Embryo Health

8/3/2019 Embryo Health

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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

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Embryo Health

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