Identification of Bacterial Profiles and Their Interactions with Selected Quality, Oxidative, and Immunological Parameters of Turkey Semen

Abstract

This study focused on the identification of naturally occurring bacteria in the reproductive fluid and impact on the quality of ejaculates obtained from the turkey breed British United Turkeys (BUT) Big 6 (n = 60). We determined possible relationships between the bacterial load and advanced sperm quality parameters that are important for effective artificial insemination and high fertility, as well as the concentration of selected antimicrobial proteins and pro-inflammatory markers of turkey semen. Sperm motility was assessed with computer-assisted sperm analysis (CASA), while the membrane and acrosome integrity were examined with smearing and staining methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL assay, and the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cell lysates were prepared to investigate the extent of lipid and protein oxidation. Furthermore, levels of interleukins 1 and 6 (IL-1, IL-6), C-reactive protein, cathelicidin, and β-defensin were quantified in the seminal plasma using the ELISA method. The most dominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was Escherichia coli, Proteus mirabilis, Staphylococcus lentus, and Citrobacter braakii. The bacterial load had a negative effect on the sperm motility (p < 0.001), as well as membrane (p < 0.05) and acrosome integrity (p < 0.01). A strong positive relationship between the bacterial load and DNA fragmentation (p < 0.001) was detected as well. Positive associations were recorded between the increasing presence of bacteria, ROS overgeneration (p < 0.001), and a subsequent oxidative damage to the proteins (p < 0.001) and lipids (p < 0.01). It was revealed that the antimicrobial peptides β-defensin (p < 0.001) and cathelicidin (p < 0.001) had a positive relationship with the motility. In contrast, pro-inflammatory markers, such as IL-1 (p < 0.001) and IL-6 (p < 0.001), had a negative impact on the motion behavior of turkey spermatozoa. Our results suggest that the semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia is associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for a failed artificial insemination in turkey breeding.

The Efficiency of Selected Extenders against Bacterial Contamination of Boar Semen in a Swine Breeding Facility in Western Slovakia

“…To assess the potential of selected boar semen extenders to prevent potential ROS overproduction as a result of bacteriospermia, we used a luminometric approach using luminol as the probe, which has been extensively used to study the global ROS production by sperm in mammalian and avian semen [ 15 , 18 , 19 ] ( Table 7 ). At 24 h the amount of ROS significantly decreased in the experimental group 3 in comparison with the control ( p < 0.05).…”

“…In comparison to conventional microbiological tools for bacterial screening, MALDI-TOF MS has emerged as a time-effective and reliable analytical approach for the identification of bacterial profiles in animal semen [ 15 , 18 , 19 , 21 , 22 ]. The advantage of such method lies in an early identification of pathogens, even at the level of strains or serotypes, which can significantly shorten the time of isolation and eventual initiation of treatment [ 23 ].…”

“…In order for the system to differentiate between spermatozoa and bacteria, the samples were stained using the IDENT stain (Hamilton-Thorne Biosciences, Beverly, MA, USA) and analyzed under fluorescent illumination. Each sample was loaded into the Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) and a minimum of 1000 spermatozoa were evaluated [ 19 ].…”

“…The resulting pellet was left to dry freely and subsequently resuspended in 30 μL 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA) and the same amount of acetonitrile (Sigma-Aldrich, St. Louis, MO, USA). Following a second round of centrifugation (3500 RPM, laboratory temperature 2 min), 1 μL of the supernatant was placed on a 96-point MALDI identification plate and left to dry freely [ 15 , 18 , 19 ].…”

“…Bacterial identification was performed with the Microflex LT instrument equipped with the flexControl software version 3.4. The spectra measured by mass spectrometry were compared with the MALDI Biotyper Bruker Taxonomy database (Bruker Daltonics, Bremen, Germany) [ 15 , 18 , 19 ].…”