Hello, everyone. I am Charmaine Espinosa, a postdoctoral research associate working with
Dr. Hans Stein. Today, I will discuss one of the research we have conducted at the
University of Illinois, where we determined the interactive effects of dietary copper and
fiber on nutrient digestibility and intestinal microbial protein.

Copper has been recognized as an important micronutrient needed for maintenance, growth,
and optimum health. And that is because copper is involved in several metabolic reactions
including cellular respiration, tissue pigmentation, and hemoglobin formation. Copper is
also an essential component of several metalloenzymes including cytochrome oxidase and
lysyl oxidase and is involved in oxidation-reduction reactions and protection against
oxidative stress.

It is well established that copper may be included at growth promoting levels in diets for
weanling and growing pigs. Research demonstrated that copper included at a range of 75 to
250 mg/kg in diets for pigs usually improve average daily gain and gain to feed ratio.
Several modes of action for the improved growth performance have been proposed, and one of
these is copper’s ability to reduce bacterial populations and activity in the intestine,
which may affect growth and community structure of microorganisms in the cecum and colon.
The hypothesized ability of copper to alter microbial activity may also subsequently
affect fermentation and total tract digestibility of nutrients.

In the previous research we have presented at the Midwest meeting last year, we
demonstrated that the apparent total tract digestibility of fat was improved if copper
hydroxychloride was supplemented to high-fiber diets. Where here, blue bars represent
diets without copper supplementation and the orange bars represent diets with copper
hydroxychloride at 150 mg/kg. And this observed improvement in the apparent total tract
digestibility of fat may be due to the effect of copper in modulating microbial activities
in the intestinal tract coupled with a subsequent reduction of fecal fat from microbial
origin. However, the effect of Cu on the digestibility of other nutrients is still
limited, and the effect of copper on microbial protein concentration has not yet been
elucidated.

Therefore, the objective of this experiment was to test the hypothesis that
supplementation of 150 mg/kg of copper from copper hydroxychloride improves the apparent
ileal digestibility and total tract digestibility of nutrients in diets without or with
distillers dried grains with solubles or DDGS, and also reduces the concentration of
microbial protein in the intestinal tract.

Twenty-four pigs were surgically fitted with a t-cannula in the distal ileum and were
randomly allotted to a 2 × 2 factorial design with two levels of DDGS at 0 or 45% and two
levels of copper from copper hydroxychloride at 0 or 150 mg/kg. This experiment consisted
of two periods for a total of 12 replicate pigs per diet.

For each period, pigs adapted to their diets for 9 days, and from day 10 to day 12, fecal
samples were collected. On day 13 and 14, ileal digesta samples were collected for 8
hours. The collected fecal and ileal digesta samples were analyze, to calculate for
nutrient digestibility, and they were also used for the analysis of volatile fatty acid
concentration and microbial protein concentration.

For the microbial protein analysis, samples were fractionated using differential
centrifugation. Samples were centrifuged first at 250 RCF for 15 minutes at 4ºC, and this
resulted in fractions that were expected to contain undigested feed particles in the
precipitate and porcine cells in the supernatant. The supernatant from the 250-RCF
centrifugation was then extracted, and this sample was centrifuged at 14,500 RCF for 30
minutes to result in a precipitate that was expected to contain microbial cells. These
cells were then subjected to a lysis buffer, and the protein concentration of the lysed
microbial cells was then analyzed.

Moving on with the results.

This graph shows the digestibility of gross energy in 4 diets; blue bars represent the
apparent ileal digestibility of gross energy, and orange bars here represent the apparent
total tract digestibility of gross energy. Lighter bars represent diets without
supplemental copper, whereas dark colors represent diets containing supplemental copper.
And this setup will be the same in the following slides. Interactions between the DDGS and
copper were observed, where inclusion of 45% DDGS in the diet without added copper
resulted in a greater reduction in apparent ileal digestibility and total tract
digestibility of gross energy compared with the diet containing 45% DDGS and 150 mg/kg of
copper from copper hydroxychloride. In addition, the observation that the apparent ileal
digestibility and total tract digestibility of gross energy were reduced as the
concentration of total dietary fiber increased in the diets is likely due to the insoluble
portion of dietary fiber in DDGS, which are poorly utilized by pigs. Fermentation of
dietary fiber results in synthesis of methane and microbial biomass, and this may reduce
the efficiency of energy utilization from dietary fiber.

For crude protein digestibility, no interaction was observed between the two factors, no
effect of copper, however inclusion of 45% DDGS in diets resulted in a reduction in both
the apparent ileal digestibility and total tract digestibility of crude protein. Inclusion
of high concentration of DDGS leads to an increased concentration of total dietary fiber
in diets, and the observed reduction likely is a result of increased endogenous
secretions, or faster gastric emptying, which subsequently reduces the time that feed
proteins are exposed to proteolytic enzymes.

Before I go and dive in to discuss the effect of DDGS and copper on fat digestibility, I
want to point out here that the apparent total tract digestibility of fat was less
compared with the apparent ileal digestibility of fat regardless of DDGS or copper
hydroxychloride inclusion because lipids cannot be absorbed in the hindgut, and therefore
this results in an increased synthesis of microbial fat. And this graph demonstrates that
inclusion of DDGS resulted in an improvement in the apparent ileal digestibility and total
tract digestibility of fat due to the greater concentration of fat in diets containing
DDGS. Also, inclusion of copper hydroxychloride resulted in an improved ileal and total
tract digestibility of fat. Dietary copper reduces a number of bacterial species in the
small intestine and in the cecum and colon of pigs, and this may have reduced fermentation
of fiber in the hindgut, with a subsequent reduction in the endogenous loss of fat.

To indirectly confirm if copper hydroxychloride changes microbial activity in the
intestinal tract, concentrations of microbial protein in ileal digesta and fecal samples
were analyzed. Here we can observe that the concentration of microbial protein in ileal
digesta was not affected by DDGS or copper concentrations.

However, if we take a look into this graph, fecal microbial protein concentration
increased if diets contained DDGS, whereas copper supplementation reduced the
concentration of microbial protein in feces. Increased concentration of fiber in diets
upon DDGS inclusion results in increased fermentation by microbes in the intestinal tract.
Therefore, the observed increase in the concentration of microbial protein upon DDGS
inclusion is likely due to an increased growth of bacteria in the hindgut with the
undigested portion of fiber serving as substrate for microbes to metabolize. On the
contrary, the observed reduction on fecal microbial protein concentration upon copper
supplementation likely a result of the bacteriostatic properties of dietary copper, by
which copper has the ability to disrupt enzyme structures and functions of bacteria. 

The observed response in microbial protein further supports the results we have observed
in the concentration of volatile fatty acids, where no differences were detected in the
concentration of volatile fatty acids in ileal digesta.

However, copper supplementation tended to reduce the concentration of volatile fatty acids
from carbohydrate fermentation in the feces due to its effect on altering the growth,
activity, and metabolism of microbes. 

In conclusion, inclusion of 45% DDGS in diets resulted in a reduced apparent ileal
digestibility and total tract digestibility of gross energy and crude protein, and this is
likely due to increased endogenous loss of nutrients and reduced utilization of energy.
And addition of copper hydroxychloride to the diets did not ameliorate this effect.
Supplementation of copper hydroxychloride to diets reduced the concentration of total
volatile fatty acids and microbial protein in the feces, indicating reduced microbial
activity in the hindgut of pigs fed diets containing copper hydroxychloride, and these
reductions were independent of the presence of fiber from DDGS in the diets. And in this
research, we demonstrated that copper supplementation in diets improved the apparent ileal
digestibility and total tract digestibility of fat, which is likely caused by reduced
endogenous loss of fat due to reduced excretion of microbial fat.

I would like to take this opportunity to acknowledge Micronutrients and Agrispecialist for
their financial support,

and of course, to all members of the Stein Monogastric Nutrition Laboratory for helping us
in executing this research. Thank you for listening, and if you would like to learn more
about other topics in nutrition, you can visit our website at
nutrition.ansci.illinois.edu.