Red wolf (Canis rufus) sperm quality and quantity is affected by semen collection method, extender components, and post-thaw holding temperature.

Cryopreserving genetic resources is becoming increasingly important for species management. In the zoo-based red wolf (Canis rufus) population, inbreeding continues to increase in the absence of new founders. Through banking sperm, we preserve genetic diversity and create the ability to decrease inbreeding accumulation in the future. The quality and quantity of banked sperm can be affected by cryopreservation media and semen collection methods. This study's objectives were to further optimize semen extender used for red wolf sperm cryopreservation, investigate effects of post-thaw holding temperature, and to determine if urethral catheterization is an effective method for semen collection in this species. Semen collection via electroejaculation (EE) was performed on 39 adult red wolf males (ages 1 to 11) from 15 institutions. Urethral catheterization (UC) was attempted on a subset (n = 14) of those males, prior to EE. Thirteen different semen extenders were used for cryopreservation, which varied in osmolarity (HI or NORM), sugar source (glucose, fructose, or a combination), and cryoprotectant (glycerol or DMSO). Significant decreases in percent motility, forward progressive status (FPS), and acrosomal integrity were observed over time across all extenders (P < 0.0001). Among the extender components examined, post-thaw sperm motility and FPS were lower in DMSO versus glycerol based treatments (P < 0.005). Therefore, DMSO should be considered unsuitable as a cryoprotectant when freezing red wolf sperm. Effects of osmolarity and sugar source were minimal and temporally variable, however notably, a higher percentage of morphologically normal sperm were observed in the fructose-based extenders compared to glucose-based extenders post-thaw (P < 0.05). Additionally, post-thaw sperm motility and FPS declined more rapidly in samples maintained at 37 °C compared to samples held at room temperature (P < 0.05). Greater volumes of semen were collected using EE compared to UC (P = 0.041), and sperm samples collected using EE also had greater motility and FPS (P < 0.05). Additionally, though no gross morphological differences were observed, there were fewer sperm with intact acrosomes in the samples collected via UC (P = 0.0443). Thus, UC should not be considered sufficient for semen collection in red wolves when the desired fate of sperm is cryopreservation and/or AI. However, UC does provide an opportunity for a basic reproductive evaluation of a red wolf male.