Effects of Composition of Diluent, Method of Addition of Glycerol, Freezing Rate, and Storage Temperature on the Revival of Ram Spermatozoa After Deep-Freezing

Abstract

The addition of 6% (vJv) egg yolk to a synthetic diluent [247 mM glucose, 49 mM NaCI, 5 mM KCI, 5 mM phosphate buffer, and 7·5% (vJv) glycerol] improved the survival of ram spermatozoa after deep·freezing. Judging by the activity of spermatozoa after thawing, a single addition of glycerol to the diluted semen at 5°C was as effective as multiple additions giving a graded increase of glycerol concentration over a period of 20 min. Reciprocal replacement of the sodium chloride of the diluent by an increase in the concentration of the phosphate buffer showed that motility of the spermatozoa after thawing was depressed when the level of phosphate exceeded 10 mM.A rapid rate of freezing of l·ml aliquots of semen in glass ampoules in the neck of a liquid nitrogen container gave lower survival of spermatozoa than a slower rate in dry ice (average rates to -40°C of 1 and 2 degrees Celsius per minute respectively).Spermatozoa survived a period of 2 weeks of storage in liquid nitrogen better than when stored in dry ice. There was a significant decline in the revival rate of samples prepared and frozen from four successive ejaculates collected by electro· ejaculation at intervals of 3 days.

Effects of Egg Yolk, Glycerol and the Freezing Rate on the Viability and Acrosomal Structures of Frozen Ram Spermatozoa

“…No advantage was gained by this modification in freezing (expts 1 and 2) and it may therefore be concluded that a temperature fluctuation coinciding with the formation of ice crystals was not detrimental to the spermatozoa. Entwistle and Martin (1972) found that less than 6% egg yolk in the diluent used for freezing ram spermatozoa in ampoules resulted in reduced survival. In experiment 2, however, no difference was observed between the survivals of spermatozoa in diluents with egg yolk levels ranging from 3 to 12 %, and the degree of acrosome disruption was similar in all diluents.…”

“…However, uncontrolled variations in the freezing rate and pattern of crystallization occur within the sample because of its volume and dimensions, and this has hindered more critical studies of the freezing conditions. In addition, the freezing curve recorded by a thermocouple within the sample has a characteristic deviation from linearity (Entwistle and Martin 1972).…”

“…Matching samples were also frozen in ampoules following method B of Entwistle and Martin (1972). Egg yolk was included in the diluent at 6% (v/v).…”

“…In general, slower freezing rates have given better results than faster rates when semen was frozen in ampoules (Hill et al 1959;Entwistle and Martin 1972), but Salamon (1970) obtained good revival with pelleted semen frozen at quite high rates. In the experiments reported here, variations in diluent composition have been studied in relation to the freezing rate for diluted ram semen frozen in straws.…”

Επιδραση Των Εμφυτευματων Μελατονινης Στα Χαρακτηριστικα Του Σπερματος Κριων Καραγκουνικης Φυλης Και Στην Αντοχη Των Σπερματοζωαριων Τους Στην Καταψυξη

“…Οι βλάβες αυτές αφορούσαν κυρίως το ακρόσωμα και την κυτταρική μεμβράνη. Όμως, οι Watson και Martin (1972) συγκρίνοντας τις βλάβες που προκαλούνται εξαιτίας της κατάψυξης στο ακρόσωμα σπερματοζωαρίων ταύρου και κριού διαπίστωσαν ότι η μικρή υπεροχή των καταψυγμένων σπερματοζωαρίων του ταύρου, ως προς την ακεραιότητα του ακροσώματος, δε δικαιολογεί από μόνη της τη μεγάλη διαφορά στη γονιμότητα του καταψυγμένου σπέρματος μεταξύ των δύο ειδών. Η τεχνική της σπερματέγχυσης και ιδιαίτερα το σημείο της τοποθέτησης του καταψυγμένου σπέρματος διαπιστώθηκε ότι επηρεάζουν σοβαρά το ποσοστό συλλήψεων.…”