Journal of Reproduction and Development, Vol. 61, No 2, 2015

—Original Article—

Addition of D-penicillamine, hypotaurine, and epinephrine (PHE)
mixture to IVF medium maintains motility and longevity of bovine
sperm and enhances stable production of blastocysts in vitro
Sung-Sik KANG1), Keisuke KOYAMA2), Weiping HUANG1), Yinghua YANG1),
Yojiro YANAGAWA1), Yoshiyuki TAKAHASHI1, 3) and Masashi NAGANO1)
1)Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine,

Hokkaido University, Sapporo 060-0818, Japan

2)Dairy Cattle Group, Konsen Agricultural Experiment Station, Hokkaido Research Organization, Nakashibetsu 086-1135,

Japan3)Genetics Hokkaido Association, Sapporo 060-0040, Japan

Abstract. The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF)
that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm
concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 106 cells/ml) on fertilization and blastocyst
developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 wereobtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 106 cells/ml using spermfrom 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at
12 h after IVF with a sperm concentration of 1 × 106 cells/ml. Moreover, high linearity, high flagellar beat cross frequency, andlow amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combinationof the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes.
Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase ofcAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF
medium at a sperm concentration of 2 × 106 cells/ml, we can achieve stable normal fertilization and blastocyst development
in any bull.
Key words: computer-assisted sperm analysis (CASA), In vitro fertilization (IVF), Oocyte, PHE, Theophylline(J. Reprod. Dev. 61: 99–105, 2015)

For in vitro fertilization (IVF) of bovine oocytes, IVF medium
supplemented with heparin was conventionally used [1]. Bovine
sperm incubated with heparin in vitro can be induced to undergo
the acrosome reaction and penetrate oocytes more effectively thansperm incubated without heparin [2]. However, IVF with heparin
was shown to require optimization of the sperm concentration for
individual bulls [1], and it exhibited large variations in penetration[3], cleavage and blastocyst rates in each bull [4]. For the practicaluse of a small number of valuable sperm such as sex-sorted sperm
and cryopreserved sperm from dead bulls [5], it is difficult to optimize
the sperm concentration in each bull. Therefore, we should establish
an effective IVF system without the need for optimization of the
sperm concentration for individual bulls.
A mixture of D-penicillamine, hypotaurine, and epinephrine
Received: July 16, 2014
Accepted: November 17, 2014
Published online in J-STAGE: December 13, 2014©2015 by the Society for Reproduction and Development
Correspondence: M Nagano (e-mail: mnaga@vetmed.hokudai.ac.jp)
This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License<http://creativecommons.org/licenses/by-nc-nd/3.0/>.

(PHE) [6] has often been added to IVF medium with heparin. This
supplementation shortened the time required for oocyte penetration[7] and increased the cleavage rate compared with the results without
PHE [8]. In addition, it was reported that the addition of PHE to
IVF medium improved motility and sperm parameters associatedwith bull fertility examined by computer-assisted sperm analysis(CASA) [9]. Some reports have demonstrated that D-penicillamine
in IVF medium extended the lifespan of bovine sperm [10] and that
hypotaurine played a role as an inhibitor of reactive oxygen species
and induced motile sperm reactivation in the golden hamster [11].
Epinephrine, a kind of catecholamine, might stimulate soluble adenylyl
cyclase and increase the concentration of cAMP in the cytoplasmof sperm [12], and increased cAMP might accelerate the beating offlagella to promote motility in mice [13]. However, the effects of a
PHE mixture on sperm motility parameters and cAMP productionin spermatozoa are not clear.
Theophylline also increases the concentration of cAMP in thecytoplasm of sperm by inhibiting phosphodiesterase (PDE) andstimulates sperm motility [14]. It has been demonstrated that astable and high fertilization rate [15] and a blastocyst developmental
rate of about 40% [16–18] were achieved by adding theophylline
to bovine IVF medium at a fixed sperm concentration of 5 × 106

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KANG et al.

cells/ml recovered by a Percoll gradient (45 and 90%) procedure.
However, in the case of IVF with a low dose of sperm, such as
sex-sorted sperm (2.1 × 106 cells/straw) [19], the number of motile
sperm might be insufficient for an IVF protocol using medium
supplemented only with theophylline. In a previous study, we reported
that the combination of PHE and theophylline resulted in high normal
fertilization, cleavage, and blastocyst rates (84.6, 81.1, and 51.6%,
respectively) for bovine IVF using 2 × 106 cells/ml of sperm [20].
However, in our previous report [20], we used sperm derived fromonly one bull. Also the effect of the combination of a PHE mixture
and theophylline on fertilization was not examined in detail.
In the present study, to establish an efficient system for bovine
embryo production by IVF without optimization of the sperm
concentration for sperm derived from any bull, we examined theeffects of a PHE mixture, theophylline, and sperm concentration(1, 2 or 5 × 106 cells/ml) on fertilization, cleavage, and blastocyst
development. In addition, we evaluated the effects of the PHE
mixture and theophylline on sperm motility and the intracellularcAMP concentration of spermatozoa.

Materials and Methods
Chemicals

All the chemicals used in this study were purchased from SigmaAldrich (St. Louis, MO, USA), unless otherwise stated.

In vitro maturation and fertilization

IVM of bovine oocytes was performed as described previously [17].
In brief, cumulus-oocyte complexes (COCs) aspirated from follicles(2 to 8 mm in diameter) of slaughterhouse-derived ovaries were
cultured for 22 h in a droplet (about 10 COCs/50 μl) of maturation
medium under a humidified atmosphere of 5% CO2 in air at 39 C.
The maturation medium consisted of HEPES-buffered TCM-199(Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal
calf serum (FCS) (Invitrogen), 0.2 mM sodium pyruvate, 0.02 units/
ml follicle-stimulating hormone (from porcine pituitary), 1 μg/ml
estradiol-17β and 50 μg/ml gentamicin sulfate. IVF was conductedaccording to a procedure described previously [21]. Briefly, after
the thawing of frozen semen from 9 bulls (A to I), motile sperm
were separated using a Percoll (GE Healthcare, Buckinghamshire,UK) gradient (45 and 90%). Matured COCs were co-incubated withmotile sperm in droplets (10–13 COCs/100 μl) of modified Brackett
and Oliphant (mBO) isotonic medium [21] containing 3 mg/ml fatty
acid-free BSA and supplemented with 2.5 mM theophylline and/or PHE (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM
epinephrine) [8] at 39 C under a humidified atmosphere of 5% CO2,
5% O2 and 90% N2. All culture droplets were covered with paraffinoil (Nacalai Tesque, Kyoto, Japan). The final sperm concentration (1,2, or 5 × 106 cells/ml), co-incubation time (12 or 18 h), and treatment
with PHE and theophylline are described in the experimental design.

Examination of spermatozoon penetration and in vitro cultureof presumptive zygotes after IVF

In vitro culture (IVC) of presumptive zygotes was performed using
procedures that were basically the same as described previously [21].
After co-incubation with sperm, zygotes were freed from cumulus

cells by vortexing. To evaluate sperm penetration, presumptive
zygotes of each experiment group were fixed with ethanol:acetic
acid at a ratio of 3:1 and stained with 1% aceto-orcein solution as
described previously [22]. Oocytes having an enlarged sperm head(s)or male pronucleus(ei) were defined as penetrated by sperm, and thefollowing categories of oocytes penetrated by sperm were recorded:
1) oocytes with male and female pronuclei with a corresponding
sperm tail (2PN), 2) oocytes with more than two enlarged sperm
heads or male pronuclei (polyspermy), and 3) oocytes other than
2PN and polyspermy, such as oocytes with an enlarged sperm head
and anaphase II/telophase II chromosome or female pronucleus, oroocytes with a male pronucleus and telophase II chromosome (others).
To evaluate the rates of development to blastocysts, cumulus-freepresumptive zygotes were cultured for 150 h in droplets (25–30
presumptive zygotes/30 or 40 μl) using modified synthetic oviductfluid, which contained 1 mM glutamine, 12 essential amino acids
for basal medium Eagle, 7 nonessential amino acids for minimum
essential medium and 10 μg/ml insulin and was supplemented with
5 mM glycine, 5 mM taurine and 1 mM glucose [21], and 3 mg/
ml fatty acid-free BSA instead of polyvinyl alcohol at 39 C under ahumidified atmosphere of 5% CO2, 5% O2 and 90% N2. Cleavage
and blastocyst rates were assessed after 30 h and 150 h of IVC,
respectively. All embryos that developed to blastocysts were subjected
to counting of their cell numbers using an air-drying method [22].

Evaluation of sperm motility and sperm motility parameters
by CASA

Motile sperm separated using a Percoll gradient (45 and 90%) andrecovered motile sperm were incubated in 100-μl droplets of IVF
medium (final concentration of 10 × 106 cells/ml in 100-μl droplets)
at 39 C in 5% CO2, 5% O2 and 90% N2. After incubation, 3 μl of
IVF medium from droplets was placed onto 4-chamber slides witha depth of 20 µm (Art. No. SC 20-01-04-B, Leja, Nieuw-Vennep,
Netherlands) on a micro warm plate (Kitazato, Shizuoka, Japan) at
37 C for counting. Sperm in three fields (at least 100 spermatozoa)
in a chamber were divided into motile and dead sperm, and thepercentages of motile sperm and sperm motility parameters were
evaluated using a CASA system (SMAS, DITECT, Tokyo, Japan).
The evaluated sperm motility parameters were straight line velocity(VSL), curvilinear velocity (VCL), average path velocity (VAP),
linearity (LIN = VSL/VCL × 100), flagellar beat cross frequency(BCF) and amplitude of lateral head (ALH).

Evaluation of intracellular cAMP concentration of
spermatozoa

The intracellular cAMP concentration of sperm was measured
using the cAMP Biotrak enzyme immunoassay system kit (RPN2251,
Amersham, GE Healthcare, Life Sciences, UK) according to theprotocol provided by the manufacturer. The microplate contained 12
× 8-well strips coated with donkey anti-rabbit IgG. In brief, motile
sperm recovered using a Percoll gradient (45 and 90%) were incubatedin 400 μl of IVF medium (final concentration of 10 × 106 cells/ml)
in a 1.5-ml tube for 2 h at 39 C under 5% CO2, 5% O2 and 90% N2.
After incubation, each aliquot was centrifuged at 500 × g for 5 min,and the supernatant was removed. The sperm pellet was resuspended
in 200 μl of lysis buffer and mixed on a microplate shaker for 10

101

PHE IMPROVES SPERM LONGEVITY

min at room temperature. One hundred microliters of lysate and 100
μl of antiserum were added to the wells and incubated at 4 C for
2 h. After incubation, 100 μl of cAMP-peroxidase conjugate wasadded to each well and incubated at 4 C for 1 h. All supernatant was
aspirated, and all wells washed four times with 400 μl of wash bufferusing a microplate washer (Model 1575, ImmunowashTM, Bio-Rad
Laboratories, Tokyo, Japan). One hundred and fifty microliters of
enzyme substrate was added to all wells and mixed on a microplateshaker at room temperature for 1 h. One hundred microliters of 1.0
M sulfuric acid was added to each well, the optical density wasdetermined with a plate reader (iMarkTM, Bio-Rad Laboratories)
at 450 nm, and cAMP concentrations in each well were evaluated.

Fig. 1.

Distribution of motile sperm based on straight line velocity (VSL)
at 0 h of incubation. Sperm motility was analyzed immediately
after treatment with a Percoll gradient (bull I).

Experimental design

In experiment 1, to examine the effect of the PHE mixture anddifferent sperm concentrations on sperm penetration and blastocyst
development rates in sperm derived from various bulls, IVF medium
containing theophylline was used. Some of the matured COCs
were co-incubated with sperm derived from bulls A, B, and C at aconcentration of 1, 2 or 5 × 106 cells/ml for 18 h, and fertilization
status was examined. Other presumptive zygotes were cultured, andcleavage and blastocyst development were examined. As controls forsperm penetration and blastocyst development, oocytes incubatedat a sperm concentration of 5 × 106 cells/ml supplemented onlywith theophylline were used. In addition, to confirm blastocyst
development after IVF using a sperm concentration of 2 × 106 cells/
ml in IVF medium containing a combination of theophylline andPHE, presumptive zygotes fertilized with sperm from six bulls (Dto I) were cultured.
In experiment 2, to examine the effect of the PHE mixture andtheophylline on sperm fertilizability in detail, matured COCs wereco-incubated with sperm at a concentration of 1 × 106 cells/ml(bulls A and I) for 12 h in IVF medium with or without PHE and/or theophylline. Sperm of bull A showed similar 2PN rates among
sperm concentrations of 1, 2 and 5 × 106 cells/ml for 18 h of IVF,
irrespective of PHE and theophylline addition, in experiment 1. In
addition, to confirm the effect of the PHE mixture and theophylline
on sperm activity, after incubation (0, 2, 4, 6, and 8 h) of sperm ofbull I, sperm motility was evaluated by CASA. For evaluation of
sperm motility parameters, motile sperm with a VSL of ≥ 25 μm/
sec (50.49 to 75.32%) were selected, as it has been recognized that
motile sperm having a VSL of less than 25 μm/sec (25.51 to 40.51%)
are probably not related to fertilization (Fig. 1).
To examine the effect of PHE and theophylline on the intracellularcAMP concentration of spermatozoa (bull I), the intracellular cAMP
concentration of sperm was evaluated after 2 h of incubation in IVF
medium with or without PHE and theophylline. As a control, sperm
immediately after Percoll treatment (0 h) were used. All experimentsin each group were carried with at least 3 replicates.

Statistical analysis

Sperm penetration rates (total penetration, 2PN, and polyspermy
rates) were compared among groups by Chi-square test. The percentages of cleavage and development to blastocysts, mean cellnumbers in blastocysts, average sperm motility parameters, andthe concentration of intracellular cAMP after 2 h of incubation

were compared by one-way ANOVA followed by Tukey-Kramer’s
HSD test as a post hoc test. Intracellular cAMP concentrations at
0 h and 2 h in each treatment were compared by Dunnett’s test.
All analyses were performed using JMP Pro (version 10.0.2, SAS
Institute, Cary, NC, USA).

Results
Experiment 1

The effects of PHE and sperm concentrations on penetration are
shown in Table 1. In bull A, the 2PN rates were similar among different
sperm concentrations (1, 2 and 5 × 106 cells/ml), irrespective of PHE
addition. In bulls B and C at a sperm concentration of 1 × 106 cells/
ml without PHE, the total penetration and 2PN rates were the lowest
among all experimental groups. In bull C, at a sperm concentrationof 1 × 106 cells/ml with the PHE mixture, the total penetration rate
was significantly lower than that of the control (sperm concentrationof 5 × 106 cells/ml without PHE; P < 0.05). In all bulls, the highest
polyspermy rates were observed when PHE was added to IVF medium
including theophylline at a sperm concentration of 5 × 106 cells/ml.
The effects of PHE and different sperm concentrations on embryonicdevelopment are shown in Table 2. At sperm concentrations of 1
and 2 × 106 cells/ml (bulls A, B, and C), there were no significant
differences in cleavage rate, blastocyst rate and mean cell numberin blastocysts compared with those at 5 × 106 cells/ml without
PHE (control). However, in bull C, the cleavage rate at a sperm
concentrations of 1 × 106 cells/ml tended to be lower (P = 0.08)
than those at 2 × 106 cells/ml and in the control. In addition, themean cell numbers of blastocysts at a sperm concentration of 1 ×
106 cells/ml tended to be lower (P = 0.13) than those at 2 × 106
cells/ml and in the control in bull C. As shown in Table 3, after
fertilization with a sperm concentration of 2 × 106 cells/ml in thepresence of PHE and theophylline, cleavage rates of 78.3 to 92.4%
and blastocyst development rates of 31.9 to 62.0% were obtained
from 6 bulls (D to I).

Experiment 2

In bulls A and I, after 12 h of co-incubation of COCs and sperm(1 × 106 cells/ml), the total penetration and 2PN rates in the presence of PHE and theophylline were higher than those in the otherexperimental groups (Table 4; P < 0.05). The effects of PHE and102

KANG et al.
Table 1. The effects of the PHE mixture and sperm concentration on sperm penetration rate at 18 h after IVF using medium
including theophylline
Bulls

Sperm concentration(×106 cells/ml)

A

1

No. of oocytes(Replicates)

Total penetration

2PN

Poly

Others*

+
−
+
−
+
−

31 (3)
31 (3)
31 (3)
31 (3)
42 (3)
37 (3)

100.0a84.2b97.0ab
96.7ab
97.8ab100.0a

74.2
75.1
68.2
77.0
75.9
87.0

9.4ab
0b
13.0ab
10.0ab21.8a
13.0ab

16.4a
9.1ab
15.8a
9.7ab
0b
0b

+
−
+
−
+
−

31 (3)
32 (3)
34 (3)
33 (3)
31 (3)
42 (4)

95.8a
59.4b
93.9a
97.0a
100.0a
95.0a

78.7a
50.0b
82.5a
84.8a
73.6ab
81.7a

4.2a
0a
11.5ab
6.4a
23.0b
9.2ab

13.0a
9.4ab
0b
5.8ab
3.3ab
4.2ab

+
−
+
−
+
−

40 (3)
40 (3)
51 (4)
48 (4)
35 (3)
32 (3)

81.7ab69.5a
97.9c
89.2bc97.0bc100.0c73.3ab49.0b
82.4a
79.2ab
53.6b
80.8a

3.3a
6.7a
15.7ab3.9a
30.6b
15.9ab

5.0ab
13.8a
0b
9.4ab
12.8a
3.3ab

2
5
B

1
2
5

C

Percentages of

PHE

1
2
5

a, b, c Values with different letters within each bull differ significantly (P < 0.05). *Others: an enlarged sperm head with ananaphase II/telophase II chromosome or a male pronucleus was observed. 2PN, two pronuclei; poly, penetrated with more than
two sperm.

Table 2. The effects of PHE and sperm concentration on blastocyst developmental rate when using IVF medium including theophylline
Sperm concentration(×106 cells/ml)

PHE

No. of oocytes(Replicates)% cleavage% blastocysts

Mean cell numbers inblastocysts (n)

A

1
2
5

+
+
−

72 (3)
71 (3)
70 (3)

81.9 ± 2.4
86.2 ± 8.8
87.5 ± 12.5

44.5 ± 8.7
50.8 ± 11.3
54.6 ± 11.5

144.9 ± 50.6 (33)148.9 ± 62.3 (36)165.9 ± 64.4 (38)

B

1
2
5

+
+
−

112 (4)80 (3)
157 (6)72.2 ± 6.3
78.3 ± 4.4
81.5 ± 9.7

36.6 ± 6.6
50.0 ± 13.3
39.2 ± 10.0

174.8 ± 60.3 (41)183.9 ± 63.8 (40)
169.1 ± 69.6 (24)

C

1
2
5

+
+
−

65 (3)
91 (3)
189 (8)68.0 ± 4.8
86.8 ± 3.1
80.5 ± 11.5

24.8 ± 15.2
31.9 ± 5.0
30.4 ± 8.0

148.3 ± 55.4 (16)191.2 ± 77.0 (29)
188.1 ± 76.1 (58)

Bulls

Values are means ± SD. Embryos derived from bulls A to C were cultured in 40-μl droplets.

Table 3. Blastocyst development after IVF with a sperm concentration of 2 × 106 cells/ml in IVF
medium containing a combination of theophylline and PHE
Bulls

No. of oocytes(Replicates)% cleavage% blastocysts

Mean cell numbers inblastocysts (n)

D
E
F
G
H
I

74 (3)
80 (3)
87 (3)
200 (7)114 (4)125 (5)84.1 ± 4.4
92.4 ± 4.1
86.1 ± 3.9
80.8 ± 9.2
91.3 ± 10.3
87.9 ± 11.3

50.6 ± 5.2
62.0 ± 12.2
39.2 ± 5.1
38.0 ± 10.9
37.9 ± 3.7
39.8 ± 7.7

153.5 ± 62.6 (37)143.1 ± 54.1 (50)161.7 ± 59.4 (34)
170.3 ± 69.3 (76)127.2 ± 68.9 (33)153.0 ± 62.5 (49)

Values are means ± SD. Embryos derived from bulls D to F and bulls G to I were cultured in 40-μl
and 30-μl droplets, respectively.

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PHE IMPROVES SPERM LONGEVITY
Table 4. The effects of PHE and theophylline on sperm penetration rate of bulls A and I at 12 h after IVF with a sperm
concentration of 1 × 106 cells/ml
Bull
A

Treatment
Theophylline

PHE

Total penetration

2PN

Poly

Others*

+

+
−
+
−

35 (3)
33 (3)
33 (3)
32 (3)

77.1a
45.5bc
54.5b
28.1c

42.9a
12.1b
9.1bc
0c

14.3
0
6.1
0

22.7
33.3
39.4
27.9

+
−
+
−

55 (5)
57 (5)
56 (5)
57 (5)

60.3a
39.0b
8.8c
1.7c

28.1a
10.8b
1.8c
0c

0
0
0
0

32.2a
28.6a
6.7b
1.7b

−
I

Percentages of

No. of oocytes(Replicates)+−a, b, c Values with different letters within each bull differ significantly (P < 0.05). *Others: an enlarged sperm head withan anaphase II/telophase II chromosome or a male pronucleus was observed. 2PN, two pronuclei; poly, penetrated withmore than two sperm.

theophylline on sperm motility and sperm motility parameters areshown in Fig. 2. The total percentage of motile sperm decreased withincreasing incubation period in all experimental groups; in particular,
those at 6 and 8 h with only theophylline supplementation weresignificantly lower than those in the nontreatment (Fig. 2A; P < 0.05).
On the other hand, in terms of the percentages of sperm moving at
more than 25 µm/sec in the VSL, only PHE addition showed stablemotility (~70%) during the experimental period, with low variationcompared with the percentages in the nontreatment, theophylline
addition, and combination of PHE and theophylline groups (Fig.
2B). The average VAP levels of sperm incubated with theophylline
regardless of PHE addition were significantly increased at 2 h anddecreased at 6 h compared with those in the nontreatment group(Fig. 2C; P < 0.05). Regardless of PHE addition to IVF medium, theaverage LIN at 2 h with theophylline was significantly higher thanthose in the nontreatment group and the PHE addition only group(Fig. 2D; P < 0.05). The average BCF with PHE and theophylline
at 6 h was higher than those in the nontreatment group and the PHE
addition only group (Fig. 2E; P < 0.05). The average ALH levels at
6 and 8 h with PHE and theophylline were significantly lower thanthose in the nontreatment group and the PHE addition only group(Fig. 2F; P < 0.05).
As shown in Fig. 3, the mean intracellular cAMP concentration of
sperm incubated with theophylline (167.5 ± 54.5 fmol/5 × 106 cells;
mean ± SD) at 2 h was significantly higher than for those incubatedwith PHE (53.9 ± 54.7 fmol/5 × 106 cells) and in the nontreatmentgroup (45.1 ± 47.9 fmol/5 × 106 cells; P < 0.05). However, thecAMP concentration of sperm incubated with PHE and theophylline(117.3 ± 92.9 fmol/5 × 106 cells) showed an intermediate value
between those of sperm incubated with theophylline or PHE. The
mean intracellular cAMP concentration of sperm incubated withtheophylline only showed a significantly higher value than that at 0h (68.2 ± 19.9 fmol/5 × 106 cells; P < 0.05).

Discussion
In bull A, addition of PHE to the IVF medium at different sperm
concentrations (1, 2, and 5 × 106 cells/ml) did not affect 2PN rates at

Fig. 2.

The sperm motility and sperm motility parameters during
incubation in IVF medium supplemented with or without PHE
mixture and theophylline. A: % of total motile sperm; B: % of
motile sperm with a VSL of  ≥ 25 µm/sec; C: mean VAP (µm/sec);
D: mean LIN (%); E: mean BCF (Hz); F: mean ALH (µm). Values
in panels C to F were calculated by using sperm that showed a VSLof  ≥ 25 µm/sec. a,b,c Values with different letters differ significantly(P < 0.05). The error bar indicates the SEM. Nontreatment, mBO
medium only; PHE, mixture of D-penicillamine, hypotaurine,and epinephrine; Theo, theophylline. VSL, straight line velocity;
VAP, average path velocity; LIN, linearity (VSL/VCL [curvilinear
velocity] × 100); BCF, flagellar beat cross frequency; ALH,
amplitude of lateral head.

104

Fig. 3.

KANG et al.

Mean intracellular cAMP concentration in sperm at 2 h after
incubation. Lines of the boxes delineate the 25th, 50th, and 75th
percentiles, and the whiskers depict the 10th and 90th percentilesof a population. a, b Different letters indicate a significant
difference among groups (P < 0.05). * The asterisk indicates asignificant difference from the control. Six replicates per groupwere performed. Control and nontreatment, mBO medium only;
PHE, mixture of D-penicillamine, hypotaurine, and epinephrine;
Theo, theophylline.

18 h post insemination but did at 12 h post insemination. However,in bull B at 18 h post insemination, PHE addition was necessary
to enhance total penetration and normal fertilization at a sperm
concentration of 1 × 106 cells/ml. Meanwhile, in bull C, the total
penetration rate at a sperm concentration of 1 × 106 cells/ml was
not enhanced compared with that at a sperm concentration of 2 ×
106 cells/ml even though PHE was added to the IVF medium. In
bulls A, B and C, addition of PHE to the IVF medium at a sperm
concentration of 5 × 106 cells/ml showed the highest polyspermy
rate. These results indicate that, when the combination of PHE andtheophylline is added to IVF medium, the sperm concentration canbe reduced from 5 × 106 cells/ml to 2 × 106 cells/ml, which canobtain a high and stable 2PN rate. In addition, after fertilization in thepresence of PHE and theophylline with a sperm concentration of 2 ×
106 cells/ml, we obtained a relatively high rate of normal fertilization(68.2 to 82.4%) from 3 bulls and high cleavage (78.3 to 92.4%) andblastocyst rates (31.9 to 62.0%) from 9 bulls. From these results, asperm concentration of 2 × 106 cells/ml is recommended for use in
IVF in the presence of PHE and theophylline in IVF medium, which
can stably provide high rates of normal fertilization and blastocyst
development using sperm collected from any bull.
In this study, we found a synergistic effect of PHE and theophylline
on the fertilizability of sperm; namely, PHE and theophylline enhanced
the sperm penetration ability and shortened the time to penetration,
especially at a low concentration of sperm. In agreement with thepresent results, it was reported that sperm penetration ability [7]and cleavage rate [8] were enhanced by the addition of PHE to IVF
medium including heparin. In the present study, analysis with the
CASA system revealed that, in the presence of only theophylline,

the percentage of total motile sperm became lower than that of thenontreatment group after 6 h of incubation; however, addition of PHE
to the IVF medium with theophylline prevented the decrease in thepercentage of total motile sperm. Sperm incubated in IVF medium
with only PHE maintained a stable percentage (about 70%) of motile
sperm with a VSL of ≥ 25 µm/sec based on total sperm during the 8h incubation period. We speculated that the addition of PHE to IVF
medium maintains sperm motility. The averages of VAP and LIN
at 2 h were increased in the presence of theophylline regardless of
PHE addition compared with those in the nontreatment group. These
results indicate that theophylline enhanced the progressive motilityof sperm within a short period.
In the presence of PHE and theophylline at 6 h after incubation,
average BCF was significantly higher compared with that in the
nontreatment group. On the other hand, the average ALH of sperm
incubated with the combination of PHE and theophylline at 6 and
8 h was significantly lower than that in the nontreatment group.
This result is not consistent with a previous report describing that
ejaculated sperm incubated in Tyrode’s HEPES-buffered medium withheparin showed significantly high average ALH and BCF levels at 1
h to 4 h compared with those incubated in Tyrode’s HEPES-bufferedmedium without heparin supplementation [23]. Chamberland et al.
[23] suggested that the increases in BCF and ALH of sperm incubatedwith heparin in vitro could be related to the hyperactivation of
sperm [23]. In addition, theophylline induced hyperactivation andthe acrosome reaction in sperm of dogs [24] and enhanced the rateof sperm penetration into oocytes in humans [25]. The increase in
BCF and decrease in ALH observed in the present study probably
indicate that sperm incubated with PHE and theophylline acquire ahigh level of activity without hyperactivation.
It was previously reported that adenosine and catecholamineagonists might increase cAMP in the cytoplasm of sperm andaccelerate the beating of flagella related to BCF in mice [12]. In
the present study, theophylline, one of the PDE inhibitors, increased
intracellular cAMP. In humans, pentoxifylline, another PDE inhibitor,
also increased the intracellular cAMP content of sperm in a mannercorrelated to increases of BCF and ALH [26]. However, in the present
study, the ALH of sperm incubated with PHE and theophylline at
6 and 8 h was suppressed compared with that of sperm incubatedwith only theophylline. Because sperm incubated with PHE andtheophylline at 2 h showed a relatively low intracellular cAMP
concentration compared with that of sperm incubated with only
theophylline addition, we speculated that PHE activated an unknown
signaling pathway to prevent the increase in intracellular cAMP and
subsequently maintained sperm motility.
According to these hypotheses, sperm incubated with PHE andtheophylline maintain high progressive motility. Therefore, these sperm
have increased opportunities to encounter oocytes and subsequently
penetrate them. Further study is needed to clarify the relationshipbetween sperm motility parameters, intracellular cAMP content ofsperm and not only IVF medium including theophylline and PHE
mixtures, but also D-penicillamine, hypotaurine and epinephrine
separately.
In conclusion, the combination of a PHE mixture and theophylline
synergistically accelerates sperm motility and sperm penetration ofbovine oocytes. Theophylline activates sperm motility with increasing

PHE IMPROVES SPERM LONGEVITY

intracellular cAMP. However, PHE prevents an excessive increase
in cAMP and maintains sperm motility without hyperactivation.
When the combination of PHE and theophylline is added to IVF
medium at a sperm concentration of 2 × 106 cells/ml, we can obtainstable normal fertilization and blastocyst development rates using
sperm from any bull.

Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to M.
Nagano (No. 25450441). We thank the Genetics Hokkaido Association for the donation of frozen bull sperm.

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