D Rath’s research while affiliated with Friedrich Loeffler Institute and other places

The study of the interactions between biofunctionalized gold nanoclusters (Au NCs) and spermatozoa is highly relevant to evaluate the potential of Au NCs as imaging probes and transfection agents in reproductive biology. In this work, confocal laser scanning microscopy (CLSM) was used to investigate the distribution of Au NCs bioconjugated with peptide (nuclear localisation sequence, NLS) and oligonucleotide (locked nucleic acid, LNA) ligands in bovine spermatozoa. Fluorescence lifetime imaging (FLIM) was employed to detect changes in the NC's chemical environment. We observed a pronounced regio‐selective accumulation of the bioconjugates in spermatozoa with high concentration at the equatorial segment. Furthermore, 3D‐CLSM showed successful non‐endosomal cellular uptake of the conjugates by intact sperm cells and the distribution of the bioconjugates was found to be influenced by the ligand types. Interestingly, the FLIM data showed differences in lifetime depending on membrane integrity. Furthermore, ligand‐dependent changes in lifetime between NC bioconjugates carrying peptide and oligonucleotide ligands were found, probably attributed to specific interactions with sperm cell compartments.

The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro, and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone, or in the presence of bovine blastocysts (n=25) or murine blastocysts (n=25). Following culture, explants were snap frozen and stored at -80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon tau-induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced, changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe’s cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing. The proportion of motile spermatozoa was significantly higher in SLC – selected samples in comparison to control (not SLC – selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC – selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC – selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC – selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.

The aim of this in vitro study was to evaluate the effects of taurine (TAU) supplementation on boar spermatozoa motility, viability, acrosome integrity and morphology. Eighteen boar semen samples were diluted with the Androhep PlusTM extender containing no TAU (control) or supplemented with 1.5 mM, 7 mM, 12.5 mM TAU and cultured at 4 °C for 18 days. Sperm motility was evaluated using the computer-aided sperm analysis (CASA) system. Furthermore, the samples were fixed and assessed for the occurrence of morphological abnormalities using phase contrast microscopy. Fluorescent dyes SYBR-14 and propidium iodide were used to determine the sperm viability. Acrosome integrity was examined using PNA-Alexa Fluor 647 and flow cytometry. A gradual decrease of the semen quality was detected in all experimental groups over the course of the study. CASA revealed no selective advantage of TAU supplementation on the spermatozoa motility (p > 0.05). TAU administration showed to be ineffective in preserving spermatozoa viability as well as acrosome integrity as measured by flow cytometry (p > 0.05). Under the conditions of this study, no significant positive effect of TAU was recorded following its administration to the Androhep PlusTM boar semen extender with respect to spermatozoa quality. © 2018 Mendel University of Agriculture and Forestry Brno. All right reserved.

The Food and Agriculture Organization of the United Nations has recognised that the production of pre-sexed livestock by sperm or embryo sexing as a useful breeding tool to increase production efficiency, especially for traits that are sex-related. In this chapter, we briefly explain sex determination in mammals, review approaches to identifying X and Y chromosome-bearing sperm and their practical implications for semen handling and artificial insemination (AI) and compare their importance and success in the main farm animal species. The problems associated with current technology for sperm sexing, as reflected in the damage caused to mammalian sperm are then considered, followed by an assessment of the potential for replacing this technology by other methods.

Standardized phospholipid liposomes are promising candidates to replace egg yolk in semen extenders. In this study liposomes were used in catch fluid (2% liposomes) during sex sorting and during cryopreservation (20% liposomes) of sorted and unsorted sperm. In a thermo-tolerance test, compared to controls containing egg yolk, sperm quality was not diminished at 0 h and 6 h, respectively (i.e. motility: 91.6%, 86.0%; membrane integrity: 82.6%, 82.4%; morphology: 94.3%, 92.9%). Cryopreservation in extenders Sexcess® andOptixcell® containing liposomes was conducted with unsorted sperm and with spermatozoa that were sorted into the liposomes containing catch fluid. All samples were evaluated in a 6 h lasting thermo-tolerance-test (n = 14). Sexcess® supplemented with egg yolk resulted in a higher post-thaw motility (56.6%, 43.8%, 37.4%), membrane integrity (52.0%, 49.1%, 50.1%) and morphology (87.1%, 78.7%, 69.3%) at 0 h, 3 h and 6 h respectively of sorted sperm cells compared to the supplementation with 20% liposomes (motility: 45.0%, 26.7%, 17.2%; membrane integrity: 30.0%, 21.2%, 28.9%; morphology: 75.3%, 68.1%, 61.4%) or Optixcell® (motility: 56.4%, 34.1%, 27.0%; membrane integrity: 33.6%, 23.6%, 25.7%; morphology: 75.3%, 67.0%, 60.1%). In case of unsorted semen, even at 6 h motility (57.9%, 44.2%, 50.2%), membrane integrity (64.9%, 38.9%, 47.2%), and morphology (85.5%, 83.1%, 83.2%) of the mentioned diluents had been on a higher quality level. In conclusion, with the chosen setup, liposomes may serve as egg yolk replacement in catch fluid during sorting but not as supplement in Sexcess® freezing extender.

Since decades, egg yolk is used as cryoprotective in freezing protocols of bovine semen to reduce freezing injury. However, egg yolk-based extenders entail disadvantages because of the potential risk of cross species contaminations and the attendant addition of antibiotics. Supplementation of standardized liposomes composed of phospholipids appears to be a promising alternative to egg yolk. Therefore, the aim of this study was to investigate the suitability of egg yolk-free extenders supplemented with liposomes for cryopreservation. Initially, a common freezing extender was modified by replacing egg yolk with different concentrations of liposomes. Afterwards, the suitability of these modified diluents and commercial extenders were evaluated in a 6-hours thermo-tolerance-test assessing sperm post-thaw quality. Although minimal standard quality requirements for bovine semen had been fulfilled and synthetic liposomes are in principle an appropriate substitute of egg yolk in freezing extender, based on the 6 h values of the test cryopreservation with liposome-extenders resulted in reduced sperm quality compared to egg yolk containing media regarding motility (43.3 vs. 57.9%), membrane integrity (39.4 vs. 64.9%) and morphology (80.8 vs. 85.5%). On the assumption of optimising liposome-extenders, they have a great potential to contribute in avoiding components of animal origin and enables to reduce addition of antibiotics.

Gold nanoparticles (AuNPs) are widely used in biomedical applications for drug targeting and bioimaging. This often neccesitates their functionalization with biomolecules carrying a defined biological function, yielding gold nanoparticle bioconjugates. The utilization of triplex-forming oligonucleotides (TFOs) as ligands gives access to nanoconjugates as tools for specific DNA-related nanotargeting via triplex hybridization. Since triplex hybridization with nanobioconjugates has to date not been shown on biologically relevant samples, sex-specific sperm marking may be an appropriate model system to demonstrate the opportunities of this targeting method in vitro. In this study, we focused on specific labeling of repetitive target sites enriched on the bovine Y-chromosome using triplex forming oligonucleotides. First, the functionality of a specific locked nucleic acid (LNA) sequence was confirmed on bovine free DNA and on demembranated sperm heads. Thereafter, the influence of AuNPs on triplex hybridization was spectrophotometrically analyzed employing synthetic dsDNA, genomic DNA and demembranated sperm heads. Results from the SPR-peak shift indicate that TFO-AuNP hybridize to bovine gDNA in a qualitative and significant manner. These results confirm successful triplex hybridization on biologically relevant target sites as well as the establishment of a method to use gold nanoparticles as a suitable tool for sex-selective hybridization.