Dogs Spermatozoa Labelling with 99mTc-HMPAO

Abstract

The study was aimed at finding out whether dog sperm could be labelled using 99mTc-HMPAO and finding the optimum incubation temperature and amount of 99mTc-HMPAO to label spermatozoa from fresh ejaculates and from frozen-thawed semen. A radiopharmaceutical was prepared by reconstituting 0.5 mg of HMPAO with 5 ml pertechnetate solution containing 100 kBq 99mTc. Semen samples were obtained from three dogs, pooled and divided into aliquots containing 100x106 spermatozoa each. Fresh and frozen-thawed semen samples were used. In experiment 1, the fresh and frozen-thawed spermatozoa were incubated with 99mTc-HMPAO at 20oC or 37oC for 20 min. In experiment 2, 0.01, 0.1 or 0.5 ml of radiopharmaceutical was used to incubate the
spermatozoa at room temperature (20oC) for 20 min. The excess radioactivity was removed by three times washing using centrifugation at 500 x g for 6 min and resuspending with TRIS buffer. The incubation procedure with the radiopharmaceutical did not influence the membrane integrity, but sperm progressive motility was
reduced by each washing and most of the spermatozoa maintained only stationary motility. The incubation temperature was not found to have an effect on the labelling efficiency. The highest labelling efficiency, 52%, was obtained with fresh semen incubated with 0.01 ml 99mTc-HMPAO. Since less than 5% of excess radioactivity was removed from the supernatant by the third washing, it is recommended to do no more than two washings in order to reduce the loss of progressive motility. This study shows that dog spermatozoa can be effectively radiolabelled, which is of value for further studies on sperm transport in the canine female genital tract.