# Presentation: 141727

## Protein-binding polyphenols enhance nitrogen cycling and profitability of dairy farms

- John H. Grabber, Glen A. Broderick, Michael Sullivan, Ronald D. Hatfield, J. Mark Powell, C. Alan Rotz, Richard E. Muck, Nancy Ehlke, and Tom H. Misselbrook
- USDA-Agricultural Research Service
-  U.S. Dairy Forage Research Center
-  Pasture Systems & Watershed Management Research Unit
- Department of Agronomy & Plant Genetics, University of Minnesota
- Institute of Grassland and Environmental Research, United Kingdom

**Notes:**
This morning I will present initial results of component and systems research aimed at assessing the impact of forage condensed tannins on protein utilization and nitrogen cycling on dairy farms.  This research is primarily carried out by a multidisciplinary group of scientists, postdocs, and technicians at the Dairy Forage Research Center in Madison, Wisconsin. Al Rotz at the Pasture Systems & Watershed Unit in Pennsylvania is collaborating on modeling studies. Nancy Ehlke at the University of Minnesota has provided tannin-containing germplasm for our studies. 

## Forages are the major component of dairy cow diets

## Alfalfa (Medicago sativa) is the 4th major crop in the United States

- Bacteria in the rumen of cattle degrade 50 – 75% of the remaining protein in alfalfa

- The “Queen of Forages**”, **has a problem...
- EXCESSIVE proteolysis in the silo & rumen

- Plant proteases degrade ~50% of the protein in alfalfa stored as silage

**Notes:**
Why the interest in tannins? Well, alfalfa, the queen of forages (and most other forages) have a problem...

## The rumen converts alfalfa with 20+% protein into a 5% protein forage for gastrointestinal digestion 

- Extra protein is fed to compensate for poor forage protein utilization

## Why is this important?

## Feeding extra protein...

- is costly for farmers
- increases nutrient import and loss from farms
- increases dairy farm reliance on erosion prone row crops like soybeans
- 					or put another way...

## The average dairy cow producing 8,200 kg of milk...

## ...excretes more than 110 kg of N per yearin 21,000 kg of manure

- About 50% of manure N (mainly urea) is lost as NH3  and NO3 to the environment

## Condensed tannins limit proteolysis in forages

- Birdsfoot trefoil (*Lotus corniculatus*) has proanthocyanidins
- Proanthocyanidins precipitate proteins, limiting proteolysis

## Polyphenol oxidase/o-diphenols limit proteolysis in forages

- Red clover (*Trifolium pratense*) has *o*-diphenols and polyphenol oxidase
- Quinones may limit proteolysis by inactivating proteases and modifying/precipitating protein substrates

## Current knowledge: Polyphenols in forage-based livestock systems 

- Major feedstuffs, including alfalfa, have very low polyphenol levels (< 0.2% DM).
- Tannin-protein and *o*-diphenol-protein complexes limit proteolysis during ensiling and rumen fermentation. The impact of various forage harvesting and storage methods on polyphenol-protein interactions is poorly understood.
- Tannin complexes can dissociate in the gastrointestinal tract, permitting protein digestion. The digestion of *o*-diphenol-protein complexes in the gastrointestinal tract is poorly understood.

**Notes:**
Tannins bind to protein under slightly acidic to neutral pH conditions
Protein-tannin complexes dissociate under acidic or alkaline conditions, permitting digestion and absorption of forage amino acids in the gastrointestinal tract.
As a result, less protein supplementation of cattle should be needed.
Tannins may slow nitrogen release in soil so forage residues and manure may act more as “time-release fertilizer”
Most feedstuff, including alfalfa don’t have enough tannin, probably 2-3% is needed. 

## Current knowledge (continued)

- For grazing livestock, tannin levels of 2 – 4% seem ideal; higher levels depress feed intake and digestion. Optimal forage tannin levels for high grain diets is unknown. Optimal levels of              *o*-diphenols & polyphenol oxidase are unknown.
- Polyphenols reduce total N excretion in manure and shift N excretion from urinary to fecal N forms. The impact of modest polyphenol levels on manure N excretion & losses needs further characterization.
- High polyphenol concentrations slow nitrogen mineralization from decaying crop residues and possibly manure in soil. The impact of modest polyphenol levels on soil N mineralization is unknown.
- Little is known about the whole-farm impact of growing & feeding polyphenol containing forages.

**Notes:**
Tannins bind to protein under slightly acidic to neutral pH conditions
Protein-tannin complexes dissociate under acidic or alkaline conditions, permitting digestion and absorption of forage amino acids in the gastrointestinal tract.
As a result, less protein supplementation of cattle should be needed.
Tannins may slow nitrogen release in soil so forage residues and manure may act more as “time-release fertilizer”
Most feedstuff, including alfalfa don’t have enough tannin, probably 2-3% is needed. 

## Our work on polyphenols

- We are studying the impact of forage polyphenols on . . .
- Protein quality in silage and hay
- Milk production and protein needs of dairy cattle fed typical forage–grain diets
- N excretion in manure and losses during storage and land application
- Mineralization of N from manure and forage residues in soil and N uptake by crops
- N cycling and profitability of dairy farms in different regions of the U.S.
- We are also developing improved polyphenol-containing forages and production systems for these forages

**Notes:**
(After reviewing slide mention....

 In our work, trefoil is mainly a model crop for guiding bioengineering of alfalfa for optimal tannin levels but some organic or conventional producers who use or would like to use trefoil will directly benefit from our studies.  

## Forages used in polyphenol studies

**Forages used in polyphenol studies**

- Birdsfoot trefoil with ~ 2, 4, and 7% extractable condensed tannin (vanillin-HCl procedure, catechin standard)
- Red clover with polyphenol oxidase and ~1% *o*-diphenol derivatives of caffeic acid (mainly clovamide and phaseic acid)
- Alfalfa essentially free of polyphenols

**Notes:**
(After reviewing slide mention....

 In our work, trefoil is mainly a model crop for guiding bioengineering of alfalfa for optimal tannin levels but some organic or conventional producers who use or would like to use trefoil will directly benefit from our studies.  

## Protein quality studies with polyphenol-containing forages Forages harvested in 2002 & 2003

## Rumen undegradable protein (protease)

- 		 Freeze
- 		 dried 	Silage	Hay

- 	Alfalfa	23.8	17.0	23.3
- 	Low tannin trefoil	25.8	19.1	26.3
- 	High tannin trefoil 	30.6	26.5	31.1
- 	Red clover	25.1	32.7	32.9
-         with *o*-diphenols and PPO

- --------------% of crude protein---------------

- Forage X Preservation method (*P* < 0.01)

**Notes:**
In alfalfa, one-half the crude protein is soluble in buffer and would likely be rapidly degraded in the rumen. A protease assay indicates about one-quarter of the crude protein would be gradually degraded in the rumen and only about one-quarter is potentially undegraded in the rumen and available for digestion and absorption in the lower tract.  If alfalfa is the predominant forage in the diet, the rumen is flooded with much more degradable protein than can be captured by rumen bacteria and the excess, maybe 50% of the total plant N is excreted as urea.  

Tannins reduce amount of rapidly and slowly digested N in forage, shifting a greater proportion of protein digestion to the lower gastrointestinal tract.  High tannin trefoil had 50% more undegradable protein, which is a tremendous improvement IF all of that protein can be digested in the lower tract--too much tannin can interfere with lower tract protein digestion. 

## Rumen undegradable protein (RUP)

- Conventional	Macerated

- 	Alfalfa	20.1	24.9
- 	Low tannin trefoil	22.7	32.0
- 	High tannin trefoil 	28.8	40.6
- 	Red clover	32.8	27.9
-          with *o*-diphenols and PPO

- --% of total crude protein--

- Forage X Conditioning (*P* < 0.01)

-  Ongoing work: Assess gastrointestinal degradability of RUP, especially in red clover

**Notes:**
The severity of conditioning influences tannin-protein interactions. Due to time constraints, we’ll  look just at protease undegradable N.  Maceration of alfalfa only enhances the amount of protease undegradable N compared to trefoils where tannins are present. 

## Lactation studies with polyphenol-containing foragesData from the 1st of 4 trials

## Forage characteristics

- 		 	    Rumen
- 		Crude	undegradable
- 		protein 	     protein	   Fiber
- 		% of DM 	     % of CP	  % of DM

- Alfalfa	 22.0a 	26.7d	35.3b
- Low tannin trefoil	 21.7a 	30.6c	32.4c
- Mid tannin trefoil	 20.4b 	39.6b	32.4c
- High tannin trefoil	 20.1b 	42.1b	32.2c
- Red clover	18.1c	52.8a	42.7a
-     with *o*-diphenols and PPO

-  Means with the same letter are not significantly different (*P* < 0.05)

**Notes:**
For the first of several planned feeding trials, 2nd cut alfalfa and trefoils were harvested and ensiled. In this particular harvest, the trefoils were 3 percentage points lower in NDF and comparable or somewhat lower in crude protein.  When fed as 50% of total mixed rations, dry matter intake was similar among forage sources.  

## Intake & production by lactating Holstein cattle 

- 		 Feed	         Milk   	 Milk		intake	(3.5% FC)	protein	-----------------kg/day------------------

- Alfalfa	24.4a	31.4d	0.94b
- Low tannin trefoil	25.2a	33.8bc	1.04a
- Mid tannin trefoil	23.3a	36.3a	1.09a
- High tannin trefoil	24.5a	35.3ab	1.07a
- Red clover 	25.6a	32.6cd	0.96b
-    with *o*-diphenols and PPO

- Diets were 50% legume silage, 10% corn silage, and 40% concentrate (corn & some soybean meal). Dietary protein was 16–17% and NDF 27–29%.
- Means with the same letter are not significantly different (*P* < 0.05)

**Notes:**
If you then look at milk production and milk components, shifting from alfalfa to low tannin trefoil boosted the production of 3.5% fat corrected milk by 5 lbs per cow.  Part of the response may be due to tannin but other factors like differences in fiber digestibility may have played a role.  However, within trefoils, shifting from low to moderate tannin levels gave another 5.5 lb boost in milk production.  Milk production began to decline at higher tannin levels,, possibly limiting microbial protein synthesis in the rumen and/or interference of protein digestion in the gastrointestinal tract.  Forage type  and tannin levels did not affect milk composition so the yield of milk protein and fat mirrored milk production.  

## Lactation studies o-diphenol & PPO-containing red cloverData compiled from 5 feeding trials

## Forage & diet characteristics

- 		_                            __Forage              _  	_                 __Diet        _  __	_Protein	 NPN	  Fiber	Protein	 Fiber
- 		% of DM  	% of N	% of DM	% of DM	% of DM

- Alfalfa	20.9a	53.1a	 43.3a	17.7a	33.0a
- Red clover	17.9b	35.8b 	43.0a	15.8b	32.0a
-     with *o*-diphenols and PPO

- Diets averaged 63% legume silage and 36% concentrate (mostly corn with some soybean meal)
- Means with the same letter are not significantly different (*P* < 0.05)

**Notes:**
For the first of several planned feeding trials, 2nd cut alfalfa and trefoils were harvested and ensiled. In this particular harvest, the trefoils were 3 percentage points lower in NDF and comparable or somewhat lower in crude protein.  When fed as 50% of total mixed rations, dry matter intake was similar among forage sources.  

## Intake & production by lactating Holstein cattle

- 			   Milk	  Milk	Milk	Weight
- 		Intake	3.5% FC	Protein	Fat	  gain
- 		------------------------- kg/day--------------------------------

- Alfalfa	22.9a	32.0a	 0.93a	1.12a	0.02b
- Red clover	21.4b	31.0a	0.91a	1.08b	0.20a
-      with *o*-diphenols and PPO

- Diets averaged 63% legume silage and 36% concentrate (mostly corn with some soybean meal)
- Means with the same letter are not significantly different (*P* < 0.05)

**Notes:**
For the first of several planned feeding trials, 2nd cut alfalfa and trefoils were harvested and ensiled. In this particular harvest, the trefoils were 3 percentage points lower in NDF and comparable or somewhat lower in crude protein.  When fed as 50% of total mixed rations, dry matter intake was similar among forage sources.  

## Forage polyphenols & manure nitrogen Studies just initiated

## Manure slurry composition

- 		Total N	 Urinary N		    g/L	% of total N

- Alfalfa	2.42	55
- Low tannin trefoil	2.18	56
- High tannin trefoil	2.19	45

- Diets were 50% forage, 10% corn silage, and 40% concentrate (corn & some soybean meal). Dietary protein was 16–17% and NDF 27–29%.

**Notes:**
We’re looking at the fate of manure N as influenced by forage tannins.  Tannins decrease the proportion of urinary N in manure, which is the fraction most susceptable to loss from manure.. Studies with laboratory chamber indicate manures excreated from diets with  tannin-containing forage have a lower loss of N from barn floors and after surface application to soil.

## Ammonia emission after application of manure slurry to soil

## Forage polyphenols & nitrogen use by cropsStudies just initiated

## Uptake and loss of manure and forage residue N by subsequent annual crops 

**Notes:**
We’re at the intial phases of  studying the uptake of manure and forage residue N by sorghum-sudangrass. 

## Developing improved cropping systems for polyphenol-containing forages

## Focus on red clover-cereal crop rotations

**Focus on red clover-cereal crop rotations**

## Developing new polyphenolcontaining forages

## Expression of red clover PPO in transgenic alfalfa

- In alfalfa, browning is dependent on:

- A PPO transgene
- Exogenous *o*-diphenol, e.g. caffeic acid

**Notes:**
We were able to clone several PPOs from red clover. When expressed in alfalfa, the proteins are active.  Here I show extract browning. [Just work left to right]

Don’t forget acknowledgements here

## Can polyphenol oxidase & o-diphenols inhibit proteolysis in alfalfa?

**Yes**

- Ongoing work:
- Engineer alfalfa for PPO and *o*-diphenol production
- Other groups are engineering alfalfa for tannin production

**Notes:**
Together, these results indicat that PPO is capable of inhibiting post-harvest protelysis, and that this inhibition is dependent on o-diphenol PPO substrates.  We’re currently also assessing proteolysis in red clover silienced for PPO expression.

Transition: Using our alfalfa system, we’ve been able to begin addressing several aspects of PPO-mediated proteolytic inhibition, including....

## Forage polyphenol impacts on nitrogen cycling & profitability of dairy farms A preliminary modeling study 

**Notes:**
Finally we’re looking at looking at forage tannin effects on N cycling and profitability on a whole farm basis. 

## Model the impact of tannin-containing alfalfa on a 100-cow dairy farm (DAFOSYM) 

**Manure Storage & Handling**

**Soils & Crops**

**Harvest & Storage**

*Milk*

**Feeding & Herd Management**

*Purchased feed & bedding*

- Storage losses

*Fertilizer*

- Nutrient loss/buildup

-  "© Cash cow image contributed by WMMB, 2004"

**Notes:**
A dairy forage system model is our main tool for doing this.  Dafosym models all major components of farms and their interactions.  Initial simulations have been done for a 100 cow dairy farm in Wisconsin

## Predicted annual farm performance if alfalfa is fed as ~ 45% of the diet

- Normal alfalfa	 1,145	12,330	1026	2433	157
- Tannin alfalfa	 1,270	12,540	436	3060	118
- Tannin impact	 + 125	+210	- 590	+627	-39

- 	 Net 	 Milk	Soybean	 Maize	 Total
- 	return	 yield 	     fed	   fed	N loss
- 	$/cow	kg/cow	  kg/cow	kg/cow 	kg/cow

## Added value of alfalfa with tannin...(per t of dry matter)

- Silage	$ 21
- Hay	$ 10

-  ...worth >$300 million per year to the U.S. dairy-forage industry

## Long-term goals of polyphenol research

- Identify optimal polyphenol characteristics and management practices for improving protein and nitrogen use on dairy farms
- Develop improved polyphenol-containing forages