Sperm Nuclei Analysis and Nuclear Organization of a Fertile Boar–Pig Hybrid by 2D FISH on Both Total and Motile Sperm Fractions

SIMPLE SUMMARY: Mammalian hybrids frequently form in nature between closely related species, and the majority are sterile due to the production of chromosomally unbalanced gametes. In Italy, the widespread wild boar has had negative consequences for free-range pig farming, which is considered the best practice for pig welfare and is a common method of farming most autochthonous pig breeds. This study aimed to analyze the sperm DNA integrity, sperm meiotic segregation and nuclear spatial organization in a boar–pig hybrid, for the first time, to evaluate its fertilizing capacity. The results show that the hybrid presented a high frequency (64%) of motile spermatozoa with a regular chromosome composition and a specific spatial distribution. This study underlines how fertile boar–pig hybrids represent a growing problem for conserving autochthonous pig breeds. ABSTRACT: A wide range of mammalian hybrids has recently been found by chance or through population-screening programs, but studies about their fertilizing capacity remain scarce and incomplete. Most of them are assumed to be sterile due to meiotic arrest caused by the failure of chromosome pairings. In this study, we evaluated both sperm meiotic segregation, by 2D fluorescent in situ hybridization (FISH) analysis, and sperm quality (Sperm Chromatin Structure Assay) by flow cytometer in a fertile boar–pig hybrid (2n = 37,XY) originating from a Nero Siciliano pig breed (Sus scrofa domesticus) and a wild boar (Sus scrofa ferus). Spermatozoa were also separated by a dual-layer (75–60%) discontinuous Percoll gradient, resulting in two fractions with a significantly better overall quality in the motile sperm fraction. These data were confirmed by FISH analysis also, where the frequencies of spermatozoa with a regular chromosome composition were 27% in total sperm fraction and 64% in motile sperm fraction. We also evaluated the nuclear architecture in all counted spermatozoa, showing a chromatin distribution changing when chromosome abnormalities occur. Our results demonstrate that the chromosome pairing has a minimal effect on the sperm segregation and semen quality of a boar–pig hybrid, making it fertile and harmful for the conservation of autochthonous pig breeds.