2.2.2. Formula for semen storage and mixing

A more complex saline diluent formula, HHBSE Saline Formula (Tables 2 and 3), is recommended when mixing and storing semen, including storage at temperatures above freezing and in liquid nitrogen (Hopkins et al., 2012).

As a few of the components required (EDTA, glycine, and tylosin) are at very low concentrations and are difficult to measure on conventional scales. Consequently, it is necessary to make a 100x solution that can be added to the solution produced in Table 2 to make up the finished product.

  1. Add the reagents (Table 2) (excluding EDTA, glycine, and tylosin) to the volumetric flask and fill to 100 ml (final volume) with distilled water to make the base solution.
  2. The minor components, EDTA, glycine and tylosin, are made up as a 100X solution (Table 3) to be added to the solution in Table 2.  Using 10 ml of the base solution (Table 2), mix in the components from Table 3.  This results in 10 ml of a 100X solution of the three minor components.  Then add 0.9 mL of the 100X solution back to the now 90 ml remaining of the base solution. Sterilize using bacteriological filters (pore size 0.2µm).


Table 2. Recipe for base solution that is used in HHBSE saline solution for semen storage and mixing. Add all of the ingredients (excluding EDTA, glycine, and tylosin) to a volumetric flask, then add distilled water to make up a final volume of 100 ml. Use 10 ml of this solution to make the solution in Table 3. Add 0.9 ml of the resulting solution from Table 3 back to the 90 ml of the remaining base solution (presented here) to make the final product. *Tylosin, EDTA, and glycine compose only a small amount of the finished solution. Consequently, it is necessary to make a 100x solution of these ingredients to add back to the rest of the ingredients listed here. See Table 3 and section 2.2.2. for details.

Ingredients

g ingredient / 100 ml final volume  

Penicillin

0.05

Streptomysin

0.044

Kanomyosin

0.06

Tylosin*

0.0032

EDTA (ultra pure)*

0.0002923

TES (acid)

0.6879

Tris (base)

0.3635

Sodium Phosphate Dibasic  

0.0142

Sodium citrate

0.02942

Arginine

0.01

Glycine*

0.00075

Proline

0.05

Catalase

0.002

BSA (lipid rich)

0.002

KCl

0.61131

NaCl

0.4847

NaHCO3

0.042


Table 3. Recipe for 100x solution of ingredients that compose a small amount of the HHBSE saline solution (in Table 2). Pipette 10 ml of the solution from Table 2 to mix with the components listed below. Add 0.9 ml of this solution to 90 ml of the base solution (in Table 2) to create the final HHBSE saline solution.

ingredients

g ingredient /10 ml final volume  

Tylosin

0.032

EDTA (ultra pure)  

0.003

Glycine

0.0075