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  • Author: R. G. CRAGLE x
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R. M. MOOR and R. G. CRAGLE

Summary.

A total of 429 eggs was used to investigate procedures both for the removal of the zona pellucida and for the culture of sheep eggs in vitro.

The effect of pronase, papain, bromelain, trypsin and α-chymotrypsin, on the zonae pellucidae of fertilized and unfertilized eggs was studied in the first series of experiments. All the enzymes were effective in removing the zonae pellucidae from unfertilized eggs. Pronase was the only enzyme that had a significant lytic effect on the zonae of fertilized eggs denuding 34% of eggs in less than 5 min and almost all the other eggs within 90 min.

Investigation of some of the factors involved in the successful culture of normal sheep eggs in vitro constituted the second part of the study. Eggs of both the eight- to sixteen-cell and morula stages were cultured for 72 hr in either sheep serum, enriched tissue culture medium 199 or Brinster's medium. The development and viability of the eggs after culture was determined by transferring some eggs to recipient sheep and making accurate cell counts on the remainder. Eggs recovered at the eight-cell stage underwent only one or two cleavage divisions during the 72-hr culture period. These eggs did not develop into foetuses when transferred to the uteri of recipient sheep. On the other hand, the majority of morulae cultured for 72 hr in enriched M199 or sheep serum underwent cleavage and blastulation in vitro. That the cultured blastocysts were normal was demonstrated by their implantation and growth into foetuses when transferred to the uteri of suitable recipients.

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G. W. SALISBURY, C. N. GRAVES, N. T. NAKABAYASHI and R. G. CRAGLE

Summary.

This study was conducted to determine the effect of added sulphur compounds, substrates and various inorganic ions on the metabolic activity of epididymal spermatozoa from twenty-one bulls and five goats. The addition of sulphur at physiological levels in the form of sulphide, sulphite, sulphate and glutathione in several different media caused no stimulation of respiration. An apparent stimulation of oxygen uptake by the addition of cysteine was shown to be due to auto-oxidation of the added cystein. Calcium and magnesium in a Ringerphosphate medium had no obvious effect on metabolism, while phosphate decreased oxygen uptake but increased glycolysis and lactate accumulation in the presence of substrate. Substrate, present even for a short period of time before removal, stimulated metabolism markedly during the subsequent incubation period.