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Water buffalo is a very important livestock in Southeast Asian
countriesproviding milk, meat, and draft power. There are two types
of buffalo, the rivertype (2n=50) which is use mainly for milk and
meat and the swamp type (2n=48)which is use mainly for draft and
only secondary for milk and meat. In swampbuffalo-dominated
countries like Philippines, upgrading the swamp buffalo
bycrossbreeding with imported river buffaloes have resulted to
production ofcrossbreds that provided additional income and
nutrition to rural farmers throughimproved milk and meat
production. The improved production potentials haveincreased the
farmers interest to raise and own crossbreds, especially
purebredriver buffaloes. However, live animal importation is risky
and expensive, hence, toexpand the breeding program and produce
purebred river buffaloes, there is a needto develop the
reproductive biotechnology to produce river buffaloes by
embryotransfer using swamp buffaloes as surrogate mothers. In view
of the above, seriesof experiments was conducted to develop and
improve the systems for embryo invitro production (IVP) and
cryopreservation in water buffalo and the efficiency ofthese
techniques was examined for propagating water buffaloes of
superiorgenetics. In the first experiments, establishment of the
embryo IVP following thesystems used on cattle and studies on
vitrification for embryo cryopreservationwere carried out.
Viability of in vitro produced-cryopreserved embryos (2n=50)after
embryo transfer was assessed in river and swamp buffalo recipients.
Inexperiment 2, factors affectingthe production of waterbuffalo
embryos in vitro wereexamined. The effects of thelength of ovary
storage, lengthof in vitro maturation, and thecomponents of the in
vitromaturation medium foroocytes were evaluated. Theincidence of
chromosomeabnormalities among earlystage embryos was examined
Studies for the Improvement of in vitro CultureSystems of
Oocytes and Embryos in Water Buffaloes
Japanese Advisor : Yukio KANAI, Professor, University of
Tsukuba
Danilda Hufana DURAN DOST - 10418Senior Agriculturist,Philippine
Carabao Center, Reproductive Biotechnology Laboratory
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to determine factors causing failure of cleaved zygotes to
develop to the blastocyst.Experiment 3 was designed to improve the
in vitro systems for efficient productionof viable embryos.
Selection parameters of developmentally competent oocyteswere
established. Furthermore, the use of density gradients of silica
particles toisolate motile sperm cells for in vitro fertilization
(IVF), the effect ofsupplementation of energy substrates pyruvate
and lactate, and the use of stage-dependent culture system using
increasing concentrations of fetal bovine serum tocater the
changing requirement of the embryos for improved
blastocystdevelopment were assessed.
Results showed that following the systems developed in cattle,
success rate inwater buffaloes is sub-optimal with average cleavage
rate of 53.5% and blastocystsdevelopment of 11.4%, suggesting a
need to refine the systems to suit the waterbuffalo requirements.
Embryos vitrified by in-straw vitrification using EFS40(40% v/v
Ethylene glycol, 18% w/v Ficoll & 0.3 M Sucrose) as
cryoprotectant and10% ethylene glycol as pre-equilibration medium
survived at 0.5 min exposuretime but the success rate was low (25%)
and fracture damage was observed. It wasfound that using the same
cryoprotectant, pre-equilibration medium and exposuretime, the
ultra-rapid vitrification techniques using open-poled straw
resulted tosuccessful vitrification with an average of 83%
post-warming hatching rate in allpre-implantation embryo stages. In
vitro-produced-vitrified embryos transferred toriver and swamp
buffalo recipients undergoing natural estrus showed 8.1% and20.0%
full-term development, and 16.1% and 40.0% calving rates,
respectively.The results demonstrated viability of the resultant
embryos, development of thetechniques and potential application of
embryo transfer in water buffaloes.
Factors affecting the success rate of in vitro embryo production
showed thatthe length of ovary storage at 30-33 and the duration of
in vitro maturation hadsignificant effects on the developmental
competence of the oocytes. Shorterstorage period (3-4 h) of ovaries
is better than longer (4-6 h) storage period. Lengthof in vitro
maturation varies among oocyte, with optimum time appeared to
bebetween 22 to 25 hours. Examination of chromosomes of the early
stage embryosrevealed 47.7% incidence of chromosome anomalies
suggesting that ovary storage,length of IVM, components of the IVM
medium, and incidence of chromosomeanomalies are factors affecting
blastocyst development in buffaloes.
Selection of homogeneous population of oocytes showed that
thecompactness of cumulus cells, the diameter of the ooplasm, and
the size of donorfollicles are important indicators of oocytes with
developmental competence.Oocytes with loosened cumulus cells
requires shorter period (20-22 h) while thosewith compact cumulus
cells longer period (24-26 h) of in vitro maturation.
Highestdevelopmental competence were observed in oocytes surrounded
by >3 layers ofcumulus cells, with >120 m in diameter, and
from >_ 6 mm follicles. On the otherhand, density gradients
sperm separation utilizing silica particles was found
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effective in enhancing significantly higher cleavage and
blastocyst developmentwith three discontinuous layers (45/65/90%)
found most effective. On in vitroculture of embryos, results showed
that the addition of pyruvate and lactate in thein vitro culture
medium improved embryo development to the pre-implantationstage.
The stage dependent culture system utilizing increasing
concentration ofserum had no beneficial effect in improving
blastocyst development. Of the 52vitrified embryos produced by IVF
system using sperm cells separated by densitygradient and cultured
for development in a stage dependent culture system withpyruvate
and lactate, full term development of embryos after non-surgical
transferto recipient animals was 13.5% (7/52), or 23.1% (6/26)
calving rate including atwin suggesting an improved eff iciencies
in the IVP of viable embryos inbuffaloes.
Results of these studies demonstrated that method of IVP of
embryo inbovine works in water buffalo but success rate is
sub-optimal. Refinement of thesystems such that minimize long
storage of ovaries, select homogeneouspopulation of oocyte for IVM
and apply the desired IVM culture medium andperiod, use pure motile
sperm cells separated by gradients of silica particles forIVF, and
supplement energy substrates in the IVC medium improved
theproduction of embryos.
