Final Report for GW14-023
Improvements in livestock feed efficiency can translate to reducing feed usage while maintaining animal performance, and in turn, may improve profitability for producers. Mammals have sterile gastrointestinal (GI) tracts until birth that are continually colonized by microbial populations until weaning, when microbial populations become stabilized. The GI tract microbiota has been demonstrated to differ in composition and abundance with differences in host feed efficiency. Furthermore, altering GI tract microbiota has been shown to improve overall GI health and enhance nutrient uptake efficiency in humans and rodents. The objectives of this study were to: 1) determine if inoculation of lambs at birth with rumen microbiota from adult donor sheep identified as highly efficient and lowly efficient alters recipient rumen microbial profile, 2) determine if lambs inoculated with microbiota from highly efficient adults demonstrate increased feed efficiency post-weaning, and if lambs inoculated with microbiota from lowly efficient adults demonstrate decreased post-weaning efficiency, 3) determine and improve producer adoption and application of feed efficiency measures in sheep, and 4) determine the long-term economic implications of improving feed efficiency via rumen microbiota inoculation at birth. Ultimately, our goal was to improve feed efficiency in sheep through 1) development of producer-friendly tools and strategies that improve feed efficiency, and 2) improved producer knowledge and use of feed efficiency measures and applications. Mature Targhee ewes (n = 60; initial BW = 45.8 ± 2.5 kg) fed a forage-based pelleted diet were assessed for individual feed intake over a 70 d period using the GrowSafe System and residual feed intake (RFI), a measure of feed efficiency, was calculated. Low RFI animals are considered more feed efficient, and high RFI animals less efficient. Rumen fluid samples were collected from the selected ewes (donors; n = 4) at the end of the trial and stored until processing. Five sets of Hampshire twin lambs (recipients; n = 10) received rumen fluid inoculations from either a high RFI or a low RFI donor ewe at birth, and again two weeks later to help encourage establishment of appropriate microbial populations. All lambs were raised by their dams in a similar environment in one pen, weaned, and received the same forage-based pelleted diet as their donors. Individual feed intake was measured using the GrowSafe System for a 70 d trial period, RFI was estimated, and rumen fluid samples were collected at d 35 from the twin lambs. VFA concentrations were measured and DNA was extracted for sequencing from the rumen fluid of the donors and recipients. Paired-end reads were filtered, quality trimmed, and compared with a database of known 16S rDNA genes. Operational taxonomic units (OTU) were defined as sequence clusters with ≥ 97% identity. Of the 5 sets of twin lambs, 3 sets exhibited the same feed efficiency status as their donor compared with their respective twin. Results from the rumen microbiome metagenomics analyses suggest that low RFI recipients and donors exhibit greater rumen microbial diversity compared with high RFI recipients and donors, respectively. However, there were no patterns to suggest that recipients had similar rumen microbial communities to either their donor or to their respective twin. While further research is necessary to determine whether rumen manipulation may be successful, selecting for feed efficiency remains an economical trait to select on. Therefore, education materials will be developed and distributed to enhance producer awareness and understanding of feed efficiency measures and tools, including the economic analysis performed in this study. Finally, producer adoption will be determined via feedback on materials and information assessed using surveys and interviews.
Mammals are born with sterile gastrointestinal (GI) tracts which are continually colonized by microbial populations until weaning, when microbial populations become stabilized (Tannock, 2007). Microbial type and abundance are highly variable among individuals and once established in the GI tract, composition of microbiota is highly resilient to short-term changes. There is evidence that the GI tract microbiome may be genetically associated with the host. In mice, approximately 5% of the variation in taxa among individuals was attributed to family and litter, and composition of GI microbiota was not significantly different among littermates, suggesting that host genetics play a significant role in composition and variation of taxa (Ley et al., 2005; Benson et al., 2010). Similarly, in human twins it was reported that intestinal microbial profiles were 98.5% similar and they also shared 98% similarity with their mother, while unrelated individuals only shared 96% similarity in microbial profiles (Turnbaugh et al., 2009).
While there is evidence that the GI tract microbiome is genetically influenced, there is also data to suggest that changes in the composition of GI tract microbiota have been associated with several diseases in humans and mice, including obesity, coronary heart disease, diabetes and inflammatory bowel disease (Anderson et al., 2009; Benson et al., 2010; Spor et al., 2011). In response to these associations, altering the microbiome has become an area of interest, particularly in human health, in an effort to overcome GI tract diseases. In a study by Van Nood et al. (2013), infusion of healthy donor feces through a nasoduodenal tube into human patients (n = 16) with a Clostridium difficile infection allowed 15 of those patients (94%) to fully recover and stay in remission for 10 weeks. Patients were able to increase diversity of fecal microbiota over a two week period after infusion until they became undistinguishable from the fecal microbiota diversity of the donors (van Nood et al., 2013).
Similarly, the GI tract microbiota have been observed to differ in composition and abundance between obese and thin individuals, as well as differences in metabolic pathways utilized. Obese mice were reported to have a shift in relative abundance of taxa compared with their lean counterparts, and obese mice seemed to be more efficient at harvesting energy from food than lean mice (Ley et al., 2005). Furthermore, mice and rats that have been inoculated with microbiota from obese donors exhibited decreased feed intake and increased adiposity, which suggested an increased ability to utilize nutrients (Backhed et al., 2004; Turnbaugh et al., 2009; Liou et al., 2013). It is of interest as to whether similar influence can occur in the rumen, especially in domesticated livestock, to improve feed efficiency.
Including feed efficiency as a trait of selection has been gaining interest in the sheep industry because it can be an important trait to producers to reduce input costs or improve stocking ratios, both of which can translate into increased profitability. Residual feed intake (RFI) is a measure of feed efficiency defined as the difference between the actual feed intake and that predicted based on the individual’s ADG (Koch et al., 1963); a lower RFI measurement (i.e. more negative) denotes better feed efficiency. Because RFI is moderately heritable and is independent of growth and mature body size, it has become a commonly applied measure of feed efficiency for livestock (Carberry et al., 2012). Limited research has been done to determine whether inoculation with donor rumen microbiota will change microbial profiles and metabolic pathways in ruminants.
The goal of this Western SARE Graduate Student Grant was to determine the potential to improve feed efficiency of sheep through manipulation of the rumen microbiota (i.e. rumen microbial species) at birth. We hypothesized that inoculation of lambs at birth with rumen microbiota from feed efficient adult sheep would alter lamb rumen microbiota and result in improved feed efficiency post-weaning. Human and laboratory animal research have demonstrated that microbiota can be positively altered through similar inoculation processes. The specific objectives of this grant included:
Objective 1. Determine if inoculation of lambs at birth with rumen microbiota from adult sheep identified as highly efficient and lowly efficient alters the rumen microbial profile.
Objective 2. Determine if lambs inoculated with highly efficient and lowly efficient adult microbiota demonstrate increased and decreased feed efficiency, respectively.
Objective 3. Enhance producer adoption and application of feed efficiency measures in sheep through development of educational materials and generation of a feed efficiency selection index.
Objective 4. Determine the long-term economic implications of improving feed efficiency via rumen microbiota inoculation at birth.
Our long-term goal was to improve feed efficiency in sheep, and increase producer knowledge and awareness of the importance of feed efficiency in improving resource utilization and profitability. Improvements in feed efficiency can result in 1) decreased feed usage; 2) increased stocking ratios; and 3) improved producer profitability through less input costs (e.g., decreased feed usage) or greater outputs (e.g., increased stocking ratios).
Animals and Diet
All animal procedures were approved by the University of Wyoming (UW) Animal Care and Use Committee. Mature Targhee ewes (donors; n = 60; initial BW = 45.8 ± 2.5 kg) fed a forage-based pelleted diet (Table 1) were assessed for individual feed intake over a 70 d period using the GrowSafe System (Airdrie, Alberta, Canada) at UW and feed efficiency was calculated from this data. For this study, residual feed intake (RFI) was used as the measure of feed efficiency that is estimated as the deviation of true feed intake from expected feed intake; low RFI animals are considered more feed efficient and high RFI animals less efficient. Rumen fluid samples (≥ 30.0 mL) were collected from the selected ewes using a tygon tube (length: 1 m, diameter: 1.5 cm) positioned through the mouth, down the esophagus, and into the rumen and a dosing syringe (400 mL) for suction. Samples were then allocated in triplicate into 2-mL tubes for DNA extraction, snap-frozen, and stored at -80º C until processing. The remaining rumen fluid was retained for volatile fatty acid (VFA) analysis and was stored at -20º C until processing.
Five sets of Hampshire twin lambs (recipients; n = 10) from UW served as the recipients and set to receive rumen fluid inoculations from either a high RFI or a low RFI donor ewe from the study described above. Same-sex twin lambs were chosen for this study to determine whether post-weaning feed efficiency status or rumen microbial communities were influenced by the donor (environment; each twin would be more phenotypically similar to their donor) or genetically (both twins would have similar phenotypes to each other). Upon birth of each set of twin lambs, which were between March 29 and April 4, 2015, rumen fluid samples (~50 mL per donor) were collected from one low RFI donor ewe (DL) and one high RFI donor ewe (DH) using the same method described above; and the rumen fluid was filtered through cheesecloth in order to remove feed particles. One twin lamb was inoculated with the low RFI filtrate (L), and the other with the high RFI filtrate (H) within 8 hours of birth using a dosing syringe. Lambs were encouraged to swallow instead of suckle by administering a throat massage during the inoculation, in order to prevent the esophageal groove closing and subsequently bypassing the rumen into the abomasum. A second inoculation (with fresh rumen fluid filtrate collected from the same donor ewes) was performed two weeks later once the rumen had started to effectively function; this served as “booster” to help encourage establishment of appropriate microbial populations. All lambs were raised by their dams in a similar environment in one pen. Recipient lambs were weaned August 3, 2015 and received the same forage-based pelleted diet as their donors (Table 1). Individual feed intake was measured using the GrowSafe System for a 70 d trial period that started August 10, 2015. Residual feed intake was estimated and rumen fluid samples were collected at d 35 from the twin lambs and stored as previously described.
Thawed rumen fluid samples (≥ 10 mL) from both donor and recipients (n = 4 donors; n = 10 recipients) were centrifuged at 3,000 g for 10 min and supernatant was added to a solution containing 25% metaphosphoric acid that contained 2-ethyl butyric acid (2EB) as internal standard (2.0 mg/ mL) such that the ratio of the volume of rumen fluid to metaphosphoric acid was 5:1. Samples were incubated on ice for 30 min and centrifuged at 3,000 g for 30 min. One mL of supernatant was transferred to automatic sampler vials for analysis by Gas Liquid Chromatography. Preparation of samples for VFA analysis was conducted according to Goetsch & Galyean (1983). Detector response factors for acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were determined for 2-EB for calculation of molar percentages of each VFA. Volatile fatty acid concentrations were determined using an Agilent 6890 gas chromatograph with Agilent ChemStation software (Agilent Technologies, Santa Clara, CA) equipped with a Supelco Nukol 15 m × 0.53 mm capillary column (0.25 μm thickness; Sigma-Aldrich Corp., St. Louis, MO). Detector and injector temperatures were 250° C. Initial oven temperature was 80° C and was increased at a rate of 8° C per min to 150° C. Hydrogen was used as carrier gas at a flow rate of 20 mL per min.
DNA Extraction from Rumen Fluid
DNA was extracted from the rumen fluid collected from both donor and recipients (n = 4 donors; n = 10 recipients) using methods detailed by (Yu & Morrison, 2004). Zirconia (0.3 g of 0.1 mm) and silicon (0.1 g of 0.5 mm) beads and 1 mL lysis buffer were added to thawed rumen fluid samples. Samples (250 mg) were homogenized using a Mini-Beadbeater-8 at maximum speed for 3 min, incubated at 70° C for 15 min with gentle mixing every 5 min, and centrifuged at 4° C for 5 min. Supernatant was transferred to new 2-mL flat cap tubes and fresh lysis buffer (300 µL) was added to the pelleted beads. The homogenization, incubation and centrifugation steps were repeated on the remaining bead pellet, and supernatants were pooled. Final purification for removal of RNA and proteins was completed using the protocol of the QIAamp DNA Stool Mini Kit (Qiagen, Santa Clarita, CA). Samples of DNA were then quantified on the NanoDrop spectrophotometer (NanoDrop, Wilmington, DE) and determined to have acceptable quality using the manufacturer prescribed standard of the A260/280 ratio ≥ 1.8.
Illumina DNA Library Preparation and Sequencing
As previously described by Ellison et al. (2014), extracted DNA (10 µg), from rumen fluid samples from both donor and recipients (n = 4 donors; n = 10 recipients) was sent to the University of Missouri (Columbia) DNA Core Facility for library preparation and high-throughput sequencing. The library was constructed following the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq DNA PCR-Free sample preparation kit (#FC-121-3001). Briefly, 1 micrograms of genomic DNA was sheared using standard Covaris methods to generate average fragmented sizes of 350 bp. The resulting 3’ and 5’ overhangs were converted to blunt ends by an end repair reaction which uses a 3’ to 5’ exonuclease activity and polymerase activity. The desired size of fragment (~ 550 bp) was selected by sample purification beads (AMPure XP). A single adenosine nucleotide was added to the 3’ ends of the blunt fragment followed by the ligation of Illumina indexed paired-end adapters. The adaptor ligated library was purified twice with sample purification beads. The purified library was quantified with a Qubit assay and library fragment size confirmed by Fragment Analyzer (Advanced Analytical Technologies, Inc.). Library was diluted and sequenced according to Illumina’s standard sequencing protocol for the HiSeq. Libraries were then multiplexed on the Illumina HighSeq 2500 platform using a PE100 run and a 350 base-pair insert size. The resulting multiplex 100 base-pair, paired-end reads were combined within a sample and filtered by truncating each read after the first run of three bases using a Phred quality score < 15, quality-trimmed by omitting reads with < 85 base pairs or a quality score of < 25, and compared with a database of 27K known 16S rDNA genes using the Bowtie reference-based assembly tool (Johns Hopkins, Baltimore, MD). Operational taxonomic units (OTUs) were defined as clusters of database sequences from which members shared pairwise sequence identity of ≥ 97% (see Ellison et al. 2014 for details). A read pair was considered to derive from a particular OTU if both reads matched to sequences from that OTU and only that OTU with ≥ 97% identity. For the purposes of this study, OTUs were considered to be generally equivalent to microbial species. From this data, an OTU table was compiled and used for further analysis.
The GLM procedure of SAS (SAS Inst. Inc., Cary, NC) was used to determine the effects of donor feed efficiency status on BW characteristics, RFI, ADFI, ADG, and G:F and on VFA concentrations for twin lambs (n = 10). Rumen microbiome metagenomics analyses were performed using the core diversity analysis command in QIIME (Caporaso et al., 2010) using a sampling depth of 559 (minimum number of sequence reads per sample) to account for variation in total number of sequence reads per sample. From these analyses, a phylum level rumen microbiome taxonomy summary, an alpha diversity chao1 rarefaction curve, and a beta diversity Bray Curtis Emperor PCoA Plot were generated to compare the rumen microbial communities among the recipients (L = lambs inoculated with low RFI donors; H = lambs inoculated with high RFI donors) and donors (DL = low RFI donor; DH = high RFI donor).
Objectives 1 and 2
The effects of donor feed efficiency status on performance traits are listed in Table 2 for recipient lambs. There were no differences (P ≥ 0.196) in RFI, ADG, ADFI, G:F, MMWT, d 0 BW, d 35 BW, or d 70 BW between H and L recipient lambs. There were also no differences (P ≥ 0.139) in VFA concentrations, including acetate, propionate, butyrate, isobutyrate, isovalerate, and valerate between H and L recipient lambs (data not shown). Recipient RFI values are depicted in Figure 1. Of the 5 sets of recipient twin lambs, 3 sets exhibited the same feed efficiency status as their donor compared with their respective twin.
Results generated from the rumen microbiome metagenomics analyses are included in Figure 2. The most predominant phylum present across all animals was Bacteroidetes, followed by Firmicutes (Figure 2A). More specifically, Prevotella was the most predominant genus across all animals, both within the Bacteroidetes phylum and overall (data not shown). The alpha diversity analyses, in which the chao1 rarefaction measure was used to adjust for variation in total number of sequence reads per sample, suggests that L recipients exhibited greater rumen microbial diversity than H recipients, and the DL donors exhibited greater rumen microbial diversity than DH donors (Figure 2B). However, results from the beta diversity analyses suggest that rumen microbial communities of H recipient lambs did not cluster closely with rumen microbial communities of DH donors, and similarly, rumen microbial communities of L recipient lambs did not cluster closely with rumen microbial communities of DL donors (Figure 2C). Furthermore, the rumen microbial communities of each twin did not cluster closely with their respective twin in any of the twin sets (data not shown).
While the rumen microbial communities of recipient lambs were not similar to either their donor or their respective twin, there was a trend in low RFI animals exhibiting greater microbial diversity over high RFI animals. Moreover, 3 of the 5 sets (60%) of recipient twins exhibited the same feed efficiency status as their donor compared with their respective twin. These data taken all-together, suggest that there may be increased success in manipulation of feed efficiency if a larger group of recipient twins are inoculated with donor rumen fluid. Successful manipulation of feed efficiency may also rely more on microbial diversity of the donor compared with the specific composition of the microbial community.
Objectives 3 and 4
While further research is necessary to determine whether rumen manipulation may be successful, selecting for feed efficiency remains an economical trait to select on. Therefore, education materials will be developed and distributed to enhance producer awareness and understanding of feed efficiency measures and tools. Educational materials will include mailings of informational pamphlets to area producers (e.g., University of Wyoming Ram Test producers) regarding the importance of feed efficiency, testing for feed efficiency, ways to include feed efficiency in selection decisions (e.g., selection index), and an economic evaluation to detail the ways that selecting on feed efficiency can affect the bottom line. An update to the current selection index used at the University of Wyoming Ram Test has also been implemented (Spring 2016); producers have an interest in having feed efficiency as a part of that index. Finally, producer adoption will be determined via feedback on materials and information assessed using surveys and interviews.
Education and Outreach
We talked with producers involved in the UW Ram Tests to determine their opinions on feed efficiency and the incorporation of it into the selection index. Many producers were fairly knowledgeable about feed efficiency and were open to adopting RFI as a standard measure of feed efficiency. In spring 2016, RFI was incorporated into the selection index for the UW Ram Tests. Over the last few years, producer understanding of feed efficiency has grown. Education materials will continue to be developed and distributed to enhance producer awareness and understanding of feed efficiency measures and tools. Educational materials will highlight the importance of feed efficiency, testing for feed efficiency, ways to include feed efficiency in selection decisions (e.g., selection index), and an economic evaluation of financial benefits that could come from selecting for feed efficiency. Moreover, while results from this study were indefinite, the data from this research will be used as a baseline to seek funding to determine if trends from this study will become clearer with greater numbers of lambs.