36 Evaluating the toxicity of several sperm diluents over time and at varying osmolalities in an internally fertilising salamander species
D. Chen A , C. Kouba B and A. Kouba AA Department of Wildlife, Fisheries, and Aquaculture, Mississippi State University, Mississippi State, MS, USA
B Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS, USA
Reproduction, Fertility and Development 35(2) 143-144 https://doi.org/10.1071/RDv35n2Ab36
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
The eastern tiger salamander (Ambystoma tigrinum) is an internally fertilising amphibian that has been used as a model species for salamander sperm cryopreservation. Due to the concentrated nature and small volume of spermic milt that is typically produced from tiger salamanders, a diluent is needed to analyse the sperm for quality and quantity metrics. Techniques for diluting salamander sperm in preparation for cryopreservation have so far been modelled off anuran sperm cryopreservation protocols. While this approach may be appropriate for externally fertilising amphibians like frogs and toads, whose sperm is activated by lower osmolality, the internal environment of the tiger salamander female reproductive tract is very different from an aquatic environment. Thus, application of mammalian sperm extenders could maintain salamander sperm motility and morphology due to the similar nature of internal fertilisation in both taxa. We hypothesised that higher osmolality diluents would result in samples maintaining higher forward progressive motility (FPM), total motility, and normal morphology over time due to the internal fertilisation environment of tiger salamanders. Several mammalian sperm diluents were tested, including: Multipurpose handling medium, sperm washing medium, modified Ham’s F-10 basal medium (HAM), and 10% Holtfreter’s solution as a control. The experimental treatments were divided into two groups: isosmotic (90 mOsm/kg, diluted with purified water) and hyperosmotic (280–320 mOsm/kg, undiluted) relative to salamander milt. Milt samples were collected from male tiger salamanders (n = 11) and diluted 1:50 with each of the media treatments. Immediately after mixing, diluted samples were measured for FPM, total motility, and normal morphology every 10 min for 1 h. The rate of loss for each of these parameters, as a measure of toxicity, was calculated for each treatment and analysed with generalised additive modelling (see Table 1). When comparing the isosmotic versus hyperosmotic treatments of the same diluent medium with paired t-tests, there was no difference (P > 0.05) for any of the response variables (e.g. total motility for HAM vs diluted HAM; P = 0.27). Our results suggest that 10% Holtfreter’s solution is best for maintaining FPM, while diluted HAM is best for total motility and morphology, thus they are recommended as diluents for maintaining sperm quality over time. Moreover, we found that osmolality is not a key driver influencing tiger salamander sperm, but rather other factors such as ionic composition could be affecting sperm form and function.
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