Research articleSelected sperm traits are simultaneously altered after scrotal heat stress and play specific roles in in vitro fertilization and embryonic development
Introduction
The structural and functional integrity of spermatozoa have been analyzed by several laboratory techniques to determine the potential relationship between semen parameters and fertility [1], [2], [3], [4], [5]. As a complement of these techniques, the scrotal insulation model is a useful experimental tool that has the potential to be applied in several fields of research [6], [7], [8], [9]. Thus, the thermal insult produces remarkable variety of morphological sperm alterations which can be evaluated and associated to male reproductive performance [10], [11], [12].
Traditionally, computer-aided methods have been used to analyze primary (area, perimeter, width, and length) and derived measurements (ellipticity, shape factor, and Fourier harmonics) to provide information on sperm-head dimensions [13], [14], [15], [16]. Moreover, chromatin integrity analyses [17], [18] have also been applied and reported a significant association with embryo quality [19], [20], embryonic development [21], and pregnancy rates [22]. However, improvements in the estimation of male fertility indicators require advances in the development of laboratory tests for sperm assessment [23], better conditions for fertility evaluation [24], and use of elaborate statistical techniques [25].
It is becoming increasingly evident that conventional approaches to sperm quality assessments, which consider only specific spermatozoal traits [26] or evaluate male fertility based just on average values of sperm parameters after single functional tests [27], are poor predictors of fertility [28], [29]. As a result, researchers have sought methods to incorporate multiparametric assessments of sperm-head traits in a clinically useful manner [30], [31], [32]. Previous reports have used a combination of experimental techniques and multivariate statistical analyses (e.g., principal component analysis [PCA], exploratory factor analysis, and clustering analysis) to reduce several variables to a few factors and explore the relationships between different sperm traits [33], [34], [35], [36].
After scrotal insulation, semen collection, cryopreservation, and computational image analysis; the aims of the present work were (1) to apply multivariate analyses to examine the set of alterations and interactions in sperm morphology and (2) to evaluate the importance of sperm parameters on the outcome of standard IVF and embryonic development.
Section snippets
Scrotal insulation, semen collection, and cryopreservation protocol
All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Federal University of Uberlândia (research protocol; #017-12). Three crossbred bulls (Bos taurus × Bos indicus), 30 to 36-month old, kept under grazing on an experimental farm and with previous fertility evaluation (≥70% morphologically normal sperm, ≥60% progressively motile sperm, and ≤20% of maximum sperm abnormalities) were used [37], [38]. The bulls were subjected to a 72-hour scrotal
Results
A total of 54 ejaculates were evaluated, and a mean of 2,250.0 ± 155.1 sperm heads were analyzed for each variable and experimental time (before and after scrotal insulation). The effect of scrotal insulation throughout the experimental period was observed in all evaluated parameters compared with the preinsulation time (Fig. 1, Fig. 2).
The sperm-head morphometric variables (area, perimeter, width, length, ellipticity, anterior-posterior symmetry, and Fourier, F0) decreased (P < 0.05) at Day 28
Discussion
Scrotal insulation was used in bulls as an experimental model to study the morphological alterations of the sperm head. Measurements of sperm-head shape and chromatin integrity were evaluated by a computational image analysis. In addition, a statistical procedure (PCA) on the basis of a variable reduction technique was performed. Thus, this strict and objective assessment of sperm heads allows for the identification of subtle morphological alterations.
In general, our results indicated that
Acknowledgments
Research was supported by Federal University of Uberlândia, Foundation for Research Support of the State of Minas Gerais (FAPEMIG; grant CVZ-APQ-01499-12), and National Council for Scientific and Technological Development (CNPq; grant 479256/2012-8). ACL was the recipient of a Postdoctoral fellowship from Foundation for Research Support of the State of Minas Gerais, Brazil.
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