Transitioning from conventional to organic milk production has allowed dairy farmers financially threatened to abandon the dairy business to become economically viable (O’Hara and Parsons, 2013). Typical northeastern organic dairies are small in size (~56 milking cows/herd; Pereira et al., 2013) and are more vulnerable to fluctuations in milk and grain prices than large confinement dairies, which rely on economies of scale (MacDonald and McBride, 2009). This problem is exacerbated as the climatic conditions in the Northeast require feeding conserved forages (i.e., hay, silage, baleage) during the winter months in addition to increased use of expensive grains (Marston et al., 2011; Pereira et al., 2013). In fact, organic dairies in the Northeast spend approximately 7 months of the year feeding exclusively conserved forages or conserved forages supplemented with grains. Thus, improving the nutritive value of baleage, the predominant conserved feed used in the region, to meet lactation requirements while reducing grain costs is crucial to improve the economic sustainability of northeastern forage-based dairies.
One of the major limitations of feeding high-forage rations is the reduction in milk production and increased output of nitrogen (N) and enteric methane to the environment (Brito et al., 2008; Hristov et al., 2013; see Figure 1). Therefore, forage-based diets must be formulated to increase the supply of energy (i.e., sugars and starch) relative to N (energy:N ratio) as this approach potentially increases the essential amino acids supply for milk and milk protein production, and reduces methane emissions by increasing forage digestibility. However, there is sorely needed, scientific-based information about the tradeoffs between milk production, nutrient utilization, and output of N and methane to the environment in forage-based dairy systems. Specifically, little is known about amino acids requirements and rumen microbiota diversity (e.g., methanogens, cellulolytic and proteolytic bacteria, protozoa) to guide farmers’ decisions on how to best optimize the use of forage-based diets. This project will fill these knowledge gaps by integrating research and educational approaches to improve the economic and environmental sustainability of dairy farms in the region.
Our recent (Pereira et al., 2013) and ongoing (USDA-NIFA-OREI award #2015-51300-24137) assessment of the research and educational needs of northeastern organic dairy farmers via surveys and focus groups revealed that improving forage quality is one of the top priorities in the region. We are proposing to grow alfalfa and red clover in combination with meadow fescue and timothy to increase the energy:N ratio within each mixture (1 legume and 2 grasses) by capitalizing on individual and complementary forage characteristics. Alfalfa has become the “gold standard” for dairy conserved forages and the alfalfa-grass mixture treatment will be used as the control. Alfalfa and red clover are known to have different concentrations of soluble N as a large proportion of alfalfa proteins undergo proteolysis during conservation (Muck, 1987; Pelletier et al., 2010). In contrast, red clover proteins are protected against proteolysis in the rumen by the presence of the polyphenol oxidase enzyme in red clover tissues resulting in less N output to the environment compared with alfalfa (Brito et al., 2007; Broderick et al., 2007). It is important to note that meadow fescue and timothy grew in mixtures with alfalfa resulted in high energy:N ratio (da Silva et al., 2014), and red clover was ranked first in energy concentration among 8 forage crops (Pelletier et al., 2010). We also demonstrated that alfalfa (Brito et al., 2008) and red clover (Antaya et al., 2015) with elevated energy:N ratio improved organic matter digestibility by an average of 2%-units in dairy cows.
This project will enable organic dairy farmers to improve forage quality by growing legume-grass mixtures selected to yield high energy:N ratio. We expect to increase organic matter digestibility by at least 2-% units, which would be equivalent to 1.7-lb of additional milk/cow as 1% increase in digestible organic matter of grass silage can enhance milk production by 0.83 lb daily (Keady et al., 2012). A 1.7-lb increase in milk production/cow would result in additional 20,000 lb of milk or 6,265$ in gross revenue (at 35$/hundredweight for milk price) assuming a herd size of 56 milking cows (Pereira et al., 2013) fed baleage with increased energy:N ratio over 7 months during the winter season. We will provide insights about rumen microbiota diversity and amino acids requirements, which are key to understand nutrient use efficiency as cows fed red clover-grass mixtures are expected to excrete less N to the environment than the alfalfa-grass counterpart.
This proposal is part of a recently funded USDA-NIFA-ORG project (award #2016-51106-25713) entitled “Developing advanced perennial legume-grass mixtures harvested as stored feeds to improve herd productivity and mitigate greenhouse gas emissions in organic dairies in the Northeast”. The scope of this larger USDA-NIFA-ORG project includes integrated research and educational approaches to mitigate greenhouse gas emissions through dairy cattle nutrition and agronomic trials in synchrony with outreach activities such as workshops and eOrganic webinars. In the current project, we are proposing 2 complementary research objectives and we will capitalize on the established outreach platform to deliver our Legume-Grass Mixture Feeding Guide, which is the direct educational product of the present submission to Northeast SARE. Our proposal addresses the Northeast SARE priorities: “reduction of environmental and health risks in agriculture” and “improved productivity, the reduction of costs, and the increase of net farm income”.
Specific objectives of this proposal include:
Objective 1: IDENTIFY AND QUANTIFY RUMEN MICROOBIOTA
Identify and quantify ruminal microorganisms (i.e., bacteria, protozoa, and methanogens) in cows fed baleage harvested from different legume-grass mixtures designed to yield high energy:N ratio.
Objective 2: MEASURE BLOOD AMINO ACIDS
Measure the concentration of amino acids in blood plasma in cows fed baleage harvested from different legume-grass mixtures designed to yield high energy:N ratio.
We hypothesize that: (1) baleage harvested from red clover-grass or alfalfa-grass mixture may result in different organic matter digestibility ultimately affecting methane emissions in lactating dairy cows; (2) Compared with red clover, cows fed alfalfa-grass baleage would have more proteolysis in the rumen resulting in less amino acids available for milk production and milk protein synthesis as alfalfa lacks polyphenol oxidase.
Our research will provide insights about rumen microbiota diversity and amino acids requirements, which are key to understand nutrient use efficiency as cows fed red clover-grass mixtures are expected to excrete less N to the environment than the alfalfa-grass counterpart. We also expect differences in methane emissions between alfalfa-grass and red clover-grass mixtures because these forage sources vary in organic matter digestibility in the rumen.
Twenty mid-lactation Jersey cows housed at the University of New Hampshire Burley-Demeritt Organic Dairy Research Farm will be assigned to 1 of 2 treatments: 1) alfalfa-meadow fescue-timothy mixture or 2) red clover-meadow fescue-timothy mixture fed as baleage. Seeding rates were done to target botanical composition levels at 70:15:15 for legume-grass-grass ratio. Cultivar selection was based on recommendations from forage evaluation programs at Cornell University and the University of Wisconsin, as well as from commercial plant breeders. A randomized complete block design (n = 10 cows/treatment) with a 2-week covariate period and a 10-week data collection period will be used. Cows will be blocked based on parity and days in milk, and will be fed individually twice daily using the electronic recognition Calan doors system. Animals will be milked 2 times daily with milk production recorded at every milking event. Body weight and body condition score (1 to 5 scale) will be recorded in the last week of the covariate period and monthly during the 10-week data collection period. Diets will be fed as total mixed ration consisting (dry matter basis) of 70% baleage and 30% of a corn meal/soybean meal-based grain mixture. Dry matter intake and milk production will be measured during the 2-week covariate period, and feeds, milk, blood, feces, urine, and rumen samples will be taken during the last week of each month thereafter (weeks 4, 7, and 10).
Feed and refusal samples will be collected weekly, pooled by period (i.e. monthly), freeze-dried, ground (1-mm screen), and later analyzed for dry matter, ash, total N, neutral detergent fiber, acid detergent fiber, and minerals using wet chemistry and mass spectrophotometric methods by a commercial laboratory (Dairy One, Ithaca, NY). In addition, feeds will be shipped to the Pennsylvania State University (University Park, Pennsylvania) for individual fatty acids analysis by gas liquid chromatography after direct methylation as reported in Resende et al. (2015). Ammonia, pH, organic acids, and ethanol will be measured on baleage extracts. Milk samples with preservatives will be collected during 4 consecutive milkings and analyzed for fat, protein, lactose, total solids, and milk urea-N using mid-infrared reflectance spectroscopy (Dairy One). Milk samples without preservative will be collected concurrently and stored at -80°C until fatty acids analysis. Samples will be extracted and analyzed for milk fatty acids by gas liquid chromatography at the Pennsylvania State University following procedures outlined by Resende et al. (2015). Blood samples will be collected from the tail vein using vacutainer tubes for 2 consecutive days approximately 4 hours after the morning feeding, pooled, and later analyzed for plasma urea-N (Brito et al., 2008).
Fecal grab samples (~200 g) will be collected once daily for 3 consecutive days at early morning (day 1), noon (day 2), and late afternoon (day 3) by stimulating defecation or collected directly from the rectum. Samples will be pooled by cow based on fresh weight over the 3 days to obtain a single composite and stored at -20°C. At the end of each sampling week, composited fecal samples will be thawed, oven-dried at 55°C, ground (1-mm screen), and sent to a commercial laboratory (Dairy One) to be analyzed for the same nutrients done for feeds (see above). Approximately 0.5-g of total mixed ration, individual feed ingredients, and feces will be placed in 100-cm2 bags (n = 3 replicates/sample) and incubated in the rumen of a rumen-fistulated lactating dairy cow for 12 days to determine the concentration of indigestible acid detergent fiber as reported by Resende et al. (2015). Indigestible acid detergent fiber will be used as an intrinsic marker to estimate fecal output of N and nutrient digestibility (Resende et al., 2015). A conventional, rumen-fistulated cow housed at the University of New Hampshire Fairchild Dairy Teaching and Research Center will be used as fistulation surgery usually requires the use of antibiotics, which are not allowed under the National Organic Program rules.
Spot urine samples will be taken once daily for 3 consecutive days concurrently with the fecal samples by massaging the pudendal nerve located below the vulva. Samples (~200 mL) will be diluted with 0.072 N sulfuric acid to prevent N losses, pooled over the 3 days by cow, and stored at -20°C until analyzed for creatinine, allantoin, uric acid, urea, ammonia, and total N using colorimetric methods (Broderick et al., 2007). Daily urinary volume will be estimated based on the concentration of creatinine assuming a constant creatinine excretion rate of 29 mg/kg of body weight (Valadares et al., 1999). Urinary excretion of total N and total purine derivatives (allantoin plus uric acid) will be calculated based on their concentration in the urine multiplied by the urinary volume. Intake of N, and N output in milk, feces, and urine will be used to calculate N balance and N use efficiency. Total purine derivatives will be used as intrinsic microbial markers to estimate microbial protein synthesis (Broderick et al., 2007).
Methane and carbon dioxide emissions will be measured using the GreenFeed system (C-Lock Inc., Rapid City, South Dakota; see Facilities and Equipment) throughout the 2-week covariate and 12-week data and sample collection periods. The GreenFeed operates by releasing a bait feed up to 5 times per feeding event with a minimum of 45 s apart triggered by a radio frequency ear tag and the cow’s head located inside the feed manger resulting in accurate breath sampling and near real-time analysis of methane and carbon dioxide emissions using built-in non-dispersive infrared gas sensors. Gas sampling will be conducted according to the voluntary visits of the cows to the GreenFeed unit, usually 4 to 5 times/day.
Samples of rumen fluid (~250 mL) will be collected once at weeks 4, 7, and 10 from all cows via stomach tubing approximately 4 h after the morning feeding. During sample collection, cows will be restrained using a squeeze chute to minimize movement. After collection, samples will be squeezed through 4 layers of cheesecloth with pH immediately measured using a portable pH meter. A 10-mL aliquot of rumen fluid will be mixed with 10 mL of 50% formalin and stored in centrifuge tubes at room temperature for later enumeration of total protozoa. A second 10-mL aliquot of strained rumen fluid will be added to 0.2 mL of 50% sulfuric acid and stored at -20°C for later analysis of ammonia-N (Resende et al., 2015). A third 10-mL aliquot of strained rumen fluid will be processed as done for ammonia-N and stored at -20°C until volatile fatty acids (acetate, propionate, butyrate) analysis by gas liquid chromatography (Resende et al., 2015). The concentration of volatile fatty acids and ammonia-N will be used for characterizing the fermentation profile in response to feeding baleage with different legume-grass botanical composition.
A forth aliquot of rumen fluid samples (2 mL) will be stored at -80°C in cryovial tubes until sent to the Molecular Research DNA laboratory (Shallowater, TX) for DNA extraction, quantitative real-time polymerase chain reaction (qPCR), and PCR amplification and sequence analysis. Microbial DNA will be extracted using the PowerSoil DNA isolation method (kit #12888-100; MO BIO Laboratories Inc., Carlsbad, CA) following the manufacturer’s guideline. DNA will be eluted in 100 µL C6 solution and quantified using a NanoDrop 2000 UV-VIS spectrophotometer, and qPCR will be conducted in 1 µL of template DNA using the TaqMan 2x Universal PCR Master Mix Kit (Thermo Fischer Scientific, Waltham, MA) in a StepOnePlus Real-Time PCR System. The estimated bacterial count per µL of DNA elution will be estimated by comparison against a known standard with serial 10-fold dilutions. Amplification of V4 variable region of the bacterial and methanogen 16S rRNA gene will be conducted using the primer pair 515F (5’GTGCCAGCMGCCGCCCTA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The protozoa primer pair GIC1080F (5’-GGGRAACTTACCAGGTCC-3’) and GIC1578R (5’-GTGATRWGRTTTACTTRT-3’) will be used by PCR to amplify the V6-V8 and signature regions 3 and 4 of 18S rRNA gene. The PCR amplifications will be performed using the HotStarTaq Plus Master Mix DNA polymerase kit (QIAGEN, Germantown, MD). All PCR products will be checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples will be pooled in equal proportions based on their molecular weight and DNA concentrations and then purified using calibrated Ampure XP beads (Beckman Coulter, Brea, MA). The purified PCR products will be used to prepare DNA libraries following Illumina TruSeq (Illumina Inc., San Diego, CA) DNA library preparation protocol. Sequencing will be performed on a MiSeq (Illumina Inc.) following the manufacturer’s guideline. Sequence data will processed using Molecular Research DNA laboratory analysis pipeline. Operational taxonomic unit (OTU) will be defined by clustering at 3% divergence (97% similarity). Final OTU will be taxonomically classified using BLASTn against curated databases. After sequencing, bioinformatics will be performed using the software MOTHUR.
Rumen protozoa enumeration will be done as described by Dehority (1993). In brief, 2 drops of brilliant green dye will be added to 1 mL of preserved rumen fluid and allowed to stand overnight for protozoa cell staining. After staining, 9 mL of 30% glycerol solution will be added to the mixture and 1 mL of the diluted sample pipetted into a Sedgewick-Rafter counting chamber (1 cm3 volume). Further dilutions will be made as needed to bring total protozoa number to a range of 100-200 counts per 50 grids. Protozoa cells will be counted in duplicate at a 100× magnification (Olympus HB2, Tokyo, Japan).
Blood samples from all 24 cows will be collected as describe above. After collection, samples will be centrifuged (1,200 × g, 20 min, 4°C) and 4-mL aliquot of plasma will be placed in test tubes containing 1.0 mL of 15% of sulfosalicylic acid followed by a second centrifugation. An aliquot of 1.8 mL of deproteinized plasma will be then removed and analyzed for amino acids by high performance liquid chromatography. Feeds will be hydrolyzed using hydrochloric acid and performic acid oxidation and analyzed for amino acids using the same procedure done for plasma.
Data will be analyzed using the MIXED procedure of SAS according to a randomized complete block design with repeated measures over time. The statistical model will include treatments, block, week, interactions, and the covariate period. Block and block × treatment effects will be considered random, whereas all other model terms will be considered fixed.
We were able to establish 3 legume-grass cropping systems as originally proposed: 1) alfalfa-grass mixture, 2) red clover-grass mixture, and birdsfoot trefoil-grass mixture. Last fall all 3 fields were harvested as baleage and yielded 197 bales distributed as: 68 alfalfa-grass bales, 75 red clover-grass bales, and 54 birdsfoot trefoil-grass bales. The botanical composition of the alfalfa-grass and red clover-grass baleage was as expected with a greater proportion of legumes than grasses. However, the birdsfoot trefoil-grass botanical composition was not was expected and resulted in much less proportion of legume than grass. In addition, field was “spotty” with some parts where birdsfoot trefoil were well established and other points in which birdsfoot was present at all. Therefore, we decided not to use the birdsfoot trefoil-grass baleage in the feeding trial this time. The experiment will begin in mid-February, 2019 with cows fed 2 treatments: alfalfa-grass mixture versus red clover-grass mixture. We will continue monitoring the birdsfoot trefoil field this year as this legume is more slowing to establish compared with alfalfa and red clover.
Education & Outreach Activities and Participation Summary
Dr. André F. Brito will supervise and mentor Mohammad Ghelich Khan to complete the proposed research successfully. Specifically, Dr. Brito will assist Mohammad to write scientific and farmer-oriented articles and Northeast SARE reports. Dr. Brito will be actively involved in preparation and dissemination of the Legume-Grass Mixture Feeding Guide as well. He will oversee the project and will be responsible for disseminating results through farmer-oriented conferences (e.g., Vermont Organic Dairy Producers Conference, NODPA Field Days and Annual Meeting, NOFA-NY Organic Dairy and Field Crop Conference), as well as local, regional, and national scientific meetings. Dr. Brito is currently leading projects funded by USDA-NIFA-OREI, USDA-NIFA-ORG, and Northeast SARE and will use this established network to educate farmers about perennial legume-grass mixtures via workshops, field days, pasture walks, and eOrganic webinars.