-corrected milk yield (kg) = milk yield (kg) × (313+11·2 × fat content (g/kg))/704(Reference Sauvant51).
Table 7 Effect of replacing calcium salts of palm oil fatty acids in the diet with supplements of conjugated linoleic acid (CLA) on milk fatty acid composition in lactating goats (Experiment 2; g/100 g fatty acids)*
CaCLA, calcium salts of conjugated linoleic acid.
* Milk fatty acid profile on day 10 of treatment.
† Diets based on lucerne hay supplemented with concentrates containing calcium salts of palm oil fatty acids that were replaced incrementally with calcium salts of conjugated linoleic acid to supply 0, 30, 60 or 90 g CaCLA/d (CP0, CP1, CP2 and CP3, respectively).
‡ Standard error of the mean for sixteen measurements; error df = 6.
§ Significance of linear (L), quadratic (Q) and cubic (C) responses to CaCLA supplements in the diet.
∥ Containing trans-17-18 : 1 as a minor component.
¶ Coeluting with cis-14-18 : 1 as a minor isomer.
** Fatty acid content of milk fat calculated assuming that lipid in milk is secreted as TAG.
Table 8 Effect of replacing calcium salts of palm oil fatty acids with supplements of conjugated linoleic acid on milk conjugated linoleic acid isomer concentrations in lactating goats (Experiment 2; mg/100 g fatty acids)*
CaCLA, calcium salts of conjugated linoleic acid.
* Milk fatty acid profile on day 10 of treatment.
† Diets based on lucerne hay supplemented with concentrates containing calcium salts of palm oil fatty acids that were replaced incrementally with calcium salts of conjugated linoleic acid to supply 0, 30, 60 or 90 g CaCLA/d (CP0, CP1, CP2 and CP3, respectively).
‡ Standard error of the mean for sixteen measurements; error df = 6.
§ Significance of linear (L) and quadratic (Q) responses to CaCLA supplements in the diet. Cubic responses to CaCLA were NS (P>0·05).
Replacement of CaPO with CaCLA decreased linearly (P < 0·05) milk fatty acid output, changes that were attributable to a decrease in ≤ C14 and C16 fatty acids, while the secretion of >C18 long-chain fatty acids in milk was independent of experimental treatment (Fig. 1 (b)). Inclusion of CaCLA in the diet increased linearly (P < 0·001) trans-10, cis-12 CLA secretion in milk from 14·7 to 139, 278 and 219 mg/d for treatments C1, C2 and C3, responses associated with a mean apparent efficiency of transfer from the diet into milk of 2·22, 2·39 and 1·69 % (se 0·381, P = 0·634), respectively.
Discussion
Post-ruminal infusion studies have established a central role of trans-10, cis-12 CLA in the regulation of milk fat synthesis in the lactating cow(Reference Shingfield and Griinari33, Reference Bauman, Perfield, Aarvatine and Baumgard34). Reductions in milk fat in response to abomasal infusions of trans-10, cis-12 CLA are known to occur in a predictable dose-dependent manner(Reference deVeth, Griinari, Pfeiffer and Bauman32, Reference Shingfield and Griinari33). Further research has also demonstrated that post-ruminal infusions of a mixture of CLA isomers containing trans-9, cis-11(Reference Perfield, Lock, Griinari, Sæbø, Delmonte, Dwyer and Bauman35) or cis-10, trans-12(Reference Sæbø, Sæbø, Griinari and Shingfield36) as major components also exert anti-lipogenic effects in the bovine. Use of lipid-encapsulated supplements containing cis-9, trans-11 CLA and trans-10, cis-12 CLA has also provided evidence to suggest that the inhibitory effects of trans-10, cis-12 CLA are comparable in the lactating bovine and ovine(Reference Lock, Teles, Perfield, Bauman and Sinclair37, Reference Sinclair, Lock, Early and Bauman38). By contrast, short-term administration of trans-10, cis-12 CLA in the peripheral circulation(Reference Schmidely and Morand-Fehr10) or at the duodenum(Reference de Andrade and Schmidely11) was reported to have no effect on milk fat synthesis in the lactating goat, suggesting that the anti-lipogenic activity of trans-10, cis-12 CLA differs between ruminant species.
Numerous experiments in lactating cows have established that dietary supplements of calcium salts of a mixture of CLA isomers containing trans-10, cis-12 CLA inhibit milk fat synthesis during early or established lactation over a short or extended period(Reference Shingfield and Griinari33). Several studies have demonstrated that the anti-lipogenic potential of trans-10, cis-12 CLA supplied as CaCLA in the diet is lower immediately post-partum in the lactating cow(Reference Moore, Hafliger, Mendivil, Sanders, Bauman and Baumgard6, Reference Bernal-Santos, Perfield, Barbano, Bauman and Overton16, Reference Gervais, Spratt, Leonard and Chouinard39). Reduced inhibition of mammary lipogenesis does not appear to be related to variations in mammary supply and incorporation of trans-10, cis-12 CLA in milk fat(Reference Moore, Hafliger, Mendivil, Sanders, Bauman and Baumgard6, Reference Castañeda-Gutiérrez, Overton, Butler and Bauman7, Reference Bernal-Santos, Perfield, Barbano, Bauman and Overton16) or specific changes in plasma glucose, insulin, leptin or NEFA concentrations(Reference Castañeda-Gutiérrez, Overton, Butler and Bauman7, Reference Kay, Roche, Moore and Baumgard40). It is possible that the coordinated reduction in the expression genes encoding for key lipogenic enzymes to trans-10, cis-12 CLA is prevented due to the attenuation of cellular signalling systems during the onset of lactation(Reference Moore, Hafliger, Mendivil, Sanders, Bauman and Baumgard6, Reference Bernal-Santos, Perfield, Barbano, Bauman and Overton16). A lower sensitivity of mammary lipogenesis to trans-10, cis-12 CLA during early lactation in the bovine is analogous to the response to trans-10, cis-12 CLA infusion in the goat(Reference Schmidely and Morand-Fehr10, Reference de Andrade and Schmidely11), suggesting some common features in the regulation of mammary lipid metabolism. However, there are also several distinct differences between species: (i) decreases in milk fat synthesis in the bovine during early lactation involve a reduction in fatty acids synthesised de novo and preformed fatty acids, whereas reductions in milk fat synthesis to CaCLA in the goat were confined to ≤ C14 and C16 fatty acids (Fig. 1), (ii) comparisons between experiments indicated that the inhibitory effects of CaCLA on milk fat synthesis in the goat are dependent on the supply of long-chain fatty acids (Fig. 2), whereas limited data in the lactating cow suggest that responses to trans-10, cis-12 CLA are independent of dietary fatty acid content(Reference Loor and Herbein41) and (iii) a lower sensitivity to trans-10, cis-12 CLA in the bovine also coincides with alterations in several key enzymes and biochemical pathways at the onset of lactation and is a transitory phenomenon, whereas the lower sensitivity in the goat occurs in established lactation during periods of positive energy and nitrogen balance (Fig. 3).
Fig. 2 Relationship between the percentage reduction in (a) milk fat yield and (b) milk fat content in lactating goats relative to a control diet in response to trans-10, cis-12 CLA intake. Treatments comprised calcium salts of CLA (●; experiment 1) or incremental replacement of calcium salts of palm oil fatty acids in the diet with calcium salts of CLA (○; experiment 2). For both experiments, symbols represent least-squares means (n 4).
Fig. 3 Relationship between the percentage reduction in (a) milk fat yield and (b) milk fat content with milk fat trans-10, cis-12 CLA concentration in lactating goats relative to responses determined in lactating cows. Treatments comprised calcium salts of CLA (●; experiment 1) or incremental replacement of calcium salts of palm oil fatty acids in the diet with calcium salts of CLA (○; experiment 2). Symbols for experiments 1 and 2 represent least-squares means (n 4). Data in lactating cows (▲) derived from (n 11) studies reported in the literature(Reference Giesy, McGuire, Shafii and Hanson5–Reference Castañeda-Gutiérrez, Overton, Butler and Bauman7, Reference de Veth, Gulati, Luchini and Bauman15–Reference Piperova, Moallem, Teter, Sampugna, Yurawecz, Morehouse, Luchini and Erdman18, Reference Selberg, Lowe, Staples, Luchini and Badinga52–Reference Huang, Schoonmaker, Bradford and Beitz55) evaluating responses to calcium salts of CLA during early or established lactation.
The present experiment provided further evidence that CaCLA supplements also decrease milk fat content and yield in the lactating goat in a dose-dependent manner (Fig. 2). A specific reduction in milk fat secretion in the absence of changes in milk yield, milk protein content or lactose concentrations is consistent with typical milk production responses to CaCLA reported for lactating cows over short or extended periods(Reference Bauman and Griinari42). However, the changes in milk fat synthesis were dependent on CaCLA supplementation regimen. Increases in trans-10, cis-12 CLA intake from CaCLA in Experiment 1 were associated with a curvilinear decrease in milk fat yield and content, whereas in Experiment 2, CaCLA treatments resulted in a linear decrease in milk fat yield and a curvilinear reduction in milk fat content (Fig. 2).
Relative reductions in milk fat secretion were larger when CaCLA replaced CaPO in the diet than when offered alone, which can be attributed to incremental replacement of CaPO with CaCLA resulting in marginal linear reductions in total fatty acid intake (Table 6), arising at least in part, from non-significant decreases in DM intake. Inclusion of CaCLA in the diet enhanced linearly dietary fatty acid intake, with the implication that the supply of 18:0 available for absorption was also increased due to extensive metabolism of the CaCLA supplement in the rumen as inferred from a low transfer of trans-10, cis-12 CLA into milk. In marked contrast to the bovine, increases in the supply of dietary fatty acids are known to enhance milk fat content in the goat(Reference Chilliard, Ferlay, Rouel and Lamberet8, Reference Chilliard, Glasser, Ferlay, Bernard, Rouel and Doreau9), indicating that alterations in dietary fatty acid supply may also contribute to between-experiment variation in milk fat responses to CaCLA. Milk fat content was higher (2·2 g/kg) for C0 than for CP0 treatments. Accounting for measured differences in milk composition between animals assigned to experiments indicated that CaPO in the diet elicited a mean 4·3 g/kg increase in milk fat concentrations consistent with responses of 3·7–5·2 g/kg reported in the literature(Reference Chilliard, Ferlay, Rouel and Lamberet8). Due to the critical role of fatty acid supply in the regulation of mammary lipogenesis in the caprine, the changes in milk fat content and secretion to substituting CaPO for CaCLA in the diet can be considered to be a close reflection of the anti-lipogenic potential of CaCLA supplements in the lactating goat.
CaCLA supplements contained several CLA isomers including trans-10, cis-12 CLA and cis-9, trans-11 CLA as major components. Due to a lack of experimental data in the goat, inferences on the possible contribution of constituent isomers to the observed reductions in milk fat have been drawn based on evidence from studies in the lactating cow. Post-ruminal infusion experiments have established that cis-9, trans-11 CLA, cis-11, trans-13 CLA, trans-8, cis-10 CLA, trans-9, trans-11 CLA and trans-10, trans-12 CLA are not involved in the regulation of milk fat synthesis in the bovine(Reference Shingfield and Griinari33). Inclusion of CaCLA in the diet was also associated with linear increases in milk fat trans-18:1 (Δ6–16) and cis-18:1 (Δ11, 12 and 15) concentrations. Administration of trans-9, -10, -11 and -12-18:1 and cis-11 and -12-18:1 at the duodenum have been shown to have no effect on milk fat synthesis in the lactating cows(Reference Shingfield and Griinari33), while recent evaluations have in some(Reference Kadegowda, Piperova and Erdman43), but not all, cases(Reference Roy, Ferlay, Shingfield and Chilliard25) suggested trans-7-18:1 as a putative milk fat inhibitor. Overall, a detailed analysis of the lipid composition of the CaCLA supplement coupled with the evidence from studies in lactating cows provides support for trans-10, cis-12 CLA being responsible for the reductions in milk fat synthesis in the goat determined in the present study, consistent with the well-established anti-lipogenic activity of this CLA isomer determined in other mammalian species(Reference Bauman, Corl, Peterson, Sébédio, Christie and Adlof1, Reference Bauman, Perfield, Aarvatine and Baumgard34).
Experimental CLA treatments also resulted in a dose-dependent increase in milk fat trans-9, cis-11 CLA concentrations. Supplements were devoid of trans-9, cis-11 CLA, indicating that this isomer was derived from metabolism of CaCLA during transit through the gastrointestinal tract, absorption or in recipient tissues. Previous studies in lactating cows have also demonstrated that CaCLA supplements enrich trans-9, cis-11 CLA concentrations in milk fat(Reference Shingfield and Griinari33). Calcium salts of fatty acids have been developed to minimise the impact of supplemental lipid on ruminal digestion and microbial protein synthesis but do not afford complete protection from biohydrogenation in the rumen(Reference Ferlay, Chilliard and Doreau44, Reference Jenkins and Bridges45). Trans-9, cis-11 CLA is a transitory intermediate of 18 : 2n-6 metabolism in the rumen(Reference Shingfield and Griinari33, Reference Wallace, McKain, Shingfield and Devillard46). However, inclusion of CaCLA in the diet for both experiments decreased 18 : 2n-6 intake, suggesting that trans-9, cis-11 CLA incorporated into milk originated from metabolism of fatty acids in the CaCLA supplement. Studies in the lactating cow have shown that trans-9, cis-11 CLA inhibits milk fat synthesis with an estimated efficacy of 50 % compared with trans-10, cis-12 CLA(Reference Perfield, Lock, Griinari, Sæbø, Delmonte, Dwyer and Bauman35). While significant, the small increases in milk fat trans-9, cis-11 CLA content to CaCLA treatments would tend to suggest a relatively minor contribution to the observed reductions in milk fat synthesis in the present study in goats.
The apparent discrepancy in milk fat responses to CaCLA determined in the present experiment compared with a lack of effect to intravenous(Reference Schmidely and Morand-Fehr10) or duodenal(Reference de Andrade and Schmidely11) infusions of trans-10, cis-12 CLA reported in earlier studies in goats may have several causes. Both intravenous and abomasal infusions were established over a short interval (2 and 3 d, respectively), while the incorporation of trans-10, cis-12 CLA in milk fat was much lower compared with the present data. It is possible that both the duration and amount of trans-10, cis-12 CLA infused were insufficient for the expression of anti-lipogenic activity in the caprine mammary gland. However, 72 h abomasal infusions have been shown to exert significant anti-lipogenic potential in the lactating bovine(Reference Baumgard, Corl, Dwyer, Saebø and Bauman2, Reference Baumgard, Sangster and Bauman3), suggesting that differences in the amount of trans-10, cis-12 CLA infused or from the diet in rumen-protected form are the more probable explanation. Previous studies examining the effect of a lipid-encapsulated supplement of CLA containing trans-10, cis-12 CLA in lactating goats provided tentative evidence indicating that the amount of supplement required to inhibit milk fat synthesis is higher compared with the lactating cow when species comparisons are made on the basis of live weight(Reference Erasmus, Bester, Fourie, Coertze and Hall12, Reference Rovai, Lock, Gipson, Goetsch and Bauman13).
Irrespective of dietary fatty acid content, increases in trans-10, cis-12 CLA intake from CaCLA supplements resulted in curvilinear decreases in milk fat content (Fig. 2). Previous studies in lactating cows have established that the reduction in milk fat secretion to CaCLA(Reference Giesy, McGuire, Shafii and Hanson5–Reference Castañeda-Gutiérrez, Overton, Butler and Bauman7) or post-ruminal infusions of relative pure preparations of trans-10, cis-12 CLA(Reference deVeth, Griinari, Pfeiffer and Bauman32, Reference Shingfield and Griinari33) also occurs in a dose-dependent non-linear manner. Such observations suggest that trans-10, cis-12 CLA acts via common mechanisms in ruminant species. The molecular mechanisms involved in the regulation of milk fat synthesis are not well defined, but studies in lactating cows have provided evidence that trans-10, cis-12 CLA decreases mammary tissue abundance of mRNA for lipogenic genes encoding for key enzymes involved in milk fat synthesis(Reference Shingfield and Griinari33, Reference Bauman, Perfield, Aarvatine and Baumgard34, Reference Bernard, Leroux, Chilliard and Bösze47). Due to a coordinated reduction in the expression of genes that encode for enzymes involved in de novo fatty acid synthesis, fatty acid uptake and transport, and triacylglycerol synthesis, it has been suggested that these changes are mediated via a pathway-specific central regulation of lipogenic gene expression with the SRBEP-1 transcription factor being identified as a possible candidate(Reference Bauman, Perfield, Aarvatine and Baumgard34). Studies in lactating cows(Reference Giesy, McGuire, Shafii and Hanson5–Reference Castañeda-Gutiérrez, Overton, Butler and Bauman7, Reference Bauman and Griinari42) and sheep(Reference Lock, Teles, Perfield, Bauman and Sinclair37, Reference Sinclair, Lock, Early and Bauman38) have established that rumen-protected sources of trans-10, cis-12 CLA result in a reduction in the secretion of fatty acids derived from both de novo synthesis and circulating plasma lipids. By contrast, changes in milk fat composition in the lactating goat to CaCLA supplements determined in the present experiment revealed that the decrease in milk fat secretion was due to a reduction in fatty acids synthesised de novo, while the output of long-chain fatty acids was maintained, irrespective of dietary fatty acid intake. Differences in milk fatty acid composition point towards a lower inhibition of the uptake and incorporation of preformed fatty acids in response to trans-10, cis-12 CLA in the goat compared with the cow or sheep.
The mean apparent efficiency of transfer of trans-10, cis-12 CLA from CaCLA supplements into caprine milk averaged 2·03 %, which is within the range of values (1·9–7·4 %) reported for studies in lactating cows(Reference de Veth, Gulati, Luchini and Bauman15). A lack of difference between experiments suggests that the incorporation of trans-10, cis-12 CLA into caprine milk is independent of the supply of essentially saturated fatty acids at the mammary gland. Furthermore, comparable efficiencies of transfer determined in the goat and cow would tend to suggest that the digestion, absorption and partitioning of trans-10, cis-12 CLA supplied in rumen-protected form as calcium salts is comparable among ruminant species. Indirect comparisons of the relationship between reductions in milk fat content and yield with milk trans-10, cis-12 CLA enrichment (Fig. 3) determined in the present study for the goat with data from studies in lactating cows fed diets containing CaCLA supplements point towards species differences being related to reduced sensitivity of mammary lipogenesis to trans-10, cis-12 CLA rather than metabolism of CaCLA in the rumen. Post-ruminal infusion studies in lactating cows have established that trans-10, cis-12 CLA induces a maximal inhibition of milk fat synthesis of approximately 50 %(Reference deVeth, Griinari, Pfeiffer and Bauman32–Reference Bauman, Perfield, Aarvatine and Baumgard34). Assuming that the incorporation in milk fat reflects the supply at the mammary gland, comparisons of milk trans-10, cis-12 CLA concentrations between ruminants fed CaCLA supplements causing a predicted 25 % reduction in milk fat secretion (Fig. 3) suggest that the goat is between 4·1- and 4·8-fold less sensitive to the anti-lipogenic effects relative to the cow. Additional in vitro and in vivo studies examining the role of trans-10, cis-12 CLA on mRNA abundance on key lipogenic enzymes in caprine and bovine mammary tissue are required to identify the underlying causative mechanisms for these differences.
CaCLA supplements also induced decreases in the concentration ratios of product:substrate for Δ9-desaturase in milk fat. Milk fat cis-9-12:1:12:0, cis-9-14:1:14:0, cis-9-16:1:16:0 and cis-9-18:1:18:0 concentration ratios are known to be highly correlated with mRNA abundance and activity of Δ9-desaturase in the mammary gland of goats(Reference Bernard, Leroux, Chilliard and Bösze47, Reference Bernard, Rouel, Leroux, Ferlay, Faulconnier, Legrand and Chilliard48). Previous studies have established that trans-10, cis-12 CLA decreases mRNA abundance for Δ9-desaturase in bovine mammary tissue(Reference Baumgard, Matitashvili, Corl, Dwyer and Bauman49, Reference Harvatine and Bauman50). Measurements of milk fatty acid composition responses to CaCLA supplements also suggest that trans-10, cis-12 CLA down-regulates transcripts encoding for Δ9-desaturase in the caprine mammary gland. Earlier studies in the lactating goat reported that duodenal infusions of trans-10, cis-12 CLA decreased milk fat desaturase indices in the absence of an effect on milk fat synthesis(Reference de Andrade and Schmidely11), consistent with the view that inhibition of Δ9-desaturase activity is not, in isolation, sufficient to induce a reduction in milk fat synthesis(Reference Shingfield and Griinari33).
In conclusion, CaCLA supplements containing trans-10, cis-12 CLA reduced milk fat synthesis in lactating goats in a dose-dependent manner. Reductions in milk fatty acid output were due to reductions in the secretion of fatty acids synthesised de novo rather than fatty acids derived from circulating plasma lipids, leading to a shift in milk fat composition containing higher proportions of long-chain fatty acids. Differences in the extent of inhibition on milk fat content and yield between experiments indicated that anti-lipogenic activity in the goat is dependent on both the supply of trans-10, cis-12 CLA and long-chain fatty acids available to the mammary gland. Indirect comparisons of the relationship between reductions in milk fat secretion and milk fat trans-10, cis-12 CLA content in response to CaCLA supplements point towards the sensitivity of mammary lipogenesis to the inhibitory effects of trans-10, cis-12 CLA being several-fold lower in the goat compared with the cow.
Acknowledgements
The authors gratefully acknowledge and appreciate the staff of the ‘Les Cèdres’ Animal Nutrition and Metabolism Unit, André Combeau, Christophe Mathevon and Denis Roux, in particular, for the diligent care of experimental animals and the technical assistance of Pierre Capitan, Didier Bany, Cyril Labonne, Piia Kairenius and Vesa Toivonen. All authors have contributed to the preparation of the paper and agree with the submitted manuscript content. There are no conflicts of interest.
