![]() ![]() ![]() A small aliquot of the chilled, extended sample should be kept by the collecting veterinarian in an Eppendorf tube in a cup of water in the fridge. It is also commonly believed that even if the semen has arrived early relative to time of fertilisation then it is better in the bitch than the fridge. It is therefore best to collect the semen as close as logistically possible to time of insemination. With increasing time of storage of chilled semen samples there is increased DNA damage to the sperm 1, so it would be assumed that the pregnancy rate from samples chilled for a long time will be reduced, and the rate of early embryonic death (resorption) will be increased. These claims are not based on pregnancy studies, and although the quality of samples is based on motility, there is a poor correlation between motility and fertility. The length of chill time is based on % motility ofĢ the sample, with the manufacturers claiming the sperm to have at least 80% of their initial motility for up to 10 days. There are some extenders on the market which claim to be able to extend semen for long periods of time (up to 10 days). 20% fresh egg yolk is added to most chilled semen extenders before adding it to the semen, except for the MOFA Andropro chilling range (Andropro ST and Andropro LT) which contains a synthetic protein, however egg yolk may still be added if the clinician wishes. The manufacturer s instructions should be followed, however most extenders require a 1:3 dilution with 1 part semen to 3 parts extender. The raw sample must be analysed for total sperm numbers, motility and morphology. Should semen fractionation not be ideal, then the sample should be washed and centrifuged in either a semen extender/thaw media or the chill media. ![]() Semen collection should be performed ensuring good fraction separation so as to provide a ready to use semen rich fraction free of prostate fluid. ![]() Sample preparation Centrifugation of samples should be avoided. Commonly use antibiotics are streptomycin, benzyl penicillin and gentamycin. Chilled semen extenders also contain buffers to prevent sudden changes in ph, an energy substrate for the sperm such as dextrose, glucose or fructose and antibiotics to inhibit bacterial growth in the sample. The LDL s, glycolipids and cholesterol reduced the tendency of the phospholipids to change from liquid to gel form during cooling. Low density lipoprotein (LDL) is believed to either stabilise the sperm membrane or forms a protective layer over the surface of the sperm or replaces membrane phospholipids that have become damaged. Heat treating the milk results in inactivation of lactenin. Whole milk contains lactenin which is spermicidal. This constitutes cold shock Cold shock is prevented by the addition of egg yolk, skim milk or synthetic proteins. The changes in the membrane structure and leakage of product leads to altered sperm function. Ultimately the sperm cell membrane becomes more permeable and leaks potassium enzymes, lipids, cholesterol, lipoprotein and ATP. Phospholipids in the sperm cell membrane change from liquid to gel phase as the sperm are cooled and as different phospholipids cool at different rates, phase separation can occur resulting in membrane proteins clustering and losing their function. This maintains the semen in liquid form, not solid, however the sperm must be protected from cold shock. Chilled semen extenders Chilled semen extenders allow for the safe cooling of semen to 4 C. Whilst being more successful in attaining pregnancy, chilled semen can be difficult as it requires accurate ovulation timing, whilst allowing time for the collecting veterinarian to collect, prepare and ship the sample. Chilled semen has similar pregnancy rates to fresh semen if used soon after collection, and a higher pregnancy rate than frozen semen insemination. 1 Canine chilled and frozen semen preparation and artificial insemination Dr Stuart Mason BVSc (Hons) MANZCVS DACT Monash Veterinary Clinic 1662 Dandenong Road, Oakleigh East, Victoria, Chilled Semen Fresh chilled semen is for short term storage and transport of canine semen required for artificial insemination. ![]()
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