Morphology, morphometry and chromatin distribution in llama sperm nuclei

Abstract

Contents The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 μm2, perimeter 13.16 μm, length 5.12 μm, width 2.81 μm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.

A brief history of the Feulgen reaction

“…Focusing on other animal cell investigations, Feulgen staining was applied to evaluate several nuclear parameters, such as shape and size, DNA content, and chromatin compaction, in lymphocytes and epithelial cells of canine origin (Dos Santos et al 2019 ). Similar morphometric aspects were also investigated by Giuliano and colleagues in llama sperm nuclei, with specific attention dedicated to chromatin distribution and measurement of the haploid DNA content (Giuliano et al 2018 ).…”

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase

“…In recent years, new evaluation techniques for llama and alpaca sperm were developed by allowing a more complete evaluation of raw and cryopreserved semen samples, such as membrane function, sperm viability, acrosome status, DNA integrity and chromatin condensation (Carretero et al., 2015; Cheuquemán et al., 2013; Fumuso et al., 2019, 2020; Kershaw et al., 2017; Santiani Acosta et al., 2013, amongst others). Some studies in SACs evaluated the sperm head and nuclei morphometry in raw semen (Buendía et al., 2002; Casaretto et al., 2012; Evangelista et al., 2015; Giuliano et al., 2018; Soler et al., 2014). The use of computer assisted semen analysis (CASA) allows to objectively measure the shape and dimensions of the sperm head and can reveal subtle differences between individuals that cannot be detected with subjective methods.…”