In Vitro Production of Embryo

IN VITRO PRODUCTION OF EMBRYOS
FROM HIGH PERFORMANCE
COWS
GOH CHEONG TATT
MUHAMMAD MUSLIM BIN MOHD RODI
MUHAMMAD AZAM BIN HASSAN
MOHAMMAD HIDHIR BIN MOHD RAZALI
MOHD ZURAINI BIN MD RASDI
ABDUL HAYYAN BIN MOHD AKHIR
ABA11029957
ABA11030199
ABA11029864
ABA11030201
ABA11030236
ABA11029916
THE DEVELOPMENT OF
FROZEN THAWED EMBRYOS
AFTER TRANSFER
&

1. INTRODUCTION
2. PROBLEM STATEMENT
3. OBJECTIVES
4. LITERATURE REVIEWS
5. METHODOLOGY
6. RESULTS
7. DISCUSSION
8. CONCLUSION
9. REFERENCES
PRESENTATION OUTLINE

Reproductive biotechnologies are widely used for
farm animal breeding to utilize the genetic potential
of male and female animals more effectively
The efficiency of usable oocyte collection,
transferable embryo production and embryo
survival after cryopreservation are required for in
vitro production of embryos from oocytes.
INTRODUCTION

Up to now, the production of embryos from
oocytes of selected cows has not been very
effective in comparison with the production of
embryos from oocytes of populations of
ordinary slaughter cows.
PROBLEM STATEMENT

1. To determine the number of oocytes from infertile, genetically
valuable cows that will be recovered in the growth phase.
2. To determine the development of these oocytes will be resulted in
more embryos for transfer and cryopreservation.
3. To determine the pregnancy rates that resulted in the transfer of
frozen-thawed and fresh embryos.
OBJECTIVES

LITERATURE REVIEWS
Authors / years Statement
Enright et al., 2000; Dobrinsky,
2002
In vitro produced embryos have lower
cryotolerance than in vivo produced embryos.
Fahning and Garcia, 1992 Embryo survival after cryopreservation has
been shown to depend on the quality, age and
developmental stage of an embryo at freezing.
Rizos et al., 2002; Sirard et al., 2006;
Lonergan, 2007
Both the embryo quality and the kinetics
of embryo development are related to the
oocyte and, most importantly, to its meiotic
and developmental competence.

1.BULLS
• The frozen-thawed sperm of 21 elite bulls of
Czech Siemmental (n = 6), Holstein Dairy (n =
8) and Beef Cattle (n = 7) were used as oocyte
fertilization
2.COWS
• A total of 52 cows, between 5 and 10 years of
age, of Czech Siemmental (n = 15), Holstein
Dairy (n = 29) and Beef Cattle (n = 8) breeds,
were used as oocyte donors
MATERIALS

1
• Isolate the Oocyte-cumulus complexes by total slicing of the ovarian cortex.
2
• Then, Oocytes are develop in TCM-199 medium (Earle’s salt), supplemented with
antibiotics, 0.20mM sodium pyruvate (Sigma-Aldrich), gonadotropins (P.G. 600, 15
IU/ml) and 5% oestrus cow serum (ECS) for 24 hours.
3
• Isolate the motile spermatozoa by the swim-up method from frozen-thawed sperm using
modified Tyrode’s medium (SP-TALP).
4
• Next, both are co-incubate with the oocytes at a concentration of 1 × 106 spermatozoa per
ml in modified Tyrode’s medium (IVF-TALP) supplemented with 10 μg/ml heparin for 20
hours.
EMBRYO PRODUCTION

1
• The embryo then placed in freezing medium, consisting of 10% glycerol (v/v) in
TCM-199 medium with 10% ECS and equilibrated for 5 min at room temperature.
2
• Next, each embryo was loaded into 0.25 ml straw in a column of freezing medium
and allowed to stand for another 10–15 min at room temperature.
3
• Straws were placed in the programmable freezer at –7°C and, after 10 min, were
seeded.
4
• After another 10 min the embryos were cooled to –35°C at a rate of 0.3°C/min; they
were then plunged into liquid nitrogen.
EMBRYO
CRYOPRESERVATION

1
• The embryos were thawed by holding the straws for 10 s in the air and then placing
them in a 30°C water bath for 30 s.
2
• The cryoprotective was removed using a three-step procedure (6.6% glycerol [v/v] and
0.3M sucrose; 3.3% glycerol and 0.3M sucrose; 0.25M sucrose in culture medium, TCM-
199 with 10% ECS).
3
• Subsequently, the embryos were washed with culture medium and transferred as soon
as possible to heifers on Day 7 after their oestrus.
4
• The pregnancy rate was assessed by palpation per rectum on Day 90 after transfer.
EMBRYO TRANSFER

• Regardless of the breed, the mean number of usable oocytes
collected per donor and the mean percentage of usable
oocytes were significantly higher (P < 0.01) for the
synchronized than for the nonsynchronized donors.
• An evaluation of each breed revealed that the mean number of
usable oocytes per donor was higher for the synchronized
donors of Czech Siemmental, Holstein Dairy and Beef Cattle
breeds, but that only in Czech Siemmental cows was the
increase significant (P < 0.05).
• The difference in the mean percentage of usable oocytes
between the synchronized and nonsynchronized donors was
significant in cows of all breeds (P < 0.01, P < 0.05 and P < 0.05,
respectively).

• Regardless of the breed, the mean number of transferable
embryos produced per donor and the mean percentage of
transferable embryos were significantly higher (P < 0.01) for
the synchronized than for the nonsynchronized donors.
• The mean number of transferable embryos per donor was
higher for the synchronized donors of all three breeds, but
only in Czech Siemmental cows was the value significant (P <
0.05).
• The difference in the mean percentage of transferable
embryos was significant in cows of Czech Siemmental and
Holstein Dairy breeds (P < 0.01).

Efficiency Of Freezable Embryo

• Regardless of the breed, the mean number of freezable
embryos produced per donor and the mean percentage of
freezable embryos were significantly higher (P < 0.01) in the
synchronized than the non-synchronized donors.
• A separate evaluation of each breed showed that the mean
number of freezable embryos per donor was higher for the
synchronized donors of all three breeds, but that the
difference was not significant.
• The difference in the mean percentage of freezable embryos
was significant in Holstein Dairy cows (P < 0.01).

• The frozen-thawed viability of the embryos was
determined by their development after transfer
into heifers on Day 7 after their oestrus.
• From the synchronized donors, 41 frozen-thawed
embryos were transferred and 20 recipients
became pregnant (48.8%). The transfer of 43 fresh
embryos derived from the synchronized donors
resulting in 24 pregnancies (55.8%) served as a
control.
Embryo development after transfer

• Source of oocyte (culled genetically value animal and transvirginal
aspiration) is almost the same
• The number of transferable and freezable embryo of Czech
Siemmental Cow and Hoilstein Dairy Cow is twice higher than Beef
Cattle Cow.
• Selection of oocytes in the growth phase of first follicular wave will
improve:
– Accelerated development of embryo
– More embryos produced
– Better morphological quality
– Efficiency of embryo transfer increase
– Pregnant rate increase
DISCUSSION

• Factors to improve the survival rate of in vitro
production of embryo
– Selection based on kinetics of embryo development
– Modification of condition
– Maturation
– Cultivation
– Cryopreservation
– Metabolic manipulation with embryo before
cryopreservation
DISCUSSION

• Higher numbers of usable oocytes from infertile,
genetically valuable cows can be recovered in the growth
phase compared with any other phase of follicular
development;
• Greater development of these oocytes results in more
embryos for transfer and cryopreservation;
• The pregnancy rate can be improved after the transfer of
frozen-thawed embryos derived from oocytes with greater
meiotic and developmental competence.
CONCLUSION

1. Cho S.R., Cho S.K., Lee S.L., Lee H.J., Choe S.Y., Rho G.J. (2002): Enhanced cryosurvival of bovine blastocysts produced in vitro in
serum-free medium. Journal of Assisted Reproduction and Genetics, 19, 487– 492.
2. de Wit A.A., Wurth Y.A., Kruip T.A. (2000): Effect of ovarian phase and follicle quality on morphology and developmental capacity
of the bovine cumulus-oocyte complex. Journal of Animal Science, 78, 1277–1283.
3. Dinnyes A., Lonergan P., Fair T., Boland M.P., Yang X. (1999): Timing of the first cleavage post-insemination affects cryosurvival of
in vitro-produced bovine blastocysts. Molecular Reproduction and Development, 53, 318–324.
4. Dobrinsky J.R. (2002): Advancements in cryopreservation of domestic animal embryos. Theriogenology, 57, 285–302.
5. Enright B.P., Lonergan P., Dinnyes A., Fair T., Ward F. A., Yang X., Boland M.P. (2000): Culture of in vitro produced bovine zygotes
in vitro vs in vivo: Implications for early embryo development and quality. Theriogenology, 54, 659–673.
6. Fahning M.L., Garcia M.A. (1992): Status of cryopreservation of embryos from domestic animals. Cryobiology, 29, 1–18.
7. Galli C., Duchi R., Crotti G., Turini P., Ponderato N., Colleoni S., Lagutina I., Lazzari G. (2003): Bovine embryo technologies.
Theriogenology, 59, 599–616.
8. Hagemann L.J., Beaumont S.E., Berg M., Donnison M.J., Ledgard A., Peterson A.J., Schurmann A., Tervit H.R. (1999): Development
during single IVP of bovine oocytes from dissected follicles: interactive effects of estrous cycle stage, follicle size and atresia.
Molecular Reproduction and Development, 53, 451–458.
9. Hasler J.F. (2001): Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle. Theriogenology, 56, 1401–1415.
10. Hernandez-Fonseca H.J., Sirisathien S., Bosch P., Cho H.S., Lott J.D., Hawkins L.L., Hollett R., Coley S., Brackett B. (2002): Offspring
resulting from direct transfer of cryopreserved bovine embryos produced in vitro in chemically defined media. Animal
Reproduction Science, 69, 151–158.
REFERENCES

In Vitro Production of Embryo

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