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* Department of Animal Science, University of Vermont, Burlington 05405
E (Kika) de la Garza American Institute for Goat Research, Langston University, Langston, OK 73050
BASF AG, Nutrition Research Station, Neumuehle 13, 76877, Offenbach/Queich, Germany
Department of Animal Science, Cornell University, Ithaca, NY 14853
3 Corresponding author:deb6{at}cornell.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: conjugated linoleic acid • milk fat depression • goat • lactation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Trans-10, cis-12 CLA has also been found to reduce milk fat yield or content, or both, in other lactating nonruminant and ruminant species in addition to the dairy cow (Bauman et al., 2008). In lactating sheep, administration of a supplement containing trans-10, cis-12 CLA decreases milk fat production, with the magnitude of the reduction being similar to dairy cows when the dose is expressed on a metabolic BW basis or when compared on the basis of the trans-10, cis-12 content of milk fat (Lock et al., 2006; Sinclair et al., 2007; Weerasinghe et al., 2007). In contrast, recent investigations involving goats reported that trans-10, cis-12 CLA had little or no effect on milk fat yield (Erasmus et al., 2004; Schmidely and Morand-Fehr, 2004; de Andrade and Schmidely, 2006). These findings contrast from the consistent effects of CLA on decreasing milk fat production observed with cows and sheep, and may be a result of species differences and the fact that the goat may be unresponsive or less sensitive to trans-10, cis-12 CLA (Sanz Sampelayo et al., 2007). Alternatively, it may be a consequence of experimental design or study differences in the aforementioned goat investigations, or both.
Our objective was to determine if a lipid-encapsulated supplement containing trans-10, cis-12 CLA would inhibit milk fat synthesis in lactating goats. We based our approach on published studies with cows and sheep in terms of CLA dose, duration of treatment, and experimental design. A secondary objective was to examine the effects of trans-10, cis-12 CLA on milk fatty acid composition and transfer of this CLA isomer to milk fat. In some cow and sheep studies, the CLA-induced reduction in milk fat has been associated with an increase in milk yield and milk protein yield, although this appears to coincide with situations when energy or protein was limiting (Griinari and Bauman, 2006); thus, responses in these variables were also of interest.
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MATERIALS AND METHODS |
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). The diet was initially offered at approximately 3 kg/d, (DMI represented ~3.5% of BW) and was then adjusted daily to allow for an ad libitum intake. Daily feed intakes were recorded using the Calan headgate system (American Calan Inc., Northwood, NH). In addition, salt blocks (American Stockman, North American Salt Co., Overland Park, KS) and fresh water were available ad libitum.
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The experimental design was a 3 x 3 Latin square with 14-d treatment periods separated by 14-d wash-out intervals. The CLA supplement was a lipid-encapsulated formulation (LE-CLA; marketed as Lutrell by BASF AG, Ludwigshafen, Germany) comprised of 2 CLA isomers in equal proportions: cis-9, trans-11 and trans-10, cis-12 CLA. Dietary treatments were as follows: A) unsupplemented (control), B) supplemented with 30 g/d of LE-CLA (low dose; CLA-1), or C) supplemented with 60 g/d of LE-CLA (high dose; CLA-2). The LE-CLA supplement had a lipid content of 65% and a fatty acid composition of 15% trans-10, cis-12 CLA; 15% cis-9, trans-11 CLA; 9% 16:0; 42% 18:0; 12% cis-9 18:1; and 1% cis-9, cis-12 18:2. Thus, the CLA-1 and CLA-2 treatments provided trans-10, cis-12 CLA at a rate of 3 and 6 g/d, respectively. The LE-CLA supplement was mixed with dry molasses on a daily basis and given before feeding to assure it was consumed totally.
Goats averaged 50 ± 7.4 kg of BW and 2.5 BCS (1 to 5 scale by 0.5 increments; Villaquiran et al., 2005) at the start of the study. They were weighed, and condition was scored at the beginning and end of each 14-d treatment period. Milk was sampled at each milking and analyzed for fat, protein, lactose, solids, and SCC using a CombiFoss 5000 (Foss Food Technology, Eden Prairie, MN) at Langston University DHI Laboratory. On the final day of each treatment period, additional milk samples were collected and stored at –20°C until subsequent fatty acid analysis.
Chemical Analysis
Feed samples were collected twice during each treatment period. Composites were formed and analyzed according to AOAC (2000) wet chemistry procedures by Dairy One Cooperative Inc. (Ithaca, NY) with values reported for CP, ADF, NDF, and NEL.
For analysis of milk fatty acids, the a.m. and p.m. milk samples from the last day of each treatment period were composited based on volume, and fat was extracted using the method of Hara and Radin (1978). Fatty acid methyl esters were prepared by base-catalyzed transmethylation according to Christie (1982) with modifications by Chouinard et al. (1999). Fatty acid methyl esters were quantified using a gas chromatograph (GC system 6890+ with flame-ionization detector; Agilent Inc., Wilmington, DE) equipped with a CP-SIL 88 fused-silica capillary column [100 m x 0.25 mm (i.d.) with 0.2-µm film thickness; Varian Inc., Walnut Creek, CA] as described previously (Lock et al., 2006). Fatty acid identification and recoveries were determined using pure methyl ester standards (Nu-Chek Prep, Elysian, MN; Natural ASA, Hovdebygda, Norway) along with comparisons of the elution order for trans-18:1 isomers reported in the literature. A butter oil reference standard (CRM 164; Commission of the European Community Bureau of References, Brussels, Belgium) was used as a routine quality control and as a check for recoveries and correction factors for individual fatty acids.
The LE-CLA supplement was analyzed for lipids following the extraction and methylation procedures described by Perfield et al. (2004). Analysis of fatty acid methyl esters was as detailed above for milk fatty acids.
Statistical Analysis
Milk production and DMI data for the final 4 d of each treatment period were averaged. Intake, milk production and composition, BW, BCS, and milk fatty acid composition were analyzed as a 3 x 3 Latin square design using JMP (Version 5, SAS Institute, Cary, NC). The mixed model to test treatment means included the random effect of goat and fixed effect of period and treatment, and results are presented as treatment means with a SEM. Treatment effects were considered significant at P < 0.05.
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RESULTS |
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. The DMI did not differ among treatment groups and averaged 2.5 ± 0.1 kg/d (mean ± SE) for d 11 to 14 of the treatment period (Table 2). Similarly, BW and BCS were not affected by the treatments and averaged 50.5 ± 1.4 kg and 2.2 ± 0.1 (mean ± SE), respectively, for the experimental periods (data not presented).
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). Similarly, milk lactose yield did not differ among treatments, although milk lactose content was slightly lower (~1%) in the CLA-2 treatment as compared with control and CLA-1 treatments (Table 2). Examination of the temporal pattern indicated that milk yield and the milk content of protein and lactose were relatively constant over the treatment period (Figure 1). In contrast, the temporal pattern for milk fat content and yield demonstrated a progressive decline over the 14-d treatment period (Figure 2). Compared with control, milk fat yield by d 11 to 14 was reduced by 8 and 21% for the CLA-1 and CLA-2 treatments, respectively. Similar values for milk fat content were a reduction of 5 and 18%, respectively (Table 2).
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). Relative to control, the greatest effects were observed for the CLA-2 treatment with numerical values for the CLA-1 treatment being intermediate. Changes in the composition included a reduction in the proportion of short- and medium-chain fatty acids (C6 to C14) and an increase in most long-chain fatty acids (
18 carbons). When grouped according to source (Bauman and Griinari, 2003), those fatty acids synthesized de novo by the mammary gland (<C16) were decreased from 29% of the total in control treatment to 24% in the CLA-2 treatment; a reciprocal increase occurred in those fatty acids derived by mammary uptake from circulation (>C16). Palmitic acid and palmitoleic acid came from both sources and were modestly increased in the milk fat of LE-CLA-supplemented goats (Table 3).
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9-desaturase, also known as stearoyl coenzyme A desaturase. Using the procedure of Kelsey et al. (2003), we calculated the desaturase index based on these fatty acid pairs, and results are presented in Table 3. Relative to the control treatment, the high dose of CLA resulted in a significant reduction in the desaturase index with intermediate values for the CLA-1 treatment.
Examination of the effects of LE-CLA supplements on the secretion of fatty acids (molar basis) demonstrated that the CLA treatments resulted in a decrease in the secretion of milk fatty acids arising from both de novo synthesis and direct uptake from circulation (Figure 3). Effects on de novo synthesis, however, were more pronounced, and this is the basis for the observed increase in the proportion of long-chain fatty acids in milk fat when goats received LE-CLA supplements (Table 3).
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). Trans-10, cis-12 CLA was of particular interest because of its inhibitory effects on milk fat synthesis, and its content in milk fat increased from 0.03 g/100 g of fatty acids in the control treatment to 0.09 and 0.19 g/100 g of fatty acids for the CLA-1 and CLA-2 treatments, respectively. Transfer efficiencies for trans-10, cis-12 CLA into milk fat were estimated by comparing dietary intake and milk fat secretion. Correcting for values observed during the control treatment, transfer efficiencies were similar (1.80 vs. 1.89%; P = 0.44) for the 2 CLA treatments. |
DISCUSSION |
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The LE-CLA supplement contained 2 CLA isomers in equal proportions, trans-10, cis-12 and cis-9, trans-11. Previous studies have shown that the cis-9, trans-11 CLA isomer had no effect on milk fat synthesis in dairy cows (Baumgard et al., 2000, 2002; Loor and Herbein, 2003), and based on this, the milk fat reduction we observed in goats was due to the trans-10, cis-12 CLA in the supplement. In the present study in which goats were in late lactation, the CLA supplement had no effect on the yield of milk, milk protein, or milk lactose (Table 2). As reviewed by Griinari and Bauman (2006), similar results have generally been observed in studies with dairy cows. In some situations, however, the reduction in milk fat has resulted in a repartitioning of nutrients to support increased milk and milk protein yields in sheep (Lock et al., 2006; Weerasinghe et al., 2007) and cows (Bernal-Santos et al., 2003; Mackle et al., 2003; Odens et al., 2007); this has been most often seen in early lactation or in situations in which nutrient intake to meet requirements tends to be less than optimal.
The mechanism for the trans-10, cis-12 CLA-induced reduction in milk fat synthesis involves a coordinated reduction in the mRNA expression for key enzymes involved in milk fat synthesis (Bauman et al., 2008), and a role for sterol response element-binding protein 1 and Spot 14 has been indicated in the cellular signaling (Harvatine and Bauman, 2006). Consistent with this, investigations with cows and sheep have established that trans-10, cis-12 CLA results in a reduction in the secretion of milk fatty acids arising from both de novo synthesis and preformed uptake from the circulation, although de novo synthesis is more dramatically affected (Griinari and Bauman, 2006). A similar pattern of response in milk fatty acids occurred in the present study with lactating goats (Figure 3). Likewise, effects on milk fat desaturase indices observed with the CLA-2 treatment (Table 3) were as reported previously at greater doses of trans-10, cis-12 CLA in studies with lactating cows and sheep.
The CLA-induced reduction in milk fat we observed contrasts with the lack of effect reported in 2 previous reports with lactating goats. Differences appear to relate primarily to design and duration, because these investigations involved jugular infusion of trans-10, cis-12 CLA for 2 d with effects compared with the day (2 milkings) immediately before the infusion (Schmidely and Morand-Fehr, 2004) or duodenal infusion of trans-10, cis-12 CLA for 3 d followed immediately by a 2-d control infusion of skim milk (de Andrade and Schmidely, 2006). Two of the key lipogenic enzymes involved in the coordinated mechanism of the CLA-induced reduction in milk fat are acetyl coenzyme A carboxylase and fatty acid synthase, and they have a half-life of 48 to 76 h (Craig et al., 1972; Volpe and Vagelos, 1973). Consistent with this, the pattern of the reduction in milk fat is one of a progressive decline over the first few days of CLA treatment, and termination of CLA treatment results in a similar progressive recovery that is a mirror image of the CLA-induced decline. Thus, results in the previous goat study in which the control period immediately followed the treatment period with no interceding washout interval would be confounded, and this would be further exacerbated when the treatment period is of short duration.
Interestingly, the investigation by de Andrade and Schmidely (2006) observed that the 3-d CLA infusion increased milk fat content of trans-10, cis-12 CLA and altered desaturase indices, although milk fat yield was unaltered. The desaturase indices are a proxy for 9-desaturase, and these results are not unexpected; although studies show that changes in 9-desaturase are not a prerequisite for the CLA-induced reduction in milk fat yield (see discussion in Perfield et al., 2004), this enzyme is also coordinately regulated by CLA and has a short half-life of only 2 to 4 h (Oshino and Sato, 1972; Toyama et al., 2007). It is important to note that in the present study the effect of LE-CLA on milk fat production was not apparent until after 3 d of supplementation with a continuing decline in milk fat for the next few days. Furthermore, Erasmus et al. (2004) conducted 2 studies with lactating goats involving different doses of a rumen-protected CLA supplement, but unfortunately details in their preliminary report are inadequate to allow for comparisons. Nevertheless, in their second study, they observed a significant reduction in milk fat content at their greatest dose and suggested that on a BW basis, goats may require a greater dose of CLA to achieve milk fat depression comparable to cows.
Our results also suggest a greater dose of CLA is required for a comparable inhibition of milk fat synthesis in goats. In lactating sheep, 25 g/d of LE-CLA provided about 2.4 g/d of trans-10, cis-12 CLA and gave a 23% and 16 to 20% reduction in the content and yield of milk fat, respectively (Lock et al., 2006; Sinclair et al., 2007); accounting for the portion metabolized in the rumen and using the dose-response relationships summarized by de Veth et al. (2004), this was comparable to a 23 and 21% reduction in milk fat content and milk fat yield in cows when the dose was expressed on the basis of metabolic BW. In the present study, we observed that a similar amount of trans-10, cis-12 CLA supplied as a lipid-encapsulated supplement to lactating goats gave a reduction of only 5 and 8% in milk fat content and yield, respectively. Values comparable to sheep and cows were only observed in lactating goats when the amount of CLA supplement was doubled; the 60 g/d of LE-CLA supplement to lactating goats gave a 18 and 21% reduction in content and yield of milk fat, respectively (Table 2). Consistent with this difference in milk fat response, the transfer efficiency of trans-10, cis-12 CLA from the lipid-encapsulated supplement to milk fat was somewhat less in goats (1.80 to 1.89%) as observed for the supplement administrated to lactating sheep (2.5 to 3.8%; Lock et al., 2006; Sinclair et al., 2007).
The reduced effectiveness of dietary supplements of CLA in goats as compared with sheep and cows could be due to differences in processes occurring in the digestive tract or in postabsorptive use of CLA. Possibilities related to digestive processes would include a greater metabolism of the CLA supplement in the rumen or a lower bioavailability of the CLA supplement in the small intestine of goats, or both. Differences in postabsorptive use could include a greater use of CLA by nonmammary tissues or a reduced uptake and availability in the mammary epithelial cell. The present study did not directly examine these possibilities, but indirect evidence provides some insight. Although we found that the transfer efficiency of trans-10, cis-12 CLA provided as a dietary supplement was only about 50% of the value observed in cows and sheep, de Andrade and Schmidely (2006) observed a 17.8% transfer efficiency of trans-10, cis-12 CLA provided by duodenal infusion in goats, which is comparable to the average 21.7% found in a summary of multiple doses in 7 studies with lactating cows (de Veth et al., 2004). That suggests that the basis may predominantly relate to a more extensive biohydrogenation of the CLA supplement in the rumen of goats, whereas bioavailability of CLA in the small intestine and the uptake of CLA by the mammary gland are similar among ruminant species. We examined this further by considering the observed reduction in milk fat in relation to the content of trans-10, cis-12 CLA in milk fat. Using the dose-response equation for dairy cows developed by de Veth et al. (2004), the incremental increase in the concentration of trans-10, cis-12 CLA in milk fat observed in the present study for the CLA-1 and CLA-2 treatments (Table 3) would correspond (95% prediction interval) to a predicted decrease in milk fat yield of 6 to 25% and 23 to 42%, respectively; the observed reduction was 8 and 21% for the CLA-1 and CLA-2 treatments, respectively. Additional investigations are required to more fully explore the basis for the lower response in milk fat synthesis observed for goats.
Overall, the present study provides the first evidence that feeding of lipid-encapsulated supplements containing trans-10, cis-12 CLA was effective in reducing milk fat synthesis in lactating goats. Similar to other species, CLA effects involved a reduction in milk fatty acids of all chain lengths, although those arising from de novo synthesis were most dramatically reduced. As a consequence, the CLA supplementation of lactating goats resulted in a shift in milk fat composition to a greater proportion of unsaturated, longer chain fatty acids. Especially noteworthy, dose-response comparisons suggest that goats are less responsive in the magnitude of milk fat depression as compared with dairy cows and sheep.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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2 Present address: Balchem Corporation, New Hampton, NY 10958.
Received for publication February 1, 2008. Accepted for publication May 9, 2008.
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) or supplemented with lipid-encapsulated conjugated linoleic acid (CLA) at a low dose (CLA-1; 
) or supplemented with lipid-encapsulated conjugated linoleic acid (CLA) at a low dose (CLA-1; 