Effect of processing and measuring procedures on estimated sizes of bull sperm heads

Reported estimates of sperm head size within a species vary considerably, partly due to procedural effects. A simple India ink method was developed that provided good contrast without inducing artifacts. Semen from five fertile bulls was smeared on replicate slides and left unfixed or fixed in Carnoys solution, with ink added for background. Other slides were fixed, and sperm were stained by the Feulgen procedure. Sperm head area was measured four ways. These were linear measurements made with the aid of an ocular micrometer and an oil immersion objective, plus three methods of measuring sperm heads projected at magnification 5,000 x. The areas of unfixed and fixed sperm heads did not differ (41.5 microm(2) versus 41.6 microm(2), respectively, P>0.05). The Feulgen-stained head area was smaller (26.2 microm(2), P<0.05). Sperm head areas calculated from ocular micrometer measurements were slightly smaller (P<0.05) than areas measured using projection. Identical results obtained by two technicians were treated as duplicates and approximately half of the variation was biological, due to source of semen. There was an interaction (P<0.05) between the sample source and fixation procedures. Thus, preparative techniques must be carefully controlled, and experiments designed to partition possible interactions between the biological material sampled and procedures used.