The Effects of L-Carnitine and Omega-3 Fatty
Acids on Porcine Embryonic Muscle Cell
Proliferation and Differentiation
Amber E. Brazle
Project Advisor: Dr. Brad Johnson
Academic Advisor: Dr. Miles McKee
May 12, 2004
The Effects of L-Carnitine and Omega-3 Fatty Acids on Porcine Embryonic Muscle
Cell Proliferation and Differentiation
Introduction
L-Carnitine is a water-soluble vitamin-like compound that is synthesized
primarily in the liver and kidney and is involved in protein and lipid metabolism.
According to recent studies, supplementing sows with L-Carnitine resulted in larger-cross
sectional areas of the semitendinosus muscle (Musser et al., 2001). The offspring also
had larger loineye areas and higher percentages of lean tissue (Musser et al., 1999).
Additional studies by Eder et al., 2001, suggest that the number of non-viable pigs will
decrease and the fetuses will have heavier birth weights.
Omega-3 fatty acids are essential polyunsaturated fatty acids (PUFAs) that cannot
be synthesized in the body. Alpha-linolenic acid is an eighteen-carbon fatty acid with
three double bonds. Omega 3 fatty acids make the blood less likely to form clots that
cause heart attacks and protect against irregular heartbeats that cause sudden cardiac
death (Harris et al., 2002).
This study was designed to determine the interactive effects of Omega-3 fatty
acids, specifically, linolenic acid, on porcine embryonic muscle cell proliferation and
differentiation in cells isolated from fetal pigs at mid gestation from sows fed L-Carnitine
as compared to cultures established from sows receiving no supplemental L-Carnitine
during gestation. Using muscle cultures for research is beneficial to the scientific world
by reducing the number of live research subjects required to determine the effects of a
compound on muscle growth and differentiation. Therefore, evaluating the effect of L-
Carnitine and Omega-3 fatty acids on porcine embryonic muscle growth and
differentiation will be used to determine more efficient swine production methods as well
as increase the nutritional value of pork.
Materials and Methods
Porcine embryonic muscle cells were obtained by completing hysterectomies on
sows at mid-gestation. The fetal pigs were removed under aseptic conditions and
transported to a laminar hood. Myogenic muscle cells were isolated, suspended in
solution, and stored in liquid nitrogen. Ann Waylan performed this portion of the
procedure in her Ph.D. research on the effects of supplementing L-Carnitine on the IGF
systems in porcine embryonic myoblasts.
Each week for six weeks, the same procedures were used each day. The cells
from one control and one L-Carnitine sow were plated and processed each week.
Prior to plating the cells, it was necessary to provide a foundation for the cells to
be plated on. On Day 1, a mixture of matri gel and serum free Dulbecco’s Modified
Eagle Medium (SF/DMEM) with antibiotics and gentamicin was plated at 0.25 mL on
three wells of two-four well plates, 0.25 mL on fifteen wells of a twenty-four well plate,
and 0.50 mL on six wells of two-six well plates. The antibiotics and gentamicin were
added to prevent unwanted contamination. The matri gel solution was allowed to set up
for one hour in an incubator. Excess solution was aspirated off and warm SF/DMEM
media was added.
The four well plates were to establish a 24 hour count, the six well plates were for
the differentiation studies, and the twenty-four well plate was to measure thymidine
incorporation with and without the fatty acid in the sows fed L-Carnitine as well as those
not receiving the L-Carnitine supplementation.
On Day 2, cells were taken from the liquid nitrogen tank and added to 15 mL of
10% Fetal Bovine Serum (FBS)/DMEM with the antibiotic and gentamicin. Fetal Bovine
Serum contains all of the necessary growth factors and nutrients. This solution was
centrifuged for ten minutes. The media was aspirated off of the pellet and 25 mL of the
10% FBS/DMEM media was added. This was vortexed and triturated to ensure even
distribution of the cells during plating. Two mL of the cell solution was plated on the
two-six well plates, one plate per sow, and 1 mL to three wells of the four well plates as
well as six wells per sow on the twenty-four well plate. All plates were incubated at
thirty-seven degrees Celsius in a carbon dioxide incubator.
Day 3 involved rinsing each of the wells and adding fresh media, providing
nutrients to the cells. The twenty-four hour count was taken on the four well plates. The
test media with the alpha-linolenic acid was added to three wells of the six well plates
and to three wells of each sow on the twenty-four well plates. Control media was added
to the remaining wells.
Tritiated thymidine was added to fifteen wells of the twenty-four well plate on
Day 5. Tritiated thymidine is an indicator of the rate of cell proliferation. The two sixwell plates were rinsed and swine serum/DMEM with the additional antibiotic and
gentamicin was applied with the test and control media to the respective wells. (Porcine
embryonic muscle cells differentiate better in swine serum.) After three hours, the
twenty-four well plate with the tritiated thymidine was rinsed with cold SFDMEM, and 1
mL of cold 5% trichloroacetic acid (TCA) was added. The TCA releases the
unincorporated tritiated thymidine so it can be removed. The seventy-two hour count
was taken from three fields of the three control wells for each animal. The muscle cells
on the wells with alpha-linolenic acid were too numerous to count.
On the next day, 1 uL of Ara-C was applied directly to the wells on the two-six
well plates. Ara-C inhibits all cell division, but differentiation could still occur. The
twenty-four well plate was rinsed with 5% TCA, and 0.5 mL of NaOH was added to each
well. The plate was incubated for thirty minutes. Afterwards, the contents of each well
were added to a vial with 10 mL of scintillation fluid. The wells were rinsed with 1 mL
of the scintillation fluid that was also added to the vials. Vials were vortexed and
counted for thymidine incorporation.
The last stage of the process was to stain the two-six well plates. This enabled
pictures to be taken of the myotube formation and fusion. Five pictures were taken for
each well, and each picture was counted for multi-nucleated cells. Fusion is a marker of
differentiation. It is standard procedure to use this measure to estimate the degree of
differentiation.
Results and Discussion
The following results are numerically based, not statistical. These data suggest
numerically that supplementing L-Carnitine to the gestating sow increased the
proliferation of cells at twenty-four hours. (See Figure 1.) These data are consistent
with previous studies in our laboratory (Waylan, 2003). It also appeared that Omega-3
fatty acids may increase myotube numbers at 120 hours in both control and L-Carnitine
cells. However, the degree of differentiation in L-Carnitine cells with and without the
fatty acid was higher than the control with the fatty acid added. The L-Carnitine with the
fatty acid treatment had a 15.8% increase over the control wells with the fatty acid. The
L-Carnitine wells without the fatty acid added had an 11.4% increase over the control
wells lacking the fatty acid. (See Figure 2.) An increase in the number of doublings
between twenty-four and seventy-two hours was observed in the sows fed L-Carnitine.
(See Figure 3.)
This too is consistent with Ann’s data. Thymidine incorporation
measures the rate of proliferation. The measure of thymidine incorporation suggested
that the control cells may have had a greater rate of proliferation at seventy-two hours.
(See Figure 4.)
Conclusions
The data show numerical differences in proliferation of the cells isolated from the
sows supplemented with L-Carnitine during gestation as well as an increase in the
number of doublings between twenty-four and seventy-two hours in the sows fed LCarnitine. It also appeared, numerically, that Omega-3 fatty acids may increase the
degree of differentiation at 120 hours in both the control and treatment groups. These
findings contribute to our understanding of porcine fetal growth and development.
Acknowledgements
The author would like to thank Ann Waylan for the cell collection work and for
guidance provided throughout the study. Thanks are also extended to the College of
Agriculture Honors Program and to the Department of Animal Science.
References
Eder, K., A. Ramanau, and H. Kluge. 2001. Effect of L-Carnitine supplementation on
performance parameters in gilts and sows. J. Anim. Physiol. a. Anim. Nutr.
85:73-80.
Harris, William S. and Lawrence J. Appel. Nov. 2002. American Heart Association,
New guidelines focus on fish, fish oil, omega-3 fatty acids.
Musser, R.E., R.D. Goodband, M.D. Tokach, K.Q. Owen, J.L. Nelssen, S.A. Blum, S.S.
Dritz, and C.A. Civis. 1999b. Effects of L-carnitine fed during gestation and
lactation on sow and litter performance. J. Anim. Sci. 77:3289-3295.
Musser, R.E., R.D. Goodband, K.Q. Owen, D.L. Davis, M.D. Tokach, S.S. Dritz, and J.L.
Nelssen. 2001. Determining the effect of increasing L-carnitine additions on sow
performance and muscle fiber development of the offspring. J. Anim. Sci.
79(Suppl. 2):65 (Abstr).
Waylan, Ann. 2003. Effects of L-carnitine on fetal growth and the IGF system in pigs.
(Dissertation).
Figures
Effect of Treatment on Average Number
of Cells per cm2 at 24 Hours
2500
2000
1500
1000
500
0
Control
L-Carnitine
Figure 1: Number of cells at twenty-four hours.
Effect of Treatments on Myotube
2
Numbers per cm at 120 Hours
600
500
400
300
200
100
0
With Fatty Acid
Figure 2: Number of myotubes at 120 hours.
Without Fatty Acid
Effect of Treatment on
Number of Doublings
2.25
2.2
2.15
2.1
2.05
2
1.95
Control
L-Carnitine
Figure 3: Number of doublings.
Effect of Treatments on
3
[ H]thymidine Incorporation per Cell
0.4
0.3
50.3
0.2
50.2
0.1
50.1
0.0
5 0
With Fatty Acid
Figure 4: Thymidine incorporation per cell.
Without Fatty Acid
Pictures of Cell Cultures
24 Hours
L-Carnitine with Fatty Acid
72 Hours
L-Carnitine without Fatty Acid
Control with Fatty Acid
Control without Fatty Acid