Papain and its inhibitor E-64 reduce camelid semen viscosity without impairing sperm function and improve post-thaw motility rates

Abstract

In camelids, the development of assisted reproductive technologies is impaired by the viscous nature of the semen. The protease papain has shown promise in reducing viscosity, although its effect on sperm integrity is unknown. The present study determined the optimal papain concentration and exposure time to reduce seminal plasma viscosity and investigated the effect of papain and its inhibitor E-64 on sperm function and cryopreservation in alpacas. Papain (0.1 mg mL–1, 20 min, 37°C) eliminated alpaca semen viscosity while maintaining sperm motility, viability, acrosome integrity and DNA integrity. Furthermore E-64 (10 µM at 37°C for 5 min after 20 min papain) inhibited the papain without impairing sperm function. Cryopreserved, papain-treated alpaca spermatozoa exhibited higher total motility rates after chilling and 0 and 1 h after thawing compared with control (untreated) samples. Papain treatment, followed by inhibition of papain with E-64, is effective in reducing alpaca seminal plasma viscosity without impairing sperm integrity and improves post-thaw motility rates of cryopreserved alpaca spermatozoa. The use of the combination of papain and E-64 to eliminate the viscous component of camelid semen may aid the development of assisted reproductive technologies in camelids.

Sperm kinematic characterization of alpaca (Vicugna pacos L.) during the reproductive season

“…For a long time, this fact was associated with the particular rheological characteristics of their seminal plasma, such as a high structural viscosity (Casaretto et al, ). Treatment with different enzymes was used to reduce the effect of viscosity on sperm motility (collagenase and trypsin, Bravo et al, ; glycosaminoglycan and proteases: Kershaw‐Young, Stuart, Evans, & Maxwell, ; papain: Kershaw‐Young, Evans, Rodney, & Maxwell, ). Nevertheless, there is evidence that the effect of seminal plasma on sperm motility is not only limited by the physical limitation imposed by viscosity, but also by some chemical component (Carretero, Fumuso, Miragaya, Herrera, & Giuliano, ).…”

New protocol to separate llama sperm without enzymatic treatment using Androcoll‐E™

“…In addition, some methods for semen evaluation based on flow cytometry cannot be applied in samples that contain SP. For all these reasons, high‐speed centrifugation of ejaculates and semen incubation with enzymes have been performed to separate spermatozoa from SP and to reduce thread formation, respectively (Bravo, Callo, & Garnica, 2000; Carretero et al., 2015; Fumuso et al., 2018; Giuliano et al., 2010; Kershaw, Evans, Rodney, & Maxwell, 2017; Kershaw‐Young & Maxwell, 2011; Morton, Gibb, Leahy, & Maxwell, 2012). However, these techniques could damage spermatozoa or modify their membrane composition, reducing their resistance to the cryopreservation process.…”

Evaluation of the cryoprotective effect of seminal plasma on llama ( Lama glama) spermatozoa

“…Most cooling and freezing techniques used so far in camelids have been adapted from those used in bulls and rams, and include diluting spermatozoa without removing seminal plasma (SP) before performing the freezing curves (Aller et al, ; Bravo, Skidmore et al, ; Carretero, Neild et al, ; Giuliano et al, ; Santiani et al, ; Vaughan ). Some reports mention difficulties in homogenising the diluted samples and in loading straws prior to freezing (Carretero, Neild et al, ), all probably due to the SAC semen rheological properties, such as thread formation and the high structural viscosity (Bravo, Callo, & Garnica, ; Giuliano et al, ; Kershaw, Evans, Rodney, & Maxwell, ). The low pregnancy rates in SACs obtained to date with frozen–thawed semen could partly be due to these rheological characteristics interfering with cryoprotectants reaching spermatozoa and preventing, or hampering, an efficient homogenisation of the ejaculate with the diluents.…”