Effect of a surfactant on the integrity of frozen ram spermatozoa

Abstract

In order to test the effect of the addition of the commercial surfactant Equex STM to TRIS and Na-Citrate extenders on the survival of ram semen, four trials using factorial designs were conducted freezing separate ejaculates collected from 2 Austral and l Border Leicester rams. Common for all trials was a monophasic dilution at room temperature, with 20% egg-yolk containing extenders split in fractions with 0.5% and without surfactant, packing of extended semen in 0.25 Minitüb-straws, an adaptation period at 5°C for 3-4 h, a freezing rate of about 5°C/min performed 10 cm above level of liquid nitrogen, and thawing of straws for 15 seconds in a 40°C water bath. Quality criteria for thawed semen were motility index (MI) and normal intact acrosomes (NA). With the use of STATGRAF 5.1, results were submitted to ANOVA and Duncan test, complemented with "t"-test (TWOSAM), assuming P < 0.05. 
In the 1st trial, the comparison of TRIS-extender (T) vs. 2.9% Na-Citrate extender (C), MI was significantly lower in C-extenders, specially in C-extender without surfactant, which caused high rates of bent tails indicating osmotic damage. This was not observed in C with surfactant nor in T- fractions. The effect of surfactant on NA was significant with both extenders. In the 2nd trial, Citrate extender was comparatively modified, adding 0.3% (w/v) Sulphanilamide to increase osmolarity and pH. The results were basically the same as in the 1st trial: addition of surfactant gave significantly higher NA-rates in TRIS and Citrate extenders and significantly higher MI in both Citrate-extenders. In the 3rd trial, designed to compare two concentrations of Na-Citrate (2.9% vs. 3.1% w/v), and to compare a fast vs. a slow dilution, no differences between extenders nor between fast and slow dilution were found. Again, the effect of the surfactant on NA and MI was significant. In the 4th trial, conducted mainly to compare fast and slow dilution with TRIS extender, no difference was found. Again there was a significant effect of the surfactant on NA. There was no interaction between tested variables. It was concluded that Na-Citrate extenders without surfactant are deleterious to ram spermatozoa, as was evident by bent tails immediately after dilution. The addition of surfactant to TRIS extender yielded about 60% more intact spermatozoa after freezing. This would make it possible to reduce spermatozoa concentration per frozen straw in order to obtain a better sperm survival.