PRELIMINARY STUDY OF BUFFALO SPERM PENETRATION INTO ZONA-FREE HAMSTER EGGS AFTER TREATMENT WITH CALCIUM IONOPHORE A23187
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PRELIMINARY STUDY OF BUFFALO SPERM
PENETRATION INTO ZONA-FREE HAMSTER EGGS
AFTER TREATMENT WITH CALCIUM IONOPHORE
TAKAHASHI, Yoshiyuki; NIHAYAH, Mohammad;
HISHINUMA, Mitsugu; JAINUDEEN, Mohammed Razeen;
ABAS, MAZNI Othman; MORI, Yuji; KANAGAWA, Hiroshi
CitationJapanese Journal of Veterinary Research, 37(3-4): 161-166
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
lPn. l. Vet. Res., 37, 161-166 (1989)
PRELIMINARY STUDY OF BUFFALO SPERM PENETRATION
INTO ZONA-FREE HAMSTER EGGS AFTER
TREATMENT WITH CALCIUM IONOPHORE A23187
Yoshiyuki TAKAHASHI!), Mohammad NIHAYAH2), Mitsugu HISHINUMA1),
Mohammed Razeen JAINUDEEN2), Othman ABAs MAZNIl), Yuji MORI3)
and Hiroshi KANAGAWA1)
(Accepted for publication August 11, 1989)
Key words: hamster egg, sperm, penetration, river buffalo, calcium ionophore
In cattle, the study of in vitro fertilization (IV F) has greatly progressed, and it
has become possible to obtain calves after the transfer of embryos which are matured,
fertilized and cultured in vitro (Goto et al., 1988; Eyestone & First, 1989).
er, the study of IVF in the buffalo is very limited, even concerning the induction of
sperm fertilizing capacity (Singh et al., 1989).
Zona-free golden hamster eggs permit the entry of spermatozoa of many mamma-
lian species provided the spermatozoa have completed capacitation and acrosome
reactions, thus allowing them to be used in assessing the fertilizing capacity of
spermatozoa of various species including the bull (Yanagimachi, 1984).
there is no literature about the penetration of hamster eggs by buffalo spermatozoa.
A previous study (Takahashi & Hanada, 1984) reported the penetration of zona-free
hamster eggs by ejaculated bull spermatozoa after treatment with calcium ionophore
A23187 (Ca-IA) in the presence of caffeine.
accomplished not only in cattle (Hanada, 1986b) but also in goats and sheep (Hanada,
1986a) using spermatozoa treated with Ca-IA.
of Ca-IA treatment in inducing the fertilizing capacity of the river buffalo (Bubalus
Bubalis) spermatozoa using zona-free hamster eggs, was examined.
Modified Krebs-Ringer bicarbonate solution (mKRB; Toyoda & Chang, 1974) was
used for handling hamster oocytes. This medium was also used to make a solution of
0.1% hyaluronidase (lyophilised powder from bovine testes, Sigma Chern. Co., St.
Based on this study, successful IVF was
In the present study, the effectiveness
1) Department of Theriogenology, Faculty of Veterinary Medicine, Hokkaido University, Sap-
poro 060, Japan
2) Department of Clinical Studies, Faculty of Veterinary Medicine, Universiti Pertanian
Malaysia, Serdang, Selangor, Malaysia
3) Laboratory of Veterinary Reproduction, Faculty of Agriculture, Tokyo University of Agricul-
ture & Technology, Fuchu, Tokyo 183, Japan
TAKAHASHI, Y. et al.
Louis, USA) and 0.05% trypsin (1: 250, Difco, Detroit, USA).
Co., St. Louis, USA) was dissolved in a dimethyl sulfoxide-ethylalcohol mixture (3: 1)
at a concentration of 10 mM and stored in a freezer (-20°C).
medium (DM; Brackett & Oliphant, 1975) supplemented with 15 mM theophylline
without bovine serum albumin (DM + Theo) was used for washing sperm, and DM
containing 20 mg/mt of crystalline bovine serum albumin (BSA; Fraction V, Sigma
Chern. Co., St. Louis, USA) was used for the insemination droplet.
Fresh semen was collected from a fifteen-year-old river buffalo bull using an
artificial vagina. One ml of the fresh ejaculated semen was diluted with 4 ml of DM +
Theo and washed 3 times by centrifugation at 500 g for 10 min.
centrifugation and aspiration of the supernatant, the sperm pellet was resuspended in
DM + Theo and the sperm concentration was adjusted to 1 X 107 cells/ml.
justed sperm suspension was exposed to 0.1 -1. 0 fl M Ca-IA.
Ca-IA for 0.5-2.0 min, 50 fll of sperm suspension was introduced into a droplet of
50 pI of DM supplemented with BSA under paraffin oil.
spermatozoa in the insemination droplet was 5 X 106 cells/ml.
Mature female golden hamsters were induced to superovulate by intraperitoneal
injection of 30 IU of pregnant mare's serum gonadotrophin followed 54 hr later by
30 IU of human chorionic gonadotrophin (hCG).
ampullar region of the oviducts 15 -16 hr after the hCG injection, and were freed
from cumulus cells by hyaluronidase. Cumulus-free oocytes were washed 3 times in
fresh mKRB and treated with trypsin to remove the zonae pellucidae.
oocytes were washed 3 times in fresh mKRB and immediately put into a droplet
containing Ca-IA-treated spermatozoa and incubated at 3rC in humidified air with 5%
In experiment 1, sperm motility was examined after the introduction of sperm into
a insemination droplet under an inverted-microscope at 1 hr intervals.
shown in Table 1.
The motility of spermatozoa was impaired with the increase in the
concentration and the length of the treatment period with Ca-IA. It was observed
that sperm motility was lost within 1 hr when they were treated with 1.0 fl M Ca-IA.
In experiment 2, the eggs were inseminated with spermatozoa that were treated
with 0.1-1.0 p M Ca-IA for 1 min. The eggs were fixed with neutral formalin
3.5-4.0 hr after the insemination, and then stained with 0.025% aceto-Iacmoide
solution. Penetration was examined using a phase-contrast microscope, which de-
tected the presence of a swollen sperm head or a male pronucleus with a tail within
the egg cytoplasm as described previously (Takahashi & Hanada, 1984). Penetration
results are shown in Table 2.
The spermatozoa which were not treated with Ca-IA
(control) attached heavily to the surfaces of zona-free hamster eggs as shown in Fig.
1a. However, they did not penetrate into the eggs. After the treatment with
0.1-0.25 fl M of Ca-IA, spermatozoa also attached heavily to the eggs as in the
Ca-IA (Sigma Chern.
A chemically defined
After the third
After the exposure to
The final concentration of
Oocytes were recovered from the
Buffalo sperm penetration into zona-free hamster eggs
Table 1. Motility of buffalo spermatozoa after treatment with calcium
Motility score; time after insemination
Time(min) 1 2 3(hrl
Moti I i ty score : -, ±, +, + + and + + + ; none, less than 25, 25 - 50, 50 - 75 and more
than 75%, respectively.
All the ionophore-treated spermatozoa were introduced into insemination medium with 10 mg BSA/ml
at the final concentration.
control. The removal of these attached spermatozoa from the eggs was quite diffi-
cult, and some of the inseminated eggs were broken during the removal of attached
spermatozoa by pipetting. The examination of eggs was not possible due to the
presence of attached spermatozoa after the treatment with 0.1 p. M Ca-IA, and thus
the penetration data could not be determined.
6 enlarged sperm heads and/or male pronuclei (mean ± SD = 1. 7 ± 1.4) after insemina-
tion with spermatozoa treated with 0.25 and 0.5 p.M Ca-IA (Fig.lb). Two of 13
eggs were penetrated after insemination of sperm treated with 1.0 p. M Ca-IA,
probably due to the loss of sperm motility in a short period as shown in Experiment l.
To induce the fertilizing capacity of cattle, sheep and goat spermatozoa with Ca-IA, it
had been recommended that spermatozoa should be exposed to 0.1-0.5 p. M Ca-IA for
1.0 to 2.5 min in the presence of 2-10 mM caffeine (Hanada, 1986a, b).
results are in close agreement with the above reports.
In the human, phosphodiesterase inhibitors such as caffeine and theophylline
Seventeen of 31 eggs (54.8%) had 1 to
TAKAHASHI, Y. et al.
Table 2. Effect of calcium ionophore A23187 concentration on the
penetration of buffalo sperm into zona-free hamster eggs
calcium ionophore CuM)
No. of eggs
No. of eggs
Whole-mount preparations of zona-free hamster eggs after insemination
with river buffalo sperm. (a) An egg showing a heavy attachment of sperm. The
egg chromosomes (1rrow) remained at metaphase II. (b) An egg showing the
polar body (PB), a female pronucleus (FPN) and three enlarged sperm heads
(arrows). Scale line = 20