Investigating media that support red wolf (Canis rufus) sperm viability and capacitation in vitro

The red wolf is a critically endangered canid, with similar to 250 and similar to 20 individuals in the ex situ and reintroduced wild populations, respectively. Assisted reproductive technologies such as sperm cryopreservation and in vitro fertilization therefore represent critically-needed tools to manage these populations. However, the motility of post-thaw red wolf sperm rapidly declines during in vitro incubation, hindering the ability to develop these technologies. In this study, we evaluated the influence of several culture media (a modified canine capacitation medium (mCCM), a modified North Carolina State University-23 medium (mNCSU-23), a synthetic oviductal fluid (SOF), a fertilization Tyrode's medium base or Fert-TALP (FERT), and a TRISbased buffer (TRIS)) on the survival and capacitation of red wolf sperm during extended (18 h) incubation at 38.5 degrees C and 5% CO2. Red wolf sperm motility averaged (+/- S.E.M.) 73.8 +/- 7.1% at the time of collection, and was better maintained over 4 h incubation in mCCM (55.0 +/- 9.8%) and mNCSU-23 (54.7 +/- 10.4), compared to mSOF (43.8 +/- 8.3%), FERT (30 +/- 10.5), and TRIS (16.4 +/- 4.1%) solutions. Patterns of tyrosine phosphorylation signal, as assessed via immunocytochemistry, indicated induction of capacitation between 2 and 4 h in vitro culture. Tyrosine phosphorylation signal was particularly robust in mCCM and mNCSU-23 incubated sperm, although significant acrosome exocytosis was not observed in response to progesterone supplementation after 3 h incubation in any of the media. In sum, results indicate mCCM and mNCSU-23 are promising base media for the in vitro incubation and capacitation of red wolf sperm, for assisted reproduction applications.