Cutting Management Approaches to Understand Phytoestrogens Accumulation in Forage Legume Species Used in Dairy Production Systems

Progress report for GNE22-289

Project Type: Graduate Student
Funds awarded in 2022: $14,997.00
Projected End Date: 08/30/2024
Grant Recipient: University of New Hampshire
Region: Northeast
State: New Hampshire
Graduate Student:
Faculty Advisor:
Dr. Richard Smith
University of New Hampshire
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Project Information


Organic dairies rely on perennial forage legumes for their high nutritional value and ability to supply biological nitrogen to the soil. However, some forage legumes have been reported to concentrate phytoestrogens in their tissues. Phytoestrogens are secondary metabolites that mimic the sex hormone estrogen, and if consumed in high quantities, may impair the reproductive performance of dairy cows. Previous research conducted outside of the US revealed that phytoestrogen concentrations differ among legume species and are influenced by environmental stresses, including the frequency and extent of defoliation, suggesting that forage harvest practices may alter phytoestrogens. Unfortunately, little is known about how phytoestrogen concentrations vary across forage legumes grown in the Northeastern US. We will fill this knowledge gap using 4 perennial legumes included in a field experiment established in 2018 to examine how cutting frequency (3 vs. 5 times per season) and cutting height (5 vs. 10 cm residual forage height) affect legume productivity, persistence, and phytoestrogen concentrations. Tissue samples of red clover, white clover, alfalfa, and birdsfoot trefoil will be collected at each cutting time. Phytoestrogen concentrations will be quantified using high-performance liquid chromatography. This study will provide farmers with new information about phytoestrogen concentrations of perennial forage species commonly used in dairy operations in the Northeast and the degree to which cutting practices can be used to manage phytoestrogens levels. Results will be disseminated to stakeholders through presentations at field days, a “Managing Phytoestrogens in Forage Legumes” fact sheet, and peer-reviewed publications.

Project Objectives:

The overall goal of this research is to investigate variation in phytoestrogen concentrations among forage legumes.

The specific objectives are to:

  1. Collect and process forage legume tissue samples from an existing field experiment.
  2. Quantify effects of cutting frequency and cutting height on variation in phytoestrogen concentration in red clover, white clover, alfalfa, and birdsfoot trefoil.
  3. Disseminate phytoestrogen data to stakeholders.

The Northeastern organic dairy industry is critical to meeting the increasing demand for organic milk [1]–[3]. Organic dairies in the region rely on perennial legume forages such as red clover (Trifolium pratense), alfalfa (Medicago sativa) and white clover (Trifolium repens) to make up a significant portion of cow diets due to their high protein concentration and palatability [4], [5]. Compared to grass alone, legume forages increase feed intake and milk production [6], [7]. Forage legumes also built soil fertility through their ability to fix atmospheric N2 [6], [8], [9].

Despite their benefits to organic dairy production systems, some tradeoffs must be considered when feeding forage legumes, particularly red clover. Previous research demonstrated that many forage legume species accumulate phytoestrogens in their tissues, and if consumed at concentrations of approximately 40 mg/kg of dry matter may impair animal reproductive performance [10]–[12]. Phytoestrogens are plant-derived nonsteroidal, secondary metabolites that are biochemically similar to estrogen, the primary female sex hormone [13]. Phytoestrogens in animal tissues have been associated with impaired reproductive systems and infertility [14]. Previous research has also demonstrated that phytoestrogens are transferred to milk of dairy cows [15], [16].

Phytoestrogens accumulation in plant tissues depends on genotypic, management, and environmental factors [10], [15]–[19]. Most phytoestrogens belong to 2 categories of polyphenolic compounds: flavonoids and lignans. Flavonoids are divided into 3 subgroups: (i) isoflavones, (ii) flavones, and (iii) coumestans [20]. Generally, the types and concentrations of phytoestrogens vary from species to species and different cultivars of the same species, but are often most abundant in legumes [21], [22]–[27]. For example, previous research suggests that red clover may be particularly prone to accumulating high concentrations of phytoestrogens, in contrast to birdsfoot trefoil, which accumulates less phytoestrogens [10], [27]. Forage harvest practices, including cutting frequency and cutting height can influence the nutritional quality of forage legumes [28] and possibly the concentration of phytoestrogens. Forage cutting frequencies are very similar to artificial defoliation or topping of plants. After each cutting, plants become stressed and alter their physiological and biochemical activity in response [29]. In a study in which Subterranean clover plants were topped, the total isoflavone concentration increased in the remaining mature leaves [30]. This leads us to believe the concentration of phytoestrogens is also expected to be higher in related legume plants after each cutting. Dairy farmers in the Northeast are utilizing perennial forage legumes without adequate information about their potential to accumulate phytoestrogens. This project aims to quantify concentrations of phytoestrogens in 4 common perennial forage legumes (red and white clover, alfalfa, and birdsfoot trefoil) and determine if and how phytoestrogens are influenced by cutting frequency and cutting height over the growing season. Data generated in the project will be used to provide management recommendations to dairy farmers interested in managing and/or minimizing the accumulation of phytoestrogens in their legume forage crops and mixtures.


Materials and methods:

Objective 1: Collect and process forage legume tissue samples from an existing field experiment.

Experimental setup and design: This project will leverage an ongoing field experiment examining the effects of cutting management (cutting frequency and height) on forage legume productivity and stand persistence that was established at the UNH Kingman Research Farm (Madbury, NH) in 2018. Compost was applied to the site in summer 2018 and treatments were sown in early September 2018. Four species of perennial forage legume were grown as bicultures with orchardgrass (‘Latar’). The legume species are red clover (‘Freedom’), white clover (‘Alice’), alfalfa (‘406AP2’), and birdsfoot trefoil (‘Bruce’). The proportion of sown legume and orchardgrass seed was 70:30. Whole-plot factors are factorial combinations of cutting frequency [3 vs. 5 times (3X vs. 5x) over the growing season] and cutting height (5 vs. 10 cm residual forage height) in a randomized complete block design. The sub-plot factor is legume species which is randomized within each whole-plot. Subplots measure 2.3 m x 7.6 m. All treatments are replicated 5 times. The forage legume samples will be collected immediately prior to each time for 3 growing seasons (2022 and 2023) and will be analyzed in the laboratory (see objective 2).

Sample collection: My work is proceeding according to plan with a few changes. In the summer of 2022, I collected legume tissue samples in plots subjected to two different harvest frequency treatments (3 and 5 cuts per season). Tissue samples were collected prior to each cutting event. A 0.5-m2 quadrat was placed randomly in three locations in each plot and all legume herbage was clipped from a height of either 5 or 10 cm (depending on cutting height treatment assigned to that plot). Immediately after harvesting, plant material was mixed, and a representative portion was placed in a plastic 50 ml centrifuge tube and immediately transferred to coolers with ice until transported to the laboratory, where they were stored at -80°C. After that, samples were freeze-dried at approximately -50°C and stored at -20°C. This step ensures that the samples remain stable for subsequent analyses.

For the 2023 growing season, I was unable to collect legume tissue samples due to insufficient legume abundance in all experimental plots. However, as an alternative, I conducted a growth chamber experiment from July 2022 to October 2023 at the Macfarlane Greenhouses at the University of New Hampshire to evaluate the influence of elevated temperature and CO2 on phytoestrogen concentration in two forage legume species. The legume species were red clover (cultivar ‘Freedom’) and cowpea (cultivar ‘Red Ripper’). The experimental treatments were combinations of temperature and CO2: (1) ambient temp + elevated CO2 [24/18 °C (D/N) and ~ 750 ppm CO2]; (2) high temperature + ambient CO2 [35/26 °C (D/N) and ~ 400 ppm CO2]; (3) elevated temperature + elevated CO2; and (4) a control treatment with ambient temperature and CO2 [24/18 °C (D/N) and ~ 400 ppm CO2]. Legume tissue samples were collected at the end of the experimental period and were processed as described above in preparation for analysis of phytoestrogen concentrations. I chose to focus on red clover because this species appears to have the highest phytoestrogen concentrations based on the results from the field experiment conducted in 2022 (see update for Objective 2, below)

Objective 2: Quantify effects of cutting frequency and cutting height on variation in phytoestrogen content in red clover, white clover, alfalfa, and birdsfoot trefoil.

This component of the project is proceeding according to plan with one minor modification. No problems were encountered during sample collection, processing, or shipping and receiving of the processed samples. However, due to problems with the HPLC I had planned to use in-house, I had to send the legume tissue samples to an outside lab (Virginia Tech Chromatography Center) for phytoestrogen analysis. This resulted in an unanticipated added expense; therefore, I only sent the samples of red clover and white clover for phytoestrogen analysis. Previous research suggests these species represent the highest risk for elevated phytoestrogen levels. The alfalfa and birdsfoot trefoil samples have all been processed and archived and can be analyzed for phytoestrogens if additional funding becomes available.

I recently received the phytoestrogen (formononetin, biochanin A, daidzein, genistein, glycitein, coumestrol, and prunetin) results from the red clover and white clover tissue samples collected from the field study 2022 and am now analyzing those results. Based on my preliminary analyses, red clover defoliated five times in a growing season (5-cut system) produced 34.5% more average total phytoestrogen than when defoliated three times (3-cut system); however, white clover did not exhibit this difference. Defoliation severity (cutting to a 5 or 10 cm residual height) did not influence the phytoestrogen level in either red clover or white clover. Importantly, the average total phytoestrogen concentration was 140 times higher in red clover compared to white clover. In both red clover and white clover, formononetin and biochanin A were the major phytoestrogens; cumulatively, they make up 85% and 65% of the total phytoestrogen concentration, respectively. The concentration of phytoestrogens in both clover species varied by harvest time in the 5-cut system but not in the 3-cut system.

Legume tissue samples collected from my 2023 growth chamber study were sent to the Virginia Tech Chromatography Center for analysis of phytoestrogens (formononetin, biochanin A, daidzein, genistein, glycitein, coumestrol, and prunetin) via high-performance liquid chromatography (HPLC). I anticipate receiving the phytoestrogen data for these samples in March 2024 and will statistically analyze and report the results soon thereafter.

Statistical analysis: All statistical analysis and plots will be performed in GraphPad Prism software (version 8.2.1). An analysis of variance (ANOVA) will be carried out after checking for normality and homogeneity of the variances. Significant differences among treatments will be considered at P < 0.05.      

Objective 3: Disseminate phytoestrogen data to stakeholders.

This project will result in quantitative data on the phytoestrogen concentrations of 4 common perennial legumes and information on how cutting management influences these concentrations over 2 growing seasons. Results of this project will be disseminated to relevant stakeholder groups including organic dairy farmers, forage growers, and the scientific community. We will partner with Mr. Carl Majewski (Dairy, livestock and forage crop field specialist UNH Cooperative Extension; see Letter of Collaboration) to help disseminate project information in line with his extension and educational program targeting dairy, livestock, and forage production and management. In addition to organizing a field day at the UNH Kingman Research Farm, we will also work with Mr. Majewski to summarize our research results in a farmer-friendly fact sheet that will be made available on the UNH Extension website. We will also coordinate with Mr. Majewski to publish an article in the E-newsletter of the Northeast Organic Farming Association of New Hampshire summarizing our results and linking to the factsheet E-newsletter. Finally, we will prepare a manuscript reporting our results for submission to a peer-reviewed journal (e.g., Agronomy or Crop Science). Data will be also disseminated through presentations in local, regional, national, and international conferences attended by our team.


Research results and discussion:



Research conclusions:



Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

The third objective of this research project is focused on outreach. We will synthesize our research results and share them with our stakeholders in collaboration with Mr. Carl Majewski (UNH Cooperative Extension; see Letter of Support). Our outreach effort will involve 4 different approaches to technology transfer to disseminate our research results to farmers and the agricultural science community: 1) A field day to be held in Year 2 with organic dairy farmers and forage growers at the UNH Kingman Research Farm, 2)  Preparation of a farmer-friendly fact sheet explaining phytoestrogens, why they are a concern, how they vary among the 4 perennial forage legume species studied, and if and how cutting strategies or other factors can be used to manage their concentrations, 3) An E-newsletter article to be published in the Northeast Organic Farming Association of New Hampshire website, and 4) A peer-reviewed article to be published in a high-ranked journal. Based on the information we provide, dairy farmers will be able to make better-informed management decisions aimed at managing the potential risk of phytoestrogens to become problematic in their dairy production systems. The proposed project builds upon a more extensive research and extension program focused on legume species persistence, nutritive value, phytoestrogen metabolism, and dairy cow production and health in collaboration with the University of Minnesota and University of Maine funded by the USDA-NIFA-Organic Agriculture Research and Extension Initiative. Here we are proposing to leverage SARE funding to further investigate phytoestrogen metabolism in response to different cutting frequencies and cutting heights to understand better how stressful conditions affect accumulation of phytoestrogens in 4 legume species.   

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.