T.C. Jenkins

Technical note: Common analytical errors yielding inaccurate results during analysis of fatty acids in feed and digesta samples1

The basic rules governing the proper fatty acid analysis of feed and digesta samples are sometimes overlooked, leading to potential errors in reporting the fatty acid content or composition of feed and digesta samples. The direct transesterification procedure of Sukhija and Palmquist (1988, J. Agric. Food Chem. 36:1202-1206) has become popular in analyzing fatty acids in feed and digesta samples obtained from animal feeding trials. One shortcoming of the Sukhija and Palmquist transesterification procedure is inaccurate analysis of fatty acids with conjugated double bonds. Digesta and milk samples from ruminant species typically contain a multitude of conjugated linoleic acid (CLA) isomers that easily undergo isomerization and epimerization following prolonged exposure to methanolic HCl. Modifications to the Sukhija and Palmquist procedure are given in this paper that allow successful determination of CLA isomers. Errors in fatty acid analysis also occur from misuse of internal standards; use of an internal standard is recommended in the Sukhija and Palmquist procedure as the preferred method to quantify total fatty acid content. The choice of internal standard may sometimes be important for obtaining accurate results. As an example, applying the direct transesterification procedure to a fat supplement high in saturated fatty acids yielded 613 mg/g of total fatty acids when C17 was used as the internal standard compared with 930 mg/g total fatty acids when C19 was used as the internal standard. Fatty acid content further increased to 952 mg/g when a unique unsaturated fatty acid (C13:1) was used as the internal standard.

Effects of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: I. Uterine and metabolic responses, reproduction, and lactation

The objectives were to evaluate the effects of differential timing of supplementation of different Ca salts (CS) of fatty acids (FA) on FA profiles of cotyledonary-caruncular tissues, metabolic status, uterine health, pregnancy, pregnancy losses after 2 artificial inseminations (AI), and milk yield. Holstein cows (n=1,380) were assigned randomly to be fed either CS of palm oil (PO) or safflower oil (SO) from 30 d prepartum until 30 d postpartum (dpp) and further randomized to receive either CS of PO or fish oil (FO) from 30 to 160 dpp. Supplementation of CS of FA was at 1.5% of dietary dry matter. Tissues (n=23) and blood (n=32) were collected from a subsample of cows. Blood was collected daily from parturition to 10 dpp and three times weekly thereafter until 30 dpp for analyses of PGF2α metabolite, nonesterified FA, β-hydroxybutyric acid, blood urea nitrogen, and glucose. Cows were evaluated once between 8 to 10 dpp for cervical discharge type. At 43 dpp, cows received 2 injections of PGF2α 14 d apart, followed 14 d later by injections of GnRH at 7 d before and 56 h after an injection of PGF2α with AI at 16 h after the second GnRH injection. All cows received intravaginally a controlled internal drug-releasing device, containing 1.38 g of progesterone, at 18 d after the first AI followed 7 d later by removal of the device and injection of GnRH. Nonpregnant cows at 32 d after AI were injected with PGF2α, followed 56 h later with a GnRH injection and second AI 16 h thereafter. Cows diagnosed pregnant after both AI were re-examined at 60 d of pregnancy to determine pregnancy losses. Milk weights were recorded monthly for all cows. Caruncular n-6:n-3 FA ratio was greater in cows fed SO. Plasma concentrations of metabolites and frequency of cervical discharge type did not differ between PO- and SO-fed cows. Plasma PGF2α metabolite was greater in SO-fed cows at 4 and 7 dpp. Pregnancy per AI at 32 and 60 d post first AI was not affected by diets, but pregnancy loss was less in FO-fed cows. At second AI, pregnancy was greater in FO-fed cows at 32 d and in SO-FO-fed cows at 60 d post AI. Pregnancy loss after second AI was not affected by diets. Overall pregnancy per AI was greater in cows fed SO followed by FO at 60 d of pregnancy and pregnancy loss was reduced in FO-fed cows. Monthly milk yield was greater (0.7 kg/d) in SO-fed cows. In conclusion, strategic feeding of CS of FA during transition and breeding periods can benefit fertility and milk production of lactating dairy cows.

Biohydrogenation of Linolenic Acid to Stearic Acid by the Rumen Microbial Population Yields Multiple Intermediate Conjugated Diene Isomers

The current literature suggests that linolenic acid biohydrogenation converts to stearic acid without the formation of CLA. However, a multitude of CLA were identified in the rumen that are generally attributed to linoleic acid biohydrogenation. This study used a stable isotope tracer to investigate the biohydrogenation intermediates of (13)C-linolenic acid, including CLA. A continuous culture fermenter was used to maintain a mixed microbial population obtained from the rumen of cattle at pH 6.5 for 6 d. The mixed fermenter contents were then transferred to batch cultures containing either (13)C-labeled or unlabeled linolenic acid, which were run in triplicate for 0, 3, 24, and 48 h. The (13)C enrichment was determined by GC-MS. After 48 h of incubation, 8 CLA isomers were significantly enriched, suggesting that these CLA isomers originated directly from linolenic acid. The cis-10, cis-12 CLA isomer exhibited the highest enrichment (21.7%), followed by cis-9, cis-11 and trans-8, trans-10 CLA. The enrichment of these 2 CLA isomers ranged from 20.1 to 21.1% and the remaining 5 CLA including cis-9, trans-11 CLA were <15.0%. A multitude of nonconjugated and partially conjugated 18:2 and 18:3 isomers was enriched during the 48 h of incubation. The results of this study confirm that mixed ruminal microbes are capable of the formation of several CLA and 18:3 isomers from linolenic acid, indicating that linolenic acid biohydrogenation pathways are more complex than previously reported.

Serum hormone and metabolite concentrations in fasted young bulls and steers

The effect of dietary energy restriction on serum insulin, insulin-like growth factor I (IGF-I), growth hormone, (GH), cortisol, plasma urea nitrogen (PUN) and nonesterified fatty acid (NEFA) concentrations was examined. Angus bulls and steers (10 mo) were allotted to two groups of 12 animals and assigned a treatment order. In a switchback design, animals in order 1 were fed a high grain diet, then fasted, while order 2 animals were fasted, and then fed. Animals were allowed 60 hr to acclimate between treatments. Serum and plasma were obtained at 20 min intervals and 60 min, respectively, for 6 hr after feeding and for the last 6 hr of a 30 hr fast. Serum was assayed for insulin, IGF-I, GH, and cortisol (total and free). Plasma was assayed for PUN and NEFA. Mean insulin (ng/ml) differed between fed (.95 +/- .08) and fasted (.26 +/- .08) animals (P less than 01). Both mean total and free cortisol (ng/ml) were lower in fed (11.48 +/- .99) (1.06 +/- .12) than in fasted (17.10 +/- .93) (1.62 +/- .12) animals, respectively (P less than .01). Animals in order 1 differed in mean IGF-I (ng/ml) between fed (199.0 +/- 8.0) and fasted (116.5 +/- 7.2) treatments (P less than .01). Mean IGF-I for animals in order 2 was 146.7 +/- 7.2 in fed and 213.9 +/- 7.2 in fasted animals (P less than .01). Mean GH did not differ between treatments. Mean PUN and NEFA were higher in fasted than in fed animals (P less than .01). Except for % free cortisol (P less than .05), the hormones did not differ between bulls and steers.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: II. Neutrophil fatty acids and function, and acute phase proteins.

The objectives were to evaluate the effects of differential supplementation of Ca salts (CS) of fatty acids (FA) on plasma acute phase proteins and both FA composition and function (i.e., activity and cytokine production) of neutrophils, during the peripartum and breeding periods. Holstein cows were assigned randomly to receive either CS of palm (PO) or safflower (SO) oils from 30 d prepartum until 35 d postpartum (dpp) and CS of PO or fish oil (FO) from 35 to 160 dpp. Supplementation of CS of FA was at 1.5% of dietary dry matter. Cows (n=32) were sampled three times weekly from parturition to 35 dpp for analyses of plasma concentrations of haptoglobin and fibrinogen. Cows (n=47) were sampled for neutrophil phagocytic and oxidative burst activities toward Escherichia coli and Staphylococcus aureus, and neutrophil abundances of L-selectin and β(2)-integrin assessed by flow cytometry at 32 d prepartum, within 7h after parturition, and 4 and 7 dpp. Profiles of FA in neutrophils and cytokine production (i.e., tumor necrosis factor alpha, TNF-α, and IL-1β) were assessed prepartum (n=14), 35 (PO vs. SO; n=26) and 85 dpp (PO vs. FO; n=28). Plasma concentrations of haptoglobin and fibrinogen were greater for cows fed SO compared with PO. The percentage of neutrophils with phagocytic and oxidative burst activities was not affected by transition diets, but activities per neutrophil were greater in SO compared with PO diets at 4 (phagocytosis and oxidative burst) and 7 dpp (oxidative burst). Neutrophil abundance of L-selectin, but not β(2)-integrin, was greater in SO compared with PO at 4 and 7 dpp. Neutrophil productions of TNF-α and IL-1β were increased at 35 dpp in SO compared with PO diets, but production of TNF-α was attenuated in FO compared with PO at 85 dpp. Neutrophil ratios of n-6:n-3 FA were greater at 35 dpp in the SO diet and less at 85 dpp in FO compared with PO diets. In conclusion, cows supplemented with CS of SO had improved innate immunity (i.e., acute phase response and neutrophil function) to better cope with the bacterial challenges in the postpartum period. Conversely, CS of FO attenuated neutrophil cytokine production.

Invited review: palmitic and stearic acid metabolism in lactating dairy cows.

Energy is the most limiting nutritional component in diets for high-producing dairy cows. Palmitic (C16:0) and stearic (C18:0) acids have unique and specific functions in lactating dairy cows beyond a ubiquitous energy source. This review delineates their metabolism and usage in lactating dairy cows from diet to milk production. Palmitic acid is the fatty acid (FA) found in the greatest quantity in milk fat. Dietary sources of C16:0 generally increase milk fat yield and are used as an energy source for milk production and replenishing body weight loss during periods of negative energy balance. Stearic acid is the most abundant FA available to the dairy cow and is used to a greater extent for milk production and energy balance than C16:0. However, C18:0 is also intimately involved in milk fat production. Quantifying the transfer of each FA from diet into milk fat is complicated by de novo synthesis of C16:0 and desaturation of C18:0 to oleic acid in the mammary gland. In addition, incorporation of both FA into milk fat appears to be limited by the cows requirement to maintain fluidity of milk, which requires a balance between saturated and unsaturated FA. Oleic acid is the second most abundant FA in milk fat and likely the main unsaturated FA involved in regulating fluidity of milk. Because the mammary gland can desaturate C18:0 to oleic acid, C18:0 appears to have a more prominent role in milk production than C16:0. To understand metabolism and utilization of these FA in lactating dairy cows, we reviewed production and milk fat synthesis studies. Additional and longer lactation studies on feeding both FA to lactating dairy cows are required to better delineate their roles in optimizing milk production and milk FA composition and yield.

Effects of Feeding a Dietary Antioxidant in Diets with Oxidized Fat on Lactation Performance and Antioxidant Status of the Cow

The objective of the study was to evaluate the effect of feeding the dietary antioxidant Agrado Plus (AOX; Novus International, St. Louis, MO) in diets that contained 2% fresh fat (FF) or oxidized fat (OF) on milk production and composition and antioxidant status of cows during mid to late lactation. Forty-four mid to late lactating primiparous cows housed in a tie-stall barn were fed a diet that contained 2% FF for 15 d as adaptation period and then randomly allocated to 1 of the 4 dietary treatments (FF, FF+AOX, OF, OF +AOX) for 6 wk. Feeding AOX increased dry matter intake, 3.5% fat-corrected milk, and milk fat yield, and decreased milk protein content but not yield. Feeding OF increased milk fat yield, but decreased dry matter intake and body weight gain. Milk fat composition changed with treatments: AOX increased cis 18:1 and decreased trans-11 18:1, whereas OF decreased trans-9 and trans-11 18:1 and cis-9, trans-11 18:2 in milk. Plasma antioxidant enzymes and status were affected by treatments. Feeding OF increased superoxidase dismutase activity but decreased plasma antioxidant status, whereas AOX supplementation increased glutathione peroxidase activity across fat types and increased the antioxidant status and superoxidase dismutase activity when feeding OF diets. It can be concluded that feeding AOX improved lactation performance and the antioxidant status of the cow across fat types, and feeding OF increased milk fat yield but decreased dry matter intake, body weight gain, and antioxidant status. The negative effects of feeding OF were partially alleviated by AOX.

Evaluation of protein fractionation and ruminal and intestinal digestibility of corn milling co-products

Novel corn milling co-products developed from technological advancements in ethanol production vary widely in chemical composition and nutrient availability. The objectives of this study were to characterize feed protein fractions and evaluate differences in rumen-undegradable protein (RUP) and its digestible fraction (dRUP), amino acid concentration, and in vitro gas production of 7 corn milling co-products. The crude protein (CP; % of dry matter) of co-products was 12.7 for germ, 26.9 for dried distillers grains plus solubles that had no heat exposure before fermentation (DDGS1), 45.4 for high-protein dried distillers grains (HPDDG), 12.7 for bran, 30.2 for wet distillers grains plus solubles (WDGS), 23.1 for wet corn gluten feed (WCGF), and 26.0 for dried distillers grains plus solubles that had heat exposure before fermentation (DDGS2). Two ruminally and duodenally fistulated Holstein steers weighing 663+/-24 kg were used to determine RUP and dRUP with the in situ and mobile bag techniques. Samples of each feed were ruminally incubated for 16 h, and mobile bags were exposed to simulated abomasal digestion before insertion into the duodenum and subsequent collection in the feces. Protein fractions A, B(1), B(2), B(3), and C were characterized as follows (% CP): germ=30.0, 15.0, 38.1, 13.5, 3.4; DDGS1=17.0, 7.0, 67.0, 4.8, 4.2; HPDDG=7.4, 0.6, 82.4, 8.8, 0.8; bran=33.5, 4.0, 54.3, 6.0, 2.2; WDGS=18.6, 2.4, 53.1, 11.0, 14.9; WCGF=36.6, 15.9, 33.2, 10.1, 4.1; and DDGS2=17.9, 2.1, 41.1, 11.1, 27.9. The proportions of RUP and dRUP were different and are reported as follows (% CP): DDGS2=56.3, 91.9; HPDDG=55.2, 97.7; WDGS=44.7, 93.1; DDGS1=33.2, 92.1; bran=20.7, 65.8; germ=16.5, 66.8; and WCGF=11.5, 51.1. The concentrations of Lys and Met in the RUP were different and are listed as follows (% CP): germ=2.9, 2.0; DDGS1=1.9, 2.0; HPDDG=2.0, 3.2; bran=3.2, 1.5; WDGS=1.9, 2.3; WCGF=3.5, 1.6; and DDGS2=1.9, 2.4. In vitro gas production (mL/48h) was highest for germ (52.1) followed by bran (50.1), WDGS (40.7), DDGS2 (40.1), WCGF (39.0), DDGS1 (38.6), and HPDDG (37.5). Comparison of co-products defined differences in chemical composition, protein fractionation, ruminal availability, and microbial fermentation.

Docosahexaenoic acid elevates trans-18:1 isomers but is not directly converted into trans-18:1 isomers in ruminal batch cultures

Pathways of docosahexaenoic (DHA) biohydrogenation are not known; however, DHA is metabolized by ruminal microorganisms. The addition of DHA to the rumen alters the fatty acid profile of the rumen and milk and leads to increased trans-18:1 isomers, particularly trans-11 18:1. This study included 2 in vitro experiments to identify if the increase in trans-11 C18:1 was due to DHA being converted into trans-11 18:1 or if DHA stimulated trans-11 products from biohydrogenation of other fatty acids. In each experiment, ruminal microorganisms collected from a lactating Holstein cow were incubated in 10-mL batch cultures for 0, 6, 24, and 48 h and a uniformly (13)C-labeled DHA was added to the cultures at 0 h as a metabolic tracer. Experiment 1 tested 0.5% DHA supplementation and experiment 2 examined 1, 2, and 3% DHA supplementation to determine if the level of DHA effected its conversion into trans-11 18:1. In both experiments, any fatty acid that was enriched with the (13)C label was determined to arise from DHA. Palmitic (C16:0), stearic (C18:0), all trans-18:1, eicosanoic (C20:0), and docosanoic (C22:0) acids were examined for enrichment. In experiment 1, the amount of trans-18:1 isomers increased 0.415 mg from 0 to 48 h; however, no label was found in trans-18:1 at any time. Docosanoic acid was highly enriched at 24h and 48 h to 20.2 and 16.3%. Low levels of enrichment were found in palmitic and stearic acids. In experiment 2, trans-18:1 isomers increased 185, 256, and 272% from 0 to 48 h when DHA was supplemented at 1, 2, and 3%, respectively; however, as in experiment 1, no enrichment occurred of any trans-18:1 isomer. In experiment 2, low levels of label were found in palmitic and stearic acids. Enrichment of docosanoic acid decreased linearly with increased DHA supplementation. These studies showed that trans-18:1 fatty acids are not produced from DHA, supporting that DHA elevates trans-18:1 by modifying biohydrogenation pathways of other polyunsaturated fatty acids.

Kinetics of Ruminal Lipolysis of Triacylglycerol and Biohydrogenation of Long-Chain Fatty Acids: New Insights from Old Data

Previous investigations into ruminal lipolysis of triacylglycerol and ruminal biohydrogenation (BH) of unsaturated long-chain fatty acids have generally quantified these processes with either zero-order or first-order kinetics. This investigation examined if Michaelis-Menten and other nonlinear kinetics might be useful for quantifying these processes. Data from 2 previously published in vitro experiments employing rumen fluid from sheep to investigate the lipolysis of trilinolein, the BH of cis-9, cis-12 linoleic acid (LA), and the BH of fatty acids derived from the lipolysis of trilinolein were used for the development of a multi-compartmental model. The model described the lipolysis of triacylglycerol well. The model also provided a good mathematical description of the resulting production of nonesterified fatty acids, the isomerization of nonesterified LA, and subsequent production of rumenic acid (RA), vaccenic acid (VA), and stearic acid (SA). However, the model described poorly the patterns of the concentrations of LA, RA, VA, and SA after incubation of trilinolein in rumen fluid. The model is consistent with known stoichiometry and biochemistry and is parsimonious in that it employs a minimal number of parameters to describe all of the major aspects of lipolysis and BH. The first step in the lipolysis of trilinolein was described by Michaelis-Menten kinetics (Vmax = 529 +/- 16 mg/L per h; Km = 698 +/- 41 mg/L). Both subsequent lipolysis steps were approximated by a first-order (linear kinetics) rate constant (k = 2.64 +/- 0.041 /h). Isomerization of LA to RA was modeled by simple Michaelis-Menten kinetics (Vmax = 2,421 +/- 83 mg/L per h; Km = 440 +/- 22 mg/L). The kinetics of the BH of RA to VA was described by a Michaelis-Menten-type process involving competitive inhibition by VA (Vmax = 492 +/- 6.5 mg/L per h; Km = 1 mg/L). The final step, the BH of VA to SA, was modeled by a quasi-first-order process (k = 0.533 +/- 0.021 /h), but as the concentration of VA increased, its BH appeared to be self-inhibited such that when the concentration of VA acid exceeded 517 +/- 10.4 mg/L, BH was completely inhibited. The major new insights and benefits afforded by this model are 1) lipolysis and BH are described by nonlinear kinetics; 2) high concentrations of VA appear to inhibit its own BH; and 3) BH of RA appears to proceed at a much greater rate when triglyceride is present in the incubation medium. This model provides a conceptual framework for researching ruminal lipolysis and BH.