BIOLOGY OF REPRODUCTION 13, 154-157

L-Arginine Stimulation of Human Sperm Motility in vitro

DAVID W. KELLER and KENNETH L. POLAKOSKI

Department of Obstetrics and Gynecology, Washington University School of Medicine,

St. Louis, Missouri 63110

ABSTRACT

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Human semen specimens with low motility were incubated with L-arginmne to determine its
effect on sperm motility. Stimulation of forward motility over control values occurred in a dose
response relationship, with optimal activity occurring at an L-arginine concentration of 0.004 Al.
L-arginine increased the forward motility of sperm at either 23 or 37#{176}C in all samples tested. The
increase in forward motility over control values was 81.6 ± 9.96 percent SEM (n=12). The percent
stimulation of sperm motility was in general inversely related to the initial semen motility. The
physiologically inactive isomer D-arginine. the amino acid L-lysine and the arginine analogs
L-homoarginine and L-nitroargmnmne did not stimulate sperm motility. L-ornithine, an arginine
metabolite, was also ineffective in stimulating sperm motility. The observation that L-arginine
enhances sperm motility in vitro suggests that it may have clinical value when human semen with
subnormal motility is utilized for artificial insemination.

INTRODUCTION All semen specimens were examined by the au-

thors. Samples were randomized in regard to the
Schachter et al. (1973), have reported that temporal sequence of their examination from experi-
oral arginine administration to oligospermic ment to experiment. Multiple compounds were tested
men results in an improvement in both sperm during the same experimental time period so that the
nature of the test compounds were not obvious to the
count and motility in the majority of individu-
examiner.
als treated. In order to evaluate the possible The standard deviation of intra-sample variation of
action of arginine upon sperm motility, in vitro percent motility (S.D.) was calculated for both ob-
studies were performed. The results indicate servers from the expression
that L-arginine has a specific role in increasing
sperm motility in oligospermic men.
S.D.= ________

MATERIALS AND METHODS V (a - b - 1)

L-arginine hydrochloride, D-arginine hydrochloride where L = individual motility value of each observa-
and L-lysine hydrochloride were purchased from tion in each sample, L = mean motility value of each
Nutritional Biochemicals Corporation. L-homoarginine sample, a = total number of samples evaluated.
was obtained from Cyclo-Chemical Division, Travenol The semen specimens were diluted in modified
Laboratories, Inc. L-nitroarginine was purchased from
Baker’s buffered glucose saline (glucose 3.0 g percent;
Eastman Organic Chemicals. L-ornithine hydrochlor-
Na2 HPO4 0.6 g percent; NaCI 0.2 g percent; KH2 P04
ide was obtained from Sigma Chemicals Company. All
0.01 g percent; pH 7.85) to a final concentration of 1
other chemicals were reagent grade. X sperm/mI.
10’ The incubation mixtures contained
After a minimum of 72 h continence, semen
50 of diluted semen and an equal volume
M’ of reagent
specimens were collected in glass vials by masturba-
dissolved in Baker’s solution. Unless otherwise noted,
tion. A complete semen analysis was performed on the final concentration of all test compounds was
each specimen, which included total sperm count,
0.004 M. Control incubations were performed, uti-
motility, morphology, pH and volume. Oligospermia
lizing an equal volume of the buffered glucose saline
was defined as less than 50 X iO sperm/mi. Forward
solution in place of the test compound. All specimens
motile sperm were defined as those which exhibited
were incubated at 37#{176}Cfor one hour at pH 7.4 prior
forward progression over two 0.005 mm squares in a
to examination.
Neubauer counting chamber. All semen specimens
examined exhibited less than 60 percent total initial
motility. The percent motility of each specimen was RESU LTS
determined after counting a total of 200 to 250
Semen specimens used for the incubation
sperm.
studies exibited sperm counts ranging from 8 to
95 X 106 sperm/mI, with initial motilities of
8.4 to 55.2 percent. Dilution of semen speci-

Accepted April 23. 1975. mens to 1 x i07 sperm/mi had no effect on the
Received July 30, 1974. motility of the specimen, as compared to the

154
 ARGININE AND SPERM MOTILITY 155

motility of the undiluted specimen. forward motility of the semen specimen is

The standard deviation of intra’sample varia- illustrated in Figure 2. In general, there was a
tion of percent motility (S.D.-defined previ- greater degree of increase in sperm motility in
ously) was calculated for both observers as ± specimens incubated which exhibited lower ini-
1.92 percent on the basis of 66 observations of tial motility. However, an increase in sperm
23 individual semen specimens. motility over initial motility values was observ-
Dose response curves of three different ed in all twelve experiments utilizing L-arginine.
subjects are illustrated in Figure 1. Increasing Table 1 illustrates the response of forward
concentrations of L-arginine resulted in in- sperm motility to a final 0.004 concentration

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M
creased forward motility over control values of L-arginine. The data in Table 1 were ana-
from a concentration of 0.00 1 M reaching a lyzed for significant differences by the paired t
maximum at 0.004 M. A diminution of re- test. Incubation with L-arginine resulted in a
sponse was observed at higher arginine concen- significant improvement in percent sperm mo-
trations. The degree of increase in motility was tility over control values (P<0.O01). In ten of
in general related to the initial motility of the the twelve experiments, the motility of the
specimen. The lowest percent increase in motili- diluted semen specimen incubated 1 h later
ty occurred with an initial motility of 51 at 37#{176}Cwith L-arginine was greater than the
percent, represented by the open triangles. The initial motility of the same semen specimen.
specimens with initial motilities of 23 and 24 The effect of various amino acids and
percent, represented by closed circles and open arginine analogs on human sperm motility in
rectangles respectively, exhibited higher in- comparison to L-arginine is illustrated in Table
creases in motility. 2. No significant improvement in sperm motili-
The percent increase in forward sperm motil- ty was observed in specimens incubated with
ity over control levels relative to the initial the arginine analogs (L-homoarginine, D-argi-
nine, L-nitroarginine), L-lysine or L-ornithine.
120 The time course of the effect of L-arginine
in vitro was also examined. In three individual
experiments, control motility was observed to
decrease to 50 percent of the initial value in a
100
160

80
0

121
0
C

60 C

a,
‘A
a)
0 U)
4) 0
a)

#{149} 40 C.,
C

20

0 20 40
0.5 I 2 3

(Arginine Concentration) M2 % Initial Motility

FIG. 2. Percent increase in forward sperm motility
FIG. 1. in vitro effect of increasing concentrations vs initial sperm motility in semen specimens incubated
of L-arginine on forward sperm motility of three with 0.004 Al L-arginine. The details of the experi-
subjects. Initial motilities of the semen specimens were mental procedures are given in the Materials and
23 percent (.), 24 percent (0), and 51 percent (A). Methods section.
156 KELLER AND POLAKOSKI

TABLE 1. Effect of L-arginine on human sperm motility.

. % Motility
h %
.
Increase
.
% Increase
. . -
Sperm count “/ Initial in in vitro over initial
Expt.a X 106/ml motility Control Test motility motility

1 14 8.4 6.4 13.5 111 61

2 72 15.1 13.0 25.0 92 66
3 24 20.4 16.2 31.6 95 55

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4 60 23.3 19.4 28.6 47 23
5 8 26.0 20.0 46.0 130 77
6 95 34.2 21.4 44.5 108 30
7 41 36.6 22.9 51.0 123 39
8 40 45.2 23.8 51.0 114 13
9 26 50.0 26.5 44.5 68 -12
10 68 40.0 28.7 40.8 42 0
11 50 42.0 33.4 57.8 73 38
12 29 55.2 51.3 63.7 24 15

816c5996d 338c+793d

aLarginine in each experiment was at a final concentration of .004 Al.

bFOrward motility was measured after one hour incubations at 3 7#{176}C.

cM

dSEM

period of 2 to 4 h of observation. In samples ry data by assuming that their therapeutic

incubated with 0.004 M L-arginine, this de- success was attributable to higher oral doses of
crease in sperm motility with time did not arginine administration. Depending upon differ-
occur. ences in both intestinal absorption and rate of
synthesis of the amino acid, is possible
it that
DISCUSSION individuals may have significantly different
levels of arginine concentration in their seminal
Previous clinical reports of the value of
plasma. The observation that arginine concen-
arginine administration to oligospermic men
tration in seminal plasma in men with normal
have been contradictory. Schachter et al.
sperm counts is greater than that measured in
(1973), attempted to reconcile the contradicto-
oligospermic or aspermic men supports this
hypothesis (Krampitz and Doepfmer, 1962).
Our data demonstrate that in vitro sperm
TABLE 2. Effect of various amino acids and arginine
analogs on human sperm motility. motility follows an arginine dose response curve
which indicates that free arginine levels have a

Increase in Number of positive correlation with sperm motility. Tani-

sperm motilityb individual mura (1967) has reported positive in vivo
Test compounda (%) experiments correlation between sperm count and arginine
bound to spermatozoa and sperm motility and
L-arginine 82c ± 100d 12 free arginine present in the seminal fluid.
L-homoarginine 12 ± 7.3 4
Specificity of arginine action is strongly
L-Iysine 7 ± 10.8 4
2 4 suggested by the inability of D-arginine, the
L-ornithine ± 9.5
D-arginine 1 ± 14.2 4 arginine analogs L-homoarginine and L-nitro-
L-nitroarginine -25 ± 18.8 3 arginine, and L-ornithine to increase sperm
motility in vitro. This finding indicates that the
aAII compounds tested were at a final concentra-
terminal guanidino group as well as carbon
tion of .004 M.
chain length are essential requirements for
bFd motility was measured after one hour
arginine action. Tyler and Rothschild (1951)
incubation at 37#{176}C
at pH 7.4.
have demonstrated that the addition of amino
cM
acids to diluted semen specimens preserves the
dSEM
motility of the specimen. It is unlikely that this
 ARGININE AND SPERM MOTILITY 157

is the sole mechanism of arginine action ob- motility in vitro raises the possibility of its
served in these experiments, since in 10 of 12 potential clinical usefulness when human semen
experiments the motility of the diluted speci- with subnormal motility is utilized for artificial
men incubated with arginine was greater than insemination. The fact that L-arginine increases
that observed in the original undiluted speci- sperm motility in vitro, however, does not
men. necessarily indicate that a corresponding im-
It is apparent that with an initial forward provement in in vivo fertility of arginine treated
sperm motility of greater than 40 percent, the ejaculates will occur. This possibility will re-

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response to incubation with L-arginine is less quire further investigation.
than that observed with lower initial forward
ACKNOWLEDGMENTS
motilities. This may be the result of a fixed
The authors appreciate the generous support of a
percentage of non-viable, non-motile sperm
grant from the Rockefeller Foundation. They also
present in the ejaculates. If this is the case, then wish to thank Dr. Philip Miller for his assistance with
semen specimens with relatively high initial the statistical analysis of the data and Miss Faye
motilities would be expected to show less Zvibleman for her help in typing the manuscript.
response to arginine incubation than those with
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Amer. Phys. 58, 143-156.
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difficult to explain why a non-essential amino tion of Free Amino-Acids in Human Ejaculate by
acid should have a similar effect in vivo. Rose Ion Exchange Chromatography. Nature, London
(1938) has shown in the immature rat that 194, 684-686.
Rose, W. C. (1938). The Nutritive Significance of the
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the animal. Holt and Albanese (1944), however, B. (1973). Treatment of Oligospermia with the
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