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Effect of Japanese Kampo medicines on in vitro preservation of bovine spermatozoa and in vitro fertilisation

N. Yumiko,1 Zh. Rong,2 X. Yongmei3 and F. Noboru1

1. Animal Resource Science Section, Faculty of Agriculture, Graduate School Kyushu University, Fukuoka 8128581 Japan
2. Department of Animal Science, Gansu Agricultural University, Lanzhou 730070, Gansu, P.R. China
3. Captive Feeding and Breeding Cantre for Crested Ibis, Yangzian 723300, Shanxi, P.R. China


In this experiment, the potentials of four kinds of Japanese Kampo medicines (JKMs), syohangekabukuryoto, hotyuekkito, ogikentyuto, ninjin-eiyoto and mixed JKMs to improve physiological function of frozen-thawed bovine sperm motility when semen samples were incubated for 22 h at 37°C in medium 199 supplemented with JKMs were investigated. Following the experiment, in vitro fertilisation (IVF) and subsequent embryonic development of bovine oocytes treated with JKMs-processed sperm were examined. Results show that some of the JKMs slightly improve motility of frozen-thawed bovine spermatozoa and increase IVF rate. Slightly increased percentage of IVF was also observed by adding the JKMs to the medium phosphate buffered saline (PBS).

Keywords: Bovine oocytes, in vitro fertilisation (IVF), Japanese Kampo Medicines (JMKs), sperm motility, wild animals


It has been reported that endangered and/or extinct wild animals such as the giant panda, golden monkey and takin have very weak libido, which lead to lower rates of fertilisation and conception (Feng et al. 1997).

In our previous studies, the possibility of employing JKMs to improve frozen-thawed bovine sperm activity and to enhance in vitro fertilisation of cattle oocytes have been demonstrated (Zhao et al. 1997a; Zhao et al 1997b). On the other hand, one of the components of JKMs, frulic acid, has been shown to strongly stimulate human sperm activity in vitro (Zheng and Zhang 1996). These experimental results suggest possible enhancement of mammalian sperm activity by JKMs. Hitherto, most of the experiments with JKMs were carried out for curing human female infertility (Mizutani et al. 1988; Yamano et al. 1990).

Materials and methods

Preparation of JKMs

In this study, we used the following four sorts of JKMs, syohangekabukuryoto, hotyuekkito, ogikentyuto, ninjin-eiyoto, and mixed JKMs (Dongchongxiacao:Shaoyao:Guipi:Danggui: Huangqi: Gancao = 3:2:2:2:2:1). These JKMs were boiled for 1.5 hours to extract crude components and then filtered. The extracts were then diluted with PBS in a ratio of 1:10 and stored at 4°C.

Preparation of semen samples

Frozen bovine semen was thawed in a water bath at 37°C and washed twice in PBS by repeated centrifugation at 300 g for 5 minutes at room temperature (2225°C). The precipitated sperm pellets were suspended in PBS to obtain a final concentration of 4 × 107 sperm cells/mL.

In the preliminary experiments, fresh semen and/or higher motile spermatozoa were shown not to be suitable for present experiments due to no difference in sperm activity between JKMs and control group. This observation suggested that JKMs should not be used for highly active spermatozoa but for slightly immotile sperm cells. To do this, sperm suspension was incubated in shaking water bath for 10 minutes at 46°C for leading to slightly faint spermatozoa.

In vitro maturation (IVM) of bovine oocytes

The ovaries obtained from cows at a local slaughterhouse were brought to the laboratory in warm (37°C) physiological saline (0.85% NaCl) within 2 h after collection. The cumulus-oocytes complexes (COCs) were collected from follicles 2 to 8 mm in diameter. COCs were washed twice in PBS and three times in maturation medium, which is 199 medium supplemented with 10% FCS, 1 μg/mL estradiol 17β and antibiotics. Approximately 15 to 20 COCs were introduced into a 100 mL drop of maturation medium in 35-mm petri-dishes under mineral oil. The oocytes were incubated at 39°C for 22 h under 5% CO2 in air in the incubator.

In vitro fertilisation (IVF) of in vitro-cultured oocytes

The oocytes following IVM culture were washed three times in PBS and introduced into 100 μL drop containing 2 μL of sperm suspension, 10 μL JKMs solutions and cultured for 6 hours at 39°C in the same incubator as for the IVM.

In vitro culture (IVC) of fertilised oocytes

After 6 h of culture for insemination, presumptive zygotes were washed twice in PBS and culture medium (199) containing 10% FCS and antibiotics. Fifteen to twenty zygotes were transferred into culture medium (100 μL drop) and cultured for 3648 h at 37°C in the CO2 incubator. Following the culture, zygotes were washed twice in PBS to remove cumulus cells. Embryonic development was examined under a light microscope.

Results and discussion

Some of the JKMs slightly improved sperm motility and increased the rates of fertilisation. Similar results to this have been reported in cattle spermatozoa using other kinds of JKMs (Zhao et al. 1997b). However, much better results have been demonstrated in the cattle semen when proteinaceous substances such as fetal calf serum or cow serum were added to the medium (Zhao et al. 1997a).

On the other hand, the JKMs used in our laboratory have been considered to be alternatives to the above-mentioned materials when these chemicals are expensive or difficult to obtain, especially in some Asian countries. In this experiment, special attention was also paid to the researches in China, regarding giant pandas and crested ibis, which are at present among the most critically endangered animals in the world.

In case of human spermatozoa, one of the most important components of JKMs, ferulic acid, has been reported to remove lipid peroxidatives as a scavenger, making it an effective factor for improving physiological function of spermatozoa (Cummins et al. 1994; Plante et al. 1994).

These series of experiments may also suggest the possibility of restoring impaired sperm function in human and animals by in vivo treatment. An interesting piece of research on JKMs would be the clarification of mechanism of physiological function of these medicinal herbs. This is planned for the future.


The authors wish to express their gratitude to people at the Chengdu Research Base of Giant Panda Breeding for doing co-operative research work and obtaining some of the Chinese medicinal herbs used in the present experiment. Parts of the present experiments were financially supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, Japan Society for the Promotion of Science, Sumitomo Foundation and Nissan Science Foundation.


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