The authors have declared that no competing interests exist.
Selenium (Se) is an essential trace mineral important for immune function and overall health of cattle. The nasopharyngeal microbiota in cattle plays an important role in overall respiratory health, especially when stresses associated with weaning, transport, and adaptation to a feedlot affect the normal respiratory defenses. Recent evidence suggests that cattle diagnosed with bovine respiratory disease complex have significantly less bacterial diversity. The objective of this study was to determine whether feeding weaned beef calves Se-enriched alfalfa (
Selenium (Se) is an essential trace mineral important for immune function and overall health of cattle. Se deficiency can cause nutritional myodegeneration, also known as white muscle disease, characterized by muscle weakness, heart failure, unthriftiness, and death. Insufficient Se intake can cause subclinical diseases resulting in poor livestock performance. The role of Se in animal health is based primarily on the functions of selenocysteine-containing proteins, many of which have antioxidant activities [
In Se-deficient areas, several means of Se supplementation are available, e.g., organic Se-yeast can be added to feed, or inorganic Na selenite can be added to mineral mixes for free-choice consumption. Agronomic biofortification is an alternative approach, whereby the Se concentration of forage is increased through the use of Se-containing fertilizer amendments [
Conversely, Se toxicity and livestock death can occur when cattle consume excessive amounts of Se in their diet. For example, consumption of Se-accumulator plants, including species of
Dietary glutamine supplementation has been shown to regulate intestinal immunity, for example though enhancement of secretory IgA production, and studies in mice show that glutamine supplementation acts in part by altering the intestinal bacterial community [
Similar to the role of the intestinal microbiome in gastrointestinal health, the respiratory microbiome is a strong determinant of respiratory health [
The objective of this study was to determine whether feeding weaned beef calves Se-enriched alfalfa (
The experimental protocol was reviewed and this study was approved by the Oregon State University Animal Care and Use Committee (ACUP Number: 4705). This was a prospective clinical trial of 9-week duration (October 10, 2015 through December 10, 2015) involving 45 weaned beef calves, primarily of Angus breeding. The study design consisted of 3 treatment groups, with three pens of five animals per treatment. The study was conducted at the Steer Metabolism barn on the Oregon State University campus (Corvallis, OR, USA).
Corvallis is located at an elevation of 72 m within the Marine West Coast climate zone. Temperatures are mild year round, with warm, sunny summers and mild, wet winters with persistent overcast skies. Because of its close proximity to the coast range, temperatures dropping below freezing are uncommon. Average monthly temperatures for November are 10.8°C (high) and 3.3°C (low). Rainfall total is 110.9 cm/yr. Typical distribution of precipitation includes about 50% of the annual total from December through February, lesser amounts in the spring and fall, and very little during summer.
The weaned beef calves ranged in age from 6.5 to 9 months (222 ± 6.4 days; mean ± SEM) and originated from the Oregon State University beef ranch, Corvallis, OR, USA. Body weights at weaning ranged from 240 to 334 kg (286 ± 9.3 kg, mean ± SEM), and body condition scores ranged from 6 to 7 (1 to 9 scale). There were 17 heifers and 28 steer calves in the study. Routine farm management practices, including vaccinations and deworming, were the same for all treatment groups.
Using a randomized complete block design, calves were blocked at the time of weaning by sex and body weight and then assigned to one of 3 treatment groups of 15 calves each. Ear tags were used to identify calves. Calves were then placed by treatment group into pens (3 pens of 5 calves/treatment group). The pens were 14 m2/calf; concrete flooring in open lots that were strip cleaned once weekly; shavings in loafing area with 4.5 m2/calf; concrete bunks with 98 cm of feeder space/calf; all measurements exceeded requirements [
Calves were fed 70% alfalfa hay and 30% grass hay twice daily. The amount of hay fed was adjusted weekly to ensure that calves had all they wanted for consumption yet with minimal wastage. The ration was formulated for growing beef calves in the 250 to 350 kg weight range to achieve a target average daily gain of 0.5 kg/day. The goal was to feed hay at a rate of 70% alfalfa and 30% grass hay. Calves were transitioned to their respective alfalfa hay sources over a 7 day period. Alfalfa hay was fed as follows: 0.68 kg/head/day 1; 1.14 kg/head/day 2; 1.59 kg/head day 3; 2.27 kg/head/day 4; 2.95 kg/head/day 5; 3.41 kg/head/day 6; and 3.86 kg/head/day 7. During this first week, grass hay was added to achieve a total hay intake of 5.45 kg/head/days 1 and 2; 5.91 kg/head/days 3 and 4; 6.36 kg/head/days 5 and 6; and 6.82 kg/head/days 7 and 8. Thereafter, calves were fed 70% alfalfa hay and 30% grass hay with increases as needed to provide all the hay they wanted for consumption yet with minimal wastage. By week 9, calves were consuming between 8.18 to 9.09 kg hay/head/day.
Prior to this study, calves had free-choice access to a mineral supplement containing 120 mg/kg Se from sodium-selenite. The mineral supplement (dry matter basis) was in loose granular format and contained 57.0 to 64.0 g/kg calcium; 30.0 g/kg phosphorus; 503 to 553 g/kg salt (NaCl); 50.0 g/kg magnesium; 50 mg/kg cobalt; 2,500 mg/kg copper; 200 mg/kg manganese; 200 mg/kg iodine; 6,500 mg/kg zinc (Wilbur-Ellis Company, Clackamas, OR). During this feeding trial, mineral supplement was added as a top dressing to the hay for each pen of calves at the rate of 0.1 mg Se/kg of dry matter intake [
The soil was enriched with Se by mixing inorganic sodium-selenate (RETORTE Ulrich Scharrer GmbH, Röthenbach, Germany) with water and spraying it onto the soil surface of an alfalfa field at application rates of 0, 45.0, or 89.9 g Se/ha immediately after the second cutting of hay in July 2014. The application rates were chosen based on a previous study [
Nutrient | Alfalfa Hay | Grass Hay | ||
---|---|---|---|---|
Control | Med-Se | High-Se | ||
Dry matter, g/kg | 874 | 874 | 878 | 954 |
Crude protein, g/kg | 170 | 183 | 157 | 78 |
Acid detergent fiber, g/kg | 377 | 386 | 373 | 380 |
Neutral detergent fiber, g/kg | 402 | 417 | 430 | 604 |
Nonfiber carbohydrates, g/kg | 316 | 289 | 286 | 243 |
Fat, g/kg | 13.7 | 12.4 | 12.0 | 0.7 |
Ash, g/kg | 98.3 | 98.6 | 115.0 | 74.3 |
TDN, g/kg | 600 | 596 | 564 | 588 |
Calcium, g/kg | 13.3 | 13.1 | 13.4 | 4.1 |
Phosphorus, g/kg | 2.4 | 2.6 | 2.5 | 2.1 |
Magnesium, g/kg | 3.2 | 3.5 | 3.4 | 1.8 |
Potassium, g/kg | 23.3 | 23.3 | 22.5 | 14.5 |
Sodium, g/kg | 0.98 | 1.14 | 1.11 | 1.91 |
Copper, mg/kg | 10 | 11 | 11 | 5 |
Iron, mg/kg | 292 | 447 | 261 | 73 |
Manganese, mg/kg | 39 | 48 | 41 | 311 |
Zinc, mg/kg | 20 | 19 | 20 | 22 |
Selenium, mg/kg | 0.34 | 2.42 | 5.17 | 0.36 |
1Alfalfa hay samples were submitted to Cumberland Analytical Services, Maugansville, MD for routine nutrient analysis and to Utah Veterinary Diagnostic Laboratory, Logan, UT for Se analysis.
2Alfalfa hay DM determination was completed at a temperature of 105°C for 12 to 14 h in a forced draught oven. Methods for CP, ADF, ash, and minerals were performed according to the Association of Official Analytical Chemists [
3Alfalfa hay samples were prepared for Se analysis as described by Davis et al. [
Blood samples were collected from the jugular vein of weaned beef calves, at baseline and after 2, 4, 6 and 8 weeks of alfalfa hay consumption, into evacuated EDTA tubes (2 mL; final EDTA concentration 2 g/L; Becton Dickinson, Franklin Lakes, NJ) and stored on ice until they were frozen at -20°C to measure whole blood Se (WB-Se) concentrations. Selenium concentrations were determined by a commercial laboratory (Utah Veterinary Diagnostic Laboratory, Logan, UT) using an inductively coupled argon plasma emission spectrometry method. Selenium was measured using an ICP-MS (ELAN 6000, Perkin Elmer, Shelton, CT) method as previously described [
During week 9 of the feeding trial, nasal swabs were collected from 5 or 6 weaned beef calves in each of the three treatment groups, by random selection of calves. One or two calves were selected from each pen (3 pens of 5 calves each per treatment group). Sterile, individually wrapped, polyester tipped applicators (Puritan®, Guilford, ME) were inserted approximately 10 cm into the nares, twirled to collect a mucosal swab, and then placed into individual sterile containers (10 mL, red topped, BD Vacutainer collection tubes; Becton Dickinson, Franklin Lakes, NJ) avoiding any contamination or contact with the wooden stick. Swabs in tubes were subsequently frozen at -80°C within 4 hours of collection. Negative control (sterile) swabs were similarly processed.
Microbial DNA was extracted from nasal swab samples using MoBio Power soil DNA isolation kit (MoBio Laboratories, USA) as per the manufacturer’s instructions. The V4 region of the 16S rRNA gene was amplified with primers 515F (
Statistical analyses were performed using SAS version 9.2 [
Nasal swabs were collected randomly from 5 or 6 calves in each of the three treatment groups and treated as individual samples. Differences in bacterial communities between the three groups were determined using the phylogeny based unweighted UniFrac distance metric and PCoA plots were generated with QIIME. ANOSIM (Analysis of Similarity) test within PRIMER 6 software package (PRIMER-E Ltd., Luton, UK) was performed on the weighted and unweighted UniFrac distance metrics to find significant differences in microbial communities between the groups.
All the datasets were tested for normality with Shapiro-Wilk test (JMP Pro 11, SAS Software Inc.). Since most of the datasets did not meet the assumptions of normal distribution, non-parametric Kruskal-Wallis tests were performed. The resulting
To determine if there were any changes in microbial function in the nasal microbiota, PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was applied to make functional gene content predictions on the 16S rRNA gene sequencing data [
Fertilizing fields with increasing amounts of Na selenate increased the Se-concentration of third cutting alfalfa hay from 0.34 mg Se/kg dry matter (non-fertilized control) to 2.42 and 5.17 mg Se/kg dry matter for Na selenate application rates of 45.0 and 89.9 g Se/ha, respectively. The relationship between amount of Se applied by fertilization (g Se/ha) and observed forage Se concentration (mg Se/kg dry matter) was y = 0.054x + 0.228,
Prior to agronomic biofortification, calves had WB-Se concentrations (mean, 204 ng/mL; range, 42–307 ng/mL), which were mostly within the reference interval of adult cows (120–300 ng/mL). Whole blood-Se concentrations after 8 weeks of feeding Se-fertilized alfalfa hay were dependent upon Se-application rates (0, 45.0, or 89.9 g Se/ha) and were 175 ± 11, 335 ± 11, and 494 ± 11 ng/mL (
The normal reference interval for WB-Se concentrations of beef cattle is 120 to 300 ng/mL.
The sequence analysis yielded 1,137,749 quality sequences for all analyzed samples (n = 16; mean ± SD, 70,859 ± 10,178). Rarefaction analysis was performed at a depth of 7,214 sequences.
Alfalfa hay was harvested from fields with no Se fertilization (control) or from fields fertilized with sodium-selenate at application rates of 45.0 (medium) or 89.9 (high) g Se/ha.
Control (Low-Se) | Med-Se | High-Se | ||
---|---|---|---|---|
Chao1 (mean ± SD) | 2123.1 ± 557.4 | 2253.7 ± 405.2 | 2611.6 ± 423.4 | 0.1838 |
Observed OTU (mean ± SD) | 887.4 ± 322.3 | 997.6 ± 241.9 | 1236.0 ± 131.0 | 0.1439 |
Shannon Index (mean ± SD) | 5.9 ± 1.3 | 6.2 ± 1.2 | 7.4 ± 0.7 | 0.1259 |
1Control calves consumed alfalfa hay harvested from fields with no Se fertilization (n = 5); Med-Se calves consumed alfalfa hay from fields fertilized with sodium-selenate at application rates of 45.0 g Se/ha for 8 weeks (n = 6); High-Se calves consumed alfalfa hay from fields fertilized with 89.9 g Se/ha for 8 weeks (n = 5)
Calves fed high Se-biofortified alfalfa hay clustered separately when compared with healthy control calves in the PCoA plot (
Data obtained from nasal samples of randomly chosen healthy control calves (n = 5) and calves fed either medium (n = 6) or high (n = 5) Se-enriched alfalfa hay for 8 weeks. Alfalfa hay was harvested from fields with no Se fertilization (control) or from fields fertilized with sodium-selenate at application rates of 45.0 (medium) or 89.9 (high) g Se/ha.
Control, Med-Se | 0.155 | 0.093 |
Control, High-Se | 0.216 | 0.04 |
Med-Se, High-Se | 0.067 | 0.225 |
Control, Med-Se | 0.240 | 0.069 |
Control, High-Se | 0.332 | 0.056 |
Med-Se, High-Se | -0.136 | 0.907 |
1Control calves consumed alfalfa hay harvested from fields with no Se fertilization (n = 5); Med-Se calves consumed alfalfa hay from fields fertilized with sodium-selenate at application rates of 45.0 g Se/ha for 8 weeks (n = 6); High-Se calves consumed alfalfa hay from fields fertilized with 89.9 g Se/ha for 8 weeks (n = 5)
Relative abundances of the most abundant bacterial phyla in all three groups are shown in
Alfalfa hay was harvested from fields with no Se fertilization (control) or from fields fertilized with sodium-selenate at application rates of 45.0 (medium) or 89.9 (high) g Se/ha.
Only a LDA score of >3 is shown.
Diet | LDA | |
---|---|---|
k__Bacteria|p__Proteobacteria|c__Gammaproteobacteria|o__Pasteurellales|f__Pasteurellaceae|g__Mannheimia | Med-Se | 3.04 |
k__Bacteria|p__Proteobacteria|c__Gammaproteobacteria|o__Oceanospirillales|f__Halomonadaceae|g__CandidatusPortiera | High-Se | 3.14 |
k__Bacteria|p__Bacteroidetes|c__Flavobacteriia|o__Flavobacteriales|f__Cryomorphaceae|g__ | Med-Se | 3.19 |
k__Bacteria|p__Proteobacteria|c__Betaproteobacteria|o__Burkholderiales|f__Alcaligenaceae|g__ | Med-Se | 3.25 |
k__Bacteria|p__Verrucomicrobia | High-Se | 3.26 |
k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Lachnospiraceae|g__ | High-Se | 3.36 |
k__Bacteria|p__Firmicutes|c__Bacilli|o__Bacillales|f__Planococcaceae|g__Sporosarcina | Med-Se | 3.40 |
k__Bacteria|p__Firmicutes|c__Clostridia|o__Clostridiales|f__Lachnospiraceae | High-Se | 3.57 |
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Rikenellaceae | High-Se | 3.63 |
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Rikenellaceae|g__ | High-Se | 3.63 |
k__Bacteria|p__Bacteroidetes|c__Bacteroidia|o__Bacteroidales|f__Bacteroidaceae|g__5_7N15 | High-Se | 3.70 |
k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales | Control | 4.03 |
k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Carnobacteriaceae|g__Trichococcus | Control | 4.04 |
k__Bacteria|p__Firmicutes|c__Bacilli|o__Lactobacillales|f__Carnobacteriaceae | Control | 4.04 |
k__Bacteria|p__Tenericutes|c__Mollicutes | Med-Se | 4.51 |
k__Bacteria|p__Tenericutes | Med-Se | 4.52 |
k__Bacteria|p__Bacteroidetes|c__Flavobacteriia|o__Flavobacteriales|f___Weeksellaceae|g__ | Control | 4.53 |
k__Bacteria|p__Bacteroidetes|c__Flavobacteriia|o__Flavobacteriales|f___Weeksellaceae_ | Control | 4.57 |
1Control calves consumed alfalfa hay harvested from fields with no Se fertilization (n = 5); Med-Se calves consumed alfalfa hay from fields fertilized with sodium-selenate at application rates of 45.0 g Se/ha for 8 weeks (n = 6); High-Se calves consumed alfalfa hay from fields fertilized with 89.9 g Se/ha for 8 weeks (n = 5)
A total of 8 differentially abundant bacterial functions were observed between the three groups after LEfSe analysis (LDA score > 2.5) of the PICRUSt data (
Only a LDA score of >2.5 is shown.
Diet | LDA | |
---|---|---|
Metabolism|LipidMetabolism|Fatty acid biosynthesis | Control | 2.52 |
Metabolism|GlycanBiosynthesisandMetabolism|Peptidoglycan biosynthesis | Control | 2.60 |
Metabolism|CarbohydrateMetabolism|Starch and sucrose metabolism | High-Se | 2.59 |
GeneticInformationProcessing|Transcription | High-Se | 2.87 |
Unclassified|CellularProcessesandSignaling|Sporulation | High-Se | 2.91 |
CellularProcesses|CellMotility|Bacterial chemotaxis | High-Se | 2.97 |
Metabolism|CarbohydrateMetabolism|Glycolysis_Gluconeogenesis | Med-Se | 2.57 |
GeneticInformationProcessing|Transcription|Transcription machinery | Med-Se | 2.71 |
1Control calves consumed alfalfa hay harvested from fields with no Se fertilization (n = 5); Med-Se calves consumed alfalfa hay from fields fertilized with sodium-selenate at application rates of 45.0 g Se/ha for 8 weeks (n = 6); High-Se calves consumed alfalfa hay from fields fertilized with 89.9 g Se/ha for 8 weeks (n = 5)
Bovine respiratory disease complex, also known as shipping fever, is one of the most common causes of morbidity and mortality in cattle entering into feedlots [
Rarefaction curves comparing the species richness (observed OTU number) and overall diversity (Chao1, Observed OTU, and Shannon index) among weaned beef calves fed Se-biofortified alfalfa hay compared with healthy control calves showed that calves fed the highest concentration of Se-biofortified alfalfa hay had more (1236 vs 887) OTUs identified at a depth of 7,214 sequences. This number of OTUs was comparable with results previously reported in feedlot cattle [
In our study, a total of 16 bacterial phyla were identified compared with 8 in the previous report [
The beta diversity was measured using the phylogeny-based unweighted and weighted UniFrac distances. ANOSIM of unweighted UniFrac distances showed that this clustering was significant (
The linear discriminant analysis of bacterial taxa showed that bacterial orders were altered between the control calves and calves fed Se-fortified hay. The bacterial orders
Analysis of bacterial functions induced by feeding Se-biofortified alfalfa hay revealed differences in metabolic pathways. For example in calves fed high levels of Se-biofortified hay there were enriched metabolic pathways involved in bacterial chemotaxis, transcription, sporulation, and starch and sucrose metabolism. The overall significance of these findings requires further study.
Calves likely have greater Se requirements during periods of stress such as during weaning and movement to a feedlot, which is one of the most stressful times for beef calves [
Research in the last 10 years has demonstrated that the microbiota is important in priming and orchestrating innate and adaptive immune responses of the host (reviewed in [
In conclusion, feeding Se-enriched alfalfa hay to weaned beef calves for 8 weeks during a preconditioning period to prepare them for the feedlot resulted in enrichment of the nasal microbiome. Calves fed the high-Se alfalfa hay had more OTUs, and beta diversity was different compared with control calves. The bacterial orders
Mean percentages of the most abundant bacterial groups on the various phylogenetic levels (phylum, class, order, family, genus) identified in nasal samples of randomly chosen healthy control calves (n = 5) and calves fed either medium (n = 6) or high (n = 5) Se-enriched alfalfa hay for 8 weeks. Alfalfa hay was harvested from fields with no Se fertilization (control) or from fields fertilized with sodium-selenate at application rates of 45.0 (medium) or 89.9 (high) g Se/ha.
(DOCX)
acid detergent fiber
analysis of similarity
bovine respiratory disease
crude protein
false discovery rate
inductively coupled argon plasma emission spectroscopy
linear discriminant analysis
linear discriminant analysis effect size
neutral detergent fiber
operational taxonomic units
phylogenetic investigation of communities by reconstruction of unobserved states
reactive oxygen species
selenium
whole blood-selenium