Post on 06-Apr-2018
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Dr. Taruna Anand, Scientist
VTC, NRC Equine, Hisar
(Haryana)-India,
Dr. Dharmendra Kumar, Scientist
CIRB, Hisar (Haryana)- India
&
Aditi Ray (M. Sc.)
EVALUATION OF EMBRYO QUALITY
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WHY IS EVALUATION OF EMBRYO HEALTH
REQUIRED?
Adverse effects may lead to incapability of establishing a viablepregnancy.
Fig: 16 - cell stage embryo Fig: Blastocyst with distinct ICM
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VARIOUS METHODS USED:
Morphological and morphometric parameters
Ultra structural features
Age and developmental stage attained
Fluorescent staining procedures
Cell numbers
Hatching
Secretion of hormones and growth factorsMetabolic tests
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MORPHOLOGICAL AND MORPHOMETRIC PARAMETERS
Classification involved identification of various
morphological features, including shape,color,size of
previtelline space, the number of extruded and degenerate
cells , number and nature of vesicles.
Classified as:
CODE 1 Excellent or Good
CODE 2 Fair
CODE 3 PoorCODE 4 Dead or Degenerating
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CODE 1 Excellent or good Symmetrical,spherical embryo
mass, individual blastomeres, uniform size, color , density, embryo
is consistent with development stage, irregularities minor & 85%
cellular material intact, zona pellucida smooth
Diagrams of good quality embryos
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CODE 2 Fair Moderate irregularities in overall shape of embryo
mass, size,color,density of individual cells. 50% of cellular material
intact, viable.
Diagrams of fair quality embryos
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CODE 3 Poor Major irregularities in overall shape of embryonic
mass, size, color, density of individual cells. 25% of cellular material
intact, viable embryonic mass.
Diagrams of poor quality embryos
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ULTRA STRUCTURAL FEATURES
Reduced volume of total mitochondria in IVC compact morulae
Junctional complexes between cells were less developed in low-
quality embryos
Microvilli well developed in high quality embryosFair & poor quality embryos contained nuclei with low
transcriptional activity, large number of lipid droplets and immature
mitochondria
IVP embryos lack desmosomal junctions, reduced microvilli
population , increased debris in perivitelline space and intercellularcavities.
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AGE AND DEVELOPMENTAL STAGE ATTAINED
Two factors considered
i)general appearance
ii) stage of development in relation to the time since fertilization.
Time of first cleavage is correlated directly with developmentalpotential
Close correlation between the time interval from insemination to
first cleavage and subsequent development.
.Compaction in IVP embryos occurs to a lesser degree than morulae
produced in vivo, and some morulae proceed directly from non-compacted stage to blastocyst
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Fig. Development ofin vivo vs in vitro in bovine embryos (fromVan
Soom,1996)
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FLUORESCENT STAINING PROCEDURES
Differential fluorochrome technique useful in counting cells of
the ICM & of trophectoderm of blastocysts, morphology of ICM
cells of blastocyst produced in vivo& in vitro.
Usage of dyes such asFDA -fluoresces ICM of blastocyst
DAPI - has affinity for DNA of non-viable or dead blastomeres.
Calcein AM - measures intracellular esterase activity.
Ethedium homodimer - DNA
binding dye impermeable to intactcell membranes.
Fluorescein -used in evaluating functional cell-to-cell
communication
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CELL NUMBERS
Bovine blastocysts developed in vivo contain 100,120 & 160 cells at the
early, expanding and expanded blastocyst stages respectively .
The average cell number of embryos , from super ovulated cattle in late-
stage blastocyst is about 140 (93-trophoblastic cells and 47 ICM cells).
Table: Cell number in in vitro embryos (from Lonergan,1992)
-------------------------------------------------------------------------------------------DAY STAGE CELL NUMBER
-----------------------------------------------------------------------------------------------------------------
Day 6 morula 50.37 17.65Day 7 early blastocyst 96.85 47.30
blastocyst 120.35
Day 8 expanded blastocyst 169.4- 18.95
Day 9 hatched blastocyst 204.40 59.14
-----------------------------------------------------------------------------------------
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HATCHING
Occur 8-10 days after ovulation and fertilization in live cow, if it fails tooccur then pregnancy also doesnt occur.
2 factors mediate hatching:
i) lysis of zona pellucida by substances secreted either by embryo or
female reproductive tract
ii) pressure exerted on zona by the expansion of the blastocyst.
Significant in development of laboratory produced embryos as a measure
of their ability to progress normally after various treatments or
manipulation.
Fig:embryos at different hatching stages.
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SECRETION OF HORMONES AND GROWTH
FACTORS BY EMBRYO
Quality of embryo is affected by the differences in the
prostaglandin E ( PGE/PGF) ratio secreted by cultured endometrial
cells.
Certain embryos also secrete PA
F or PA
F-like substances in vitro.Day 10 bovine embryo can synthesize and secrete both steroids
and prostaglandins
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METABOLIC TESTS
ENZYME ASSAYS:
Embryos release metabolic end-products from metabolic substrates
from the medium.
3 main conditions:
I) assay should not be applicable to individual embryos
ii) assay should not have adverse effect on subsequent embryonic
survival
iii) assay should be simple and effective.
More reliable indicator than morphological score.
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GLUCOSE, GLUTAMINE AND PYRUVATE UPTAKE
EMP activity rise with increasing embryo development.
Glucose essential for hatching bovine blastocysts
Utilization of sources such as amino acids for oxidative
metabolismHigh concentrations of hexose sugar are detrimental to blastocyst
development and may induce apoptosis.
ATP content increased during oocyte maturation,maximum at 8
celled stage, declining to minimum at hatched blastocyst stage.
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THANKS