The effect of feeding serum on bovine blastocyst development

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Intemabonal Asia Llnk Symposium "Reproductive Biotechnology for Improved An~malBrecdug In Southeast Asia"

The effect of feeding serum on bovine blastocyst development
~ . ~ r i w l a n r ~ i n ~~s .i ~h '. ,~ o e l i h e r~e.' ~, . ~ u t l eT.L.YUSU?,
d ~ e ~ , ~ . ~ u r w a n t a r a~' , . ~ i w ~ a n t o '
'~csearchInstitute for Animal Production, PO BOX 221, Bogor Indonesia
' ~ t ~ n n m e of
n tClinic, Reproduction, and Pathology, Faculty of Vetennary Medlc~ne.
Bogor Agr~culturalUniversity, Darrnaga, Bogor Indonesia

'university of Wisconsin, USA

main objective of this experiment is to obtain information on proper technique for in vitro
production of Holstein x Bali cattle hybrid (Fl) embryos using USA Holstein oocytes collected
from the slau&terhouse and fertilized with spermatozoa of Bali or Bus sortdaicus bulls. Holstein
x Brahman ck1e hybrid embryos using sperkatozoa of USA Brahman bulls and prebred Holstein embryo production. All viable embryos were frozen and brought to Indonesia and some of
them had been transferred to FH recipients in Indonesia.

These embryos may be used for improving the genetic value of the Indonesian dairy cattle. There
is transportation constraint in importing oocytes from USA. It takes more than 24 hours to bring
it to Indonesia. In fact, maturation of oocytes and readiness to be fertilized normally requires only
24 hours in 5% COz incubator at 38.S0C.
Therefore, this research is needed to study the effect of
gonadotropin hormone treatmens and time for oocytes maturation and readiness to be fertilized at
a p e r i d more than 24 hours. If this problem could be solved lhen the importation of oocytes
would be cheaper and easier than importation of live animals or embryos.
Owrirb were ~:uiiectedhorn lht: siau~hterhousein Wisconsin, USA. -inr r w c y t r s \rthre rnatilred in
TCM-199 medium enriched with FSH 10 pllml, oestradiol 17 P 1 pllml and 10 % FCS for 20
hours in COr incubator at 39 "C and RH 90%. The oocytes were fertilized in vrrro with motile
spem selected by 90% and 45% of Percoll gradient. The spem pellet was obtained after seven
minutes of the first centrifugation. The sperm pellet was washed with 1 ml TL Hepes and were
centrifugatated again for two minutes, the supernatant was discarded and spermatozoa in the pellet were directly counted using haemacytometer.The semen with I to 2.5 x lo6 sperm concentration was used for fertilization of matured oocytes. Sperm and oocytes were incubated in fertilization medium (TALP) for 20 hours. All zygotes were cultured in CRlaa medium and fed on day 4,
5 and 6 (as treatment A, B and C, respectively) with 5 p1 s e w n 150 p1 culture up to blastocyst.
Counting of morula, blastocyst, expanded blastocyst, degenerated embryo and cleavage followed
the method of Wosik and Stubbings (1 994).
Results oi this experimellts showed that the percentages of cleavage, blaslocysts and unfertilized
ova were not different among treatments lp>O.OS), but morula, degenerated embryos and expanded blastocysts were significantly different among treatment A vs C (p0.05) (Table 1 and Figure I). B a e d on results of this study, it is
concluded that the best method for 1VP (in vilro production) of embryos is feeding serum on day

6 (early blastocyst).
Some o f the embryos have been transferred to F1I recip~entsin Indonesia. Twenty two cows
were used as recipients, after estrous synchronization using CIDR (Controlled Internal Drug Release) implanted for 12 days, but only seven cows which had good qual~tyof corpora lutea were
transplanted with the IVF embryos (three embryos per recipient) and resulted in pregnancy of
three recipients (pregnancy rate 42.86 %). Unfortunately two of them aborted and only one (fs
male) was born alive with 45 kg birth weight (hybrid FH x Brahman). In the second trial, 32
cows were prepared as recipients after synchronization with ClDR implanted for 12 days, but
only nine cows had good qualiQ of corpora lutea. Three recipients were transplanted with IVF
embryos and two of them became pregnant (pregnancy rate 66.7%) but one of them was aborted
at seven months of pregnancy and one (male purebred FH)was born alive. Six other recipients

I

Intemattonai Asia Link Symposium "Reproducttvz Biotechnology for Improved Aninial Breedlng ~nSoutheast Asia"

Inm&imJ

were transplanted with the in ~ v v oembryos(two embryos per cow) and three of them became
pregnant (pregnancy rate 50%). Three days after thcy were born. however. the calves died due
to mastitis problem of the dams.

Ananim.

Trtntrnrnt

Momla

Cleavage

Clastocyst

E.ipatlded
hldslocysr

--Day4 (n=S24)

445(853)'

31Y(G25)b

235(478j1

--

- - .-

b ~ ( l 3 . 2 ) ~7 9 ( 1 4 8 ) '

I

Biggers JD,McGinnis LK,Raf'fin

-

Utlferi~ll- Dccenerated
zed o\ a
embryo

126(228)'

i

oviductal

prtgnant

992.\

--

I,nrleveloped oocytes
... . -

GB, BonDurant RH.
pregnant

--.

Devclapmcnt of oocytes (%)

Statistik Pertanian. Departemen Pertanian

Bthboodi E,

Table 1. The effect of feeding serum on In virrn embryo development
Feeding serum

"Rcpductive Biotechnobgy Cor Improved Animal

prtimplantal

63(1):28

1

Chian

[PubMed

Sirard

Choi

Takagi

p

Karnashita H,

T

follicular

dz'
1

vifro.

Dowson
tion.

Elliot M=,
15

JL, Ealy

preimplantation

tion

48:25-33.

Gandolfi

paracrine
b!astocyst. Theriogmology

Han

blastocyst

ing
C teawage

Morula

01astocyst

Expblast

+Day-5

+Day-&

Figure I . The effect of feeding serum on In vltro ernbryos dcvcl~pment

42:645-654.

24, McCauley

Hasler
Trimar

1995.

bryos

Embryos development
+Day4

influ
:95-

Koyama

the

I

Onlogeny
embry

Monterroso

protein

freezing
Thenogenolagy 43:1

lwamatsu T,

In vifro
224:9

licular

IH,

Van Soom

tt

Vanroose G, Casi AL,
v,

It is generally concluded Ihar USA originated oocytes could be preserved, matured and fertilized
in virro after a period o f more than 14 hours up to 36 hours with or without hormone supplement
in of its medium. Embryos produced by this process could reach the blastocyst and expanded
blastocyst stages and will be transferred to recipients in Indonesia.
It would be appropriate to recommend that further cooperarcd action research and implementation in this reproduction biotechnology (IVF} should be conducted using genetically superior oocytes of Holstein cows from USA and fertilized with spermatozoa from developed countries in
the effort to increase production and improve the dairy industry in Indonesia.

owes.

Mitsumino

S2:537-547.

Fuj ita

Utsurni

mcytes

follicu
bovi

ogy 45:787-799.

Kreysing

Nagai

1997.
vifra

(CPPs).Reprod.Fertil.Dev.9:465-474.
Liu Z

ftrtilization
l O(l1):2985-299 PMID:8747059[PubMed

tl