Hello, my name is Charmaine Espinosa, and I am a postdoctoral research associate working
with Dr. Hans H. Stein. Ever wonder how pharmacological levels of dietary copper improve
growth performance of pigs? Well here in this podcast, I’ll be discussing one of the
research we have conducted at the University of Illinois looking at the effect of copper
hydroxychloride supplementation on growth performance and its relation on the abundance of
genes involved in lipid metabolism of growing pigs.

Several research demonstrated that supplementing copper to diets fed to pigs at 75 to 200
mg/kg reduces postweaning scouring and also improve average daily gain and gain to feed
ratio of pigs. And one of the hypothesized mechanisms of copper in improving growth
performance is that copper may have the ability to improve animal fat utilization and
enzymatic activity. As you may know, copper is involved in metabolic reactions, and also a
component of several metalloenzymes, which may stimulate enzyme activities involved in
nutrient digestion. Addition of high concentrations of Cu may therefore increase lipase
and phospholipase A activities in the small intestine, which may result in increased
absorption of fatty acids and improved growth performance.

And just to give an example, we conducted a growth performance experiment at the
University of Illinois where we wanted to determine if copper hydroxychloride exerts some
positive effect in our swine research facility. And here we demonstrated that Cu
hydroxychloride, represented here in orange bars, resulted in a greater average daily
gain, and also in an improved gain to feed ratio. We then hypothesized that these observed
improvements in growth performance were due to the effects of copper in improving energy
and fat digestibility.

However, we demonstrated in our previous research that yes, copper supplementation
improves the apparent total tract digestibility of fat. But if we take a look at this
data, we can observe that supplemental copper did not improve the true total tract
digestibility of fat. Therefore, the improvement in the apparent total tract digestibility
of fat upon copper supplementation was only due to the ability and bacteriostatic property
of copper in reducing the endogenous loss of fat from 11 to 7 g/kg DMI. 

The lack of effect of copper on the true total tract digestibility of fat further supports
the results that Coble and others demonstrated in their research, where here we can
observe that copper supplementation in diets for finishing pigs did not increase the mRNA
expression of proteins involved with digestion, such as copper transport protein-1 and
fatty acid binding protein, in the mucosal layer of small intestine in pigs.

So, if it’s not due to digestibility, copper may have some effects on the post-absorptive
metabolism of lipids, since in other species, it was demonstrated that inclusion of 45
mg/kg of dietary copper in diets for rabbits improved body mass gain by upregulating the
mRNA transcription of fatty acid transport protein and fatty acid binding protein in
skeletal muscle. Supplementation of copper to diets also increased lipogenesis and fatty
acid uptake in fish by upregulating the mRNA transcription of fatty acid synthase and
acetyl CoA carboxylase in intestinal tissue. Therefore, it is possible that copper will
also exert similar effects in pigs; however, the effect of supplementing dietary copper
above the requirement on postabsorptive lipid metabolism in pigs is limited and remains
inconclusive.

Therefore, the objective of this experiment is to test the hypothesis that supplementation
of copper hydroxychloride at 150 mg/kg to diets improves growth performance by
upregulating the mRNA transcription of genes involved in post-absorptive metabolism of
lipids.

Thirty-two pigs were randomly allotted to 2 experimental diets. The first diet is
formulated based on corn, soybean meal, and distillers dried grains with solubles, and the
second diet was formulated as the first diet with the exception of adding 150 mg/kg of
copper from copper hydroxychloride.

Experimental diets were fed to pigs for 28 days, and on the last day of the experiment,
one pig per pen was sacrificed for us to be able to harvest liver, skeletal muscle, and
adipose tissue. Weights of pigs and their feed intake were recorded to analyze data for
growth performance. Harvested tissues were used to analyze expression of genes involved in
lipid metabolism. Tested genes involved in lipogenesis include acetyl CoA carboxylase and
fatty acid synthase. Tested genes also include lipoprotein lipase which hydrolyzes the
triacylglycerol component of chylomicrons and very low-density lipoproteins to
monoglycerides and free fatty acids. Tested genes involved in the catabolism and oxidation
of fatty acids include hormone sensitive lipase and carnitine palmitoyl transferase 1 or
CPT1, and genes that belong to the nuclear receptor family include peroxisome
proliferator-activated receptor alpha, or PPARα, and peroxisome proliferator-activated
receptor gamma, or PPARγ. Lastly, we also tested for genes involved in the uptake and
transport of fatty acids which are fatty acid binding protein, fatty acid transport
protein, and cluster of differentiation 36, or CD36.

Moving on with the results: This figure shows the overall gain to feed ratio of pigs fed
the control diet here in the blue bar, and pigs fed the diet containing copper
hydroxychloride in the orange bar. And this representation will be the same in the
following slides. Same as what we observed in previous experiments, pigs fed the diet
containing copper hydroxychloride had greater gain to feed ratio compared with the
control. And let’s figure out, how can we possibly explain these consistent improvements?

This graph shows the mRNA abundance of genes in the subcutaneous adipose tissue. Here we
can observe that there were no differences in these tested genes among treatments.
However, the expression of lipoprotein lipase and fatty acid binding protein 4 increased
when copper hydroxychloride was added to the diet. The observed increase in the expression
of lipoprotein lipase upon copper hydroxychloride supplementation is possibly due to the
role of copper as a cofactor of the activator complex of lipoprotein lipase, where the
activation of lipoprotein lipase is influenced by apolipoprotein and divalent cations.
This then indicates an increased uptake and utilization of fatty acids from the hydrolysis
of the triacylglycerol component of chylomicrons, which is possibly the reason of the
increased expression of fatty acid binding protein 4 upon copper supplementation.
Supplementation of copper hydroxychloride also tended to increase the mRNA abundance of
CPT1, indicating an increased synthesis of ATP due to enhanced oxidation of fatty acids.

Going now to the liver, here we can observe an increased expression of CD36 and a tendency
for an increased fatty acid binding protein 1 upon copper supplementation, and this may
indicate increased uptake of fatty acids by hepatocytes. Lipids in the liver may originate
from non-esterified fatty acids and lipoprotein remnants formed from the action of
lipoprotein lipase. The increased expression of lipoprotein lipase in the adipose tissue
upon copper supplementation may have increased the concentration of non-esterified fatty
acids and remnant particles in the plasma membrane. This increased flux of fatty acids may
have activated and increased the abundance of fatty acid binding protein 1 and CD36 in the
liver because these genes have high affinities for fatty acids. However, no difference was
observed for the remaining genes.

And in the muscle, no differences among treatments were observed in these tested genes;
however, a tendency for an increased abundance in PPARα was observed upon copper
supplementation, which may indicate an increased uptake of fatty acids in skeletal muscle
by pigs fed the diet containing copper hydroxychloride. PPARα favors upregulation of genes
involved in the uptake and oxidation of fatty acids, and this may have improved fatty acid
oxidation and subsequently increase synthesis of ATP. And the bottom line here is that it
is possible that the observed improvement in growth performance of pigs fed the
copper-supplemented diets may be a result of improved lipid metabolism with a subsequent
improvement in energy utilization.

In conclusion, supplementation of 150 mg/kg of copper from copper hydroxychloride to the
control diet improves gain to feed ratio of pigs, which we have consistently demonstrated
in our research. This improvement may be partially explained by the ability of
supplemental copper in increasing the mRNA abundance of genes involved in the uptake,
transport, and oxidation of fatty acids.

With that, 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.