Embryo Transfer (ET)

	
I.	General Information

	1.	An embryo in early development (morula/early blastocyst) 
		is transferred from a donor animal to a recipient animal

	2.	First accomplished in 1890

	3.	Successful in sheep, goats, swine, cattle, 
		and horses

	Advantages

		a.	Can increase offspring from genetically 
			superior females
		b.	Obtain offspring from animals that can't carry 
			pregnancies to term

	Disadvantages

		a.	Costly
		b.	Need Trained Technician
		c.	Need to corral and restrain females



II.	Steps involved in E.T.

	1.	Estrus Synchronization of Donor 
		and Recipients

	2.	Superovulation of Donor
		A.	PMSG
		B.	Not possible in horses

	3.	Artificial Insemination of Donor

	4.	Embryo Collection of Donor
		A.	Usually non-surgical

	5.	Microscopic Examination of Embryos
		A.	Select healthy embryos
		B.	Extra Embryos can be frozen

	6.	Transfer of Embryo to Recipient
		A.	Surgical
		B.	Non-surgical

III.	Microscopic Examination of Embryos

	1.	Characteristics to observe
		A.	Compactness
			a.	Compact rather than loose mass of cells
		B.	Shape
			a.	Spherical better than Oval
		C.	Variation in Cell Size
			a.	Blastomeres uniform in size
		D.	Color and Texture
			a.	Not too light
			b.	Not too dark
		E.	Presence of Vesicles
			a.	Cytoplasm should not be granular 
				or unevenly distributed
			b.	Moderate size vesicles
		F.	Presence of Extruded Cells
			a.	No dissociated cells
		G.	Size
			a.	Normal
			b.	Learn with practice
		H.	Regularity of Zona Pellucida
			a.	Perivitelline Space Empty
			b.	Regular in diameter
			c.	No wrinkling or collapse
		I.	Presence of cellular debris
			a.	No cell fragments
		J.	Proper stage of development for age
			a.	Morula
				i.	Cow=4-7 days
				ii.	Sow=4-5 days
				iii.	Mare=4-5 days
				iv.	Ewe=3-6 days
			b.	Blastocyst
				i.	Cow=8 days
				ii.	Sow=6 days
				iii.	Mare=6 days
				iv.	Ewe=7 days

IV.	Freezing and Thawing Embryos

	1.	Similar to Semen
	2.	Ethylene Glycol used as cryoprotectant rather than glycerol
	3.	Stepwise Freezing Procedure
		A.	Embryo placed in 1.5 M Ethylene glycol in straw
		B.	Phosphate buffered saline is placed 
			at one end divided by bubble
			a.	1:3 Ethylene Glycol to PBS ratio
		C.	Straw is placed into alcohol bath freezer to -7 C
		D.	PBS side is seeded by touching a metal rod cooled 
			with liquid nitrogen
			a.	5 minute hold
		E.	Cooled 0.5 C per minute to -35 C
			a.	15 minute hold
		F.	Plunged in liquid nitrogen
			a.	Can be stored indefinitely
	4.	Thawing
		A.	30 C water bath
		B.	50% of normal pregnancy rate
		C.	Only top quality embryos should be frozen