Inoculants Maintaining Quality Forages Until Feed Out

B I O T A L Inoculants Maintaining Quality Forages Until Feed Out Tony Hall & Katelynn Robbins Lallemand Animal Nutrition 12/22/2015

Why attention to silage management is vitally important Silages form over 50% of most dairy rations. The quality of the silages fed is therefore one of the key determinants of profitability. Plus, high quality silages are easier to balance the ration around. Poor quality silages require more, and more expensive, complementary feedstuffs Badly preserved and/or spoiled silages put the dairy always struggling to get anywhere near objectives, and can lead to serious herd health and fertility problems. Thus, every crop for silage should be treated with a proven inoculant, as part of a good overall management program, to maximize profitability.

What is the basic process? Cut Forage Crop (Sugars)* [ph ~ 6.0-6.5] + Anaerobic Conditions (Covered or Sealed; No Oxygen) Ensiled Forage Crop (Acids) [ph ~ 5.0-3.5] (Preserved in Acid) *One Plant Sugar Unit -> Two Lactic Acid Units + Two Water units + Energy ph Drops.

Forages Are Not Sterile! Source R Muck USDA

What happens during ensiling is a microbial war of the worlds between the good guys and the bad guys Good guys Lactic acid bacteria heterofermentative homofermentative Bad guys Fungi yeasts molds Enterobacteria Clostridia Bacilli

While the ensiling fermentation looks like a simple process, it still needs to be controlled! ph 5.0 Time

While the ensiling fermentation looks like a simple process, it still needs to be controlled! Inefficient (heterolactic) fermentation, or fermentation by enterobacteria or growth of aerobic organisms slow ph drop Clostridial Fermentation ph 5.0 Yeast activity Ideal Fermentation Over fermented Time

While the ensiling fermentation looks like a simple process, it still needs to be controlled! ph 5.0 Inefficient (heterolactic) fermentation, or fermentation by enterobacteria or growth of aerobic organisms slow ph drop $ $ $ $ Clostridial Fermentation Yeast activity $ $ Ideal Fermentation Time Over fermented

For maximum dry matter recovery we need an initial dominant homolactic fermentation Type of fermentation End products DM recovery Homolactic LAB, e.g. P. pentosaceus, L. plantarum (glucose/ fructose) Heterolactic LAB, e.g. L. brevis (glucose) Heterolactic LAB (fructose) (%) Lactic acid 100 Lactic acid, ethanol, 76 CO 2 Lactic acid, acetic 95 acid, mannitol, CO 2 Yeast (glucose & lactic acid) Ethanol, CO 2 51 Clostridia (glucose & lactic acid) Butyric acid, CO 2 49

From Limin Kung, DuPont Lecture (2012) - Approaches For Using Silage Inoculants 1. Speeding up and altering fermentation to minimize nutrient losses - All types of silages 2. Preventing a clostridial fermentation - wet alfalfa and grasses - conventional homolactic acid bacteria - quick attainment of a low ph 3. Improving aerobic stability - HMC and corn silages - L. buchneri products acetic acid is antifungal 4. Making a good fermentation better - improvements in DM recovery

Really, this all boils down to achieving the twin goals in making good quality silage from high quality forage Front-end Rapid preservation for maximum recovery of nutrients, minimizing growth of aerobes and spoilage bacteria (clostridia, enterobacteria, etc.) Back-end Good shelf life : aerobic stability - no heating - no spoilage Adapted from Kung

Really, this all boils down to achieving the twin goals in making good quality silage from high quality forage Front-end Achieved Rapid preservation by getting the for ph maximum down below 5 recovery as of quickly nutrients, as possible minimizing growth of aerobes and spoilage bacteria (clostridia, enterobacteria, etc.) Back-end Good shelf life : aerobic stability - no heating - no spoilage Adapted from Kung

Really, this all boils down to achieving the twin goals in making good quality silage from high quality forage Front-end Achieved Rapid preservation by getting the for ph maximum down below 5 recovery as of quickly nutrients, as possible minimizing growth of aerobes and spoilage bacteria (clostridia, enterobacteria, etc.) Back-end Here we need something that will stop Good yeasts shelf from life : growing: they case > 99% aerobic of all heating stability events - no heating silages - no spoilage Adapted from Kung

We need to get the ph down below our critical control point (ph 5) as quickly as possible: 1. To shut down plant s own enzymes that are trying to return the nutrients to the soil; 2. To prevent bad fermentations, e.g. clostridia, enterobacteria; and 3. To minimize the growth of aerobic organisms (yeasts, moulds, Bacilli).

Front-end Rapid ensiling fermentation to drop ph quickly, minimize plant respiration, prevent bad fermentations and so maximize preservation of nutrients - Increased DM recovery - Increased protein recovery - Increased energy recovery Very strong feature in:

ph-driving LAB in Biotal Silage Inoculant II, Plus II and Buchneri 500: selected for best performance in the up-front ensiling fermentation The lactic acid producing bacteria (LAB) strains in Biotal came from a screening program that started with over 8,000 isolates from high quality forages. The final product strains were the very best through all the screens: Homofermentative Unlike >50% of the isolates tested Unable to break down protein Unlike > 50% of the isolates tested (including all E. faecium ) Ability to utilize a wide range of sugars (including 5C pentoses) Unlike most LAB, including most lactobacilli and pediococci Fastest growth and acid production across a range of temperatures and ph values Biotal P. pentosaceus NCIMB 12455 - the fastest of all tested!

Biotal Silage Inoculant II, Plus II and Buchneri 500 combine fast, efficient LAB with enzymes to drive the ideal fermentation process The enzyme formulation breaks down complex sugars (polysaccharides) in the plant to simple sugars to drive acid production by our LAB strains. FORAGE + ENZYMES SIMPLE SUGARS MICROBES + SUGARS Best Pickles ACID TO PICKLE THE CROP

So lets be really clear here! We need a very efficient homo-lactic bacteria in the inoculant to generate 2 units of lactic acid per unit of sugar with the minimum of dry matter loss. We need it to do it quickly, to out grow the ubiquitous Clostridia, and generate ph 5.0 or less by day 3 so the Clostridia cannot get established. So it not only has to make lactic acid to drop the ph it also needs to have a fast growth rate or doubling time Biotal P. pentosaceus NCIMB 12455 - the fastest of all tested!

Microbial math: the power of an organism (Pediococcus pentosaceus 12455) that doubles every 30 minutes! What happens in 16 generations (8 hours)? 100,000 200,000 400,000 800,000 1,600,000 3,200,000 6,400,000 12,800,000 25,600,000 51,200,000 102,400,000 204,800,000 409,600,000 819,200,000 1,638,400,000 3,276,800,000 6,553,600,000 Starting at 100,000 CFU (typical homolactic inoculant) Typical silage end-point (2 3 billion CFU/ g)

Proven in challenging conditions: 3rd cut alfalfa, 32.4% DM, highly buffered (548 meq/ kg DM) Bolsen, KSU. ph 5.9 5.7 5.5 5.3 Control Ecosyl Alltech Biotal Kemin AgMaster Pioneer ph 5.0 5.1 4.9 4.7 0.51 3 5 7 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (days) Only Biotal was below the ph 5.0 barrier within 3 days: three products and the control were still above ph 5 after seven days

ph Singo Trial - ph over first 7 days 6.3 5.8 5.3 4.8 Control AK P2 Buch 500 4.3 0 1 1.5 2 3 4 5 6 7 Time (days)

Remember this? While the ensiling fermentation looks like a simple process, it still needs to be controlled! Inefficient (heterolactic) fermentation, or fermentation by enterobacteria or growth of aerobic organisms slow ph drop ph 5.0 $ $ $ $ Ideal Fermentation Time

Faster fermentation minimizes DM loss proven in independent trials Control Treated Difference Corn silage London Universtity 9.70 8.50 1.20 USDA FRC 4.80 0.50 4.30 CEDAR 12.80 4.60 8.20 IGER 7.98 3.75 4.23 CEDAR 9.90 3.10 6.80 U Delaware 6.80 5.10 1.70 U Florida 5.50 4.40 1.10 U Delaware 10.30 4.70 5.60 U Delaware 10.30 5.40 4.90 Average difference 4.23 Haylage Kingshay 2.34 1.93 0.41 U Florida 13.30 3.10 10.20 U Delaware 13.20 2.60 10.60 Average difference 7.07 Barley silage Lethbridge 7.00 2.60 4.40 U Delaware 7.10 4.80 2.30 Average difference 3.35 Overall average 4.71

What is a 4% reduction in DM loss worth? Assuming corn silage is worth around $60/ ton on a fresh weight basis, coming out of the silo At 35% DM this would mean the value of DM $60 0.35 = $171.43/ ton So, each 1% improvement in DM recovery is worth $171 100 = $1.71 per 1% 4% improvement in DM recovery is worth 4 x $1.71 = $6.84/ ton DM = $2.40/ ton as fed (assuming 35% DM)

Effects of a Clostridial Fermentation Butyric acid production sugars or 2 lactic acid butyric acid + 2CO 2 + 2H 2 O Substantial dry matter, digestibility & energy losses; Raises ph if lactic acid is fermented

Clostridial Silage Ketosis Two possible mechanisms: cow converts excessive butyric acid from the silage to BHBA one enzymatic step in the liver any BHBA increase makes the cow sick reduced dry matter intake due to the silage toxic amines, ammonia in the silage probably not the butyric acid per se (except as it causes ketosis)

Action Plan Clostridial Silages Seven 'D's once you know you have a clostridial silage diagnosis (amount of butyric acid) decay (volatilize some butyric acid) daily intake (calculate grams of butyric) diversion (away from transition cows) dilution (down to <50 grams/cow/day) disposal (of high butyric silage) deal (with other high risk silages on the farm)

From Kung (2012)

The issue with homolactic fermentation Low ph alone does not inhibit the growth of yeasts and molds Yeasts can use lactic acid as a food source, initiating aerobic spoilage, so. Back-end Needs there to be something that is going to stop the growth of yeasts, which cause >99% of all heating events

The Domino Effect on exposure to air (oxygen): the progression of aerobic spoilage Silage is exposed to air Yeasts wake up and degrade residual sugars then lactic acid Yeast numbers increase Highly degradable nutrients are destroyed Heat is produced ph increases Molds/bacteria wake up causing massive spoilage, potential toxin production Adapted from Kung, 2010

The Domino Effect on exposure to air (oxygen): the progression of aerobic spoilage Silage is exposed to air Yeasts wake up and degrade residual sugars then lactic acid Yeast numbers increase Highly degradable nutrients are destroyed Heat is produced ph increases Molds/bacteria wake up causing massive spoilage, potential toxin production Adapted from Kung, 2010

Yeast levels in Corn Silage samples 55% of the samples in this study were 100 million yeasts per gram or higher! Only 18% were less than 1,000 yeasts per gram. (Data source: CVAS, 2009) Kung, 2011

Aerobic Stability, h Aerobic stability is directly related to yeast levels: yeasts initiate heating in silage (and TMRs) 300 250 200 The higher the yeast level is, the less stable the silage is: so we HAVE to have a low yeast level to get aerobic stability 150 100 50 0 0 2 4 6 8 10 (100) (10,000) (1,000,000) (100 million) Yeasts, log 10 cfu/g Kleinschmitt & Kung 2003

Aerobic Stability, h Aerobic stability is directly related to yeast levels: yeasts initiate heating in silage (and TMRs) 300 250 200 The higher the yeast level is, the less stable the silage is: so we HAVE to have a low yeast level to get aerobic stability 150 100 50 0 0 2 4 6 8 10 (100) (10,000) (1,000,000) (100 million) Yeasts, log 10 cfu/g Kleinschmitt & Kung 2003

Minimizing yeast growth is a good thing for production Milk (lb/ hd.day) Time Cows fed HMC from a 14 day supply removed from a silo, kept in a loose pile and fed daily. Milk yield declined as the level of yeasts in the pile rose. Hoffman and Ocker, 1997

Acetic Acid, % DM Effect of acetic acid on yeast levels in corn silage 3.5 3.0 2.5 P < 0.05 R 2 = 0.68 2.0 1.5 1.0 0.5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Schmidt et al., 2007 Yeasts, log cfu/g

Acetic Acid, % DM Effect of acetic acid on aerobic stability in corn silage 3.5 3.0 2.5 2.0 1.5 1.0 P < 0.05 R 2 = 0.95 0.5 0 50 100 150 200 250 300 Schmidt et al., 2007 Aerobic Stability, h

Biotal Plus II enzymes drive controlled production of acetic acid Xylanase produces the 5-carbon sugar xylose LAB that can use xylose do so to produce acetic acid very efficiently: 5-C xylose 1 x 3-C lactic acid + 1 x 2-C acetic acid (And nothing else: i.e. no CO 2 ) Acetic acid inhibits growth of yeasts, this improves stability at feedout Pediococcus pentosaceus 12455 (Biotal Plus II strain) was selected for its ability to utilize 5-C sugars, including xylose

Biotal Plus II: production of acetic acid improves aerobic stability Biotal Plus II treatment: USDA, Corn Silage & High Moisture Corn Reduced DM loss (0.5 % vs 4.8 %) and improved fermentation efficiency Significantly improved aerobic stability, e.g. Untreated Treated Time to heat: 57 h 161 h University of London, Corn Silage Biotal Plus II treatment gave: 12% improvement in DM recovery Less heating (36 F less after 3 days) Better ph stability (1unit after 5 days) Less DM loss after opening (162 lb/ ton)

Increases acetic acid Kills yeasts/molds Improves shelf-life Less DM losses Less spoiled silage Lactobacillus buchneri NCIMB 40788

Anaerobic conversion of lactic acid by Lactobacillus buchneri Lactic acid lactaldehyde 1,2-propanediol pyruvic acid acetyl CoA acetaldehyde ethanol acetic acid Oude-Elferink, et al., 1998

Effects of L. buchneri : corn silage studies meta-analysis Item Control LB1* LB2** Lactate, % 6.6 d 5.9 e 4.8 f Acetate, % 2.2 c 2.6 b 3.9 a Aerobic stability, h 25 b 35 b 503 a 26 published and citable comparisons (journal articles and meeting abstracts no in house reports or unpublished data) *LB1 100,000 CFU/g; **LB2 400,000 CFU/g. def Means in a row with unlike differ P < 0.10 abc Means in a row with unlike differ P < 0.05 Kleinschmitt and Kung, 2006. J. Dairy Sci.

Effects of L. buchneri : corn silage studies meta-analysis Item Control LB1* LB2** Lactate, % 6.6 d 5.9 e 4.8 f Acetate, % 2.2 c 2.6 b 3.9 a Aerobic stability, h 25 b 35 b 503 a 26 published and citable comparisons (journal articles and meeting abstracts no in house reports or unpublished data) *LB1 100,000 CFU/g; **LB2 400,000 CFU/g. def Means in a row with unlike differ P < 0.10 abc Means in a row with unlike differ P < 0.05 Kleinschmitt and Kung, 2006. J. Dairy Sci.

Spoilage reduction can be very visible: in the lab Untreated Top 6 treated with Top 6 untreated and on the dairy too!

DMI (lb/ d) Feeding spoiled silage is very bad practice! NDF-D (%) Effect of spoiled silage on dry matter intake 17 1.3 lb 16 15 Linear Actual Effect of spoiled silage on NDF-D 14 65 13 0 5.4 10.7 16 % Spoiled silage included in silage portion of ration 60 7.2 points 55 50 Linear Actual 45 Whitlock et al, 2001 40 0 5.4 10.7 16 % Spoiled silage included in silage portion of ration

So, what can feeding spoiled silage cost the producer, based on the Whitlock data? Feeding just a ration in which just 5% of the silage in was spoiled reduced dry matter intake by 1.3 lb/ hd/ day Over a 120 day finishing period that would mean intake would be reduced by 156 lb! Assuming a feed conversion ratio of 6.5:1, that would result in: 24 lb less gain over the 120 day finishing period. Worth $37.92/ head (Beef @ $158/ cwt; 03/02/2015)

On the dairy side, we can zero in on the fact that feeding a ration in which just 5% of the silage was badly spoiled, reduced total ration NDFD by 7.2 points Work at MSU, led by Mike Allen, showed that each point of NDFD is worth 0.55 lb of 4% FCM yield So, that 7.2 point reduction on total ration NDFD would cost the producer 3.96 lb of lost 4% FCM per head per day! Worth $24.82/ ton of silage DM (Milk @ $16/ cwt)

Remember this? Minimizing yeast growth is a good thing for production Milk (lb/ hd.day) Time Cows fed HMC from a 14 day supply removed from a silo, kept in a loose pile and fed daily. Milk yield declined as the level of yeasts in the pile rose. Hoffman and Ocker, 1997

And even if there is no visible spoilage, heating and the growth of yeasts still carries a significant cost! Well produced corn silage: good chop length, well-packed, covered, weighted with tires, tidy face. Feeder decided at 2 PM that he would knock down the next day s supply of silage to get an fast start in the morning. Let s see what that meant roughly 3 and a quarter hours later.

20 40

Effect of treating corn with Buchneri 40788 on microbial populations from farm silos (n = 31) Item Control L. buchneri, cfu/g equivalent (qpcr) 67,000 b 4,800,000 a Yeasts, cfu/g 320,000 a 43,000 b a,b Numbers in rows with unlike superscript differ, P < 0.05 Mari et al., 2009

Effect of treating corn with Buchneri 40788 on aerobic stability of farm silos (n = 31) a b a,b Columns with unlike letters differ, P < 0.05 Mari et al., 2009

High dose L. buchneri 40788 - FDA reviewed claim For improved aerobic stability of silage and high moisture corn stored for not less than 60 days. The FDA requires that inoculant products would supply not less than 4x10 5 (400,000) colony forming units (CFU) of L. buchneri per g fresh forage for the silages and not less than 6x10 5 (600,000) CFU of L. buchneri per gram of high moisture corn grain. The claim has been allowed following a thorough review of the large dataset of trials built by LAN using the L. buchneri NCIMB 40788 strain in high rate formulations.

Crops treated with L. buchneri 40788 products commercially since first launch in 1997 Grass, alfalfa and mixed haylages Grass, alfalfa and mixed dry baled hays Horseage Corn silage Sorghum silage Barley silage Wheatlage Oatlage Ryelage High moisture corn (all types: HMEC, HMSC, cobblage, etc.) High moisture small grains Pea silage Triticale and pea/ trit mixtures Sugar cane silage Sugar beet pulp Sweet corn waste Dry cereals ( soda grain alternative), etc., etc.??

400,000 CFU L. buchneri 40788 per gram of forage 100,000 CFU P. pentosaceus 12455 per gram of forage and the proven enzyme formulation from Biotal Plus II Combines the proven benefits of Biotal Plus II with the world-leading aerobic stability enhancement from Buchneri 40788 to control both ends of the ensiling process

ph Faster fermentation, better silage quality in 22% DM haylage Dr. Gbola Adesogan, UF, Gainesville 7 6 5 4 3 2 1 x x Untreated control Buchneri combo 0 0 10 20 30 40 50 60 Time (days) Parameter Control Buchneri 500 Control Buchneri Combo ph 4.08 a 3.50 b Crude Protein (g/ kg DM) 105 a 135 b Ammonia (g/ kg DM) 10.15 a 3.14 b DOMD (g/ kg DM) 326 a 409 b Dry matter recovery (%) 86.7 a 96.9 b Butyric acid (g/ kg DM) 23.1 a 0.0 b

Aerobic stability (h) Improving aerobic stability in 33% DM corn silage Prof. L. Kung Jr., UD 250 200 a 150 100 50 b c 0 Control Buchneri 500 Biom ax 5 Untreated Buchneri Biomax 5 control combo

Temperature (F) Effective on high moisture corn (~27% moisture) Pat Hoffman, UW-Madison Heating in aerobic stability tests 100 90 80 70 60 50 0 24 48 72 96 120 Time (h) Control Buchneri 500

Hours of Stability Treating the silage with Buchneri 500 vs. treating the TMR with propionic acid 400 320 240 Added to TMR 160 80 0 Corn Silage TMR Ctrl Buchneri 500 TMR mate 5 lb/t TMR mate 10 lb/t Schmidt and Kung, 2006

Does the Cost of Preservation Pay? Silage, lbs per cow/d $0.75/ ton Cost of the Preservative $1/ton $1.50/ ton $1.80/ton Cost (cents) per cow per day 20 3/4 1 1.5 1.8 40 1.5 2 3 3.6 60 2.2 3 4.5 5.4 80 3 4 6 7.2

Biotal L. Buchneri 40788 Milk Production Control LB 40788 TMR Stability (h) 68 100 Av Daily Milk (# s) 88.0 89.8* Milk Fat (%) 3.37 3.43 Milk Protein (%) 3.07 3.27 Milk Component Yield (# s) 5.66 6.0 (+6%) Limin Kung; University of Delaware; 2003 Ag Bagged Forages; Control & LB 40788 in Milk Cow TMR s

Biotal inoculant technologies Fast front end fermentation (rapid ph drop)

Biotal inoculant technologies Fast front end fermentation (rapid ph drop) Enzymes to drive and direct the fermentation to improve aerobic stability and enhance feed quality

Biotal inoculant technologies Fast front end fermentation (rapid ph drop) Enzymes to drive the fermentation and enhance feed quality World-leading stability at feed-out

Product Features Targets Front-end fermentation control Front-end fermentation, digestibility plus increased aerobic stability Front-end fermentation & digestibility plus worldleading, FDA-reviewed aerobic stability 1. Lower dry matter haylage(<35% DM) 2. Other silages where there is no concern over feed-out stability 1. Higher DM haylages (>35% DM) 2. Other silages where there is a concern over aerobic stability Where feed-out stability is a main issue: - problem haylages - drier corn silages - drought-affected silages - crops with field disease issues - HMC - silages that will be moved - for reduced TMR heating

Product Make-up 100,000 CFU/g of crop comprised of: 90,000 Pediococcus pentosaceus 12455 10,000 Lactobacillus plantarum 12422 Proprietary enzyme pack 120,000 CFU/g of crop comprised of: 100,000 Pediococcus pentosaceus 12455 20,000 Propionibacterium freudenreichii R2453 Patented high activity enzyme pack 500,000 CFU/g of crop on corn silage 750,000 CFU/g of crop on HMC comprised of: 400,000/ 600,000 Lactobacillus buchneri 40788 100,000/ 150,000 Pediococcus pentosaceus 12455 Patented high activity enzyme pack

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