If the bull has not been used for three or four days, the collection of a second ejaculate for freezing may be advisable. The second ejaculate seems to withstand freezing better than the first in many instances. A clean, dry artificial vagina should be used for each ejaculate collected. Repeated collections in the same artificial vagina may result in contamination of the semen with bacteria, lubricating jelly and minute particles of dirt. The semen sample should be protected from contamination and from sudden temperature drops (cold shock). =Preparation of extender.= A suitable egg yolk-citrate extender for freezing bull semen can be prepared by the following procedure. One part egg yolk (free of egg white and the membrane surrounding the yolk) is mixed with 4 parts 2.4 to 2.9 percent sodium citrate dihydrate solution. The citrate is prepared with distilled water and then boiled or autoclaved. The citrate solution should be cooled before it is mixed with the egg yolk. After the egg and citrate are mixed, 1000 units of penicillin and 1000 micrograms of streptomycin are added per milliliter of extender. Sulfanilamide should not be added. This extender can be prepared 12 to 24 hours before use if it is stored at refrigerator temperature. The portion of the extender needed for the original dilution of the semen should be warmed to room temperature before it is mixed with the semen. =Dilution after collection.= As soon as possible after collection, the semen sample should be diluted with the extender. The extender must be at the same temperature as the semen (room temperature) when the two are mixed together. At this time the semen can be partially diluted (1 part semen to 4 parts of extender) or diluted to a sperm concentration twice the final desired concentration (later in adding the glycerol for freezing, the semen is diluted further with an equal volume of glycerol containing extender). The diluted semen is slowly cooled (1-1/2 to 2-1/2 hours) to 5 deg. C. (41 deg. F.). Some units using frozen semen now allow the semen to stand at 5 deg. C. for 5 to 6 hours before glycerolization to allow the antibiotics to be more effective against any vibrio fetus organisms that may be present. This step is taken because it has been shown that glycerol inhibits the effectiveness of the antibiotics.[6] After cooling, semen can be further diluted to twice the desired sperm concentration if that were not done at the start. (Caution: Be sure semen and diluent are at the same temperature.)