Playing the Numbers Game

Like the Pioneer corn and soybean breeding programs, the process of developing new Pioneer® brand forage inoculants is a numbers game. The more bacterial strains and combinations of strains Pioneer can test, the more likely it will identify and offer new, high-performing inoculant products.

Pioneer researchers play this game well, annually subjecting thousands of bacterial strains and combinations to initial evaluations.

"Today, the maximum nutritional value of a forage is achieved the day the crop is harvested," says William Rutherford, Ph.D., Pioneer research coordinator. "The goal for the future is to make the forage higher in nutritive value than the day it was harvested."

Increasing test volumes
A key technique to pursue this goal is called high-throughput screening. High-throughput screening (HTS) is a research model - borrowed from the pharmaceutical industry - for rapid screening of bacterial strains and combinations of strains for their impact on aerobic stability.

HTS requires very small quantities of dried, ground forage, which is rehydrated to a specific dry matter level. The model simulates the conditions of field ensiling, alleviating the problem of year-round forage availability.

The rehydrated forage, along with the bacterial strain or strains being tested, is packed into individual wells - or micro-silos - on a 3- by 5-inch microtiter plate, the size of a plain index card. The plate holds 96 silos.

The primary cause of aerobic instability in silage is growth of yeast and mold, so Pioneer researchers challenge the bacteria in each well with these organisms. Strains that effectively keep the mold or yeast from growing are kept for further evaluation. Those that don't are discarded.

"HTS allows us to evaluate much larger numbers of bacterial strains than in the past, which greatly speeds the product-development process," Rutherford says. "With high-throughput screening, we quickly analyze and throw away the 50 percent of the strains that have absolutely no chance of inhibiting mold and yeast organisms."

Many possibilities
Screening efficiency is necessary due to the large numbers of bacterial strains available. "Within each genus and species of bacteria, there can be as many as 100,000 to 1 million different strains," Rutherford says. "They all have the same name, but they're all different. It's like comparing Holstein cows: They all have the same basic look, but some are much better performers than others."

The standard methodology for testing inoculants has been actual, on-farm silo trials, Rutherford explains. "We'd go to the farm, harvest the forage and apply the inoculant as the forage went into the silo," he says. "The actual number of bacterial strains we could test each year was obviously very limited."

Advancing to other trials
While high-throughput screening greatly speeds initial evaluation time, it's still just the first step. Pioneer has developed a detailed process designed to identify, characterize and isolate unique bacterial strains that improve forage fermentation, nutrient availability and bunklife.

"Keeper" strains identified through micro-silo testing advance to an additional round of evaluation trials, including "mini-silo" trials.

Mini-silos are 4- by 14-inch PVC pipes with rubber caps on each end. The forage or high-moisture corn placed in each silo is hydraulically compressed. After fermentation, researchers remove the grain or silage from the PVC silo and evaluate it for visible spoilage, fermentation acids, pH and microbial population.

After the PVC mini-silo evaluation, the silage goes into another container, which is placed in an aerobic stability chamber to determine bunklife characteristics. While the chamber temperature is held constant, any changes in silage temperature are automatically recorded.

Does it perform in livestock?
The "keeper" bacterial strains from the mini-silo and aerobic stability chamber testing then advance to live-animal feeding trials. These feeding trials use up to 80 steers. Neck transponders and a computerized feeding system allow researchers to monitor feed intake of each animal to determine individual average daily gain and feed efficiency.

Only after these live-animal trials will Pioneer make product commercialization decisions.

"Even though PVC mini-silo testing is significantly faster than live-animal feeding trials or on-farm testing, it still can't compete with high-throughput screening for initial evaluation," Rutherford says. "With conventional PVC mini-silo testing, it could take six weeks to two months to evaluate 100 bacterial strains. With high-throughput screening, we can evaluate 1,000 samples or more every day, 52 weeks a year."

Lactobacillus buchneri example
Research techniques such as high-throughput screening allow Pioneer to introduce new-generation inoculant products that promote aerobic stability.

Lactobacillus buchneri strain LN6071 and patent #6,337,068 - contained in Pioneer brand 11A44 inoculant, for example - proved to have unique bunklife properties equal to propionic acid-based forage additives. Pioneer researchers analyzed more than 50 strains of Lactobacillus buchneri before deciding strain LN6071 was best.

"Forage ensiling mismanagement - such as harvesting too dry and too mature, excessive chop length, not properly packing or not sealing during silo filling - leaves the silage prone to aerobic instability at the 'front-end' of the fermentation process," says Bill Seglar, DVM, Pioneer nutritional sciences veterinarian. "Because Lactobacillus buchneri is primarily active three to four weeks after ensiling, end products with this strain will not have a strong impact on minimizing aerobic instability the initial days just after filling."

However, the volatile fatty acid profile of L. buchneri leads to improved bunklife when feeding out silage at the 'back-end' of the fermentation process, Seglar says.

"Making good silage is more than just a matter of piling up a bunch of chopped forage," Rutherford concludes. "There's a lot of science and technology involved. At Pioneer, we use true, hard science to create silage additives. No company has more science behind its inoculants than Pioneer."