Abstract
The conservation status of the bottlenose dolphin (Tursiops truncatus) is vulnerable in the Mediterranean Sea, so it is necessary to continue working on conservation strategies from different perspectives.1 Given the ease of maintenance and training of bottlenose dolphins, aquariums and zoos can contribute to their conservation and ex-situ research. Additionally, this species is taken as an effective model to understand the health of other cetaceans and the marine ecosystem.2 Therefore, improving the knowledge related to their reproduction is very important to ensure the viability of these animals. Dolphins are polygamous species that have developed mechanisms to ensure paternity. The presence of different subpopulations in an ejaculate based on sperm traits is an effective strategy to increase the chances of fertilizing the oocyte.3 In mammals, ejaculates can be heterogeneous, presenting several sperm subpopulations with different morphometric characteristics, which may determine subsequent spermatozoa selection in the uterus, which is an effective strategy to increase the chances of fertilizing the oocyte.3,4
The aim was to determine the sperm morphometric traits of the bottlenose dolphin and to validate the clusterization of dolphin sperm ejaculate in different subpopulations according to their morphometry. The morphometry of 275 spermatozoa of two dolphin males (voluntary semen collection) were analyzed using ImageJ software. The average results obtained from both males were the following: width = 2.51±0.12 µm (mean±SD), length = 5.12±0.21 µm, area = 10.10±0.62 µm2, perimeter = 12.35±0.39 µm, ellipticity = 2.05±0.13, rugosity = 0.83±0.03, and elongation = 0.34±0.03 of spermatozoa head; width = 1.30±0.11 µm, and length = 2.99±0.23 µm of midpiece, principal piece length = 49.82±1.20 µm, terminal piece length = 12.90±0.93 µm, flagellum length = 65.71±1.17 µm, and total sperm length = 70.83±1.19 µm. Then, a principal component analysis (PCA) was performed to reduce the number of morphometry descriptors to a few standardized variables (principal components—PCs). The analysis revealed that 4 PCs accounted for more than 91% of the cumulative variance, being the PC1 with the highest explained variance (47.21%), followed by the PC2 (25.65%), PC3 (11.17%), and PC4 (7.72%). PC1 was related to sperm parameters of length (head, principal piece, terminal piece, and total sperm length); PC2 was related to parameters of shape of sperm head (width, ellipticity, rugosity, and elongation); PC3 was related to parameters of size of head, midpiece, and flagellum (head area, head perimeter, midpiece length, and flagellum length); and PC4 was related to midpiece width. Then, a statistical analysis (Hopkins Test and Visual Assessment of cluster Tendency—VAT) was conducted to determine whether the sperm population was clusterizable. Finally, two different sperm subpopulations were determined, although differed depending on the male: cluster 1 had spermatozoa similarity in PC1, PC2, and PC3 and spermatozoa dissimilarity in PC4; cluster 2 had spermatozoa dissimilarity in PC1, PC2, and PC3 and spermatozoa similarity in PC4.
Although some dolphin morphometric parameters have been previously described, this study is the first reporting full head and flagellum sperm size and shape values.5-7 The results of PCA suggest that males may produce a mix of different sperm with different features. This opens a new approach to study the role of morphometry on sperm function in polygamous species as a reproductive competition strategy.
Acknowledgments
We are grateful to the staff of the Oceanogràfic Valencia and the dolphin trainers and veterinarians for their assistance with animal care and semen collections. This research was supported by the Spanish Ministry of Science and Innovation (PID2019-106380RB-I00/AEI/10.1303/501100011033) and Fundación Oceanogràfic.
*Presenting author
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