Characterization of Bottlenose Dolphin (Tursiops truncatus) Sperm Based on Morphometric Traits

Abstract

Bottlenose dolphin (Tursiops truncatus) males follow many reproductive strategies to ensure their paternity. However, little is known about the sperm traits, including morphometric features, that contribute to their reproductive success. Our aim was to study dolphin sperm morphometry (a total of 13 parameters) in two adult males to evaluate (i) presumptive sperm subpopulations, (ii) the correlation of sperm morphometry with testosterone levels and (iii) the effect of refrigerated storage on the sperm morphometry. Sperm populations were classified into four principal components (PCs) based on morphometry (>94% of cumulative variance). The PCs clustered into two different sperm subpopulations, which differed between males. Furthermore, the levels of serum testosterone were positively correlated with the length of the midpiece but negatively correlated with head width and the principal piece, flagellum and total sperm lengths. Most of the sperm morphometric parameters changed during the storage period (day 1 vs. day 7), but only the principal piece length was affected by the storage temperature (5 °C vs. 15 °C). This is the first study to identify dolphin sperm subpopulations based on morphometry and the influence of serum testosterone and refrigeration on sperm morphometry.

Keywords: cetacean; morphology; morphometry; semen; sperm cells.

Figure 1

The main structures of bottlenose dolphin spermatozoon (Tursiops truncatus): head and flagellum (midpiece, principal piece and terminal piece). The picture was taken under phase-contrast microscopy. Scale bar, 10 µm.

Figure 2

Quality of clustering based on Visual Assessment of the cluster Tendency (VAT) in sperm population of two bottlenose dolphin (Tursiops truncatus) (Male 1—left panel; Male 2—right panel). The cuvette visually detects the clustering trend by counting the number of square orange blocks along the diagonal in a cuvette image. Orange indicates high similarity (values close to 0), while blue indicates low similarity (values higher than 2.5).

Figure 3

Bottlenose dolphin (Tursiops truncatus) sperm clustering. (i) Graphs showing two cluster areas (blue for cluster 1 and yellow for cluster 2) of sperm populations in both males of the study (a,b). The numbers indicate individual sperm classified in the clusters. (ii) Hierarchical tree/dendrogram combined with heatmap. The dendrograms indicate the relationships between the principal components (columns) and spermatozoa (rows) based on Spearman’s correlation coefficients. Each leaf corresponds to one spermatozoon. As we move up the tree, spermatozoa that are similar to each other are combined into branches, which are themselves fused at a higher height. Horizontal axis shows the height of the fusion that indicates the dissimilarity or similarity distance between two spermatozoa. Similarity decreases with the height of fusion. Heatmaps show the columns/rows of the data matrix reordered according to the hierarchical clustering result, placing similar spermatozoa close to each other based on principal components (PC1, PC2, PC3 and PC4). Spermatozoa similarity or dissimilarity is indicated by an orange scale. Dark orange indicates high similarity; light orange indicates low similarity.