Fecal microbiota colonization dynamics in dairy heifers associated with early-life rumen microbiota modulation and gut health.

Early-life modulation of rumen microbiota holds promise for enhancing calf growth, health, and long-term production in ruminants. However, limited attention has been given to the impact of rumen microbiota modulation on the establishment of hindgut microbiota. In this study, fecal microbiota development was examined in identical twin calves for 12 months. The treatment group (T-group) received adult cow fresh rumen liquid inoculum during the pre-weaning period, while the control group did not (C-group). The effects of inoculum were assessed on calf gut health and as microbial seeding route into the hindgut. The early rumen modulation had no effect on age-related fecal microbiota development. The fecal bacterial community evolved gradually following dietary changes and categorized into pre-weaning and post-weaning communities. Bacterial richness increased with age and stabilized at month 9, while between-sample variation reduced in post-weaning samples. Archaeal load in fecal samples increased after month 4, while archaeal richness increased and stabilized in both groups by month 9. Between-sample similarity was higher during the pre-weaning period, with increased dissimilarity from month 4 onward. Anaerobic fungi were detected in feces at month 4, with richness peaking at month 7. Before month 6, fungal community composition distinctly differed from mature communities. When colostrum, calf rumen, and donor inoculum were evaluated as seeding sources for hindgut colonization, the calf's own rumen was identified as the primary seeding source for fecal bacteria and fungi. Colostrum was a source for several bacteria detected in feces, but these were of temporary importance until weaning. The donor inoculum had limited impact on gut health as diarrhea rates were similar between the T-group and C-group. In conclusion, early-life microbiota modulation shows potential in ruminant development. However, a more targeted approach with bacteria adapted to the hindgut environment may be necessary to modulate hindgut effectively. This research contributes to our understanding of the complex relationship between gut microbiota and calf health and growth.

Mineral concentrations in milk from cows fed seaweed (Saccharina latissima) under different basal protein supplementation.

Sixteen multiparous Holstein cows in four blocks of 4 × 4 Latin square over 4-week experimental periods were used to study the effects of seaweed (Saccharina latissima) supplement (with/without) and protein source (rapeseed meal (RSM)/wheat distiller's grain (WDG)) on milk mineral concentrations. Dietary treatments did not affect milk production and basic composition. Feeding seaweed slightly decreased milk Ca and Cu concentrations; whilst increased (by 3.3-fold) milk iodine (I) concentration, due to a higher dietary I supply. Substitution of WDG with RSM increased feed-to-milk transfer of Ca, Na, and Se and decreased that of Mg, P, Fe, and Mn; but only reduced milk Mn and I concentrations (the latter by 27 % as a potential result of increased glucosinolate intake). Seaweed supplement can improve milk I content when cows' I supply/availability is limited, but care should be taken to avoid excess milk I contents that may pose nutritional risks for young children.

Long-term effects of early-life rumen microbiota modulation on dairy cow production performance and methane emissions.

Rumen microbiota modulation during the pre-weaning period has been suggested as means to affect animal performance later in life. In this follow-up study, we examined the post-weaning rumen microbiota development differences in monozygotic twin-heifers that were inoculated (T-group) or not inoculated (C-group) (n = 4 each) with fresh adult rumen liquid during their pre-weaning period. We also assessed the treatment effect on production parameters and methane emissions of cows during their 1st lactation period. The rumen microbiota was determined by the 16S rRNA gene, 18S rRNA gene, and ITS1 amplicon sequencing. Animal weight gain and rumen fermentation parameters were monitored from 2 to 12 months of age. The weight gain was not affected by treatment, but butyrate proportion was higher in T-group in month 3 (p = 0.04). Apart from archaea (p = 0.084), the richness of bacteria (p < 0.0001) and ciliate protozoa increased until month 7 (p = 0.004) and anaerobic fungi until month 11 (p = 0.005). The microbiota structure, measured as Bray-Curtis distances, continued to develop until months 3, 6, 7, and 10, in archaea, ciliate protozoa, bacteria, and anaerobic fungi, respectively (for all: p = 0.001). Treatment or age × treatment interaction had a significant (p < 0.05) effect on 18 bacterial, 2 archaeal, and 6 ciliate protozoan taxonomic groups, with differences occurring mostly before month 4 in bacteria, and month 3 in archaea and ciliate protozoa. Treatment stimulated earlier maturation of prokaryote community in T-group before month 4 and earlier maturation of ciliate protozoa at month 2 (Random Forest: 0.75 month for bacteria and 1.5 month for protozoa). No treatment effect on the maturity of anaerobic fungi was observed. The milk production and quality, feed efficiency, and methane emissions were monitored during cow's 1st lactation. The T-group had lower variation in energy-corrected milk yield (p < 0.001), tended to differ in pattern of residual energy intake over time (p = 0.069), and had numerically lower somatic cell count throughout their 1st lactation period (p = 0.081), but no differences between the groups in methane emissions (g/d, g/kg DMI, or g/kg milk) were observed. Our results demonstrated that the orally administered microbial inoculant induced transient changes in early rumen microbiome maturation. In addition, the treatment may influence the later production performance, although the mechanisms that mediate these effects need to be further explored.

Macromineral and trace element concentrations in milk from Finnish Ayrshire cows fed microalgae (Spirulina platensis) and rapeseed (Brassica napus).

Given the lack of research regarding the effect of microalgal supplementation in dairy cows on milk mineral concentrations, this study investigated the effect of feeding different protein supplements in dairy cow diets on milk, feces, and blood plasma mineral concentrations, associated milk and blood plasma transfer efficiencies, and apparent digestibility. Lactating Finnish Ayrshire cows (n = 8) were allocated at the start of the trial to 4 diets used in a replicated 4 × 4 Latin square design experiment: (1) control diet (CON), (2) a pelleted rapeseed supplement (RSS; 2,550 g/d), (3) a mixture of rapeseed and Spirulina platensis (RSAL; 1,280 g of RSS + 570 g of S. platensis per day), and (4) S. platensis (ALG; 1,130 g of S. platensis per day). In each of the 4 experimental periods, a 2-wk adaptation to the experimental diets was followed by a 7-d sampling and measurement period. Feed samples were composited per measurement period, milk, and feed samples (4 consecutive days; d 17-20), and blood plasma samples (d 21) were composited for each cow period (n = 32). Data were statistically analyzed using a linear mixed effects model with diet, period within square, square and their interaction as fixed factors, and cow within square as a random factor. Cows fed ALG were not significantly different in their milk or blood plasma mineral concentrations compared with CON, although feeding ALG increased fecal concentrations of macrominerals (Ca and Mg) and trace elements (Co, Cu, Fe, I, Mn, and Zn), and reduced their apparent digestibility, compared with CON. When compared with CON and ALG, milk from cows fed RSAL and RSS had lower milk I concentrations (-69.6 and -102.7 µg/kg of milk, respectively), but total plasma I concentrations were not affected significantly. Feeding S. platensis to dairy cows did not affect mineral concentrations in cows' blood or milk, but care should be taken when rapeseed is fed to avoid reducing milk I concentrations which may in turn reduce consumers' I intake from milk and dairy products.

Fresh Rumen Liquid Inoculant Enhances the Rumen Microbial Community Establishment in Pre-weaned Dairy Calves.

The development of the functional rumen in calves involves a complex interplay between the host and host-related microbiome. Attempts to modulate rumen microbial community establishment may therefore have an impact on weaning success, calf health, and animal performance later in life. In this experiment, we aimed to elucidate how rumen liquid inoculum from an adult cow, provided to calves during the pre-weaning period, influences the establishment of rumen bacterial, archaeal, fungal, and ciliate protozoan communities in monozygotic twin calves (n = 6 pairs). The calves were divided into treatment (T-group) and control (C-group) groups, where the T-group received fresh rumen liquid as an oral inoculum during a 2-8-week period. The C-group was not inoculated. The rumen microbial community composition was determined using bacterial and archaeal 16S ribosomal RNA (rRNA) gene, protozoal 18S rRNA gene, and fungal ITS1 region amplicon sequencing. Animal weight gain and feed intake were monitored throughout the experiment. The T-group tended to have a higher concentrate intake (Treatment: p < 0.08) and had a significantly higher weekly weight gain (Treatment: p < 0.05), but no significant difference in volatile fatty acid concentrations between the groups was observed. In the T-group, the inoculum stimulated the earlier establishment of mature rumen-related bacterial taxa, affecting significant differences between the groups until 6 weeks of age. The inoculum also increased the archaeal operational taxonomic unit (OTU) diversity (Treatment: p < 0.05) but did not affect the archaeal quantity. Archaeal communities differed significantly between groups until week 4 (p = 0.02). Due to the inoculum, ciliate protozoa were detected in the T-group in week 2, while the C-group remained defaunated until 6 weeks of age. In week 8, Eremoplastron dilobum was the dominant ciliate protozoa in the C-group and Isotricha sp. in the T-group, respectively. The Shannon diversity of rumen anaerobic fungi reduced with age (Week: p < 0.01), and community establishment was influenced by a change of diet and potential interaction with other rumen microorganisms. Our results indicate that an adult cow rumen liquid inoculum enhanced the maturation of bacterial and archaeal communities in pre-weaning calves' rumen, whereas its effect on eukaryotic communities was less clear and requires further investigation.

Impact of hexamine addition to a nitrite-based additive on fermentation quality, Clostridia and Saccharomyces cerevisiae in a white lupin-wheat silage.

BACKGROUND: Nitrite and hexamine are used as silage additives because of their adverse effects on Clostridia and Clostridia spores. The effect of sodium nitrite and sodium nitrite/hexamine mixtures on silage quality was investigated. A white lupin-wheat mixture was treated with sodium nitrite (NaHe0) (900 g t-1 forage), or mixtures of sodium nitrite (900 g t-1 ) and hexamine. The application rate of hexamine was 300 g t-1 (NaHe300) or 600 g t-1 (NaHe600). Additional treatments were the untreated control (Con), and formic acid (FA) applied at a rate of 4 L t-1 (1000 g kg-1 ). RESULTS: Additives improved silage quality noticeably only by reducing silage ammonia content compared with the control. The addition of hexamine to a sodium nitrite solution did not improve silage quality compared with the solution containing sodium nitrite alone. The increasing addition of hexamine resulted in linearly rising pH values (P < 0.001) and decreasing amounts of lactic acid (P < 0.01). Sodium nitrite based additives were more effective than formic acid in preventing butyric acid formation. Additives did not restrict the growth of Saccharomyces cerevisiae compared to the control. CONCLUSION: The addition of hexamine did not improve silage quality compared with a solution of sodium nitrite. © 2018 Society of Chemical Industry.

Prepartal high-energy feeding with grass silage-based diets does not disturb the hepatic adaptation of dairy cows during the periparturient period.

The liver of dairy cow naturally undergoes metabolic adaptation during the periparturient period in response to the increasing demand for nutrients. The hepatic adaptation is affected by prepartal energy intake level and is potentially associated with inflammatory responses. To study the changes in the liver function during the periparturient period, 16 cows (body condition score = 3.7 ± 0.3, mean ± standard deviation; parity = second through fourth) were allocated to a grass silage-based controlled-energy diet (104 MJ/d) or a high-energy diet (135 MJ/d) during the last 6 wk before the predicted parturition. Liver samples were collected by biopsy at 8 d before the predicted parturition (-8 d) and at 1 and 9 d after the actual parturition (1 and 9 d). The lipidomic profile of liver samples collected at -8 and 9 d was analyzed using ultra performance liquid chromatography-mass spectrometry-based lipidomics. Liver samples from all the time points were subjected to microarray analysis and the subsequent pathway analysis with Ingenuity Pathway Analysis software (Ingenuity Systems, Mountain View, CA). Prepartal energy intake level affected hepatic gene expression and lipidomic profiles prepartum, whereas little or no effect was observed postpartum. At -8 d, hepatic lipogenesis was promoted by prepartal high-energy feeding through the activation of X receptor/retinoid X receptor pathway and through increased transcription of thyroid hormone-responsive (THRSP). Hepatic inflammatory and acute phase responses at -8 d were suppressed (z-score = -2.236) by prepartal high-energy feeding through the increase in the mRNA abundance of suppressor of cytokine signaling 3 (SOCS3) and the decrease in the mRNA abundance of interleukin 1 (IL1), nuclear factor kappa B 1 (NFKB1), apolipoprotein A1 (APOA1), serum amyloid A3 (SAA3), haptoglobin (HP), lipopolysaccharide-binding protein (LBP), and inter-α-trypsin inhibitor heavy chain 3 (ITIH3). Moreover, prepartal high-energy feeding elevated hepatic concentrations of C18- (7%), C20- (17%), C21- (26%), C23-sphingomyelins (26%), and total saturated sphingomyelin (21%). In addition, cows in both groups displayed increased lipogenesis at the gene expression level after parturition and alterations in the concentration of various sphingolipids between the first and last samplings. In conclusion, prepartal high-energy feeding promoted lipogenesis and suppressed inflammatory and acute phase responses in the liver before parturition, whereas only minor effects were observed after parturition.

Compartmental flux and in situ methods underestimate total feed nitrogen as judged by the omasal sampling method due to ignoring soluble feed nitrogen flow.

The objective of the present study was to estimate ruminal feed N outflow in lactating cows using the omasal sampling, compartmental flux or in situ method. A total of five ruminally fistulated Finnish Ayrshire dairy cows were used in a 5 × 5 Latin square study with 21 d periods. Experimental silages of grass or red clover harvested at two stages of maturity in addition to a supplement of 9·0 kg concentrate/d were fed to the cows. In vivo omasal N flow was determined using the omasal sampling technique. Ruminal in situ N flow was calculated from N intake and degradability (38 µm nylon bags). The samples of ruminal contents and faeces were divided into seven particle-size fractions by wet sieving; the concentrations of indigestible neutral-detergent fibre and N were used to calculate N flow in the compartmental flux method. In vivo omasal N flow was greater for the red clover silage diets than for the grass silage diets. The N flow calculated using the compartmental flux technique and that calculated using the in situ technique were highly correlated, but both were less than and poorly correlated with the in vivo N flow. In both in situ and compartmental flux techniques, forage maturity increased the particle-associated N flow, with the increase being significantly greater for the red clover diets than for the grass silage diets. In conclusion, the compartmental flux and in situ methods described the N flow associated with the particle fractions rather than the total ruminal outflow of feed N.

Dietary fish oil supplements modify ruminal biohydrogenation, alter the flow of fatty acids at the omasum, and induce changes in the ruminal Butyrivibrio population in lactating cows.

Four lactating cows fitted with ruminal cannulae and fed a grass silage-based diet were used in a 4 × 4 Latin square with 28-d periods to investigate the effects of incremental dietary fish oil (FO) supplementation (0, 75, 150, or 300 g/d) on the flow of fatty acids at the omasum and populations of rumen bacteria capable of biohydrogenation. FO decreased silage intake and ruminal volatile fatty acid concentrations and promoted an increase in molar butyrate and propionate proportions at the expense of acetate. Extensive ruminal biohydrogenation of 20:5(n-3) and 22:6(n-3) resulted in corresponding increases in numerous 20- and 22-carbon unsaturated fatty acids at the omasum. Omasal flow of several 20-, 21-, and 22-carbon all-cis (n-3) PUFA exceeded the intake from FO. Supplements of FO also induced a dose-dependent decrease in 18:0 and increased trans 18:1 and trans 18:2 flow at the omasum. Trans-11 was the major 18:1 intermediate in digesta, while FO induced quadratic increases in trans-10 18:1 flow, reaching a maximum of 300 g/d. FO had no substantial influence on omasal flow of CLA. Results suggest that one or more fatty acids in FO inhibit the reduction of trans-18:1 and trans-18:2 intermediates by ruminal microorganisms. qPCR based on 16S rRNA genes in omasal digesta indicated that key Butyrivibrio spp. declined linearly in response to FO. Dose-dependent increases in ruminal outflow of biohydrogenation intermediates containing one or more trans double bonds in response to FO has major implications for host metabolism and the nutritional quality of ruminant foods.

Equol in milk of dairy cows is derived from forage legumes such as red clover.

The intake of isoflavones and the resulting equol contents of both plasma and milk of the same red clover-fed cows are reported for the first time in cyclic change-over design study. Cows were fed four different red clover silages and two timothy-meadow fescue silages as controls. The red clover silages contained daidzein, formononetin, biochanin A and genistein, whereas the timothy-meadow fescue silages contained no isoflavones. We found a strong association (y = 0.071x+2.75, R 2 0.71) between the formononetin intake (x) and equol concentration (y) in the plasma, while the formononetin intake and milk equol concentration were weakly associated (y = 0.0035x+0.358, R 2 0.20). This means that a small part of the total formononetin in the silage is secreted into milk as equol. The mean equol contents in plasma and milk of cows fed red clover silage diets were in the range of 4.6-8.4 mg/l and 458-643 microg/l, respectively, while the respective values for the control diets were in the range of 0.8-1.5 mg/l and 171-287 microg/l. We showed that shorter growing periods of red clover resulted in higher silage formononetin contents and plasma and milk equol contents, suggesting that the equol content of milk can be manipulated by varying the harvesting strategy of red clover. We conclude that milk equol is derived from the formononetin of red clover silage and that milk from red clover-fed cows can be considered as a source of equol in human nutrition.

Ruminal infusions of cobalt-EDTA reduce mammary delta9-desaturase index and alter milk fatty acid composition in lactating cows.

Ruminal administration of a triple indigestible marker system comprised of cobalt EDTA (CoEDTA), ytterbium acetate (YbAc), and chromium-mordanted straw (CrS) decreases product:substrate ratios for Delta9-desaturase in bovine milk fat. This experiment was designed to identify the marker(s) responsible and develop an alternative system for simultaneous determination of nutrient flow in the gastro-intestinal tract and milk fatty acid composition. Five lactating dairy cows were used in a 5 x 5 Latin square with 21-d periods to evaluate the effects of YbAc, CoEDTA, and CrS independently or as part of a triple marker system (TMS), and CrEDTA as an alternative to CoEDTA on milk fat composition. Markers were administered in the rumen over a 7-d interval and samples of milk were collected on d -1, 3, 7, and 11. Both TMS and CoEDTA alone reduced the concentrations of milk fatty acids containing a cis-9 double bond, whereas YbAc, CrS, and CrEDTA had no effect. Reductions in product:substrate ratios for Delta9-desaturase were time dependent and evident within 3 d of administration. Ruminal infusion of CoEDTA for 7 d induced mean decreases in milk cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 18:1/18:0, and cis-9, trans-11 conjugated linoleic acid/trans-11 18:1 concentration ratios of 47.7, 26.7, 40.3, and 42.6%, respectively. In conclusion, ruminal infusion of CoEDTA alters milk fatty acid composition and appears to inhibit Delta9-desaturase activity in the bovine mammary gland. Results indicate that a TMS based on CrEDTA, YbAc, and indigestible neutral detergent fiber can be used for estimating nutrient flow without altering milk fat composition in lactating cows.

Transfer of absorbed cis-9, trans-11 conjugated linoleic acid into milk is biologically more efficient than endogenous synthesis from absorbed vaccenic acid in lactating cows.

Cis-9, trans-11, the major isomer of conjugated linoleic acid (CLA) in bovine milk fat, is derived from ruminal biohydrogenation of 18:2 (n-6) and endogenous conversion of trans-11 18:1 (vaccenic acid; VA) in the mammary gland. Most evidence to date suggests that endogenous synthesis is the major source of cis-9, trans-11 CLA, but the extent of VA desaturation is less well defined. Four lactating cows were used in consecutive 4 x 4 Latin squares to examine changes in milk fatty acid composition and secretion in response to abomasal infusions of lipid supplements enriched with cis-9, trans-11 CLA (88.8%) or VA (29.4%). Treatments were infused over 4-d, followed by a 3-d washout, during 7 d experimental periods and administered to deliver 0, 3, 6, and 12 g cis-9, trans-11 CLA/d (Expt. 1) or 0, 7.5, 15 and 30 g VA/d (Expt. 2). Infusions of cis-9, trans-11 CLA increased linearly milk cis-9, trans-11 CLA concentrations from 0.68 to 1.46 g/100 g fatty acids. Abomasal infusions of VA increased linearly milk VA and cis-9, trans-11 CLA content from 1.22 to 2.72 and 0.61 to 1.24 g/100 g fatty acids, respectively. Changes in milk fatty acid secretion indicated that 28.9% of VA was converted to cis-9, trans-11 CLA. Results provide evidence that conversion by Delta9-desaturase to cis-9, trans-11 CLA in the lactating cow is independent of postruminal VA supply. In conclusion, endogenous synthesis via VA was equivalent to approximately 21% of the response to increases in cis-9, trans-11 CLA available for absorption.

Determination of digesta flow entering the omasal canal of dairy cows using different marker systems.

Four studies were conducted to compare the effect of four indigestible markers (LiCoEDTA, Yb-acetate, Cr-mordanted straw and indigestible neutral-detergent fibre (INDF)) and three marker systems on the flow of digesta entering the omasal canal of lactating dairy cows. Samples of digesta aspirated from the omasal canal were pooled and separated using filtration and high-speed centrifugation into three fractions defined as the liquid phase, small particulate and large particulate matter. Co was primarily associated with the liquid phase, Yb was concentrated in small particulate matter, whilst Cr and INDF were associated with large particles. Digesta flow was calculated based on single markers or using the reconstitution system based on combinations of two (Co + Yb, Co + Cr and Co + INDF) or three markers (Co + Yb + Cr and Co + Yb + INDF). Use of single markers resulted in large differences between estimates of organic matter (OM) flow entering the omasal canal suggesting that samples were not representative of true digesta. Digesta appeared to consist of at least three phases that tended to separate during sampling. OM was concentrated in particulate matter, whilst the liquid phase consisted mainly of volatile fatty acids and inorganic matter. Yb was intimately associated with nitrogenous compounds, whereas Cr and INDF were concentrated in fibrous material. Current data indicated that marker systems based on Yb in combination with Cr or INDF are required for the accurate determination of OM, N and neutral-detergent fibre flow. In cases where the flow of water-soluble nutrients entering the omasal canal is also required, the marker system should also include Co.