Standard Operating Procedures for Bull Management (1088.47 KB)

-Aa.
Preparation of semen specimen
Two number frozen semen straw sample is thawed (37°c for 30 sec) pooled and slowly
diluted 1:2 V/V in a 1.5 ml test tube using modified sodium citrate medium with 2% (V/V)
fetal bovine serum (FBS) albumin. The specimen is centrifuged (37°c) for 8 min at 400 x g.
The supernatant is discarded and the sperm pellet is slowly re-suspended to 2ml modified
sodium citrate medium with 2% FBS. The specimen is maintained in a water bath at 37°c and
allowed to stabilize for 3 to 5 min. The percentage of motile cells is estimated
microscopically at 37°c.
b. Preparation of Media
Hypo-osmotic solution is prepared by weighing the Fructose and sodium citrate as per the
formula.
Aliquot of 1 ml can be prepared in the test tubes and stored at – 20°c for later use.
c. Procedure for HOS test
Add 0.1 ml of the specimen containing the spermatozoa with 1 ml of hypo-osmatic solution.
The solution is then incubated at 37°c for one hour.
Similarly 0.1 ml of semen is incubated in 1.0 ml of normal saline solution (0.9% NaCl) for
one hour
d. Counting of spermatozoa
A drop is placed on the clean glass slide and covered with a cover glass and observed under
phase-contrast microscope at x 400 magnifications. A total of 200 sperm are counted in at
least 5 different fields. (Both in HOS /Normal Saline )
The initial response of the sperm to the HO solution is a small enlargement of the tip of the
tail or at the junction of the mid piece and tailpiece. Later the alterations are characterized by
the presence of a swollen area at the tip of the tail, a hairpin curvature of the tail, a shortened
and thickened tail, or a swollen area that partly or completely enveloped the curvature of the
sperm. There is good correlation between the percentage of motile spermatozoa and
spermatozoa that reacted to the HOS test and between the percentages of sperm with intact
membranes and HOS relative sperm.
Preparation the Media
Sodium citrate 2 H20 - 7.35gm
Osmolality -150 m osmol
Fructose - 13.51 gm
Dist. water - 1000 ml
Ionic strength - 0.15