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

OTHER BIOCHEMICAL TESTS

• Oxygen uptake/utilization test
• Pyruvate utilization test
• GOT
• Hyaluronidase activity
• Resazurin test
• Buffering capacity test
• Alkaline and acid phosphatase test
• Millovanov's test

OXYGEN UPTAKE OR UTILIZATION TEST

• If the sperms are very active they take up higher volume of oxygen per unit of time.
• The respiratory quotient (ZO₂) is calculated as volume of CO₂ produced by thespermatozoa per unit of time divided by the volume of O₂ consumed in the same unit of time.
• The ZO₂ value of bull semen is to be 21. This test indicates activity and live percentage of spermatozoa.

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PYRUVATE UTILISATION TEST

• Found by Melrose during 1952.
• The quality of semen samples could be graded according to the oxygen consumption after the addition of pyruvate and pyruvate plus 2:4 dinitrophenol. Flouride was added to the sample to reduce the exogenous metaboloism to a low level.
• When added with pyruvate the oxygen consumption in high fertile bull semen and low fertile bull semen is increased but on addition of 2:4 dinitrophenol the oxygen uptake is increasing two fold in high fertile bulls but not in low fertile bulls.
• The oxygen uptake was measured with the help of monometric equipment.

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GLUTAMIC OXALOACETIC TRANSAMINASE (GOT) ACTIVITY

• To record the extent of damage occurred to spermatozoa, there is no satisfactory method except to depend upon biochemical tests to estimate the presence of enzyme (GOT) in seminal plasma after freezing and thawing.
• Normally it is not found in seminal plasma. The presence of this enzyme indicates the damage to spermatozoa

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HYALURONIDASE ACTIVITY

• This enzyme is present in acrosomal system of spermatozoa.
• Integrity of acrosome is directly involved in fertilizing capacity of spermatozoa.
• Presence of hyaluranidase activity in extracellular fluid will indicate acrosomal damage.

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RESAZURIN TEST

• It is used as an indicator of metabolic activity of semen.
• On account of dehydrogenase activity of semen blue colour of resazurine changes to pink at first and finally to colourless state.
• Good semen sample reduces resazurin to pink colour in one minute and requires four minutes to become colourless.

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BUFFERING CAPACITY TEST

• It is the chemical ability of a fluid to absorb acid or alkali with little change in pH.
• Bull semen
  • highly buffered at pH below 5.5 and above 9.0
  • moderate well buffered at pH 5.5 to 6.5 and 8.0 to 9.0
  • lacks buffering capacity 6.5 to 8.0

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ALKALINE AND ACID PHOSPHATASE TEST

• Its presence reflects the functional state of accessory sex glands and metabolic activity of spermatozoa.
• Increase in conception rate in cows following inseminations with semen containing increasing order of alkaline phosphatase.

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MILLOVANOV'S RESISTANCE TEST

• This test assesses the ability of spermatozoa to withstand the action of 1 per cent sodium chloride solution.
• The resistance is denoted as the millilitre of 1per cent sodium chloride solution required to stop the progressive motility of all spermatozoa in 0.02 ml of semen.

Procedure

• Pipette out 0.02 ml of semen into a 200 ml capacity flask.
• Add 1% sodium chloride solution volumes each of 10 ml from a pipette.
• Assess the progressive motility of semen after each addition 10 ml of sodium chloride.
• Sodium chloride is added at intervals till the progressive motility of spermatozoa is ceased.
• Good quality semen shows ‘R’ value to be not less than 5000.

ml of sodium chloride solution required

R value = -------------------------------------------------

0.02

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