Source: KANSAS STATE UNIV submitted to
ESTABLISHING THE VALUE OF ALFALFA WITH HIGHLY DIGESTIBLE FIBER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1020486
Grant No.
2019-70005-30238
Cumulative Award Amt.
$478,826.00
Proposal No.
2019-02803
Multistate No.
(N/A)
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2022
Grant Year
2020
Program Code
[AFRP]- Alfalfa and Forage Program
Project Director
Min, D.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Agronomy
Non Technical Summary
Alfalfa (Medicago sativa L.) is the most common and important forage crop grown across the U.S. The value of this alfalfa forage crop exceeds $9.5 billion U.S. dollars, and the domestic dairy market is the largest single outlet for this crop. Alfalfa produces high DM yield as maturity increases, but nutritive value decreases, particularly after blossoming. In other words, fiber components such as hemicellulose, cellulose, and lignin increase as stage of maturity proceeds, which lowers digestibility. Although lignin is an important component for plant rigidity, it also has particularly negative effects on digestibility. High quality forages are especially critical in dairy production because of the tenuous balance between the dramatic nutrient requirements of modern dairy cows and their need for forage fiber to maintain rumen health. Harvesting alfalfa at the optimum stage (such as late bud to early bloom) has become common practice among alfalfa grower targeting the dairy market in the U.S. Thus, if alfalfa producers miss the optimum stage of maturity in harvesting alfalfa, nutritive values of alfalfa can be negatively affected, often resulting in the need to market hay for less demanding species, at a lower price.To deal with this inverse relationship between forage yield and quality with stage of maturity, reduced-lignin and more digestible alfalfa varieties have been developed. The use of alfalfa varieties with improved nutritive value has some potential to be beneficial to alfalfa growers. For example, reduced-lignin alfalfa potentially can achieve higher DM yields by delaying alfalfa harvest while still maintaining higher nutritive values. This could allow for a wider optimal harvest window, providing growers management flexibility to delay harvest to a later stage of maturity. Currently, reduced-lignin alfalfa varieties are commercially available, but only one peer-reviewed paper has been published, focusing on forage agronomy and laboratory-estimated nutritive value of reduced-lignin alfalfa. Many producers thus rely on "guess work" to determine the optimum time of first harvest for these new varieties. In general, dry matter yields of alfalfa from first harvest represent 1/2 to 2/3 of total annual yields. However, no data is available on how the first cutting date affects DM yield and forage quality during the rest of the growing season for reduced-lignin alfalfa varieties as compared to commercial alfalfa varieties. In addition, the agronomic and the quality performance of the popular alfalfa varieties in KS and NE and more importantly the low lignin alfalfa under different water-deficit conditions under field conditions is unknown. Globally, there has been just one study so far quantifying water-deficit stress response in alfalfa from China , hence indicating the large knowledge gap that exists in our understanding the responses alfalfa varieties of local and national interest. Similarly, estimating whole plot water use efficiency through a powerful carbon isotope discrimination approach has not been determined for environments that alfalfa is grown in the U.S. Results from the proposed research objectives will be used to inform farmers' decisions on first cutting of alfalfa; in particular, how late reduced-lignin alfalfa can be harvested after blossoming compared to other conventional alfalfa varieties under different water-deficit stress scenarios. In particular, we will conducting the very first in-depth physiological and agronomic research using rain-out shelters exposed to a wide range of water-deficit stress conditions during different stages of maturity. The information generated will provide useful information on alfalfa water use information to optimize cutting strategies. Tradeoffs between DM yield and nutritive value will also be studied, including how much these tradeoffs are altered in reduced-lignin alfalfa versus conventional alfalfa varieties in different stages of maturity under different water-deficit conditions.To an even greater extent, dairy nutritionists are forced to extrapolate and guess about the appropriate feeding strategies for reduced-lignin alfalfa varieties. Often, nutritionists make assumptions about responses to these new forages based on responses to high-NDFD corn silage. However, this is a flawed approach because these are extremely different forage types, particularly when comparing corn silage to dry alfalfa hay. The impact of reduced-lignin alfalfa on dairy cattle need to be tested systematically, rather than guessing, which almost always leads to inconsistencies and lowers farmer income.Our proposal is accompanied by several letters of support that point to the need that has been communicated to us by our industry colleagues. There is a real and urgent need to address practical questions about reduced-lignin alfalfa management and feeding strategies before these traits can offer their full advantages throughout the alfalfa value chain. After successful completion of this project, we anticipate a growing acceptance of a more modern forage evaluation index (RFQ or "Eureka"), the emergence of a price differential for high-NDFD alfalfa, and substantially greater economic and management incentives for adoption of alfalfa with improved digestibility traits. Ultimately, these changes will not only improve the bottom line for alfalfa growers and dairies, but will improve the sustainability of U.S. agriculture since less land will be devoted to growing alfalfa that simply goes through dairy cows undigested.Our research team is particularly well suited to this project and its joint research and extension goals. Our institutions straddle the border between the eastern and western dairy industries, and all three investigators routinely speak at forage and dairy industry conferences throughout the U.S. and world-wide. Kononoff (UNL) is currently serving on the committee that is revising the National Research Council's Nutrient Requirements of Dairy Cattle, which is the go-to resource for feeding dairy cattle in the U.S. Collectively, we have the research experience and industry network to help address this significant gap in knowledge and begin to resolve a market inefficiency.The basic methods and approaches to collect and produce data/results will be implemented by conducting agronomic research by growing different alfalfa varieties in the field, lab analysis for forage quality, and dairy feeding trials using low lignin alfalfa varieties and other conventional varieties. During a research period, we will have field days to invite alfalfa producers, dairy farmers, and ag-industry people to explain the research trials in both Kansas and Nebraska. Our team also is going to develop Extension Factsheets, videos, and on-demand website as anextension educational resource for alfalfa producers, dairy farmers, and ag-industry people.Ultimate goals to achieve from this project:1) assesing water use efficiency of reduced lignin alfalfa compared to conventional varieties2) determinig precise information on milk yield and feed efficiency responses of highly-productive dairy cattle fed reduced-lignin alfalfa3) determinig economic impacts of feeding highly digestible alfalfa such as reduced lignin alfalfa4) making wider adoption of changes in forage analysis and valuation in alfalfaGeneral impact expected to be if this goal is met:1) Better management practices of growing reduced alignin alfalfa with more flexible harvesting schedule while maintaining high forage quality2) A growing acceptance of more modern forage evaluation index such as relative forage quality (RFQ) in the hay marketplace3) Emergence of a price differential for highly digestible alfalfaSocietal benefits being realized1) greater economic and management incentives for adoption of alfalfa with flexible harvesting window and improved digestibility traits2) improvement of sustainability of alfalfa-based dairy production systems
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20516401060100%
Knowledge Area
205 - Plant Management Systems;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
Alfalfa (Medicago sativa L.) is the most common and important forage crop grown across the U.S. The value of this alfalfa forage crop exceeds $9.5 billion U.S. dollars (USDA-NASS, 2019), and the domestic dairy market is the largest single outlet for this crop (Putnam et al, 2001). Alfalfa produces high DM yield as maturity increases, but nutritive value decreases, particularly after blossoming. In other words, fiber components such as hemicellulose, cellulose, and lignin increase as stage of maturity proceeds, which lowers digestibility (Albrecht et al., 1987; Jung et al., 1997a, 1997b; Sewalt et al, 1997; Casler et al., 2002). Although lignin is an important component for plant rigidity, it also has particularly negative effects on digestibility. High quality forages are especially critical in dairy production because of the tenuous balance between the dramatic nutrient requirements of modern dairy cows and their need for forage fiber to maintain rumen health. Harvesting alfalfa at the optimum stage (such as late bud to early bloom) has become common practice among alfalfa grower targeting the dairy market in the U.S. Thus, if alfalfa producers miss the optimum stage of maturity in harvesting alfalfa, nutritive values of alfalfa can be negatively affected, often resulting in the need to market hay for less demanding species, at a lower price.To deal with this inverse relationship between forage yield and quality with stage of maturity, reduced-lignin and more digestible alfalfa varieties have been developed. The use of alfalfa varieties with improved nutritive value has some potential to be beneficial to alfalfa growers. For example, reduced-lignin alfalfa potentially can achieve higher DM yields by delaying alfalfa harvest while still maintaining higher nutritive values. This could allow for a wider optimal harvest window, providing growers management flexibility to delay harvest to a later stage of maturity. Currently, reduced-lignin alfalfa varieties are commercially available, but only one peer-reviewed paper has been published (Grev et al., 2017), focusing on forage agronomy and laboratory-estimated nutritive value of reduced-lignin alfalfa. Many producers thus rely on "guess work" to determine the optimum time of first harvest for these new varieties. In general, dry matter yields of alfalfa from first harvest represent 1/2 to 2/3 of total annual yields. However, no data is available on how the first cutting date affects DM yield and forage quality during the rest of the growing season for reduced-lignin alfalfa varieties as compared to commercial alfalfa varieties. In addition, the agronomic and the quality performance of the popular alfalfa varieties in KS and NE and more importantly the low lignin alfalfa under different water-deficit conditions under field conditions is unknown. Globally, there has been just one study so far quantifying water-deficit stress response in alfalfa from China (Liu et al., 2018), hence indicating the large knowledge gap that exists in our understanding the responses alfalfa varieties of local and national interest. Similarly, estimating whole plot water use efficiency through a powerful carbon isotope discrimination approach has not been determined for environments that alfalfa is grown in the U.S. Results from the proposed research objectives will be used to inform farmers' decisions on first cutting of alfalfa; in particular, how late reduced-lignin alfalfa can be harvested after blossoming compared to other conventional alfalfa varieties under different water-deficit stress scenarios. In particular, we will conducting the very first in-depth physiological and agronomic research using rain-out shelters exposed to a wide range of water-deficit stress conditions during different stages of maturity. The information generated will provide useful information on alfalfa water use information to optimize cutting strategies. Tradeoffs between DM yield and nutritive value will also be studied, including how much these tradeoffs are altered in reduced-lignin alfalfa versus conventional alfalfa varieties in different stages of maturity under different water-deficit conditions.To an even greater extent, dairy nutritionists are forced to extrapolate and guess about the appropriate feeding strategies for reduced-lignin alfalfa varieties. Often, nutritionists make assumptions about responses to these new forages based on responses to high-NDFD corn silage. However, this is a flawed approach because these are extremely different forage types, particularly when comparing corn silage to dry alfalfa hay. The impact of reduced-lignin alfalfa on dairy cattle need to be tested systematically, rather than guessing, which almost always leads to inconsistencies and lowers farmer income.Our proposal is accompanied by several letters of support that point to the need that has been communicated to us by our industry colleagues. There is a real and urgent need to address practical questions about reduced-lignin alfalfa management and feeding strategies before these traits can offer their full advantages throughout the alfalfa value chain. After successful completion of this project, we anticipate a growing acceptance of a more modern forage evaluation index (RFQ or "Eureka"), the emergence of a price differential for high-NDFD alfalfa, and substantially greater economic and management incentives for adoption of alfalfa with improved digestibility traits. Ultimately, these changes will not only improve the bottom line for alfalfa growers and dairies, but will improve the sustainability of U.S. agriculture since less land will be devoted to growing alfalfa that simply goes through dairy cows undigested.Our research team is particularly well suited to this project and its joint research and extension goals. Our institutions straddle the border between the eastern and western dairy industries, and all three investigators routinely speak at forage and dairy industry conferences throughout the U.S. and world-wide. Kononoff (UNL) is currently serving on the committee that is revising the National Research Council's Nutrient Requirements of Dairy Cattle, which is the go-to resource for feeding dairy cattle in the U.S. Collectively, we have the research experience and industry network to help address this significant gap in knowledge and begin to resolve a market inefficiency.OBJECTIVES1. Physiological and agronomic characterization of field-grown alfalfa cultivars exposed to a range of water-deficit stress conditions under different stages of maturity. 2. Determine impacts of alfalfa hay varying in NDF digestibility on productivity and energetics of lactating dairy cows.3. Develop and disseminate programing for alfalfa producers, dairy nutritionists, and dairy producers that seeks to achieve a better understanding of analytical-based valuations of alfalfa, enhanced production of high quality forages and overall farm profitability.
Project Methods
1) An agronomic field experiment will be conducted at the KSU North Agronomy Farm (39.2181° N, 96.5907° W) in Manhattan, KS. Six different alfalfa varieties (2 most popular varieties in KS: AmeriStand455TQRR and Gunner, 2 most popular varieties in NE: HybriForce-3400 and AFX469, and 2 reduced lignin varieties: HVXMegaTron and HVXHarvaTron) will be used for small plot agronomic field research under a rain-out shelter facility (Fig. 6a). A recommended seeding rate of 22.4 kg pure live seed per hectare will be used. The study will be planted in late summer or early fall of 2019 using a commercial grade planter with a recommended spacing (e.g., rows 7 inches apart). Before planting alfalfa, soil samples will be taken for basic analysis at the KSU Soil Testing Lab. Individual plot size will be 6.0 ft (1.8 m) × 20.0 ft (6.0 m). With 1,200 ft2 (111 m2) planting area in each of the rain-out shelter, we will be able to accommodate six large blocks one for each of the six varieties, in a rain-out shelter. Alfalfa forage samples will be collected to assess alfalfa yield and nutritive values respond in different stages of maturity (i.e., early bud, early bloom, and 7 days after early bloom) under 3 different water treatments (rainfed, irrigated, water-deficit stress using rain shelters). The irrigated plots will be watered regularly to ensure no water limitation throughout the experiment, while the rainfed plots will be maintained naturally, similar to growers field conditions. Stress in the rain-out shelters will be initiated about 5 days before the early-bud stage (previous information collected on these lines will be used to make a calculated estimate on the early bud stage). Before harvesting all 6 varieteis across all replicates and all three treatments will be measured using (i) portable photosynthesis system (LI-6400 XT, LI-COR) to calculate the instantaneous water use efficiency i.e., A(assimilation)/gs(stomatal conductance) and the photosynthetic rate; (2) Chloroplyll content using SPAD to determine changes in chlorophyll index (Bergkamp et al., 2018). Both these key physiological measurements will be collected from 09:30 AM to 11:30 AM following Sunoj et al., 2017, Sun et al., 2018. In addition, before harvesting the plot, the top most set of leaves will be sampled and the wet and dry weight will be recorded to determine the leaf moisture content. In addition, these collected leaves will be ground to capture the signature of the water-deficit stress using carbon isotope discrimination (CID), which is a powerful surrogate to estimate time integrated measure of whole plant/plot water-use-efficiency in C3 crops (Moghaddam et al., 2013; Zhu et al., 2019). CID will be measured over three different developmental stages (early bud, early bloom and 7 days after early bloom) experiencing varying levels of water-deficit stress in the six different alfalfa varieties will provide extensive knowledge on the alfalfa water-use-efficiency under varying conditions where alfalfa is currently grown in the Plains and the regions of the U.S. Nutritive values of alfalfa including crude protein, sugar, ash, acid detergent fiber, lignin, NDF, ash-free NDF, and NDF digestibility will be measured from each cutting during production years of 2020 and 2021, respectively.?2)Sixty lactating Holstein dairy cows will be used in a randomized complete block design study with a covariate period. Cows in early to mid-lactation (40 to 100 days in milk) from the Kansas State University Dairy Teaching and Research Center will first be acclimated to tie stalls and the CON diet during a 14-day covariate period. Cows will be fed total mixed rations twice per day, offering 110% of expected feed intake to ensure ad libitum access to feed. Water will be available free choice at all times. Cows will be milked 3 times daily in a milking parlor with automatic milk weight and sample collection equipment. During the last 4 days of the covariate period, milk samples will be collected for analysis of milk components (fat, protein, lactose). These methods are routine for co-PI Bradford (e.g., Olagaray et al., 2018).At the end of the covariate period, milk yield on CON diet during the 4-day collection period will be used along with parity and stage of lactation to block cows (n = 3 similar cows per block). Treatments will then be randomly assigned within block (n = 20/treatment).Cows will be fed their assigned treatment diets for 6 weeks, and management will continue as described for the covariate period. Milk yield and feed intake data will be collected daily throughout the study, and milk samples will be collected at all 3 milkings on one day/week for analysis of milk components. Yields of all milk components will be analyzed, but milk fat yield will be of particular interest as an indirect marker of adequate physically-effective fiber concentrations in diets.3)Twelve multiparous lactating Jersey dairy cows will be used. All cows will be housed in a temperature-controlled barn at the Dairy Metabolism Facility at the Animal Science Complex at the UNL (Lincoln, NE) and milked twice daily in individual tie stalls equipped with rubber mats. All animal care and experimental procedures will be approved by the UNL Animal Care and Use Committee. At the conclusion of the last experimental period, all cows will be less than 90 d pregnant and as a result, energy committed to fetal development will be minimal. The experimental design will be a three times replicated 3 × 3 Latin square. Cows will be randomly assigned to 1 of the 3 dietary treatments according to Kononoff and Hanford (2006).Animals will be blocked into each square by milk production and days in milk. Treatments will be alternated over 3 experimental periods and measurements will be collected on each animal consuming each dietary treatment. The study will be conducted with a total of 3 experimental periods, each being 28-d in duration. Each period included 21-d for ab libitum diet adaptation, targeting approximately 5% feed refusal during that time, followed by 4-d of collection with 95 % ad libitum feeding to reduce the amount of refusals. All dietary treatments will contain corn silage, alfalfa hay, brome hay, and a concentrate mixture that will be combined as a total mixed ration (TMR).

Progress 09/01/19 to 08/31/22

Outputs
Target Audience:Target audiences include farmers, both undergraduate and graduate students, researchers, and industry people. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?- Twograduate students were trained at the University of Nebraska and Michigan State University. How have the results been disseminated to communities of interest?Extension and outreach programs are as follows: -Title: UNL Research Updated, Paul Kononoff Nebraska Dairy Convention February 17, 2022 Columbus, NE -Title: The Penn State Particle Separator; after 25 years of use, what it tells us about forages and TMR? Penn State Dairy Nutrition Workshop November 2-3, 2022 Harrisburg, PA -Reduced lignin alfalfa: updates on agronomy and feeding (joint with Kim Cassida). Tri-State Dairy Nutrition Conference. Apr. 12-13, 2022. Ft. Wayne, IN. -Feeding low lignin alfalfa. Great Lakes Forage & Grazing Conference. March 17, 2022. St. Johns, MI. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? There is little data demonstrating the net effect of lower-lignin (LL) alfalfa (Medicago sativa L.) on dairy cattle. Specific objectives include: 1) Agronomic characterization of field-grown alfalfa cultivars exposed to a range of water-deficit stress conditions during different stages of maturity, and 2) Determine impacts of alfalfa hay varying in neutral detergent fiber digestibility (NDFD) on productivity and energetics of lactating dairy cows. The first production year results showed that the highest dry matter (DM) yield was observed in the conventional variety followed by lower-lignin alfalfa harvested 7 days after early flowering grown under irrigation at Kansas State University. In a Michigan State University feeding trial, treatment had no impact on milk yield or protein content, but lower-lignin (LL) alfalfa hay linearly decreased milk fat concentration when it replaced a conventional variety (CON; Hibriforce 3400). The LL hay was a 50:50 blend of an engineered LL alfalfa, HarvXtra 54HVX42 and a breeding-derived LL variety, HiGest 460. Total-tract NDF digestibility was also linearly decreased by LL, with no impact on total-tract crude protein (CP) or starch digestibility. In a University of Nebraska feeding trial, no difference was observed for milk fat yield and content or milk protein yield and content. No differences were observed on total-tract digestibility (%) of DM (averaging 67.2), NDF (averaging 51.0), or CP (averaging 67.2). Feeding study results from both Michigan and Nebraska suggest that replacing conventionl alfalfa with lower-lignin alfalfa has no beneficial effects on milk production, milk composition, or nutrient digestibility.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Yield Gap Analysis of Rainfed Alfalfa in the United States. Frontiers in Plant Science. DOI: 10.3389/fpls.2022.931403


Progress 09/01/20 to 08/31/21

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Further activities Forage nutritive value analysis CID analysis Fertilizer application Cost-benefit analysis of each treatment Presentabstracts at the American Dairy Science Association annual meeting, June 2022 Submitmanuscripts for publication in the Journal of Dairy Science Contribute to advisory panel meetings and co-develop Extension materials for improving alfalfa valuation in the field

Impacts
What was accomplished under these goals? Forage agronomy trial (Kansas State University) Forage dry matter yield (DMY) There is a significant yield difference among varieties, growth stages, and water treatments but replication has no effect on DMY (Table 1). Table 1. Statistical analysis of dry matter yield by varieties, treatments, cuttings, and growth stages Source DF Type III SS Mean Square F Value Pr > F TRT 2 153.2 76.6 121.8 *** REP 3 0.4 0.1 0.2 NS Cutting 4 1354.0 338.5 537.9 *** GS 2 106.6 53.3 84.7 *** Variety 4 25.8 6.4 10.2 *** TRT*Cutting 8 102.0 12.8 20.3 *** TRT*Growth_stage 4 82.0 20.5 32.6 *** TRT*Variety 8 15.6 1.9 3.1 ** TRT*Cutting*GS*Varieties 187 525.5 2.8 4.5 *** Note: TRT: Treatment, REP: Replication, GS: Growth stage, *** Level of significance at 1% and ** at 5% In the 2021 production year, the average dry matter yield of alfalfa per cutting (all varieties) grown under water drought imposed (T1), irrigated (T2) and rainfed conditions (T3) were found 3.7, 4.5, and 3.7 Mg ha-1 respectively. The average yield of alfalfa in T1 and T3 was found similar because the soil fertility and soil moisture were found higher in T1 at the beginning of the establishment year than in T3 and T3 had poor germination and growth in the establishment year. But the yield was drastically dropped down in T1 after a first cutting. After first cutting, the average dry matter yield was found 2.6, 3.8, and 2.9 Mg ha-1 in drought imposed, irrigated, and rainfed conditions respectively. Besides, water treatments, there was no significant yield difference observed among varieties. The variety HighGest AFX460 gave the highest DMY under irrigated conditions whereas variety HybriForce-3400 performed better during drought conditions. Dairy Cattle Feeding Research (Michigan State University and University of Nebraska)

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Diatta, A., D. H. Min, K. Jagadish. 2021. Drought Stress Responses in Non-transgenic and Transgenic alfalfa  Current Status and Future Research Directions. 170:1-50. Advances in Agronomy


Progress 09/01/19 to 08/31/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Agronomic and physiological research at K-State: Since this is a seeding year, our goal is to get the alfalfa stands well-established. Thus, our first cutting will be sometime in late July and possibly one more cutting in late September in 2020, depending on growth and weather condition in Manhattan, KS. Alfalfa forage samples will be collected to assess alfalfa yield and nutritive values response at different stages of maturity (i.e., early bud, early bloom, and 7 days after early bloom) under 3 different water treatments (rainfed, irrigated, water-deficit stress using rain shelters), from second cutting onwards, i.e. September 2020. Considering the smaller size plots within the rain-out shelters, at least two 2-meter rows per each cultivar and at least three replicate samples will be hand harvested from all three treatments (water-deficit stress, irrigated and rainfed) in fall 2020. Subsamples (500 g) from each harvested plot will be collected and forced-air oven dried for 72 h at 140°F (60°C) to estimate moisture content, which will be used to adjust bulk yield samples to a dry matter (DM) basis. Ground forage samples will be sent to a commercial lab in Nebraska for forage quality analysis (i.e., Moisture, Dry Matter, Crude Protein, Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF), Calcium, Phosphorous, Potassium, Magnesium, Ash, NDFD48, IVTDMD48, Fat, Lignin, Non-Fiber Carbohydrates (NFC), Starch, Ethanol Soluble Carbohydrates (ESC), Water Soluble Carbohydrates (WSC). Calculated: Relative Feed Value (RFV) Relative Forage Quality (RFQ). The irrigated plots will be watered regularly to ensure no water limitation throughout the experiment, while the rainfed plots will be maintained naturally, similar to growers' field conditions. Stress in the rain-out shelters will be initiated about 10 days before the early-bud stage (previous information collected on these lines will be used to make a calculated estimate on the early bud stage). Each tent will have a mix of readings from neutron tubes and tensiometers to record the soil moisture stress level. Counts in the experimental plots from the access tubes will be recorded once every 3 days during the stress imposition period, within each cutting interval using a neutron probe (Model 503DR Hydroprobe Moisture Depth Gauge by Campbell Pacific Nuclear International, Inc. Martinez, CA). In addition, the tensiometers will provide an additional complimentary data on soil moisture content. The access tube and neutron probe allow us to obtain the counts at five different soil depths (15, 45, 75, 105, and 135 cm) for each tube. Using the wet and dry calibrations (developed by Dr. Jagadish team) the amount of soil available water at different depths of the soil will be estimated, to help ascertain the level of stress imposed during the key developmental stages i.e., bud and bloom stages. The tensiometers will provide confirmatory soil moisture data at the top 15 cm depth. The rain-out shelters are fully automated which are programmed to cover the crop during a rain event and then moves back to the original parking position 30 min after the rain stops, exposing the alfalfa plots to natural environmental conditions. We will impose stress during the September cutting in 2020, with an aim to have well established plants following the first July 2020 cutting. Alfalfa forage samples will be collected to assess alfalfa yield and nutritive values at different stages of maturity (i.e., early bud, early bloom, and 7 days after early bloom) under 3 different water treatments (rainfed, irrigated, water-deficit stress using rain shelters). Before harvesting all three varieties across three replicates and all three treatments will be measured using (i) portable photosynthesis system (LI-6400 XT, LI-COR) to calculate the instantaneous water use efficiency i.e., A (assimilation)/gs (stomatal conductance) and the photosynthetic rate; (ii) Chloroplyll content using SPAD to determine changes in chlorophyll index. Both these key physiological measurements will be collected between 0930 AM to 1130AM. In addition, before harvesting the plot, about 5 topmost set of leaves will be sampled and the wet and dry weight will be recorded to determine the leaf moisture content. In addition, these collected leaves will be ground to capture the signature of the water-deficit stress using carbon isotope discrimination (CID), which is a powerful surrogate to estimate time integrated measure of whole plant/plot water-use-efficiency in C3 crops. CID will be measured over three different developmental stages (early bud, early bloom and 7 days after early bloom) experiencing varying levels of water-deficit stress in four different alfalfa varieties will provide extensive knowledge on the alfalfa water-use-efficiency. In October 2019, a Co-PI, Barry Bradford accepted a position at Michigan State University, and after deliberating with my colleagues on the project, we collectively agreed to work toward a sub-award for me to carry out objective 2a at MSU. Dr Bradford is working with his dairy research colleagues at MSU to reserve a time in late 2020 through early 2021 to complete the 60-cow feeding experiment; to fit within the system here, they will likely run two cohorts of 30 cows back-to-back, which will fit into our original design and statistical analysis plan without changes. We aim to have results and analysis completed by late April 2021 to enable an MS student to defend his thesis. All three stands will be harvested at 1/10th bloom throughout the 2020 growing season to capture the difference in fiber composition at the same maturity. Up to this point, the main challenge has been establishing enough stand due to some winterkill and weeds. The spring in southeastern Nebraska saw many bouts of below freezing temps and frost followed by a stretch of warm weather, which put stress on the growing plants. The stands from the first seeding are 8 to 10 inches in height and show no signs of pest damage or nutrient deficiencies. Weeds are sporadic throughout fields but should recede as the canopy closes and more so following the first cutting. Energy Metabolism and Digestibility Study. In the fall of 2020 12 multiparous lactating Jersey dairy cows will be used. All cows will be housed in a temperature-controlled barn at the Dairy Metabolism Facility at the Animal Science Complex at the UNL (Lincoln, NE) and milked twice daily. The experimental design will be a three times replicated 3 × 3 Latin square. All diets will contain approximately 50% forage, of which ~ 2/3 will be alfalfa. Treatments will be 1) a control diet with CON, 2) a treatment diet containing only RL, and 3) a 50:50 blend of CON and RL (RL50). Effects of treatment on energy utilization, nutrient digestibility, and milk production will be determined using indirect calorimetry.

Impacts
What was accomplished under these goals? Due to poor germination of newly seeded alfalfa last fall of 2019 resulting from dry period after planting, we reseeded this spring of 2020. Three different alfalfa varieties including Dairyland HybriForce-3400 (conventional variety), Pioneer 54HVX (reduced-lignin type variety), and Alforex460 (reduced-lignin type variety) were planted on April 22, 2020 for agronomic and physiological field research at the K-State North Agronomy Farm (39.2181° N, 96.5907° W) in Manhattan, KS. A recommended seeding rate of 20.0 lbs pure live seed per acre was used. Before planting alfalfa, soil samples from the research site was taken for basic soil compositional analysis at the Kansas State University Soil Testing Lab. Individual plot size is 3.0 ft × 20.0 ft. With 1,200 ft2 planting area in each of the rain-out shelter, we were be able to accommodate two replicates of the 3 varieties in each rain-out shelter. In addition, small plots for irrigated and rainfed treatments were established with the same varieties as those in rain-out shelters, in adjacent plots on same soil type. Two fields at UNL were seeded at 15 lb of live seed per acre with three different alfalfa varieties on September 5, 2019. In one field, 14.5 acres of the control variety (CON), Dairyland Hybriforce 3400 was planted. The second field was designated for the reduced-liginin (RL) varieties and divided in half with 8 acres being planted to Pioneer Hi-Bred HarvXtra (RL1) and the other 8 acres planted to Alforex 460 (RL2). All grasses and weeds present in the fields prior to planting were knocked down using Roundup PowerMax on August 29, 2019.

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