EVALUATION OF EMBRYO QUALITY

Dr. Taruna Anand, Scientist VTC, NRC Equine, Hisar (Haryana)-India, Dr. Dharmendra Kumar, Scientist CIRB, Hisar (Haryana)- India & Aditi Ray (M. Sc.)

WHY IS EVALUATION OF EMBRYO HEALTH REQUIRED?

Adverse effects may lead to incapability of establishing a viable pregnancy.

Fig: 16 - cell stage embryo

Fig: Blastocyst with distinct ICM

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 factors Metabolic tests

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 CODE 2 CODE 3 CODE 4 Excellent or Good Fair Poor Dead or Degenerating

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

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

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

ULTRA STRUCTURAL FEATURES

Reduced volume of total mitochondria in IVC compact morulae Junctional complexes between cells were less developed in lowquality embryos Microvilli well developed in high quality embryos Fair & 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 intercellular cavities.

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 developmental potential 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 noncompacted stage to blastocyst

Fig. Development of in vivo vs in vitro in bovine embryos (from Van Soom,1996)

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 as FDA -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 intact cell membranes. Fluorescein -used in evaluating functional cell-to-cell communication

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 latestage 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 -----------------------------------------------------------------------------------------------------------------

morula 50.37 17.65 early blastocyst 96.85 47.30 blastocyst 120.35 39.53 Day 8 expanded blastocyst 169.4- 18.95 Day 9 hatched blastocyst 204.40 59.14 -----------------------------------------------------------------------------------------

Day 6 Day 7

HATCHING
Occur 8-10 days after ovulation and fertilization in live cow, if it fails to occur 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.

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 PAF or PAF-like substances in vitro. Day 10 bovine embryo can synthesize and secrete both steroids and prostaglandins

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.

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 metabolism High 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.

THANKS