Exogenous FSH/LH modulates TGF beta signaling genes in granulosa cells of Simmental heifers without affecting IVP results.

Follicular wave synchronization and follicular superstimulation with FSH are commonly used in OPU-IVP programs to increase oocyte developmental competence. Factors like Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), from the TGF beta superfamily, are produced by the oocyte and modulate follicular function. The aim of this study was to analyze the FSH-induced effects on (1) embryo production in dual-purpose Simmental cattle, and (2) TGF beta-mediated effects on oocyte-granulosa cell communication. Simmental heifers (n = 12, age 484 ± 62 days) underwent two OPU-IVP cycles in a cross-over design. Follicular waves were synchronized using 0.5 mg cloprostenol on Day 0, followed by 10 µg buserelin on Day 2. Subsequently, half of the heifers were randomly assigned to receive FSH/LH (four injections of 75 IU FSHp and 75IU LHp, 12 h apart on Days 4 and 5) before the first OPU, while the remaining heifers received FSH/LH before the second OPU. At the time of OPU, i.e. 7 days after the start of synchronization, granulosa cells were collected for RT-qPCR analysis. FSH treatment did not affect the number of oocytes collected (17.3 vs. 13.3, P > 0.05), but increased the percentage of quality 1 oocytes compared to controls (45.7 % vs. 22.0 %, P < 0.001). Neither cleavage (86.4 % vs. 85.7 %), nor blastocyst (42.1 % vs. 39.3 %) rate, or the number of transferable embryos produced by IVP (4.1 vs 4.8) was influenced by FSH treatment (P > 0.05 in all cases). FSH treatment increased HIF1A and FSHR levels in granulosa cells, while STAR was decreased (P = 0.008 in all cases). FSH treatment did not affect BMP15 or GDF9 mRNA expression (P > 0.05) but appeared to modulate the expression of genes involved in the BMP signaling pathway. Transcriptional levels of BMP15 receptor (BMPR1A, P = 0.016), and its downstream signaling factor SMAD1 (P = 0.008) were affected by FSH treatment. Our results demonstrated no benefit of this FSH stimulation protocol on IVP results in Simmental heifers. Further, our results suggest that the effects of FSH on bovine oocytes during acquisition of developmental competence may be mediated through BMP, but do not involve the regulation of transcriptional availability of GDF9, providing new insights into possible paracrine effects of the oocyte on granulosa cells.

Acinetobacter in fresh faecal samples is related to sperm motility: A case report in Piétrain boars housed on different deep-litter bedding materials.

The swine gastrointestinal tract contains a great variety of microbes, forming a complex and dynamic ecosystem. Various internal and external factors (e.g. age, breed and diet) may influence its composition. This study aimed to investigate the gut microbial diversity of German Piétrain boars housed on different deep-litter bedding materials (regional wood shavings, linen, hemp, spelt husks, and wood shavings) via 16S-rDNA sequencing. Additionally, short-chain fatty acids were analysed using gas chromatography. Fresh faecal samples (n = 80) from 40 Piétrain boars were collected twice during the trial. Although it can be assumed that boars ingest bedding orally, no differences in the microbiome composition could be found. The main phyla were Firmicutes and Bacteroides. Acinetobacter was identified as a biomarker for sperm quality differences (total sperm motility) in breeding boars.

Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

Comparison of deep-litter bedding materials and analysis of semen traits in Piétrain boars: A randomized controlled field study.

External factors can affect reproductive traits of breeding boars and especially the sensitive process of spermatogenesis. The aim of this study was to investigate probable influences of bedding materials (chipsy wood shavings (CWS), hemp straw (HS), linen straw (LS), spelt husks (SH), and regional wood shavings (RWS)) on semen traits of 40 randomly selected Piétrain boars (8 boars per group, age: 2.35 ± 1.23 years). After a six-week adaptation period, 40 fresh semen samples were collected weekly for four weeks and diluted in BTS (4 consecutive ejaculates per boar, 32 samples per group, 160 samples in total). Semen samples were analyzed using an extended range of spermatological methods (e.g., computer-assisted sperm analysis and flow cytometry). Generalized linear mixed models for each sperm parameter as well as the area under the curve for total sperm motility and thermo-resistance test were calculated. Materials LS and SH exceeded the standard maximum level for pesticide residues (VO (EG) No. 396/2005). Materials HS and LS presented the highest water-binding capacity of 413 % and 357 %, respectively, while SH showed the lowest value of 250 %. There were no significant (P > 0.05) differences between groups in any sperm characteristic, therefore indicating that bedding material had no influence on sperm quality. For most semen traits, however, we found significant (P ≤ 0.001) differences between sampling weeks. Based on pesticide results, we suggest CWS, RWS, or HS as possible bedding materials for pig production farms in the future. Furthermore, we strongly recommend a quality analysis of any new bedding material before use in swine husbandry.

Reprotoxic effects of fenpropimorph on the fertilizing potential of AI boars: A case study.

This case study describes the effects of a contamination of boar bedding material with reprotoxic compounds in an AI centre in southern Germany. The origin of the investigations was an extreme decline in the production output of the boars. In July 2021, more than 54% of boars were not in production and over 45% of ejaculates had insufficient sperm quality and quantity, which is a significant drop in comparison with the other months. This drop was accompanied by oligozoospermia (azoospermia), asthenozoospermia and teratozoospermia. Through intensive troubleshooting, the changes could be attributed to fenpropimorph, an ergosterol biosynthesis-inhibiting fungicide with reprotoxic potential, which was found in the sawdust used as bedding as well as in liver samples of affected animals, reaching a concentration (mean ± SD) between 0.20 ± 0.36 mg/kg and 0.019 ± 0.001 mg/kg respectively. Furthermore, autopsy findings revealed hyperaemia of the testis, histologically focal degeneration of the germinal epithelium and signs of reduced spermatogenesis and spermiogenesis.

Coexpression of hypoxia-inducible factor-1α and glucose transporter-1 is associated with poor prognosis in oral squamous cell carcinoma patients.

AIMS: To study whether coexpression of the two hypoxia-related proteins hypoxia-inducible factor (HIF)-1α and glucose transporter (GLUT)-1 has prognostic relevance in oral squamous cell carcinomas (OSCCs). METHODS AND RESULTS: Eighty-two OSCC samples were analysed for expression levels of HIF-1α and GLUT-1 by immunohistochemistry. Protein expression was assessed with an immunoreactive score system, and the correlations between gene expression and both clinical and pathohistological parameters were examined. Overexpression of either GLUT-1 or HIF-1α was associated with poor disease-specific survival in OSCC patients. Multivariate Cox proportional-hazards regression analysis revealed that increased expression of HIF-1α was significantly associated with disease-specific survival (relative risk = 3.24, P = 0.024), as compared with the group with a low level of expression. Coexpression of HIF-1α and GLUT-1 was additively and significantly associated with adverse prognoses in patients with OSCC. Patients whose tumours had increased levels of expression of both HIF-1α and GLUT-1 were found to have a 5.13-fold increased risk of tumour-related death (P = 0.017). CONCLUSIONS: Coexpression of high levels of HIF-1α and GLUT-1 is significantly correlated with prognosis in OSCC patients, suggesting that the coexpression of these proteins can be used as both an early diagnostic and independent prognostic marker.

HIF-1alpha is a prognostic marker in oral squamous cell carcinomas.

The critical molecular regulator of hypoxia is the hypoxia-inducible factor 1 alpha (HIF-1alpha). The prognostic impact of this regulator protein in oral squamous cell carcinomas (OSCC) has not been comprehensively investigated. The aim of this study was to analyze the expression of HIF-1alpha in 82 patients with OSCC and to correlate it with their disease-specific survival. Immunohistochemical staining for HIF-1alpha was performed on 82 OSCC specimens using a standard immunoperoxidase technique. The expression of HIF-1alpha was correlated with poor disease-specific survival for OSCC patients. Patients with negatively or weakly HIF-1alpha-expressing tumors had a survival rate of 80%, whereas the survival decreased to only 33.6% in case of moderate or strong expression. In multivariate Cox regression analysis, we found a 3.5-fold increased risk of tumor-related death when HIF-1alpha was strongly expressed (p=0.016) compared to negative or weak expression of HIF-1alpha. We suggest HIF-1alpha is an independent prognostic marker in OSCC. Immunohistochemical detection of HIF-1alpha appears to be useful in the diagnosis of OSCC and to provide prognostic information in addition to TNM stage and histological grade.

Biosynthesis of Wdr16, a marker protein for kinocilia-bearing cells, starts at the time of kinocilia formation in rat, and wdr16 gene knockdown causes hydrocephalus in zebrafish.

The rat ortholog of the WD40 repeat protein Wdr16 is abundantly expressed in testis and cultured ependymal cells. Low levels are found in lung and brain, respectively, while it is absent from kinocilia-free tissues. In testis and ependymal primary cultures, Wdr16 messenger RNA appears concomitantly with the messages for sperm-associated antigen 6, a kinocilia marker, and for hydin, a protein linked to ciliary function and hydrocephalus. In testis, ependyma and respiratory epithelium, the Wdr16 protein is up-regulated together with kinocilia formation. The wdr16 gene is restricted to genera in possession of kinocilia, and it is strongly conserved during evolution. The human and zebrafish proteins are identical in 62% of their aligned amino acids. On the message level, the zebrafish Wdr16 ortholog was found exclusively in kinocilia-bearing tissues by in situ hybridisation. Gene knockdown in zebrafish embryos by antisense morpholino injection resulted in severe hydrocephalus formation with unaltered ependymal morphology or ciliary beat. Wdr16 can be considered a differentiation marker of kinocilia-bearing cells. In the brain, it appears to be functionally related to water homeostasis or osmoregulation.