American Journal of Primatology 37:l-7 (1995)

RESEARCH ARTICLES

Effect of Liquefaction Time on Semen Parameters in

Chimpanzee (Pan troglodytes) Ejaculates
L.G. YOUNG AND E.B. SMITHWICK
Department of Physiology and Yerkes Regional Primate Research Center, Emory University,
Atlanta, Georgia

Ejaculates were obtained from eight adult male chimpanzees trained to

use an artificial vagina. Spontaneously liquefied fractions were collected
a t 0-1, 1-2 and 2-4 h after ejaculation. The mean volumes of the spon-
taneously liquefied fractions did not differ significantly among the three
fractions. The total number of sperm, the percentage of motile sperm and
the percentage of live sperm decreased significantly (P < 0.005) between
the 0-1 and 1-2 h fractions. Citrate concentration and alpha-glucosidase
activity (mU/ml) decreased significantly (P < 0.005) from 0-1 to 1-2 to
2-4 h fractions. The volume of the liquefied fraction correlated positively
(P< 0.001) with total protein and with total citrate a t 0-1, 1-2 and 2-4
h. The total number of sperm correlated positively total with total alpha-
glucosidase activity a t 0-1, 1-2 and 2-4 h. Among the biochemical pa-
rameters measured, total protein and total citrate correlated positively a t
0-1,l-2 and 2-4 h. 0 1995 Wiley-Liss, Inc.

Key words: chimpanzee, ejaculate, liquefaction, cellular, biochemical

INTRODUCTION
Chimpanzee semen coagulates upon ejaculation, but a spontaneous and partial
liquefaction occurs with incubation at 37°C [Martin & Gould, 1981; Marson et al.,
19891. Semen parameters reported for the spontaneously liquefied fraction of the
ejaculate [Marson et al., 1989; Srivastava et al., 1981; Gould et al., 19931 show
large variations in values obtained for both cellular and biochemical components
of the semen. Such variations are related, a t least in part, to intra-animal varia-
tions [Martin and Gould, 1981; Marson et al., 1989; Gould et al., 19931 and to
inter-animal variations [Gould et al., 19931. However, in these previous studies,
the spontaneously liquefied fraction was collected for analyses at varying times
after ejaculation. The effect of liquefaction time on cellular and biochemical pa-
rameters measured in the spontaneously liquefied fraction of the chimpanzee ejac-
ulate has not been reported.
This study reports the effect of time on cellular and biochemical parameters of
the spontaneously liquefied chimpanzee ejaculate. For each ejaculate, fractions
which liquefied spontaneously between 0-1 hour, between 1-2 hours, and between

Received for publication August 8, 1994; accepted February 1, 1995

Address reprint requests to Dr. Leona G. Young, Department of Physiology, Emory University, Atlanta,
GA 30322.

0 1995 Wiley-Liss, Inc.

2 I Young and Smithwick
2-4 hours were collected, and cellular and biochemical parameters in each fraction
were analyzed.
METHODS
Animals
Eight adult male chimpanzees (Pun troglodytes), aged 12 to 27 years and
weighing 49.5 to 70.5 kg were used in this study. All the males in the study were
captive-born in the Yerkes Regional Primate Research Center colony. Routine
examinations of the animals in this population revealed no medical condition
potentially associated with infertility. The animals were housed in age-matched
heterosexual social groups of 2-5 animals in indoor-outdoor facilities. Outdoor
quarters were subject to ambient light and temperature. Indoor quarters received
ambient light except on days weather conditions prevented the animals from un-
restricted indoor/outdoor access. On these days, the light-dark schedule was 11 h
light and 13 h dark. Indoor quarters were maintained between 65" and 80°F. This
study was conducted between September and December of 1992.
Semen Collection and Analyses
Ejaculates were obtained from conscious chimpanzees trained to use a n arti-
ficial vagina [Fussell et al., 19731. Semen was collected between 0830 and 1030 h.
The frequency of spontaneous ejaculations between semen collections could be
neither controlled nor determined.
Ejaculates were incubated at 37°C and the fractions of ejaculate which spon-
taneously liquefied between 0-1 h, between 1-2 h and between 2-4 h were col-
lected separately. The total amount which liquefied in each time period was eval-
uated for semen volume (ml of liquefied seminal fluid containing sperm) and for
sperm count (the number of sperm/ml of seminal fluid) [Gould et al., 19931. Sperm
maintained in their seminal fluid were evaluated for morphology, percentage mo-
tile and percentage live indicated a s the percentage of sperm shown by selective
staining to have intact membranes [Young et al., 19871.
For each fraction of each semen sample, the sperm were separated from sem-
inal fluid by centrifugation for 10 min at 5,000 g at room temperature (RT). The
sperm-free seminal fluid was analyzed for protein using the BioRad (Richmond,
CA) assay [Bradford, 19761 with bovine serum albumin (BSA) as standard, for
fructose [Polakoski and Zaneveld, 19771, for citrate [WHO, 19871, and for alpha-
glucosidase activity [Cooper et al., 19901.
Statistical Analyses of Results
Mean values in tables are expressed as mean 2 standard deviation (mean ?
S.D.). Comparisons among means for the three time periods were made using
two-way analyses of the variance. Factor one was animal. Factor two was time. To
avoid violating the assumption of independence of data points, all comparable
measures from each individual animal were averaged, so that each subject con-
tributed only a single data point for each parameter. Post hoc tests to determine
the minimum difference between means to be significant were conducted using
Tukey's pairwise comparison with a level of significance of P < 0.01. Correlation
coefficients and linear regressions were calculated using the average over all of the
animals.
RESULTS
Table I shows the values (mean ? S.D.) for semen volume, percentage of the
ejaculate which liquefied at 37"C, concentration of sperm, percentage of motile
TABLE I. Mean +. S.D. and Range of Semen Parameters in Fractions Collected Between 0-1,l-2, and 2-4 h of
Eiaculate Liauefaction
Semen volume (ml) Percent liquefaction Sperm x 1o7im1 Percentage motile sperm Percentage live sperm
(hours) (hours) (hours) (hours) (hours)
Animal 0-1 1-2 2-4 0-1 1-2 2-4 0-1 1-2 2-4 0-1 1-2 2-4 0-1 1-2 2-4
~ ~

BF 0.5 2 0.2 0.3 ? 0.1 0.4 2 0.1 8?4 6+2 6?2 151 2 57 163 ? 47 112 ? 58 6 8 2 11 57 ? 12 42 5 18 8 2 ? 3 75 ? 7 67 2 12
(n = 4)" (0.3-0.9) (0.3-0.5) (0.3-0.5) (5-15) (4-8) (5-9) (72-230) (112-225) (45-205) (57-86) (40-74) (21-71) (80-86) (64-82) (51-81)
CL 0.3 5 0.1 0.3 2 0.1 0.4 ? 0.1 521 5?1 651 235 ? 170 79 2 62 72 ? 47 72 ? 17 59 2 16 49 ? 18 74 2 5 60 2 13 52 2 14
(n = 4) (0.2-0.4) (0.2-0.4) (0.3-0.5) (3-7) (3-6) (5-8) (48-505) (8-158) (22-148) (44-84) (35-79) (23-66) (65-79) (42-79) (34-73)
JS 0.9 2 0.3 0.3 2 0.1 0.3 ? 0.1 26 + 8 823 7e4 71 ? 35 67 2 59 62 ? 42 38 5 19 38 11 29 ? 21 67 2 7 64 2 14 43 2 13
(n = 4) (0.7-1.4) (0.2-0.4) (0.1-0.4) (20-39) (5-11) (3-11) (27-121) (13-165) (7-120) (16-58) (21-49) (11-66) (56-74) (41-77) (28-61)
JN 0.6 ? 0.2 0.3 ? 0.1 0.4 i 0.1 12 ? 4 7 i 2 823 142 5 81 56 ? 37 54 2 36 76 i 9 68 2 5 38 2 10 70 ? 17 54 2 24 57 e 4
(n = 3) (0.4-0.8) (0.2-0.5) (0.2-0.5) (8-17) (5-11) (4-11) (39-238) (4-83) (9-96) (65-86) (63-75) (29-51) (47-86) (20-73) (52-62)
PH 0.5 i 0.3 0.6 2 0.3 0.4 ? 0.2 10 2 5 11 ? 5 853 62 ? 9 51 2 17 56 ? 44 43 ? 17 36 2 11 25 ? 3 70 ? 22 60 ? 24 49 ? 18
(n = 4) (0.2-0.9) (0.2-0.9) (0.2-0.7) (4-18) (3-17) (4-12) (51-72) (32-76) (22-129) (22-68) (20-49) (21-29) (35-93) (20-76) (23-70)
PN 0.2 0.3 0.3 4 7 5 211 79 53 61 54 33 70 63 45
(n=l)
PD 0.2 ? 0 0.2 ? 0 0.2 i 0.1 4?1 621 622 20i4 16?1 1350 68 2 10 53 2 30 16 t 15 89 2 4 7 9 ? 10 5 6 2 18
(n=2) (0.15-0.2) (0.2-0.25) (0.18-0.3) (4-5) (5-6) (5-8) (17-24) (14-17) (12-13) (58-78) (23-83) (1-31) (85-94) (68-89) (38-74)
ZB 0.3 2 0.2 0.4 ? 0.1 0.4 ? 0.1 723 8?2 822 93?89 91267 5 1 ~ 4 5 7 0 ? 11 5 5 2 12 3 3 ? 11 80 i 9 61 2 13 66 5 20
(n=4) (0.2-0.5) (0.3 ? 0.5) (0.3-0.5) (3-11) (5-10) (5-11) (11-240) (5-157) (4-104) (59-84) (43-71) (18-48) (68-90) (49-83) (36-93)
Meanb 0.43 2 0.23 0.3 i 0.1 0.3 2 0.1 925 722 7?2 123272 75?40 59226 62 i 2 1 52 ? 10 33 2 9 75 ? 25 64 2 8 54 ? 8
(N = 8)'

an = Number of ejaculates per animal

bWeighted means.
'N = Number of animals.
4 I Young and Smithwick
TABLE 11. Mean k S.D. and Range for Concentration and for Total Amount of Protein,
Fructose, Citrate and of a-Glucosidase Activity in Fractions Collected Between 0-1, 1-2
and 24 h of Ejaculate Liquefaction
Protein Fructose Citrate a-glucosidase
Fractions (mg) (mg) (me) (mu)
0-1 h 50.0 ? 4.2 *
2.3 0.9 2.2 t 1.1 26.5 ? 13.8
per ml (27.2 - 68.4) (0.2 - 5.9) (0.1 - 4.3) (4.6 - 2.6)
Total 23.6 ? 3.1 1.1? 0.6 0.9 t 0.7 10.7 & 4.4
(5.4 - 97.6) (0.6 - 3.18) (0.2 - 5.6) (11.0 44.3)
~

1-2 h 52.0 ? 1.3 2.7 -t 0.8 1.9 t 0.6 14.6 ? 7.3

per ml (33.6 ? 70.8) (0.2 5.1)
~ (0.1 - 3.8) (2.3 - 37.9)
Total 18.0 ? 1.5 0.9 ? 0.3 0.6 2 0.3 5.2 t 2.5
(8.0 k 61.2) (0.04 - 3.1) (0.1 - 2.3) (0.23 - 19.9)
2-4 h 54.4 ? 1.2 2.7 ? 0.9 1.6 -t 0.5 8.8 ? 3.9
per ml (35.6 - 71.6) (0.3 - 3.5) (0.1 - 3.5) (1.5 - 29.0)
Total 18.8 f 3.2 0.9 t 0.3 0.6 ? 0.2 4.5 ? 1.5
(8.6 - 47.6) (0.03 - 2.5) (0.03 - 1.8) (0.15 - 14.5)

sperm, and percentage of live sperm in spontaneously liquefied semen fractions

collected between 0-1, 1-2 and 2-4 h after ejaculation for eight adult male chim-
panzees.
The total volume of semen collected between 0 and 4 h was approximately 25%
of the ejaculate. The volume of semen collected in each of the time periods did not
differ significantly among the three time periods. The concentration of sperm de-
creased significantly (P < 0.002) between the 0-1 and 1-2 h fractions, but not
between the 1-2 and 2-4 h fractions. The total number of sperm collected was 49 k
28 x lo7 sperm between 0-1 h, 28 * 14 x lo7 sperm between 1-2 and 29 2
10 x lo7 sperm between 2-4 h. The total number of sperm also decreased signif-
icantly ( P < 0.005) between the 0-1 and 1-2 h fractions, but not between the 1-2
and 2-4 h fractions. The percentage of motile sperm and the percentage of live
sperm decreased significantly (P < 0.001) in semen fractions collected between
0-1, 1-2 and 2-4 h.
Table I1 summarizes the values (mean t S.D.) obtained for concentrations and
for total amounts of protein, fructose and citrate and for concentrations and total
amounts of alpha-glucosidase activity in seminal fluid from spontaneously lique-
fied semen collected between 0-1, 1-2 and 2-4 after ejaculation from eight adult
male chimpanzees.
The concentrations and the total amounts of protein and of fructose did not
differ significantly in fractions collected between 0-1, 1-2 and 2-4 h. The concen-
tration of citrate decreased significantly (P < 0.006) between the 0-1 and 1-2 h
fractions, but not between the 1-2 and 2-4 h fractions, whereas the total amounts
of citrate did not differ significantly among the three fractions collected. The con-
centration and the total amount of alpha-glucosidase activity decreased signifi-
cantly (P < 0.001) between the 0-1 and 1-2 h fractions, but did not change sig-
nificantly between the 1-2 and 2-4 h fractions.
The volume of the liquefied fraction correlated positively with total protein at
0-1 (r = 0.936, P < 0.001), 1-2 (r = 0.916, P < 0.001) and 2-4 h (r = 0.895, P <
0.001) and with total citrate a t 0-1 (r = 0.679, P < 0.001), 1-2 ( r = 0.810, P <
0.001) and 2-4 h (r = 0.791, P < 0.001). The percentage liquefaction of the ejac-
ulate correlated positively with total protein at 0-1 (r = 0.736, P < 0.001), 1-2
(r = 0.791, P < 0.001) and 2-4 h (r = 0.617, P < 0.001) and with total citrate a t
 Ejaculate Liquefaction in Chimpanzees / 5
0-1 h (r = 0.819, P < 0.001), but not at 1-2 and 2-4 h. The total number of sperm
correlated positively with total alpha-glucosidase activity at 0-1 (r = 0.787, P <
0.001), 1-2 (r = 0.868, P < 0.001) and 2-4 h (r = 0.906, P < 0.001). The percentage
of motile sperm and the percentage of live sperm did not correlate with any of the
biochemical parameters measured.
Among the biochemical parameters measured, total protein and total citrate
correlated positively a t 0-1 (r = 0.817, P < 0.001), 1-2 (r = 0.840, P < 0.001) and
2-4 h (r = 0.780, P < 0.001). Total fructose and total a-glucosidase activity did not
correlate either with total protein or with total citrate.
DISCUSSION
Ejaculates from eight adult male chimpanzees were analyzed to establish the
effect of time on the cellular and biochemical parameters of spontaneously lique-
fied fractions. Because of the large intra- and inter-animal variations of semen
characteristics reported for this species [Marson et al., 1989; Gould et al., 19931,we
have reported data obtained for each animal as well as summary data.
The volume of ejaculate which liquefied did not change significantly among
0-1,1-2 and 2-4 fractions, whereas the number of sperm was significantly greater
in the 0-1 h fraction than in the 1-2 and 2-4 h fractions. For samples collected
*
between 0-1 h, the volume was lower (0.43 0.23 ml) and the sperm count was
higher (123 2 72 x lo7 sperdml) than values reported previously by Srivastava
et al. 119931(0.60 2 0.24 ml; 9.3 ? 3.2 x lo7sperdml) and by Marson et al. [19891
* *
(0.78 0.13 ml; 47.9 13.2 x lo7 sperdml) for the 0-1 h spontaneously liquefied
fractions of ejaculates collected from conscious adult male chimpanzees. At the
present time, we have no explanation for these differences except, perhaps, differ-
ences in the age, health and habits of the animals used in the different studies.
Moreover, as the frequency of spontaneous ejaculations between collections could
be neither controlled nor determined, this also probably contributed to the inter-
and intra-animal variability observed. The percentage of motile sperm (62 ? 21%
*
motile) and the percentage of live sperm (75 25% live) were essentially equiv-
alent to those reported by Srivastava et al. [19811(67.05 8.3%live) and by Marson
et al. [1989] (55 2 4% motile; 68 2 3%live) for 0-1 h liquefaction fractions. Data
for liquefaction longer than one hour have not been reported previously for the
chimpanzee.
Protein concentrations did not differ significantly among the 0-1 and 1-2 and
*
2-4 h liquefied fractions. The protein concentration in 0-1 h fractions (50.0 4.2
mg/ml) estimated in this study is approximately the same as that reported previ-
ously in 0-30 min (55 2 4.14 mg/ml) liquefaction fractions of chimpanzee ejacu-
lates [Gould et al., 19931.
In the chimpanzee, evidence suggests that coagulation of the ejaculate is in-
duced by secretory products of the seminal vesicles and that liquefaction of the
coagulum is induced by secretory products of the prostate [Marson et al., 19891.
Fructose provides a functional marker for secretions added by the seminal vesicles
and citrate provides a functional marker for secretions added by the prostate. In
the present study, neither total fructose nor total citrate changed significantly
among the 0-1 and 1-2 and 2-4 h fractions. However, citrate concentration, but
not fructose concentration, decreased significantly between the 0-1 and 1-2 h
fractions. In addition, total fructose showed no correlation with either the volume
of liquefied semen or the percentage of liquefaction a t 0-1, 1-2 or 2-4 h, whereas
total citrate correlated positively with the volume of liquefied semen at 0-1, 1-2
and 2-4 h and with the percentage of liquefaction a t 0-1 h. These results support
those of Marson et al. [1989]which suggest that in the chimpanzee the liquefaction
6 I Young and Smithwick
process depends on factors secreted by the prostate. Further, for samples collected
between 0-1 h, the concentrations of fructose (2.3 2 0.9 mg/ml) and of citrate
(2.2 f 1.1 mg/ml) were equivalent to those reported by Marson et al. [1989] (re-
calculated as 2.1 -+ 2.0 mg/ml fructose and 2.0 ? 0.4 mg/ml) for the 0-1 h spon-
taneously liquefied fractions of ejaculates collected from conscious adult male
chimpanzees. This suggests that there are minimal differences in semen from
ejaculates collected by masturbation (Marson et al., 1989) and by artificial vagina
after 0-1 h liquefaction.
In the human, a-glucosidase is an androgen-dependent enzyme of epididymal
origin which provides a functional marker for secretions contributed to the ejacu-
late by the epididymides [Chapdelaine et al., 1978; WHO, 1987; Cooper et al., 1988;
Guerin et al., 19901.In the chimpanzee, it is assumed that a-glucosidase in seminal
fluid is contributed by the epididymides [Gould et al., 19931. The positive correla-
tion (r = 0.817, P < 0.001) between total a-glucosidase activity and total sperm
number at 0-1,l-2 and 2-4 h supports this assumption. The highest a-glucosidase
activity was measured in 0-1 h fractions and was significantly less in subsequent
fractions. This suggests the epididymal contribution to the ejaculate does not co-
agulate at ejaculation but remains liquid; therefore, its cellular and biochemical
content predominates in semen fractions collected shortly after ejaculation.
These studies show significant changes in the cellular and biochemical param-
eters measured in the spontaneously liquefied fractions of the chimpanzee ejacu-
late at different times after ejaculation. Large inter-animal variations in semen
parameters relate to the expected biological variability of animals in the popula-
tion studied. Large intra-animal variability can be related to sociopsychological
parameters, including frequency of spontaneous ejaculations between semen col-
lections which, in this study, could not be controlled. Even with these large inter-
and intra-animal variabilities, there are still significant differences among the
cellular and biochemical parameters measured, suggesting that liquefaction time
must be considered in measuring semen parameters. Among the cellular param-
eters, there are significant decreases in the number of sperm, the percentage of
motile sperm and the percentage of live sperm between the 0-1 and the 1-2 h
fractions. Among the biochemical parameters, there are significant decreases in
total citrate and in total a-glucosidase activity between the 0-1 and 1-2 h frac-
tions. Therefore, by using that fraction of ejaculate which liquefies spontaneously
between 0-1 hour as a standard, the data reported by different laboratories will be
more comparable.

ACKNOWLEDGMENTS
This study was supported in part by NIH grants RR-05994 and RR-00165. We
thank Dr. Kenneth G. Gould of the Yerkes Regional Primate Research Center for
providing the chimpanzee ejaculates used in this study. We thank Ms. Sarah
Wilcox and Ms. Katherine Roberts for their technical assistance. We thank Dr.
George Cotsanis and Dr. Gilbert Rinard for their help in selecting the appropriate
statistical analyses for the data. The Yerkes Center is fully accredited by the
American Association for Accreditation of Laboratory Animal Care.

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