VGO 511: Veterinary Andrology and Reproductive Techniques (1+1)

POST THAW EVALUATION OF SEMEN

Post thaw motility (PTM)

• Post thaw motility is examined 24 hours after freezing the semen.
• Thaw the frozen semen straw at a water bath of 37⁰C for 30 seconds.
• Wipe the straw and cut the ends of the straw and collect the semen in a small test tube.
• Keep a small drop on a clean, grease free glass slide.
• Cover it with cover slip and examine under the phase contrast microscope.
• Motility based on progressive motile sperms is evaluated on the nearest 5 percentage.
• If motility differs by 10 per cent or more between two straws of the same batch a third straw is examined.
• The minimum post thaw motility of 50 percentage is acceptable.

The frozen semen should have a minimum of 50% progressively motile sperms after thawing at 37^oC for 30 seconds.

• In routine practice only post thaw motility is performed and it is the most reliable test when carried out by an experienced person.
• [Click here for video: PTM -10% (Buffalo semen); PTM -50-60% (Buffalo semen); PTM -60-70% (Buffalo semen); PTM-Jerky and circular (Buffalo semen); PTM -50-60% (Bull semen); PTM -60-70% (Bull semen)]

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The following three tests are done only as added tests when it is suspected that semen may be of lowered fertility but meets minimum standards for fertility.

Post thaw acrosomal integrity

• The acrosome covers 60 per cent of the anterior portion of the nucleus.
• Acrosome can be detached from sperm under the influence of different physical and chemical factors.
• The enzymes localized in the acrosome determine the sperm penetrating and fertilizing capacity. Optimum fertility depends on the acrosome being structurally and biochemically intact.

Post thaw acrosome intergrity can be seen by staining sperms with Giemsa stain. The procedure is same like staining fresh semen. The post thaw semen should have a minimum of 65% intact acrosome.

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Post thaw release of acrosome enzymes.

• Leakage of enzymes (hyaluronidase and acrosin) from the acrosome to the seminal plasma can be used as an indicator of acrosomal changes.
• This test involves special chemicals, equipments and it is time consuming.

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Post thaw incubation survival of spermatozoa at 37⁰C

• Thaw the frozen semen straw at a water bath of 37⁰C for 30 seconds
• Wipe the straw and cut the ends of the straw and collect the semen in a small test tube.
• Keep a small drop on a clean, grease free glass slide.
• Cover it with cover slip and examine under the phase contrast microscope and estimate the post thaw motility.
• Incubate the semen in the test tube in the same water bath at 37⁰C.
• Estimate the progressive motility at every half an hour interval till the motility completely ceases.
• Longer the survival, greater the chance for the sperm to survive in the female genitalia on insemination resulting in better fertility.

If the sperms are surviving for longer period of time after thawing there will be chance for sperms to survive prolonged period in female genitalia & thereby chance for higher conception rate.

• Individual variations among bulls have been observed for post thaw survival of spermatozoa.

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