Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution.
The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs-Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.
Yamashiro
Hideaki
H
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
Han
Young-Joon
YJ
Sugawara
Atsushi
A
Tomioka
Ikuo
I
Hoshino
Yumi
Y
Sato
Eimei
E
D000177:Acrosome / Q000473:pathology; D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004530:Egg Yolk; D004822:Epididymis; D005615:Freezing; D005990:Glycerol / Q000494:pharmacology; D007552:Isotonic Solutions / Q000494:pharmacology; D008297:Male; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa
D002241:Carbohydrates; D003451:Cryoprotective Agents; D007552:Isotonic Solutions; C051865:Krebs-Ringer solution; D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride; D005990:Glycerol