Veterinary Andrology and Reproductive Techniques

CHEMICAL TESTS

Chemical tests available are

• DNA estimation
• Ascorbic acid content

DNA estimation

• The available literature shows that the quantity and quality of DNA content of sperm alters during storage, seasonal variation and aging of the male.
• This alteration of DNA content may be co-related with fertility.
  
• Gledhill (1966) reported that in infertile bulls the amount of DNA content may not vary but the quality of basic protein alters.

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Ascorbic acid content

• The ascorbic acid content in semen may be co-related with the fertility of semen (Philips et al., 1940 and Reid et al., 1947).
• Hence evaluation of semen for ascorbic acid content may be desirable.

Procedure

This method was described by Roy et al., 1950.

• Take 0.2 ml of fresh semen in a sterile test tube.
• Add 0.8 ml trichlor acetic acid.
• Mix the contents well.
• Filter the mixture through Whatman filter paper No. 1.
• Add 0.05 ml of 2,6 dichlorophenol indophenol solution in a centrifuge tube.
• Titer it against protein free filtrate of semen in a micropipette.
• Check the end point where the pink color disappears.
• A blank solution of 0.2 ml of standard ascorbic acid plus 0.8 ml of 10% trichlor acetic acid is titrated against 0.05 ml of 2, 6 dichlorophenol indophenol dye.

                                                         Titration reading of standard

Ascorbic acid concentration = ------------------------------------------ x 1.2

                                                           Titration reading of unknown

 

Preparation of dye

• Extract 100 mg of 2, 6 dichlorophenol indophenol through a filter paper with 25 ml of boiling distilled water. It can be kept as stock solution. This can be stored in refrigerated temperature. On the day of estimation 5 ml of stock solution is made up to 100 ml by adding freshly boiled and cooled distilled water.

Standard ascorbic acid

• Stock solution can be prepared by dissolving 60 mg of ascorbic acid in 5% acetic acid and the volume 100 ml. For routine use 1 ml of the the stock solution is diluted with 5% actetic acid to 100 ml. Thus 1.2 gm of ascorbic acid are present in 100 ml of solution.

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