The Mechanism of Gossypol Detoxification by
Ruminant Animals1
RAYMOND REISER AND HWEI C. FU
Department of Biochemistry and Nutrition,
Texas Agricultural Experiment Station,
College Station, Texas
It has been known for many years that
cottonseed meal containing free gossypol
produces toxic symptoms if fed to swine
(Withers and Carruth, '15, '18) or chicks
(Couch et al., '55; Rigdon et al., '58) at
significant levels, and that gossypol has
been demonstrated to be toxic to other ani
mals (Withers and Carruth,'18; Schwartze,
'24; Eagle, '50). But it has also been dem
onstrated that relatively large amounts of
gossypol contained in cottonseed meal may
be fed to ruminant animals with no sign
of toxicity (Cranford, '10; Jones et al., '41;
Ramsey and Miles, '53).
The mechanism whereby gossypol is de
toxified by the ruminant animal has re
mained unknown. Since most differences
in metabolism between ruminant and non-
ruminant animals may be traced to the
activity of rumen microorganisms, the pos
sibility was considered that these organ
isms might also act upon gossypol to de
stroy it in some manner.
Previous work in this laboratory has
shown that dietary polyunsaturated fatty
acids may be hydrogenated by rumen
liquor,2 or in the rumen in vivo (Reiser and
Reddy, '56). This suggested that gossypol
might also be reduced in the highly re
ducing environment of the rumen.
EXPERIMENTAL
Preparation of soluble gossypol. About
50 mg of crystalline gossypol3 were dis
solved in 15 ml of alcohol. Twenty milli-
liters of glycerol were added and the
alcohol evaporated on a steam bath under
reduced pressure. The product is a clear
sol which may be mixed with water in any
proportion without precipitation.
Determination of free and bound gossy
pol. Gossypol assays, free and bound,
J. NUTRITION, 76: "62
were determined by the method of Pons
and Hoffpauir ('57).
Determination of free t-amino group.
This assay was made by the method of
Baliga et al. ('59).
Preparation of rumen liquor. Rumen
liquor was obtained from freshly slaugh
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tered beefs, then strained through cheese
cloth and used as soon as possible.
Aerobic and anaerobic incubation. To
determine whether anaerobic or aerobic
organisms have a different influence on
gossypol, 3.5 ml of the strained liquor were
mixed with 1.25 ml of gossypol sol. In
one set of experiments 0.5 ml of 4% glu
cose and in another set 0.5 ml of 95%
alcohol were added as a source of energy.
The results of the study are given in
table 1. There was no loss of total gos
sypol, but the free gossypol was signifi
cantly reduced, indicating a binding effect
but no destruction. There was probably
no significant difference between the aero
bic and anaerobic cultures, bringing into
question bacterial participation in the free
gossypol disappearance. Nevertheless glu
cose apparently stimulated more activity
than alcohol. However, different rumen
liquors were used and there was no con
trol run without glucose or alcohol.
Effect of energy source. The influence
of the nature of the added energy source
was further studied by anaerobic incuba
tion. The results (table 2) do not support
the superior activity of glucose. This sug-
Received for publication September 18, 1961.
1 Supported, in part, by a grant from the Oscar
Johnston Foundation.
- Reiser, R. 1951 Hydrogénation of polyunsatu
rated fatty acids by the ruminant. Federation Proc.,
10: 236 (abstract).
3 Gossypol acetate was sent to us from the Southern
Utilization Research and Development Division, USDA,
New Orleans, Louisiana by Dr. T. H. Hopper whose
kindness we gratefully acknowledge.
215
216 RAYMOND REISER AND HWEI C. FU

TABLE 1
Effect of aerobic and anaerobic incubation of rumen fluid and gossypol on the disappearance
of total and free gossypol

gossypolIncubation Disappearance of
conditions^Anaerobic

62 ±4.9s 0 29 ±2.6 0
AerobicTestFree% 75±2.1JÕ.3Total%
0TestFree% 36±4.123,4Total%
0
1 Incubation at 37°C for 48 hours.
2 Test 1. Rumen liquor, 3.5 ml; 4% glucose, 0.5 ml; and gossypol solution, 1.25 ml.
3 Each value is the result of 4 replicate tests.
4 Test 2. Rumen liquor, 3.5 ml; 95% alcohol, 0.5 ml; and gossypol solution, 1.25 ml.
••
Standard deviation.

TABLE 2
Effect of incubating gossypol and rumen liquor anaerobically with glucose or alcohol

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gossypolTest of
Mediai 1»Free% 22Free%
no.i»2»3«Disappearance

±9.0*70±1.881
85 000Test 5.233
80 ± 000
4.454±10Total%
±
±4.6Total%
1 Incubation at 37°Cfor 48 hours.
2 Each value is the result of 4 replicate tests.
3 Medium no. 1. Rumen liquor, 5 ml; gossypol solution, 0.2 ml.
4 Standard deviation.
5 Medium no. 2. Rumen liquor, 5 ml; gossypol solution, 0.2 ml; and 2% glucose, 2 ml.
6 Medium no. 3. Rumen liquor, 5 ml; gossypol solution, 0.2 ml; and 95% alcohol, 1 ml.

TABLE 3
Effect of centrifugation of rumen fluid at various speeds on the formation
of the bound gossypol

Centrifugation gossypolTest of free

speed
2»30±0«33 3»36
I»%49485855Test 4*67±9.167±369±664±0Tests»59±059±059±059±0
(20min.)None5001,0002,0002,5004,5005,0006,0006,50010,00010,500Disappearance

±2.229
±2.548 ±2.439
±2.530±545±5Test
±8.331±3.1Test

1 Test 1. Rumen liquor, 10 ml; gossypol solution, 0.2 ml; incubated aerobically at 37°C for 24
hours; one tube at each speed.
2 Test 2. Rumen liquor, 5 ml; gossypol solution, 0.2 ml; and 1% glucose, 2 ml; incubated an
aerobically at 37" C for 48 hours, in duplicate.
3 Test 3. Rumen liquor, 5 ml and gossypol solution, 0.2 ml; incubated anaerobically at 37°Cfor
48 hours, in triplicate.
*Test 4. Rumen liquor, 5 ml and gossypol solution, 0.4 ml; heated for 20 minutes at 70°C in
a water bath, in duplicate.
5 Same as test 4, except that a different sample of rumen fluid was used, in duplicate.
6 Standard deviation.
 GOSSYPOL
gests that the reaction is probably not due
to a reduction of the gossypol to a colorless
derivative by bacteria and again suggests
that the disappearance of free gossypol is
not due to microbial action.
Activity of centrifugea rumen liquor.
To determine whether the gossypol is
bound by bacteria or by soluble protein,
samples of rumen liquor were centrifuged
at various speeds up to 10,500 rpm and
then incubated with gossypol sols for 48
hours at 37°C.The results (table 3) show
clearly that centrifugation had no influ
ence on the percentage of gossypol that
was bound. Although bacteria are diffi
cult to remove completely from rumen
liquor by centrifugation, there are rela
tively few remaining in the supernatant
after 20 minutes at 10,500 rpm, and one
should expect a graded decrease in num
bers from uncentrifuged liquor. If the
gossypol had been bound by the bacteria
one should expect a decrease in the degree
of binding in the centrifuged specimens.
Because no decrease occurred, the tenta
tive conclusion was made that the gossypol
was bound by soluble protein.
Nonbacterial nature of the reaction. A
second test to determine whether the gos
sypol is bound by rumen bacteria or solu
ble protein was made by adding gossypol
sol to the liquor and immediately immers
ing in a hot water bath at 70°Cfor 10, 20,
30, or 60 minutes.
The results (table 4) show clearly that
the binding effect cannot be due to the met
abolic action of bacteria because the reac
tion was completed at 70° C in 10 minutes
or less. The reaction is thus chemical.
TABLE 4
Effect of heating gossypol-rumen fluid solutions
for varying periods of time at 70°C on
the disappearance of total and
free gossypol
gossypol'-2Free81±1.7379
of
Heating
periodminutes10203060Disappearance
±2.580
±4.482
±7.4Total0000
1 Determined in triplicate.
2 The test solutions contained 0.4 ml of gossypol
solution and 5 ml of rumen fluid.
3 Standard deviation.
DETOXIFICATION 217
Comparison of bacterial incubation and
hot water temperatures. To obtain some
comparison between the hot water bath
and incubation reactions, 10-ml samples
of rumen liquor were mixed with 4 ml of
gossypol sol and either held at 70°Cfor
20 minutes, aerobically, or incubated at
37°Cfor 48 hours anaerobically. Dupli
cate tests gave identical results, the 70°C
reaction binding somewhat more gossypol
than the 37°C incubation reactions
(table 5).
Relation between binding of free gos
sypol and disappearance of lysine ¿-amino
groups. A final test of whether the gos
sypol is bound by rumen liquor protein is
the simultaneous disappearance of free
gossypol and lysine e-amino groups. Sam
ples of rumen liquor were incubated at
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37°Cfor 48 hours with the ratios of rumen
to gossypol sol of 5 to 0.4 and 10 to 0.4.
One sample was heated at 70°Cfor 20
minutes with the ratio of rumen to gos
sypol sol of 5 to 0.4. The milligrams of
free gossypol and lysine c-amino groups,
that disappeared were determined (table 6).
The mole ratio of bound gossypol to bound
lysine was 1:2 in three of the 4 tests.
Influence of proteolytic enzymes on the
stability of rumen liquor bound gossypol.
To test the effect of digestion upon the
stability of the lysine-gossypol complex,
5 ml of rumen liquor were incubated for
24 hours at 37°Cwith 0.2 ml of gossypol
sol. One milligram of the enzymes was
added and incubation continued for an
additional 24 hours. The percentage of
free gossypol that disappeared was then
determined. The addition of enzymes had
no influence on the gossypol binding ac
tivity of the liquor (table 7).
TABLE 5
Comparison of bound gossypol formation under
different types of incubation
Disappearance of gossypol'-2
Types of
incubation Free Total
Aerobic water bath at
70°Cfor 20 minutes ±03
Anaerobic at 37°C
for 48 hours75 64±000
1 In duplicate.
2 The test samples contained 10 ml of rumen liquor
and 4 ml of gossypol solution.
3 Standard deviation.
218 RAYMOND REISER AND HWEI C. FU

TABLE 6
The simultaneous disappearance of the free c-amino group of lysine and of free gossypol
during incubation of rumen fluid-gossypol solution

ratio
Test' of free of bound
:boundlysine1»2«3»4«mg0.53
no.Disappearance lysine c-amino groupDisappearance of free gossypolMole gossypol

±0.08"1.24 1.2:
±0.260.98 ±0.161.58±01.75±0moles0.00310.00440.00310.00331111:
1.9:
±0.0450.99 2.1:
±0.0045moles0.00340.00840.00670.0068mg1.48±0.112.28 2.0
1All tests were run in duplicate.
2Test 1. Five milliliters of rumen liquor incubated anaerobically with 0.4 ml of gossypol-glycerol
solution at 37°Cfor 48 hours.
3Standard deviation.
4Test 2. Ten milliliters of rumen liquor incubated anaerobically with 0.8 ml of gossypol solution
at 37°Cfor 48 hours.
5Test 3. Ten milliliters of rumen liquor incubated anaerobically with 0.4 ml of eossypol solution
at 37°Cfor 48 hours.
' Test 4. Ten milliliters of rumen liquor incubated aerobically with 0.4 ml of gossypol solution
at 70°Cin water bath for 20 minutes.

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TABLE 7 LITERATURE CITED
Effect of proteolytic enzymes on the disappearance Baliga, B. P., M. E. Bayliss and C. M. Lyman
of free gossypol bound in rumen 1959 Determination of free lysine amino
fluid-gossypol solution1 groups in cottonseed meals and preliminary
studies on relation to protein quality. Arch.
Enzymes added Biochem. Biophys., 84: 1.
Couch, J. R., W. Y. Chang and C. M. Lyman
1'No Test 1955 The effect of free gossypol on chick
enzymePancreatinRhozyme-6Rhozyme-pfTest growth. Poultry Sci., 34: 178.
±2.538±6.125±079 Crawford, A. C. 1910 A poisonous principle in
certain cottonseed meals. J. Pharmacol. Exper.
Therap., 1: 519.
Eagle, E. 1950 Effect of repeated doses of
2«No gossypol on the dog. Arch. Biochem., 26: 68.
enzymePepsinTrypsin32±4.1338±1.684 Jones, J. H., J. M. Jones and J. J. Bayles 1941
±2.977 Utilization of home grown feeds in fattening
±4.9 steers in the trans-pecos region. Texas Agr.
1The basic incubation medium in each tube was Exp. Sta. Bull. 604, College Station, Texas.
5 ml of rumen fluid andhours
0.2 ml Pons, W. A., Jr., and C. L. Hoffpauir 1957
After incubation for 24 at of gossypol
37°C, solution.
one milligram Determination of free and total gossypol in
of the enzyme was added to the medium and incuba mixed feeds containing cottonseed meals. J.
tion continued for an additional 24 hours.
2In duplicate. Assoc. Official Agr. Chemists, 40: 1068.
' Standard deviation. Ramsey, D. S., and J. T. Miles 1953 Cotton
4 In triplicate. seed vs. cottonseed meal and corn as protein
source in a concentrate mixture for dairy cows.
SUMMARY AND CONCLUSION J. Dairy Sci., 36: 1308.
The indifference of the degree of gos Reiser, R., and H. G. R. Reddy 1956 The hydro
génationof dietary unsaturated fatty acids. J.
sypol binding of rumen liquor to aerobic Amer. Oil Chemists' Soc., 33: 155.
or anaerobic incubation, high or low tem Rigdon, R. H., G. Cross, T. M. Feguson and J. R.
perature, centrifugation or proteolytic en Couch 1958 Effects of gossypol in young
chicks with the production of a ceroid like
zymes, and the simultaneous disappear pigment. A. M. A. Arch. Pathol., 65: 228.
ance of two moles of lysine e-amino groups Schwartze, E. W. 1924 Pharmacology of gos
to each mole of gossypol, demonstrates sypol, J. Agr. Res., 28: 191.
convincingly that the mechanism of rumi Withers, W. A., and F. E. Carruth 1915 Gos
sypol, the toxic substance in cottonseed. J.
nant detoxification of gossypol is by bind Agr. Res., 12: 83.
ing to soluble proteins, and that the bond 1918 Gossypol, the toxic substance in
is permanent during protein digestion. cottonseed. Ibid., 12: 83.