Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae

Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition.     

Fatty acid composition and bacterial community changes in the rumen fluid of lactating sheep fed sunflower oil plus incremental levels of marine algae

Supplementation of ruminant diets with plant oils and marine lipids is an effective strategy for lowering saturated fatty acid (FA) content and increasing the concentration of cis-9,trans-11 conjugated linoleic acid and long-chain n-3 FA in ruminant milk. However, changes in populations of ruminal microorganisms associated with altered biohydrogenation of dietary unsaturated FA are not well characterized. Twenty-five lactating Assaf ewes were allocated at random to 1 of 5 treatments composed of dehydrated alfalfa hay and concentrates containing no additional lipid (control), or supplemented with 25 g of sunflower oil and 0 (SO), 8 (SOMA(1)), 16 (SOMA(2)), or 24 (SOMA(3)) g of marine algae/kg of diet dry matter. On d 28 on diet, samples of rumen fluid were collected for lipid analysis and microbial DNA extraction. Appearance and identification of biohydrogenation intermediates was determined based on complementary gas chromatography and Ag+-HPLC analysis of FA methyl esters. Total bacteria and the Butyrivibrio group were studied in microbial DNA by terminal RFLP analysis, and real-time PCR was used to quantify the known Butyrivibrio bacteria that produce trans-11 18:1 or 18:0. Dietary supplements of sunflower oil alone or in combination with marine algae altered the FA profile of rumen fluid, which was associated with changes in populations of specific bacteria. Inclusion of marine algae in diets containing sunflower oil resulted in the accumulation of trans 18:1 and 10-O-18:0 and a marked decrease in 18:0 concentrations in rumen fluid. At the highest levels of supplementation (SOMA(2) and SOMA(3)), marine algae also promoted a shift in ruminal biohydrogenation pathways toward the formation of trans-10 18:1 at the expense of trans-11 18:1. Changes in the concentration of biohydrogenation intermediates were not accompanied by significant variations in the abundance of known cultivated ruminal bacteria capable of hydrogenating unsaturated FA. However, certain bacterial groups detected by terminal RFLP (such as potentially uncultured Lachnospiraceae strains or Quinella-related bacteria) exhibited variations in their relative frequency consistent with a potential role in one or more metabolic pathways of biohydrogenation in the rumen.     

Effect of fish oil and sunflower oil on rumen fermentation characteristics and fatty acid composition of digesta in ewes fed a high concentrate diet

Studies in ruminants have shown that supplementing the diet with a mixture of fish oil (FO) and sunflower oil (SO) enhances the concentration of cis-9, trans-11 conjugated linoleic acid (CLA), 20:5 n-3, and 22:6 n-3 in milk because of alterations in ruminal biohydrogenation, but the intermediates formed under these conditions are not well characterized. Five ewes fitted with rumen cannula and fed a high concentrate diet were used to examine the effect of a mixture (30 g/kg of DM) of FO and SO (1:2, wt/wt) on temporal changes in rumen fermentation characteristics and the relative abundance of biohydrogenation intermediates in ruminal digesta collected after 0, 3, and 10 d on diet. Appearance and identification of biohydrogenation intermediates was determined based on complementary gas-liquid chromatography and Ag+-HPLC analysis of fatty acid methyl esters and gas chromatography-mass spectrometry analysis of corresponding 4,4-dimethyloxazoline derivatives. Inclusion of FO and SO in the diet had no effect on rumen pH, volatile fatty acid concentrations, or nutrient digestion, but altered the fatty acid composition of ruminal digesta, changes that were characterized by time-dependent decreases in 18:0 and 18:2 n-6 and the accumulation of trans 16:1, trans 18:1, 10-O-18:0, and trans 18:2. Lipid supplements enhanced the proportion of 20:5 n-3 and 22:6 n-3 in digesta and resulted in numerical increases in cis-9, trans-11 conjugated linoleic acid concentrations, but decreased the relative abundance of trans-10, cis-12 conjugated linoleic acid. Furthermore, detailed analysis revealed the appearance of several unique 20:1, 20:2, 22:1, 22:3, and 22:4 products in ruminal digesta that accumulated over time, providing the first indications of 20 and 22 carbon fatty acid intermediates formed during the biohydrogenation of long-chain unsaturated fatty acids in sheep. In conclusion, FO and SO in a high concentrate diet caused a time-dependent inhibition of the complete biohydrogenation of 16 and 18 carbon unsaturated fatty acids, and resulted in the accumulation of trans 16:1, trans 18:1, and trans 18:2, 20, and 22 carbon metabolites in ruminal digesta of sheep, with no evidence of a shift in ruminal biohydrogenation pathways toward trans-10 18:1 formation.     

Changes in milk fatty acid profile and animal performance in response to fish oil supplementation, alone or in combination with sunflower oil, in dairy ewes

Ruminant diet supplementation with sunflower oil (SO) and fish oil (FO) has been reported as a good strategy for enhancing some milk fat compounds such as conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids in dairy cows, but no information is available regarding dairy sheep. In this work, ewe diet was supplemented with FO, alone or in combination with SO, with the aim of improving milk nutritional value and evaluating its effect on animal performance. Sixty-four Assaf ewes in mid lactation, fed a high-concentrate diet, were distributed in 8 lots of 8 animals each and assigned to 4 treatments (2 lots/treatment): no lipid supplementation (control) or supplementation with 20 g of SO/kg (SO), 10 g of FO/kg (FO), or 20 g of SO plus 10 g of FO/kg (SOFO). Milk production and composition, including a complete fatty acid profile, were analyzed on d 0, 3, 7, 14, 21, and 28 of treatments. Supplementation with FO tended to reduce dry matter intake compared with the control treatment (−15%), and its use in combination with SO (SOFO) resulted in a significant decrease in milk yield as well (−13%). All lipid supplements reduced milk protein content, and FO also reduced milk fat content by up to 21% alone (FO) and 27% in combination with SO (SOFO). Although the mechanisms involved in FO-induced milk fat depression are not yet well established, the observed increase in some milk trans-FA that are putative inhibitors of milk fat synthesis, such as trans-9,cis-11 CLA, and the 63% decrease in C18:0 (consistent with the theory of reduced milk fat fluidity) may be involved. When compared with the control, lipid supplementation remarkably improved the milk content of rumenic acid (cis-9,trans-11 CLA; up to 4-fold increases with SO and SOFO diets), whereas FO-containing diets also increased milk n-3 polyunsaturated fatty acids, mainly docosahexaenoic acid (with mean contents of 0.29 and 0.38% of total fatty acids for SOFO and FO, respectively), and reduced the n-6:n-3 FA ratio to approximately half the control value. All lipid supplements resulted in high levels of some trans-FA, mainly trans-11 C18:1 (vaccenic acid) but also trans-10 C18:1.     

Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation

Small ruminants are susceptible to milk fat depression (MFD) induced by marine lipid supplementation. However, as observed in dairy cows, there is wide individual variation in the response to MFD-inducing diets, which may be due to individual differences in ruminal processes. Therefore, we compared the ruminal responses of goats and sheep with varying degrees of MFD extent to improve our understanding of this complex syndrome. Our specific aims were to attempt to elucidate whether pre-existing variations in ruminal fermentation and biohydrogenation determine a higher tolerance or susceptibility to MFD, and whether the severity of MFD depends exclusively on the response to the diet. The trial was conducted with 25 does and 23 ewes fed a basal diet without lipid supplementation for 3 wk (control period). Then, 2% fish oil (FO) was added to the same diet for 5 additional weeks (MFD period). Based on the extent of the elicited MFD (i.e., the percentage variation between milk fat concentrations recorded at the end of the control and MFD periods), the 5 most responsive (RESPON+) and the 5 least responsive (RESPON?) animals were selected within each species. On the last day of each period, ruminal fluid samples were collected to examine fermentation parameters and fatty acid profiles. In general, the individual degree of MFD in sheep and goats did not seem to be predetermined by traits related to ruminal fermentation and biohydrogenation, including fatty acids that may serve as biomarkers of microorganisms. Regarding differences in the response to FO, the results suggest no link between MFD susceptibility and concentration of biohydrogenation intermediates such as trans-10-containing C18, C20, and C22 metabolites. The explanation for individual responses based on a shortage of ruminal acetate and 18:0 for mammary uptake also seems to be dismissed, based on the lack of variation in these compounds between RESPON+ and RESPON?. However, the concentration of unsaturated fatty acids provided by FO (e.g., cis-9 16:1, cis-11 18:1, and 20:5n-3) was higher in the rumen of RESPON+ than RESPON? ewes and does. Thus, although further research is needed, the extent of biohydrogenation of these fatty acids might be associated with tolerance or susceptibility to MFD.     

Effect of inoculated corn silage enriched with sunflower oil on rumen fermentation and lipid metabolism in an artificial rumen (RUSITEC)

BACKGROUND: Some rumen isolates are able to produce conjugated linoleic acid (CLA) from linoleic acid (LA) in vitro. Effects of providing diets containing corn silage (CS) and lucerne hay to an artificial rumen (RUSITEC) in which the corn was not inoculated (CS), or inoculated with Lactobacillus plantarum CCM 4000 (CS + LP), Lactobacillus fermentum LF2 (CS + LF) or Enterococcus faecium CCM 4231 (CS + EF) and supplied with sunflower oil (SO; 30 g kg^?1; w/w) on rumen metabolism were examined. RESULTS: The SO affected the outputs of all fatty acids. TVA output of uninoculated CS with SO was lower as compared to inoculated CS. The interaction of the CS × SO in the daily output of TVA was detected (P < 0.001). The biohydrogenation of oleic, linoleic, α‐linolenic and total fatty acids was influenced by SO (P < 0.05 and P < 0.001). CONCLUSION: Inoculated silage induces changes in the rumen metabolism which might be related to differences observed in the extent of rumen BH of PUFA in RUSITEC. SO supplementation might positively enhances the production of some rumen intermediates; however, relationships between inoculated silages and oil supplementation can be presumed in the daily production of trans‐vaccenic acid. Copyright © 2009 Society of Chemical Industry     

Short communication: The effect of substituting fish oil in dairy cow diets with docosahexaenoic acid-micro algae on milk composition and fatty acids profile

The effects of substituting fish oil (FO) with docosahexaenoic acid (DHA)-micro algae on milk chemical and fatty acid composition were examined in this study. Twenty-four Holstein cows in mid lactation grazing on an alfalfa-grass based pasture were divided into 4 treatment groups (6 cows/treatment) and supplemented with 7 kg/d grain mix plus 350 g of soybean oil and one of the following: 1) 150 g of FO, 2) 100 g of FO plus 50 g of algae, 3) 50 g of FO plus 100 g of algae, or 4) 150 g of algae. Cows were fed treatment diets for 3 wk, and milk samples were collected from each cow during the last 3 d of the study. Milk production (17.96, 17.56, 17.55, and 19.26 kg/d for treatment diets 1 to 4, respectively), milk fat percentages (3.17, 3.49, 3.74, and 3.43%), and milk protein percentages (3.35, 3.50, 3.71, and 3.42%) were similar between treatment diets. Concentrations (g/100 g of fatty acids) of milk cis-9 trans-11 (c9t11) conjugated linoleic acid (CLA; 3.41, 3.69, 4.47, and 4.21 for treatment diets 1 to 4, respectively) and vaccenic acid (11.80, 12.83, 13.87, and 13.53) were similar between treatment diets. Results of this study suggest that DHA-micro algae can partially or fully substitute FO in a cow's diet without any adverse effects on milk production, milk composition, or milk c9t11 CLA content. The DHA-micro algae may be used as a viable alternative for FO in cow's diet to modify rumen biohydrogenation to increase milk c9t11 CLA content.     

High-concentrate diets and polyunsaturated oils alter trans and conjugated isomers in bovine rumen, blood, and milk

Three Holstein cows were fed a high-concentrate diet (65:35 concentrate to forage) supplemented with either 5% sunflower oil (SO), 5% linseed oil (LO), or 2.5% fish oil (FO) to examine effects on biohydrogenation and fatty acid profiles in rumen, blood plasma, and milk. Diets were fed in a 3 × 3 Latin square with 4-wk periods with grass hay as the forage. Milk yield, dry matter intake, and percentages of milk fat (2.64) and protein (3.22) did not differ. All diets resulted in incomplete hydrogenation of unsaturated fatty acids as indicated by the profiles of 18:1 isomers, conjugated 18:2 isomers, nonconjugated 18:2 isomers, and 18:0 in ruminal fluid. Percentages of 8:0-14:0 and 16:0 in milk fat were greater with FO. Percentage and yield of trans10,cis12-18:2 were small and greater in cows fed SO (0.14%, 0.57 g/d) than FO (0.03%, 0.15 g/d) or LO (0.04%, 0.12 g/d). Percentage and yield of trans10-18:1, however, increased with FO (6.16%) and SO (6.47%) compared with LO (1.65%). Dietary FO doubled percentage of cis11-18:1 in rumen, plasma, and milk fat. Despite a lack of difference in ruminal percentage of trans11-18:1 (10.5%), cows fed FO had greater plasma trans11-18:1 (116 vs. 61.5 μg/mL) but this response did not result in greater trans11-18:1 percentage in milk fat, which averaged 5.41% across diets. Percentage (2.2%) and yield (14.3 g/d) of cis9,trans11-18:2 in milk fat did not differ due to oils. Unique responses to feeding LO included greater than 2-fold increases in percentages of trans13+14-18:1, trans15-18:1, trans16-18:1, cis15-18:1, cis9,trans12-18:2 and trans11,cis15 -18:2 in umen, plasma, and milk, and cis9,trans13-18:2 in plasma and milk. Ruminal 18:0 percentage had the highest positive correlation with milk fat content (r = 0.82) across all diets. When compared with previous data with cows fed high-concentrate diets without oil supplementation, results suggest that greater production of trans10-18:1, cis11-18:1, and trans11,cis15-18:2 coupled with low production of 18:0 in the rumen may be associated with low milk fat content when feeding high-concentrate diets and fish oil. In contrast, SO or LO could lead to low milk fat content by increasing ruminal trans10-18:1 (SO) or trans11,cis15-18:2 and trans9,trans12-18:2 (LO) along with a reduction in mammary synthesis of 8:0-16:0. Simultaneous increases in ruminal trans11-18:1 with fish oil, at a fraction of sunflower oil supplementation, may represent an effective strategy to maintain cis9,trans11-18:2 synthesis in mammary while reducing milk fat output and sparing energy.     

Conjugated linoleic acid increases in milk from cows fed condensed corn distillers solubles and fish oil

Twelve lactating Holstein cows were randomly assigned to 1 of 4 experimental diets in a replicated 4 × 4 Latin square design with 4-wk periods to ascertain the lactational response to feeding fish oil (FO), condensed corn distillers solubles (CDS) as a source of extra linoleic acid, or both. Diets contained either no FO or 0.5% FO and either no CDS or 10% CDS in a 2 × 2 factorial arrangement of treatments. Diets were fed as total mixed rations for ad libitum consumption. The forage to concentrate ratio was 55:45 on a dry matter basis for all diets and the diets contained 16.2% crude protein. The ether extract concentrations were 2.86, 3.22, 4.77, and 5.02% for control, FO, CDS, and FOCDS diets, respectively. Inclusion of FO or CDS or both had no effect on dry matter intake, feed efficiency, body weight, and body condition scores compared with diets without FO and CDS, respectively. Yields of milk (33.3 kg/d), energy-corrected milk, protein, lactose, and milk urea N were similar for all diets. Feeding FO and CDS decreased milk fat percentages (3.85, 3.39, 3.33, and 3.12%) and yields compared with diets without FO and CDS. Proportions of trans-11 C18:1 (vaccenic acid), cis-9 trans-11 conjugated linoleic acid (CLA; 0.52, 0.90, 1.11, and 1.52 g/100 g of fatty acids), and trans-10 cis-12 CLA (0.07, 0.14, 0.13, and 0.16 g/100 g of fatty acids) in milk fat were increased by FO and CDS. No interactions were observed between FO and CDS on cis-9 trans-11 CLA although vaccenic acid tended to be higher with the interaction. The addition of CDS to diets increased trans-10 C18:1. Greater ratios of vaccenic acid to cis-9 trans-11 CLA in plasma than in milk fat indicate tissue synthesis of cis-9 trans-11 CLA in the mammary gland from vaccenic acid in cows fed FO or CDS. Feeding fish oil at 0.5% of diet dry matter with a C18:2 n-6 rich source such as CDS increased the milk CLA content but decreased milk fat percentages.     

High-grain diets supplemented with phytogenic compounds or autolyzed yeast modulate ruminal bacterial community and fermentation in dry cows

The feeding of concentrate-rich diets may lead to microbial imbalances and dysfermentation in the rumen. The main objective of this study was to determine the effects of supplementing phytogenic compounds (PHY) or autolyzed yeast (AY) on rumen fermentation and microbial abundance in cows intermittently fed concentrate-rich diets. The experiment was carried out as an incomplete 3 × 4 Latin square design, with 8 nonlactating rumen-fistulated Holstein-Friesian cows. The cows were randomly assigned to a concentrate diet that was either not supplemented (CON), or supplemented with PHY or AY. Each of the 4 consecutive experimental periods was composed of a 1-wk roughage-only diet (RD), 6-d gradual concentrate increase, followed by 1 wk of 65% concentrate (dry matter basis; Conc I), and 1 wk of RD and a final 2-wk 65% concentrate (dry matter basis; Conc II) phase. Digesta samples were collected from the rumen mat for bacterial 16S rRNA gene Illumina MiSeq (Illumina, Balgach, Switzerland) sequencing, and samples of particle-associated rumen liquid were obtained for measuring short-chain fatty acids, lactate, ammonia, and pH during RD (d 6), Conc I (d 19), and Conc II (d 39). The concentrate feeding caused a decrease of overall bacterial diversity indices, especially during Conc I. The genera Ruminococcus, Butyrivibrio, and Coprococcus were decreased, whereas Prevotella, Megasphaera, Lachnospira, and Bacteroides were increased in abundance. Supplementation of both feed additives increased the abundance of gram-positive and decreased that of gram-negative bacteria. Supplementation of AY enhanced cellulolytic bacteria such as Ruminococcus spp., whereas PHY decreased starch and sugar fermenters including Bacteroides spp., Shuttleworthia spp., and Syntrophococcus spp. Moreover, PHY supplementation increased butyrate percentage in the rumen in both concentrate phases. In conclusion, intermittent high-concentrate feeding altered the digesta-associated rumen bacterial community and rumen fermentation with more significant alterations found in Conc I than in Conc II. The data also showed that both feed additives had the most significant modulatory effects on the bacterial community, and their subsequent fermentation, during periods of low pH.     

Examination of the persistency of milk fatty acid composition responses to fish oil and sunflower oil in the diet of dairy cows

Based on the potential benefits of cis-9, trans-11 conjugated linoleic acid (CLA) for human health, there is a need to develop effective strategies for enhancing milk fat CLA concentrations. Levels of cis-9, trans-11 CLA in milk can be increased by supplements of fish oil (FO) and sunflower oil (SO), but there is considerable variation in the response. Part of this variance may reflect time-dependent ruminal adaptations to high levels of lipid in the diet, which lead to alterations in the formation of specific biohydrogenation intermediates. To test this hypothesis, 16 late lactation Holstein-British Friesian cows were used in a repeated measures randomized block design to examine milk fatty acid composition responses to FO and SO in the diet over a 28-d period. Cows were allocated at random to corn silage-based rations (8 per treatment) containing 0 (control) or 45 g of oil supplement/kg of dry matter consisting (1:2; wt/wt) of FO and SO (FSO), and milk composition was determined on alternate days from d 1. Compared with the control, the FSO diet decreased mean dry matter intake (21.1 vs. 17.9 kg/d), milk fat (47.7 vs. 32.6 g/kg), and protein content (36.1 vs. 33.3 g/kg), but had no effect on milk yield (27.1 vs. 26.4 kg/d). Reductions in milk fat content relative to the FSO diet were associated with increases in milk trans-10 18:1, trans-10, cis-12 CLA, and trans-9, cis-11 CLA concentrations (r² = 0.74, 0.57, and 0.80, respectively). Compared with the control, the FSO diet reduced milk 4:0 to 18:0 and cis 18:1 content and increased trans 18:1, trans 18:2, cis-9, trans-11 CLA, 20:5 n-3, and 22:6 n-3 concentrations. The FSO diet caused a rapid elevation in milk cis-9, trans-11 CLA content, reaching a maximum of 5.37 g/100 g of fatty acids on d 5, but these increases were transient, declining to 2.35 g/100 g of fatty acids by d 15. They remained relatively constant thereafter. Even though concentrations of trans-11 18:1 followed the same pattern of temporal changes as cis-9, trans-11 CLA, the total trans 18:1 content of FSO milk was unchanged because of the concomitant increases in the concentration of other isomers (Δ^4-10 and Δ^12-15), predominantely trans-10 18:1. In conclusion, supplementing diets with FSO enhances milk fat cis-9, trans-11 CLA content, but the high level of enrichment declines because of changes in ruminal biohydrogenation that result in trans-10 replacing trans-11 as the major 18:1 biohydrogenation intermediate formed in the rumen.     

Effects of different forage:concentrate ratios in dairy ewe diets supplemented with sunflower oil on animal performance and milk fatty acid profile

The aim of this study was to evaluate the effect of different forage:concentrate (FC) ratios in dairy ewe diets supplemented with sunflower oil (SO) on animal performance and milk fatty acid (FA) profile, particularly focusing on trans C18:1 FA and conjugated linoleic acid (CLA). Sixty lactating Assaf ewes were randomly assigned to 6 treatments in a 3 × 2 factorial arrangement: 3 FC ratios (30:70, 50:50, and 70:30) and 2 levels of SO addition (0 and 20 g/kg of dry matter). Both the diet FC ratio and SO supplementation affected milk yield, but differences between treatments were small. Although the proportion of concentrate induced limited changes in milk FA profile, dietary SO significantly decreased saturated FA and enhanced total CLA. Furthermore, the incorporation of SO in ewe diets decreased the atherogenicity index value by about 25% and doubled the contents of potentially healthy FA such as trans-11 C18:1 and cis-9,trans-11 CLA. However, the inclusion of SO in a high-concentrate diet (30:70) could switch linoleic acid biohydrogenation pathways, resulting in a significant increase in trans-10 C18:1, trans-9,cis-11 C18:2, and trans-10,cis-12 C18:2 milk fat percentages.     

Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.     

Changes in rumen bacterial and archaeal communities over the transition period in primiparous Holstein dairy cows

In the present study, we hypothesized that the rumen bacterial and archaeal communities would change significantly over the transition period of dairy cows, mainly as an adaptation to the classical use of low-grain prepartum and high-grain postpartum diets. Bacterial 16S rRNA gene amplicon sequencing of rumen samples from 10 primiparous Holstein dairy cows revealed no changes over the transition period in relative abundance of genera such as Ruminococcus, Butyrivibrio, Clostridium, Coprococcus, and Pseudobutyrivibrio. However, other dominant genus-level taxa, such as Prevotella, unclassified Ruminococcaceae, and unclassified Succinivibrionaceae, showed distinct changes in relative abundance from the prepartum to the postpartum period. Overall, we observed individual fluctuation patterns over the transition period for a range of bacterial taxa that, in some cases, were correlated with observed changes in the rumen short-chain fatty acids profile. Combined results from clone library and terminal-restriction fragment length polymorphism (T-RFLP) analyses, targeting the methyl-coenzyme M reductase α-subunit (mcrA) gene, revealed a methanogenic archaeal community dominated by the Methanobacteriales and Methanomassiliicoccales orders, particularly the genera Methanobrevibacter, Methanosphaera, and Methanomassiliicoccus. As observed for the bacterial community, the T-RFLP patterns showed significant shifts in methanogenic community composition over the transition period. Together, the composition of the rumen bacterial and archaeal communities exhibited changes in response to particularly the dietary changes of dairy cows over the transition period.     

Evidence for the inhibition of the terminal step of ruminal α-linolenic acid biohydrogenation by condensed tannins

Effects of condensed tannins (CT), either via extract or plant-bound, and saponin extract on ruminal biohydrogenation of α-linolenic acid (ALA) were investigated in vitro. Grass-clover hay served as basal diet (control). The control hay was supplemented with extracts contributing either CT from Acacia mearnsii [7.9% of dietary dry matter (DM)] or saponins from Yucca schidigera (1.1% of DM). The fourth treatment consisted of dried sainfoin (Onobrychis viciifolia), a CT-containing forage legume, in an amount also providing 7.9% CT in dietary DM. All diets were supplemented with linseed oil at a level contributing 60% of total dietary ALA in all treatments. Diets were incubated for 10 d (n = 4) in the rumen simulation technique system, using the last 5 d for statistical evaluation. Fatty acids were analyzed in feed, feed residues, incubation fluid, and its effluent. Data were subjected to ANOVA considering diet and experimental run as main effects. Both CT treatments reduced ruminal fiber and crude protein degradation, and lowered incubation fluid ammonia concentration. Only the CT extract suppressed methane formation and shifted microbial populations toward bacteria at cost of protozoa. The saponin extract remained without clear effects on fermentation characteristics except for increased protozoal counts. The extent of ALA biohydrogenation was 20% less with the CT plant, but this probably resulted from reduced organic matter degradability rather than from an inhibition of biohydrogenation. After incubation analysis of incubation fluid effluent and feed residues showed a considerable proportion of the 3 biohydrogenation intermediates, cis-9, trans-11, cis-15 C18:3, trans-11, cis-15 C18:2, and trans-11 C18:1, which did not occur in the initial feeds. Only the CT-extract diet led to a different profile in the effluent compared with the control diet with trans-11 C18:1 being considerably increased at cost of C18:0. This could have been achieved by suppressing protozoa and enhancing the bacterial population, thus removing potential microbes involved in biohydrogenation and increasing competition between bacteria involved in biohydrogenation and others. The elevation of trans-11 C18:1 as the precursor of cis-9, trans-11 conjugated linoleic acid formed in body tissue and mammary gland is probably favorable from a human health point of view.     

Evaluating the conjugated linoleic acid and trans 18:1 isomers in milk fat of dairy cows fed increasing amounts of sunflower oil and a constant level of fish oil

The objective was to evaluate different levels of sun-flower oil (SFO) in dairy rations to increase vaccenic (trans-11-18:1) and rumenic acids (cis-9,trans-11-18:2) in milk fat, and assess the content and composition of other trans-octadecenoic (trans-18:1) and conjugated linoleic acids (CLA) isomers. Eighty lactating Holstein cows were fed control diets for 4 wk and then placed on 4 diets for 38 d; milk fat was analyzed after 10 and 38 d. The treatments were: control, 1.5% SFO plus 0.5% fish oil (FO), 3% SFO plus 0.5% FO, and 4.5% SFO plus 0.5% FO. The forage-to-concentrate ratio was 50:50 and consisted of barley/alfalfa/hay silage and corn/barley grain concentrate. There were no differences in milk production. Supplementation of SFO/FO reduced milk fat compared with respective pretreatment periods, but milk protein and lactose levels were not affected. There was a linear decrease in all short- and medium-chain saturated fatty acids (SFA) in milk fat after 10 d (25.5, 24.1, 20.2, and 16.7%) and a corresponding linear increase in total trans-18:1 (5.2, 9.1, 14.1, and 21.3%) and total CLA (0.7, 1.9, 2.4, and 3.9%). The other FA in milk fat were not affected. Separation of trans-18:1 isomers was achieved by combination of gas chromatography (GC; 100-m highly polar capillary column) and prior separation of trans FA by silver ion-thin layer chromatography followed by GC. The CLA isomers were resolved by a combination of GC and silver ion-HPLC. The trans-11- and trans-10-18:1 isomers accounted for ∼50% of the total trans-18:1 increase when SFO/FO diets were fed. On continued feeding to 38 d, trans-11-18:1 increased with 1.5% SFO/FO, stayed the same with 3%, and declined with 4.5% SFO/FO. Rumenic acid showed a similar pattern on continued feeding as trans-11-18:2; levels increased to 0.43, 1.5, 1.9, and 3.4% at 10 d and to 0.42, 2.15, 2.09, and 2.78% at 38 d. Rumenic acid was the major CLA isomer in all 4 diets: 66, 77, 78 and 85%. The CLA isomers trans-7,cis-9-, trans-9,cis-11-, trans-10,cis-12-, trans-11,trans-13-, and trans-9,trans-11-/trans-10,trans-12-18:2 also increased from 0.18 (control) to 0.52% (4.5% SFO/FO). Milk fat produced from 3% SFO/FO appeared most promising: trans-11-18:1 and cis-9,trans-11-18:2 increased 4.5-fold, total SFA reduced 18%, and moderate levels of trans-10-18:1 (3.2%), other trans-18:1 (6.6%) and CLA isomers (0.5%) were observed, and that composition remained unchanged to 38 d. The 4.5% SFO/FO diet produced higher levels of trans-11-18:1 and cis-9,trans-11-18:2, a 28% reduction in SFA, and similar levels of other trans-18:1 (9.2%) and CLA isomers (0.52%), but the higher levels of trans-11-18:1 and cis-9,trans-11-18:2 were not sustained. A stable milk fat quality was achieved by feeding moderate amounts of SFO (3% of DM) in the presence of 0.5% FO that had 4% vaccenic and 2% rumenic acids.     

Effect of increasing amounts of olive crude phenolic concentrate in the diet of dairy ewes on rumen liquor and milk fatty acid composition

Agro-industrial by-products contain several secondary plant metabolites, such as polyphenols, tannins, saponins, and essential oils. The effects of these compounds on animal metabolism may vary significantly according to the dose, the chemical nature of the molecules, and the overall composition of the diet. In the Mediterranean area, the olive oil extraction is associated with 2 by-products: olive pomace and wastewater, both rich in polyphenols. In particular, wastewater may be further processed to obtain olive crude phenolic concentrate (OCPC). An experiment was carried out aiming to evaluate animal performance, milk fatty acid (FA) profile, diversity of rumen microbial population, and rumen liquor FA profile in dairy ewes fed diets containing extruded linseed (EL) and increasing doses of OCPC. Twenty-eight Comisana ewes in mid lactation were allotted to 4 experimental groups. The experiment lasted 5 wk after 3 wk of adaptation. Diets were characterized by lucerne hay administrated ad libitum and by 800 g/ewe and day of 4 experimental concentrates containing 22% of EL on dry matter and increasing dose of OCPC: 0 (L0), 0.6 (L0.6), 0.8 (L0.8), and 1.2 (L1.2) g of OCPC/kg of dry matter. Milk yield was daily recorded and milk composition was analyzed weekly. At the beginning and at the end of the experiment, samples of rumen liquor were collected to analyze FA profile, changes in rumen microbial population, and dimethylacetal (DMA) composition. The inclusion of OCPC did not affect milk yield and gross composition, whereas milk from L0.8 and L1.2 sheep contained higher concentrations of linoleic (+18%) and α-linolenic acid (+24%) and lower concentration of the rumen biohydrogenation intermediates. A similar pattern was observed for rumen liquor FA composition. No differences were found in the diversity of the rumen microbial population. Total amount of DMA did not differ among treatments, whereas significant differences were found in the concentration of individual DMA; in the diet with a higher amount of OCPC, DMA 13:0, 14:0, 15:0, and 18:0 increased, whereas DMA 16:0 decreased. Probably the presence of polyphenols in the diet induced a rearrangement of bacteria membrane phospholipids as a response to the rumen environment stimulus. Overall, the use of OCPC allowed a significant increase in the polyunsaturated FA content of milk, probably due to a perturbation of the rumen biohydrogenation process. Further studies are needed to understand the correlation between diet composition and the pattern of DMA in rumen liquor.     

Effectiveness of oils rich in linoleic and linolenic acids to enhance conjugated linoleic acid in milk from dairy cows

Forty Holstein dairy cows were used to determine the effectiveness of linoleic or linolenic-rich oils to enhance C_18:2cis-9, trans-11 conjugated linoleic acid (CLA) and C_18:1trans-11 (vaccenic acid; VA) in milk. The experimental design was a complete randomized design for 9 wk with measurements made during the last 6 wk. Cows were fed a basal diet containing 59% forage (control) or a basal diet supplemented with either 4% soybean oil (SO), 4% flaxseed oil (FO), or 2% soybean oil plus 2% flaxseed oil (SFO) on a dry matter basis. Total fatty acids in the diet were 3.27, 7.47, 7.61, and 7.50 g/100 g in control, SO, FO, and SFO diets, respectively. Feed intake, energy-corrected milk (ECM) yield, and ECM produced/kg of feed intake were similar among treatments. The proportions of VA were increased by 318, 105, and 206% in milk fat from cows in the SO, FO, and SFO groups compared with cows in the control group. Similar increases in C_18:2cis-9, trans-11 CLA were 273, 150, and 183% in SO, FO, and SFO treatments, respectively. Under similar feeding conditions, oils rich in linoleic acid (soybean oil) were more effective in enhancing VA and C_18:2cis-9, trans-11 CLA in milk fat than oils containing linolenic acid (flaxseed oil) in dairy cows fed high-forage diets (59% forage). The effects of mixing linoleic and linolenic acids (50:50) on enhancing VA and C_18:2cis-9, trans-11 CLA were additive, but not greater than when fed separately. Increasing the proportion of healthy fatty acids (VA and CLA) by feeding soybean or flaxseed oil would result in milk with higher nutritive and therapeutic value.     

Biohydrogenation of fatty acids and digestibility of fresh alfalfa or alfalfa hay plus sucrose in continuous culture

The pattern of biohydrogenation of fatty acids from fresh alfalfa or alfalfa hay supplemented with 3 concentrations (0, 4, and 8%) of sucrose was studied at a constant pH of 6.2. Four continuous culture fermenters were used in a 4 × 4 Latin square design to test the hypothesis that fresh forage would increase flow of vaccenic acid (VA) from the fermenters compared with the same forage in hay form and that this difference would be diminished by adding sucrose to the hay diet by changing the bacterial community profile. Effluent was collected from each of the 4 fermenters during the last 3 d of each 10-d period. Nutrient digestibility, volatile fatty acids (VFA), and fatty acids in the effluent were measured. Flow of bacterial organic matter (OM) and neutral and acid detergent fiber and acid detergent fiber digestibilities were higher for fresh alfalfa than alfalfa hay. True OM digestibility of alfalfa hay tended to linearly decrease with sucrose supplementation. However, microbial efficiency and flow of bacterial OM (g/d) linearly increased with sucrose addition. There was no change in total VFA concentration; however, proportion of acetate linearly decreased and proportion of butyrate linearly increased with sucrose addition. Fresh alfalfa increased total biohydrogenation of fatty acids compared with than hay. Vaccenic acid flow (mg/d) was much higher for fresh alfalfa compared with alfalfa hay (216 vs. 41) and VA was the predominant 18:1 isomer, followed by trans-13 18:1; however, sucrose had no effect on VA flow. The percentage of VA (of total trans-18:1) was not different between fresh alfalfa and hay, whereas percentage of trans-10 18:1 was much lower for fresh alfalfa. Therefore, the ratio of VA to trans-10 18:1 was higher for fresh alfalfa. Flow of trans-12 18:1 linearly increased, whereas flows of cis-12 and total cis-18:1 had quadratic responses to sucrose supplementation. Total biohydrogenation and biohydrogenation of linoleic and linolenic acids linearly decreased with sucrose; however, there was no effect of sucrose on total trans fatty acid flow. Sucrose may be more detrimental to the last step of biohydrogenation of VA. The effects of sucrose on biohydrogenation and concentration of VFA may have been caused by a shift in microbial population by mechanisms that are independent of pH.