Media

2.3.1. General Considerations

It is important that standards are maintained and variables reduced to a minimum. Water quality is of utmost importance and a good water purification system is required. Media and other additives should be prepared from either AR or tissue culture grade chemicals. Unless otherwise indicated, sterile techniques should be used for all procedures. All sera used are heat-inactivated (60°C for 30 min), filter sterilized, and stored at -18°C until thawed for use. Recommended materials are: Milli-Q filtration system (Millipore Corp., Bedford, MA) and Millex-GS sterilizing filters (0.22-|um pore size, Millipore Corp.).

2.3.2. Sperm Collection andAI

A simple buffer, phosphate buffered salme (PBS) with 0.4% bovine serum albumin (BSA) (pH 7.1) is all that is required for this work.

Hours

Fertilization in ampulla - 24 —

First cleavage - 48 ~ Second cleavage -

Third cleavage - 72 -

-Estradiol peak

-Onset of estrus - LH peak

-Ovulation begins

-Egg at ampulla-isthmus junction

Fourth cleavage -

Blastocyst - Q) Hatching

-Embryo leaves ampulla-isthmus junction •Embryo enters uterus

8 10 12

-oTP-1 production

Trophoblast elongation -

Fig. 1. Timing of early embryonic development in sheep.

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