10. Tweezers, gloves.
11. 0.25-mL straws (IMV, L’Aigle, France, or similar).
12. Heat sealer for straws, or use PVA powder.
13. Freezing equipment (static LN 2 vapor, ventilated LN 2 vapor,
or programmable).
14. Thermocouple equipment (thinnest possible copperconstantan Type K thermocouple wires with computer interface, Omega, Norwalk, CT).
2.2 Diluents
1. “ASG-PE”: medium composition of “ASG-PE” (¼ modified
medium “7.1.,” Lake and Ravie, 1979) is prepared as described
in Table 1.
2. “Solution 1”: medium composition of “Solution 1” [59] is
prepared as described in Table 2.
2.3 Freezing Media
1. Freezing can be done with dimethylacetamide (DMA) or glycerol as CPA (see Note 1). DMA is described at 6% (~60 g/L)
[39] or at 0.6 M [21]. The final concentration of the CPA can
be obtained by adding one volume of three times concentrated
CPA solution in diluent to two volumes prediluted semen.
Table 3 shows how to prepare three times concentrated CPA
medium (see also Note 2).
3 Methods
3.1 Semen Collection
and Processing
1. Semen is collected by the abdominal massage technique [60]
(see Notes 3 and 4).
Table 1
Medium composition of “ASG-PE” (¼ modification of medium “7.1.” from [28])
g/100 mL
mmol/L
Sodium-L-glutamate.H 2 O
1.21
64.7
Tri-potassium-citrate.H 2 O
0.102
3.14
Magnesium acetate.4H 2 O
0.064
2.97
D-(+)-glucose monohydrate
0.53
26.5
BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid)
2.43
114
NaOH
0.185
46.2
Pure water (e.g., Milli-Q)
To 100 mL
pH ¼ 7.1
Cryopreservation of Avian Semen
389