Animal Breeding - Evaluating Semen
(Adapted from "Biotechnology in Agriculture", MAVCC, Stillwater, OK.)
OBJECTIVES:
Upon completion of the activity, students should be able to:
1.
Demonstrate the use of a microscope in evaluating bull semen.
2.
Explain how to score motility and morphology.
MATERIALS AND/OR EQUIPMENT:
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Microscope with 10X, 40X, and 100X (oil immersion) objectives
Immersion oil for microscope
Several microscope slides and cover slips
Plate glass about 12" by 12"
2 bricks
1 trouble light with 75 watt bulb
fresh or frozen (or both) bull semen sample
20 ml normal saline solution (physiologic saline)
Semen stain (Hancock's Blom's Eosin-Nigrosin Stain)
Water bath at 37º C (98º F)
2 eye droppers
Several test tubes
Glass thermometer for water bath
TIME REQUIRED FOR ACTIVITY:
Two to four days
BACKGROUND MATERIAL:
The purpose of this laboratory activity is to give student hands-on
experience using a microscope to evaluate bull semen. They will score
motility and morphology. These scores help producers decide if a bull is
desirable for breeding.
PROCEDURE:
1.
Record opening entries in laboratory notebook.
2.
Set up a water bath to maintain semen at 94º to 100ºF (34º to
37ºC).
●Method 1--Saucepan on a burner
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Method 2--Thermal container
3.
Maintain a test tube of normal saline in the water bath. Maintain
the semen sample in a test tube in the water bath.
4.
Preheat microscope slides and cover slips by placing them on a
clean glass plate supported on bricks with a 75 watt bulb
underneath as a heat source. Leave slides on the glass plate for
about 10 minutes before using them.
Note: Mass motility evaluation works well with fresh semen but
not frozen since the latter is diluted.
5.
Evaluate mass motility by placing a drop of fresh semen on a
warmed microscope slide and quickly observing it under the
microscope on the 10X objective. Focus on the semen and score
mass motility using this scale:
Rapid swirling motion
20
Slow swirling motion
General motion but not swirling
10
Very slow movement
No movement
6.
Score
Score 12
Score
Score 3
Score 0
Evaluate individual motility by placing a drop of fresh semen on a
warmed slide and adding a drop of warmed saline solution. Mix
the two together and cover with a warmed cover slip.
Note: Use thawed samples of frozen semen direct without saline dilution.
a.
Focus and observe the sample at 40X.
b.
c.
Pick out an individual sperm cell and observe it move
across the visual field. A strong sperm will move in a
straight line across the field in less than 5 seconds.
Evaluate several cells and record an average score using
this scale:
Rapid straight line movement across the field
(5 sec. or less)Score 20
Moderate straight line movement across the field
(5 to 10 sec.)Score 12
Slow straight line movement of slightly erratic
motionScore 10
Very slow erratic motion
No motion
7.
Score 3
Score 0
Determine the average motility score. Total the mass motility and
the average motility scores and divide by 2.
Note: If you did not mass motility, use the individual motility score as the
average score.
Mass motility
score
_____
________
Individual motility score
_____________
Total
____________
_
Divide by 2 - Average
_____________
Record motility scores in your laboratory notebook.
motility
8.
Evaluate sperm morphology (structure). Place one drop of fresh
semen on slide (does not need to be warmed); add one drop of
saline and one drop of semen stain. Mix well and cover with a
cover slip.
Note: Use the same procedure for a thawed sample of semen but do not
add saline.
Slides can be stored for a few days after staining and covering. The rest of this
laboratory sheet may be completed during another class period.
9.
field
number
Place the slide on the microscope under oil immersion and 100X
magnification. Search the slide and find areas where you see from
5 to 20 sperm cells in a visual field. Use the attached pictures at the
end of the laboratory sheet to identify abnormal cells. Count the
total number of cells in the field and then count the number of
abnormal cells in each of the three groups. Record the information
in your laboratory notebook using a chart similar to the one below:
total
cells
primary
abnormalities
1
2
3
4
5
6
7
8
9
10
totals
100
Note:Stop counting when you reach 100 total cells.
secondary
and tertiary
abnormalities
10.
Evaluate morphology using the following scale:
Primary abnormalities
less than 10Score 40
10 to 19Score 24
20 to 29Score 10
more than 29Score 3
Secondary and tertiary abnormalities
less than 25Score 40
26 to 39Score 24
40 to 59Score 10
more than 59Score 3
11.
Determine average morphology score. Add the primary score to
the secondary and tertiary score and divide by two.
Primary abnormalities score
___________
Secondary and tertiary abnormalities score
___________
Total
___________
Divide by 2 = Average score
___________
Record this morphology score in your laboratory notebook.
12.
0 to 20
21 to 40
41 to 60
Evaluate the semen sample by adding the average motility score to
the average morphology score. Rate your sample using the
following scale:
Unsatisfactory sample
Questionable sample
Satisfactory sample
13.
Record all procedures, observations, and calculations in your
laboratory notebook.
14.
Write a laboratory report and turn it in for evaluation.