CRU Lunch Seminar, May 3rd, 2012
ASSISTED REPRODUCTIVE TECHNOLOGY IN SPOTTED BUFFALO
CONSERVATION PROGRAM
Yulnawati Yusnizar*
*CRU Member as a PhD Student, in a Collaborative Project between Dept. Animal Production and
Technology, Bogor Agricultural University, Bogor, Indonesia and Dept. Animal Breeding and
Genetics, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
ABSTRACT
Spotted buffalo is an exotic animal that is considered being a legend symbol of
pride for Torajan people who use it in culture ceremonies and treasure in Toraja, South
Sulawesi, Indonesia. Its population is very limited nowadays and close to extinction due
to low birth rate because of problems in natural mating. The research about spotted
buffalo is also very limited because of the difficulties of sample collection. Farmers are
very protective to their buffaloes, because they have very high economic value. By
persuasive explanation to the farmers and also with collaboration with local
government, we have started studies in spotted buffalo conservation program since
2007. We tried to optimize some extender composition to maintain the frozen-thawed
epididymal sperm quality. Furthermore, we have already tried to use the frozen-thawed
epididymal sperm in artificial insemination (AI) and in vitro embryo production (IVEP)
program. Embryo sexing using PCR has also been tried to check the gender of the
embryo before transferred.
Our results showed that the fresh epididymal sperm has a good quality (3578.3 ±
740.3 million sperm/ml; 74.2% ± 1.2 were motile progressive; 85% ± 2.4 were viable
with only 7.1% ± 1.2 of them were have abnormal morphology and 86.2% ± 1.9 with
the intact membranes) and suitable for ART (AI and IVEP) purpose. The average
quality of frozen-thawed epididymal sperm was differed in several extenders. The
average percentage of post-thawed progressive motility in Andromed, Tris-egg yolk,
Tris-egg yolk plus 0.4% w/v sucrose, Tris-egg yolk plus 0.8% w/v sucrose, citrate-egg
yolk and citrate-egg yolk plus fructose were 45 ± 4.1; 32.5 ± 4.8; 39.2 ±3.4; 40 ± 2.9;
32.5 ± 4.8 and 39.2 ± 5.3, respectively (P>0.05). The average percentage of post-thawed
epididymal sperm viability in several extenders (as the same order) were 64.8 ± 1.2;
57.5 ± 3.9; 65.5 ± 4.4; 66 ± 3.4; 53.6 ± 3.3 and 63.3 ± 4.0, respectively. The average
percentage of post-thawed membrane intactness of epididymal sperm were 65.8 ± 2.0;
58.6 ± 3.7; 65.3 ± 3.1; 65.4 ± 4.2; 53.4 ± 2.6 and 63.0 ± 2.6 in Andromed, Tris-egg
yolk, Tris-egg yolk plus 0.4% w/v sucrose, Tris-egg yolk plus 0.8% w/v sucrose,
citrate-egg yolk and citrate-egg yolk plus fructose extenders, respectively. Since we
have limited number of females that available to use in AI program, we decided that
only groups of epididymal sperm in Andromed and Tris-egg yolk plus 0.8% w/v
sucrose extender that were used in AI program. There were 3 of 8 females (37.5%) were
pregnant and success to deliver offspring after inseminated using frozen-thawed sperm
in Andromed extender, while 40% (4 of 10) of females in other group that inseminated
using frozen-thawed sperm in Tris-egg yolk plus 0.8% w/v sucrose extender were also
success to get pregnant and deliver healthy offspring. About 4 of 7 (57.1%) offspring in
total of AI program were spotted, but only one (14.3%) of them was male. Our
preliminary result in in vitro embryo production showed that the average number of
oocytes that were suitable to be used in each step of in vitro embryo production stage
was about 19.8; 18.2 and 13.6, respectively at maturation, fertilization and culture stage.
The average of fertilization and cleavage rates were 77.6% ± 19.6 and 44.9% ± 18.9.
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CRU Lunch Seminar, May 3rd, 2012
The interesting of the result showed that about 94.2% of the embryos were confirmed as
male embryos.
The success rate of pregnancy & parturation in AI program need to be increase
by improving the quality of frozen-thawed epididymal sperm, and synchronize the
estrous and ovulation together in one program to encounter the silent heat phenomena
that common in female buffalo. We also need to perform the further study to increase
the number of male offspring as AI product. The low success rate of fertilization and
embryo cleavage was due to lack of high quality oocytes as the gamete sources. In the
other hands, the high percentage of male embryos of IVEP program is interesting to be
studied furthermore by performing embryo transfer to check the viability of the
embryos during pregnancy.
In conclusion, we optimist that assisted reproductive technology could be used
in spotted buffalo conservation program. Some improvements in the application of ART
are absolutely needed to increase the success rates and the efficiency of AI and IVEP
using epididymal sperm in order to increase spotted buffalo population.
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