Sperm motility activation in the critically endangered booroolong frog: the effect of medium osmolality and phosphodiesterase inhibitors

Effective activation of sperm motility is fundamental to successful artificial fertilisation; however, studies investigating optimal procedures in amphibians are lacking. This study found the optimal osmolality of activation media for sperm motility activation and evaluated the effect of phosphodiesterase (PDE) inhibitors on sperm activation and longevity in the critically endangered booroolong frog, Litoria booroolongensis. To assess the effect of medium osmolality (10, 25, 50, 75, 100 and 200 mOsmol kg−1) and PDE inhibitors (control, 2.5 mM caffeine, 5 mM caffeine, 2.5 mM pentoxifylline, 5 mM pentoxifylline, 2.5 mM theophylline and 5 mM theophylline) on initial activation, percentage sperm motility and sperm velocity were quantified using computer-assisted sperm analysis. To assess the effect of PDE inhibitors (control, 2.5 mM caffeine and 2.5 mM theophylline) on sperm longevity, percentage motility and velocity were assessed hourly until 10 h after activation. High (>60%) percentage motility was achieved in a broad range of activation-medium osmolalities (10–75 mOsmol kg−1). PDE inhibitors did not have an effect on initial sperm motility or velocity, but caffeine and theophylline improved sperm longevity, significantly increasing motility and velocity at 8, 9 and 10 h after activation. Data also show that sperm longevity in L. booroolongensis is extreme, with spermatozoa remaining motile more than twice as long as those of any other anuran amphibian.

Antibiotics and oxygen availability affect the short-term storage of spermatozoa from the critically endangered booroolong frog, Litoria booroolongensis

Sperm-storage technologies aim to extend sperm longevity and increase the time available to achieve artificial fertilisation. The aim of the present study was to quantify the effects of antibiotic supplementation (4 mg mL–1 gentamicin) and altered gaseous storage environment (100%, 20% and 0% O2) on sperm longevity in the critically endangered booroolong frog, Litoria booroolongensis. A split-sample experimental design was adopted, whereby each sperm suspension (n = 10) was evenly divided among six experimental treatments (100% O2 with antibiotic, 20% O2 with antibiotic, 0% O2 with antibiotic, 100% O2 without antibiotic, 20% O2 without antibiotic, 0% O2 without antibiotic). Sperm suspensions were refrigerated at 5°C for the duration of the 21-day storage period. Percentage sperm motility and sperm velocity were quantified every 3 days using a computer-assisted sperm analysis system. Treatments aerated with either 100% or 20% oxygen, without the addition of the antibiotic gentamicin, consistently exhibited the highest percentage sperm motility. On Day 21 of storage, sperm suspensions in these two treatments (100% O2 without antibiotic, 20% O2 without antibiotic) maintained 61.3% and 52.0% sperm motility, respectively, whereas all remaining experimental treatments exhibited <30% sperm motility. Sperm velocity did not differ significantly among storage treatments, at any of the sampling periods, with the exception of day 21. Overall, the results from this study indicate that increased oxygen availability is beneficial to sperm longevity, but that gentamicin inhibits sperm motility in L. booroolongensis.

Sperm variation within a single ejaculate affects offspring development in Atlantic salmon

It is generally believed that variation in sperm phenotype within a single ejaculate has no consequences for offspring performance, because sperm phenotypes are thought not to reflect sperm genotypes. We show that variation in individual sperm function within an ejaculate affects the performance of the resulting offspring in the Atlantic salmon Salmo salar . We experimentally manipulated the time between sperm activation and fertilization in order to select for sperm cohorts differing in longevity within single ejaculates of wild caught male salmon. We found that within-ejaculate variation in sperm longevity significantly affected offspring development and hence time until hatching. Whether these effects have a genetic or epigenetic basis needs to be further evaluated. However, our results provide experimental evidence for transgenerational effects of individual sperm function.