Semen concentration

Gray Beckman Spectrophotometer

photo of beckman spestrophotometer

 Key
  1. Power switches
  2. Wavelength adjustment knob
  3. Shutter knob
  4. Cuvette compartment and holder
  5. Dark current knob
  6. Slitwidth knob
  7. Transmittance scale
  8. Absorbance scale
  9. Sensitivity knob (leave on 2, do not change)
  10. Cuvette holder selection knob


 Power Switches

photo of the on/off switch of the beckman spectrophotometer

Cuvette Compartment

cuvette holder of the beckman

Square Cuvette

 photo of the plastic cuvette

Instructions:
  1. Turn on spectrophotometer power switches (# 1.) at least 15 minutes before use.

  2. Set the wavelength adjustment knob (# 2.) to set the wavelength to 650 nm.

  3. Turn the shutter knob (# 3.) to Shtr.

  4. Put 3 ml of a 2.9% NaCitrate solution (pH 7.0) in a square cuvette and place in the first of the four slots (in the front) of the cuvette holder (# 4.). Only handle the cuvette by the frosted sides. Make sure the cuvette is properly aligned in the holder to allow light to pass from left to right through the clear sides of the cuvette. The frosted sides of the cuvette should face the front of the spectrophotometer. The cuvette holder selection knob (# 10.) should be pushed in almost completely so that the first notch in the selector is engaged. This should align the first cuvette slot with the light beam.

  5. Use the dark current knob (# 5.) to set the metter to read 0% transmittance (# 7.).

  6. Turn the shutter knob (# 3.) to open. Set the transmittance to 100% with the slitwidth knob (# 6.).

  7. Turn the shutter knob (# 3.) to Shtr.

  8. Remove cuvette and add a semen sample. The volume of semen sample added depends on the species:

    Species

    Volume of Semen to Add

    Equine 100 µl

    Bovine

    15 µl
    Ovine 15 µl 
    Porcine 40 µl
    Rooster 15 µl 


  9. Cover cuvette with a cap and mix 10-20 times.

  10. Place cuvette back in spectrophotometer. Make sure the cuvette holder is properly aligned.

  11. Turn the shutter knob (#. 3) to open and read the absorbance as soon as the reading stabilizes. This takes about 5 - 10 seconds.

  12. Use the formulas below to convert absorbance to concentration. You can also use the calibration curves that follow if you used the recommended dilutions. If you used a different dilution, then use the alternative formulas that takes into account the dilution factor. If the absorbance readings fall outside of the standard curves, which for the bull would be absorbance readings of <0.1 or >0.5 and for the stallion would be <0.1 or >0.7, then you need to either add more or less sperm to another cuvette to bring the absorbance within the specified range. Then use the alternative concentration formula below that takes into account different dilution factors.


    Absorbance to Concentration Conversion When Recommended Dilutions Used

    Species

    Volume of Semen Added

    Volume of NaCitrate
     Dilution Factor

    Concentration X 106/ml

    Equine

    100 µl

    3.0 ml
    31 

    (894*Absorbance) - 12

    Bovine

    15 µl

    3.0 ml
    201 

    (4879*Absorbance) + 22
    Ovine 15 µl  3.0 ml  201  (5220*Absorbance)-60 
    Porcine 40 µl 3.0 ml  76 (1626*Absorbance) - 83 
    Rooster  15 µl  3.0 ml   201 (11,170*Absorbance) - 90 



    Absorbance to Concetration Conversion For Arbitrary Dilution Factors

    Species

    Concentration X 106/ml

    Equine

    (Dilution Factor) * [28.84 * (Absorbance) - .39]

    Bovine

    (Dilution Factor) * [24.27 * (Absorbance) + .11]
    Ovine  (Dilution Factor) *[25.97*(Absorbance) - .30] 
    Porcine (Dilution Factor) *[21.39*(Absorbance) - 1.09]  
    Rooster (Dilution Factor) * [55.57*(Absorbance) - .45]

 

calibration curve for the spec  concentration=894*Abs - 12

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