Castration alters the ileum microbiota of Holstein bulls and promotes beef flavor compounds.

BACKGROUND: In the beef industry, bull calves are usually castrated to improve flavor and meat quality; however, this can reduce their growth and slaughter performance. The gut microbiota is known to exert a significant influence on growth and slaughter performance. However, there is a paucity of research investigating the impact of castration on gut microbiota composition and its subsequent effects on slaughter performance and meat flavor. RESULT: The objective of this study was to examine the processes via which castration hinders slaughter productivity and enhances meat quality. Bull and castrated calves were maintained under the same management conditions, and at slaughter, meat quality was assessed, and ileum and epithelial tissue samples were obtained. The research employed metagenomic sequencing and non-targeted metabolomics techniques to investigate the makeup of the microbiota and identify differential metabolites. The findings of this study revealed the Carcass weight and eye muscle area /carcass weight in the bull group were significantly higher than those in the steer group. There were no significant differences in the length, width, and crypt depth of the ileum villi between the two groups. A total of 53 flavor compounds were identified in the two groups of beef, of which 16 were significantly higher in the steer group than in the bull group, and 5 were significantly higher in the bull group than in the steer group. In addition, bacteria, Eukaryota, and virus species were significantly separated between the two groups. The lipid metabolism pathways of α-linolenic acid, linoleic acid, and unsaturated fatty acids were significantly enriched in the Steers group. Compared with the steer group, the organic system pathway is significantly enriched in the bull group. The study also found that five metabolites (LPC (0:0/20:3), LPC (20:3/0:0), LPE (0:0/22:5), LPE (22:5/0:0), D-Mannosamine), and three species (s_Cloning_vector_Hsp70_LexA-HP1, s_Bacteroides_Coprophilus_CAG: 333, and s_Clostridium_nexile-CAG: 348) interfere with each other and collectively have a positive impact on the flavor compounds of beef. CONCLUSIONS: These findings provide a basic understanding that under the same management conditions, castration does indeed reduce the slaughter performance of bulls and improve the flavor of beef. Microorganisms and metabolites contribute to these changes through interactions.

Optimized total RNA isolation from bovine sperm with enhanced sperm head lysis.

Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities.

A century of canopy kelp persistence and recovery in the Gulf of Alaska.

BACKGROUND AND AIMS: Coastal Alaska contains vast kelp habitat that supports diverse marine and human communities. Over the past century, the North Pacific Ocean has undergone oceanographic and ecological regime shifts that have the potential to influence the structure and function of kelp ecosystems strongly. However, the remoteness and complexity of the glacially carved region precludes the regular monitoring efforts that would be necessary to detect such changes. METHODS: To begin to fill this critical knowledge gap, we drew upon historical and modern surveys to analyse the change in spatial coverage and species composition of canopy kelp between two time points (1913 and the early 2000s to 2010s). We also incorporated decadal surveys on sea otter range expansion following complete extirpation and reintroduction to assess the influence of sea otter recovery on the spatial extent of canopy kelp. KEY RESULTS: We found increases in the spatial extent of canopy kelp throughout the Gulf of Alaska where there was coverage from both surveys. Kelp in Southcentral Alaska showed extensive recovery after the catastrophic Novarupta volcano. Kelp in Southeast Alaska showed persistence and spatial increase that closely matched increases in the range of sea otters. Observations of thermally tolerant kelp species increased more than observations of cold-adapted species between the two surveys. CONCLUSIONS: Contrary to trends observed at lower latitudes, the kelp forests that ring the Gulf of Alaska have been remarkably stable and even increased in the past century, despite oceanographic and ecosystem changes. To improve monitoring, we propose identification of sentinel kelp beds for regular monitoring to detect changes to these iconic and foundational canopy kelp species more readily.

Effect of autologous platelet-rich plasma on the fertility and quality of cryopreserved buffalo bull semen: a comparative study using OptiXcell® and tris egg yolk extenders.

BACKGROUND: Buffalo spermatozoa have a distinct membrane structure that makes them more vulnerable to cryopreservation, resulting in lower-quality post-thawed sperm. This decreases the success rate of artificial insemination in buffaloes. Understanding and addressing these specific vulnerabilities are essential for improving reproductive techniques in buffalo populations. The properties of cryopreserved buffalo bull semen were examined in this study regarding the impact of adding autologous platelet-rich plasma (PRP) to OptiXcell® or Tris egg yolk-based extenders. Ten buffalo bulls were used to collect semen. Each bull's ejaculate was separated into two main equal amounts, each of which was then diluted with either OptiXcell® or Tris egg yolk-based extender, supplemented with various PRP concentrations (5%, 10%, and 15%), and the control (0%), before being cryopreserved according to established protocols. Following equilibration and thawing, the quality and functionality of the sperm were evaluated, along with the antioxidant enzyme activities (GSH and TAC), malondialdehyde (MDA) content, and in vivo fertilization rate of the thawed semen. RESULTS: All PRP concentrations in both extenders, particularly 10% PRP, improved the quality and functionality of the sperm in both equilibrated and frozen-thawed semen. Additionally, the antioxidant enzyme activities in both extenders were higher in the PRP-supplemented groups compared to the control group in thawed semen (P < 0.05). All post-thaw sperm quality, antioxidant enzyme activities, and functionality aside from DNA integrity were higher (P < 0.05) in the PRP-supplemented OptiXcell® than in the PRP-supplemented Tris egg yolk-based extender. The fertility of cryopreserved semen in the extenders supplemented with 10% and 15% PRP increased (P < 0.05) significantly more than that of the control extenders, with 10% PRP being the optimum concentration in OptiXcell® (80%) compared to that of Tris egg yolk-based extender (66.67%) and control of two extenders (53.33% and 46.67%, respectively). CONCLUSIONS: Even though autologous PRP-supplemented extenders have a protective impact on equilibrated and cryopreserved semen, 10% PRP-supplemented OptiXcell® extenders are more effective at preserving post-thaw semen quality, functionality, and antioxidant capacity, which increases the in vivo fertility of buffalo bulls.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane.

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C - PKC; phospholipases A2 and Cζ - PLA2, PLCζ; soluble adenylyl cyclase 10 - sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 - Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.

Species invasion progressively disrupts the trophic structure of native food webs.

Species invasions can have substantial impacts on native species and ecosystems, with important consequences for biodiversity. How these disturbances drive changes in the trophic structure of native food webs through time is poorly understood. Here, we quantify trophic disruption in freshwater food webs to invasion by an apex fish predator, lake trout, using an extensive stable isotope dataset across a natural gradient of uninvaded and invaded lakes in the northern Rocky Mountains, USA. Lake trout invasion increased fish diet variability (trophic dispersion), displaced native fishes from their reference diets (trophic displacement), and reorganized macroinvertebrate communities, indicating strong food web disruption. Trophic dispersion was greatest 25 to 50 y after colonization and dissipated as food webs stabilized in later stages of invasion (>50 y). For the native apex predator, bull trout, trophic dispersion preceded trophic displacement, leading to their functional loss in late-invasion food webs. Our results demonstrate how invasive species progressively disrupt native food webs via trophic dispersion and displacement, ultimately yielding biological communities strongly divergent from those in uninvaded ecosystems.

Effect of access to natural shade on scrotal thermoregulatory capacity, integrity of the testicular parenchyma and sperm morphology of Nelore (Bos indicus) and Canchim (Bos taurus x Bos indicus) bulls.

The objective of this work was to evaluate the effect of using naturally shaded pastures on scrotal thermoregulatory capacity, testicular echotexture, and sperm morphology of Nelore (Bos indicus) and Canchim (5/8 Bos taurus x 3/8 Bos indicus) bulls in a tropical climate region. Sixty-four adult Nelore and Canchim bulls were used, equally allocated in Full Sun (FS, n = 32) or Crop-Livestock-Forestry (CLF, n = 32) pasture systems. During five consecutive climate seasons, the bulls underwent monthly breeding soundness evaluations and the biometeorological variables in the systems were continuously monitored. Microclimate was significantly different between systems. CLF system had lower BGHI than FS throughout the experimental period. No triple interaction (Season x Breed x Treatment, P > 0.05) was observed for any of the variables. Animals in CLF showed lower body temperature in Summer (FS:39.41 ± 0.05 vs. CLF:39.30 ± 0.05 °C; P = 0.005) and in Autumn (FS:39.54 ± 0.05 vs. CLF:39.35 ± 0.05 °C; P = 0.005). Access to shading did not determine differences in the evolution of scrotal biometry, temperatures, and scrotal thermal gradients (P > 0.05). Regardless of breed, animals in CLF showed greater right testicular volume (FS:247.5 ± 5.7 vs. CLF:259.0 ± 5.7 cm³; P < 0.05), more suitable parenchyma echotexture, and fewer microlithiasis spots in the Spring and Summer. Testosterone concentration was higher in FS (FS:2.6 ± 0.2 vs. CLF:2.1 ± 0.2 ng/mL; P = 0.035). Canchim bulls presented higher total sperm defects during the Autumn and Winter (P = 0.010), but the total defects levels for Canchim and Nelore bulls were in normal range for adult bulls. Thus, the natural shade in CLF system was effective in improving the microclimate of pastures and minimizing adverse environmental effects on some reproductive features of interest in beef cattle.

Low concentrations of soybean lecithin nanoparticles had a positive impact on Holstein bulls' cryopreserved semen.

Spermatozoa can experience negative changes when subjected to freezing and thawing, including lowered motility, viability and acrosome response. Herein, the effects of different concentrations of soybean lecithin nanoparticles on cryopreserved Holstein bull semen were examined. Semen was collected, cryopreserved and utilized for sperm kinetic parameter analysis following dilution, equilibration and thawing with 0.5% soybean lecithin (E1), the control extender, and 0.75% (E2), 0.5% (E3), 0.25% (E4) and 0.125% (E5) of lecithin nanoparticles. Results revealed that following dilution, the progressive motility (PM) at E3, E4 and E5 of lecithin nanoparticles was higher (p < .05) than it was for E2. After equilibration, compared to the E1, E2, and E3 values, the PM, vitality, normal morphology, membrane integrity and intact acrosome values at the E5 were consistently greater (p < .05). Comparing the percentages of intact acrosome and membrane integrity at E2 and E3 to E4 and E5, a substantial decrease (p < .05) was seen. Following thawing, the percentage of PM improved at E2 and E5, even though their mean PM values were similar (p > .05) compared to E1, E3 and E4. Vigour and progression parameters of sperm (DAP, DCL, DSL, VAP, VCL, VSL and STR) at E5 were higher (p < .05) than those at E1, E2, E3 and E4. In conclusion, the cryopreserved sperm from Holstein bulls revealed outstanding properties both after equilibration and after thawing with 0.125% lecithin nanoparticles, and they were sensitive to high dosages.

Characteristics of Bachaur bull semen.

The Bachaur is a mediumized draft purpose breed which has been recognized by ICAR-National Bureau of Animal Genetic Resources (NBAGR) Karnal, India, and presently is on the verge of extinction. Since there are no data regarding the seminal parameters of this breed, this work was performed to evaluate seminal parameters of freshly ejaculated semen. A total of three healthy breeding Bachaur bulls aged 2.5-5 years were selected for the study which were maintained under identical managemental conditions. Semen parameters of these bulls were studied across 10 ejaculates. The average scrotal circumference and testicular weight of the three bulls were 27.78 ± 1.2 cm and 400.67 ± 26.6 g, respectively. The average overall volume (mL), pH, concentration (million/mL), liveability (%), abnormality (%), HOST (%) and acrosome integrity (%) were 2.20 ± 0.19, 6.86 ± 0.06, 1245.60 ± 23.49, 85.09 ± 0.91, 4.13 ± 0.06, 81.16 ± 1.18 and 83.54 ± 1.32, respectively. The average overall mass motility of three Bachaur bulls was 3.57 ± 0.06 in 0-5 scale and individual motility averaged 84.78 ± 1.70 per cent. The volume of ejaculates in Bachaur bull seemed to be lower as compared to other exotic and Indian breeds. However, the semen parameters with regard to mass motility, liveability, abnormalities, hypo-osmotic swelling test (HOST) and acrosomal integrity seemed similar to other exotic and Indian breeds.

Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

Comparison of computer-assisted sperm analysis and smartphone-applied sperm analysis for evaluation of frozen-thawed bull semen.

This study aims to compare the efficacy of computer-assisted sperm analysis (CASA) and smartphone-applied sperm analysis (SASA) in assessing the quality of frozen-thawed bull semen. A total of 75 straws (n = 75) semen samples were used from different production batches of five Holstein bulls. The semen analyses were conducted in three groups: Group I (CASA-37°C), semen samples were evaluated using the CASA system at 37°C (n = 25); Group II (SASA-25°C), semen samples were assessed using the SASA system at a temperature of room heat (25°C) (n = 25); and Group III (SASA-37°C), semen samples were evaluated using the SASA system at 37°C (n = 25). The frozen-thawed bull semen samples were analysed in terms of total motility (TM), progressive motility (PM), immotile, velocity average path (VAP), velocity curve linear (VCL), velocity straight line (VSL) and sperm concentration. There was no significant difference between the groups in terms of spermatozoa concentration (p > .05). However, significant differences among the groups were observed for total motile spermatozoa values (p < .001). Values of progressive motile spermatozoa were lower in Group I and Group II compared to Group III (p < .001). The immotile spermatozoa values were significant between the groups (p < .001) and were found to be proportional to total motile spermatozoa values. Additionally, the VAP, VCL and VSL values were comparable between Group II and Group III, but lower when compared to Group I. In conclusion, the results of the study demonstrate that the Sperm Cell™ system can accurately analyse the concentration of frozen-thawed bull semen. The analyses performed at room temperature indicate a parallelism between the PM value and CASA results. However, it is thought that SASA devices require a series of standardization studies in different semen extenders, different sample concentrations and different animal species, analogous to the standardization evolution process of CASA devices in semen analysis.

Evaluation of predictive ability of linear type gonadal traits on reproductive capacity of breeding dairy bulls.

Dimensions of linear type traits facilitate selection of livestock for breeding and rearing. To date, use of linear type traits for selection of breeding bulls is highly concentric to scrotal circumference (SC), with probable overlook to other important traits. Present study reported the importance of various gonadal linear type traits on spermatozoa production, age-related changes in gonadal linear type traits of bulls and predictive ability of these traits on bulls' reproductive potentials. Among all gonadal traits, testicular density (TD), scrotal volume (SV), paired testicular weight (PWT) and SC were found most important predictor variables in order, which can discriminate between good/poor breeding bulls, that is, produced frozen semen doses (FSD) or not. Dimensions of gonadal traits increased significantly up to 36 months age and thereafter, development became slow and negligible. In contrast, TD decreased by 30%, 51%, 64%, 68% and 71% at 12, 24, 36, 48 and >49 months age, respectively, from its base value at 6 months. Bulls of lower TD (≤0.88 g/cm3) had significantly higher ejaculate volume (+9%), sperm motility, sperm concentration (+100 million/mL) and sperm output (+26%)/ejaculate as compared to bulls of higher TD (>0.88 g/cm3). Discriminant function was developed using TD, SV, PWT and SC to identify bulls of superior reproductive potentials. It was concluded that among the investigated traits, TD was the strongest to discriminate between FSD and Non-FSD bulls. Therefore, our findings suggested that TD could be more potential trait than SC for dairy bulls' breeding soundness evaluation and assessment of reproductive ability.

Death and injuries caused by cattle: A forensic overview.

Lethal episodes arising from interactions with cattle may be the result of a response of the animals to simple handling and herding, or from reactions to deliberate provocation or goading intended to incite aggressive behavior for public entertainment purposes. Deaths may be considered to be unprovoked and unanticipated, or provoked and predictable. Cattle cause significant numbers of deaths globally and are considered the most dangerous large animal in Britain. Behavior may be unpredictable even in apparently docile domesticated animals, and attacks may be by a single animal or a herd and result in injuries from kicking, head/butting/charging, stomping, goring, and crushing. Craniofacial injuries may involve fractures of the spine or skull with cerebral contusions and lacerations associated with subarachnoid, subdural, and extradural hemorrhages. Chest injuries are also characterized by fractures which may be multiple with flail chest, hemo- and pneumothoraces, and organ disruption. Injuries to the abdomen and perineum include intestinal perforations, splenic rupture, perineal and vaginal tears, urethral lacerations and avulsions, and bladder and rectal perforations. Significant vascular injuries include complete and partial transections and lacerations. Males living in rural areas are most at risk of a fatal encounter.

Bull Sperm SWATH-MS-Based Proteomics Reveals Link between High Fertility and Energy Production, Motility Structures, and Sperm-Oocyte Interaction.

The prediction of male or semen fertility potential remains a persistent challenge that has yet to be fully resolved. This work analyzed several in vitro parameters and proteome of spermatozoa in bulls cataloged as high- (HF; n = 5) and low-field (LF; n = 5) fertility after more than a thousand artificial inseminations. Sperm motility was evaluated by computer-assisted sperm analysis. Sperm viability, mitochondrial membrane potential (MMP) and reactive oxygen species (mROS) of spermatozoa were assessed by flow cytometry. Proteome was evaluated by the SWATH-MS procedure. Spermatozoa of HF bulls showed significantly higher total motility than the LF group (41.4% vs 29.7%). Rates of healthy sperm (live, high MMP, and low mROS) for HF and LF bull groups were 49% and 43%, respectively (p > 0.05). Spermatozoa of HF bulls showed a higher presence of differentially abundant proteins (DAPs) related to both energy production (COX7C), mainly the OXPHOS pathway, and the development of structures linked with the motility process (TPPP2, SSMEM1, and SPAG16). Furthermore, we observed that equatorin (EQTN), together with other DAPs related to the interaction with the oocyte, was overrepresented in HF bull spermatozoa. The biological processes related to protein processing, catabolism, and protein folding were found to be overrepresented in LF bull sperm in which the HSP90AA1 chaperone was identified as the most DAP. Data are available via ProteomeXchange with identifier PXD042286.

Sequence level genome-wide associations for bull production and fertility traits in tropically adapted bulls.

BACKGROUND: The genetics of male fertility is complex and not fully understood. Male subfertility can adversely affect the economics of livestock production. For example, inadvertently mating bulls with poor fertility can result in reduced annual liveweight production and suboptimal husbandry management. Fertility traits, such as scrotal circumference and semen quality are commonly used to select bulls before mating and can be targeted in genomic studies. In this study, we conducted genome-wide association analyses using sequence-level data targeting seven bull production and fertility traits measured in a multi-breed population of 6,422 tropically adapted bulls. The beef bull production and fertility traits included body weight (Weight), body condition score (CS), scrotal circumference (SC), sheath score (Sheath), percentage of normal spermatozoa (PNS), percentage of spermatozoa with mid-piece abnormalities (MP) and percentage of spermatozoa with proximal droplets (PD). RESULTS: After quality control, 13,398,171 polymorphisms were tested for their associations with each trait in a mixed-model approach, fitting a multi-breed genomic relationship matrix. A Bonferroni genome-wide significance threshold of 5 × 10- 8 was imposed. This effort led to identifying genetic variants and candidate genes underpinning bull fertility and production traits. Genetic variants in Bos taurus autosome (BTA) 5 were associated with SC, Sheath, PNS, PD and MP. Whereas chromosome X was significant for SC, PNS, and PD. The traits we studied are highly polygenic and had significant results across the genome (BTA 1, 2, 4, 6, 7, 8, 11, 12, 14, 16, 18, 19, 23, 28, and 29). We also highlighted potential high-impact variants and candidate genes associated with Scrotal Circumference (SC) and Sheath Score (Sheath), which warrants further investigation in future studies. CONCLUSION: The work presented here is a step closer to identifying molecular mechanisms that underpin bull fertility and production. Our work also emphasises the importance of including the X chromosome in genomic analyses. Future research aims to investigate potential causative variants and genes in downstream analyses.

New insights into posttranslational modifications of proteins during bull sperm capacitation.

BACKGROUND: Due to the unique nature of spermatozoa, which are transcriptionally and translationally silent, the regulation of capacitation is based on the formation of posttranslational modifications of proteins (PTMs). However, the interactions between different types of PTMs during the capacitation remain unclear. Therefore, we aimed to unravel the PTM-based regulation of sperm capacitation by considering the relationship between tyrosine phosphorylation and reversible oxidative PTMs (oxPTMs), i.e., S-nitrosylation and S-glutathionylation. Since reversible oxPTMs may be closely related to peroxyredoxin (PRDX) activity, the second aim was to verify the role of PRDXs in the PTM-based regulation of capacitation. METHODS: Cryopreserved bull sperm were capacitated in vitro with or without PRDX inhibitor. Qualitative parameters of sperm and symptoms characteristic of capacitation were analyzed. Posttranslational protein modifications (S-nitrosylation, S-glutathionylation, tyrosine phosphorylation) were investigated at the cellular level (flow cytometry, fluorescence microscopy) and at the proteomic level (fluorescent gel-based proteomic approach). RESULTS: Zona-pellucida binding proteins (ACRBP, SPAM1, ZAN, ZPBP1 and IZUMO4) were particularly rich in reversible oxPTMs. Moreover, numerous flagellar proteins were associated with all analyzed types of PTMs, which indicates that the direction of posttranslational modifications was integrated. Inhibition of PRDX activity during capacitation caused an increase in S-nitrosylation and S-glutathionylation and a decrease in tyrosine phosphorylation. Inhibition of PRDXs caused GAPDHS to undergo S-glutathionylation and the GSTO2 and SOD2 enzymes to undergo denitrosylation. Moreover, PRDX inhibition caused the AKAP proteins to be dephosphorylated. CONCLUSIONS: Our research provides evidence that crosstalk occurs between tyrosine phosphorylation and reversible oxPTMs during bull sperm capacitation. This study demonstrates that capacitation triggers S-nitrosylation and S-glutathionylation (and reverse reactions) of zona-pellucida binding proteins, which may be a new important mechanism that determines the interaction between sperms and oocytes. Moreover, TCA-related and flagellar proteins, which are particularly rich in PTMs, may play a key role in sperm capacitation. We propose that the deglutathionylation of ODFs and IZUMO4 proteins is a new hallmark of bull sperm capacitation. The obtained results indicate a relationship between PRDX activity and protein phosphorylation, S-glutathionylation and S-nitrosylation. The activity of PRDXs may be crucial for maintaining redox balance and for providing proper PKA-mediated protein phosphorylation during capacitation. Video Abstract.

Acute oesophageal safety and long-term follow-up of AI-guided high-power short-duration with 50 W for atrial fibrillation ablation.

AIMS: Pulmonary vein isolation using radiofrequency ablation is an effective treatment option for patients with symptomatic atrial fibrillation (AF). Application of high power over a short period of time (HPSD) is reported to create more efficient lesions and may prevent collateral thermal oesophageal injury. This study aims to compare efficacy and safety of two different HPSD ablation approaches using different ablation index settings. METHODS AND RESULTS: Consecutive patients undergoing AF ablation with HPSD (50 W; ablation index-guided) using the ThermoCool SmartTouch SF catheter were included. Patients were grouped by ablation protocol: ablation with target ablation index (AI) of 400 on the anterior left atrial wall vs. 300 at the posterior left atrial wall (AI 400/300) or AI 450/350 was performed upon the operator's preference and compared. Peri-procedural parameters and complications were recorded, and incidences of endoscopically detected thermal oesophageal lesions (EDEL) analysed. Recurrence rates after a mean follow-up of 25 ± 7 months and reconnection patterns in patients undergoing redo procedures were investigated. A total of 795 patients (67 ± 10 years; 58% male; 48% paroxysmal AF) underwent a first AF ablation with HPSD (211 in group AI 400/300 and 584 in group 450/350). Median procedure time was 82.9 ± 24.6 min with longer ablation times in patients with target AI 400/300 due to higher intraprocedural reconnection rates, increased box lesions, and additional right atrial isthmus ablations. EDEL rates among target AI 400/300 procedures were significantly lower (3% vs. 7%; P = 0.019). Correspondingly, AI 450/350 was the strongest independent predictor of post-ablation EDEL (OR 4.799, CI 1.427-16.138, P = 0.011). Twelve-month (76% vs. 76%; P = 0.892) and long-term ablation single procedure success (68% vs. 71%; log-rank P = 0.452) after a mean of 25 ± 7 months were comparable among both target AI groups; however, long-term success was significantly higher for paroxysmal AF compared to persistent AF (12 months: 80% vs. 72%; P = 0.010; end of follow-up: 76% vs. 65%; log-rank P = 0.001). One hundred three patients (16%) underwent a redo procedure during follow-up documented comparable pulmonary vein (PV) reconnection among groups. Multivariate predictors of AF recurrence were age, left atrium (LA) size, persistent AF, and extra-PV ablation targets. CONCLUSION: High-power short-duration AF ablation with target AI of 400 for non-posterior wall and 300 for posterior wall lesions resulted in comparable long-term results compared to higher AI (450/350) ablations with significantly lower risk for thermal oesophageal lesions. Older age, larger LA size, persistent AF, and extra-PV ablation targets were identified in a multivariate analysis as independent risk factors for recurrences of atrial arrhythmias.

Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa.

BACKGROUND: Being highly fragmented and low in concentration, isolation of good quality RNA from sperm cells is a big challenge. Attempts have been made to evaluate various sperm RNA isolation methods from purified buffalo bull sperm cells. METHODS: Both, non-membrane and membrane-based methods have been evaluated for isolating RNA from Murrah buffalo sperms and compared for their respective efficacies. The traditional TRIzol, TRIzol-heat lysed (H-TRIzol) and cocktail of TCEP-RLT lysis buffer (Qiagen RNeasy mini kit)-TRIzol (C-TRIzol) based isopropanol isolation methods have been evaluated. RESULTS: H-TRIzol yielded best results among conventional methods. The combined T-RLT RNA isolation protocol yielded best quality and quantity compared to other membrane-based methods, due to high lytic property of cocktail of lysis reagents, necessary for complete breakdown of sperm membrane and RNA binding membrane for RNA isolation. Combined lysis performed by treatment with RLT-T and T-RLT differing in order of reagents used were also evaluated. T-RLT combination giving better results compared to RLT-T due to high gDNA contamination and membrane clogging in later protocol steps. CONCLUSION: Overall, in terms of total RNA quantity and quality per million spermatozoa, the heat-lysed TRIzol method (H-TRIzol) performs best among RNA separation techniques employed and is also quite easy to perform. This comparative evaluation of sperm RNA isolation protocols can be useful in deciding the best protocol for isolation of good quality and high concentration sperm RNA from buffalo semen, for transcriptome and other downstream studies.

Impact of zinc, copper, manganese and chromium supplementation on growth performance and blood metabolic profile of Sahiwal (Bos indicus) male calves.

This study aimed to see the effect of oral supplementation of specific trace minerals mixture on the growth, immunity, and reproductive development of indigenous growing bull calves. Eighteen Sahiwal bull calves, with an average age of 6 months were chosen and divided into three groups. Group 1 was fed with a basal diet, Group 2 was provided with an additional specific trace mineral supplement to achieve a diet containing 70 ppm of Zn, 17.50 ppm of Cu, 65 ppm of Mn, and 1.75 ppm of Cr. Group 3 received a 25% extra supplement to achieve a diet containing 87.50 ppm of Zn, 21.87 ppm of Cu, 81.25 ppm of Mn, and 2.18 ppm of Cr. The experiment was carried out for a total of 180 days. According to the findings, there was no significant impact of specific trace minerals supplementation on the animals' body weight, morphometric parameters, dry matter intake, average daily gain, nutritional value, digestibility and nitrogen retention. However, higher levels of Zn, Cu, and Mn led to increased (p < 0.05) total retention, while Cr retention remained the same. Serum mineral concentrations of Zn, Cu, and Mn increased significantly (p < 0.05) in G2 and G3 compared to the G1 group while Ca, P, and Cr had no significant change. Blood plasma glucose, albumin, globulin, and total protein showed no significant differences. Plasma alkaline phosphatase activity improved significantly (p < 0.05) in G2 and G3 but alanine transaminase, aspartate aminotransferase, hemoglobin, hematocrit, and IGF-1 remained unchanged. Superoxide dismutase activity, ferric-reducing antioxidant power, and total immunoglobulin concentration increased significantly (p < 0.05) in G2 and G3 groups, however, catalase activity and IgG count did not change among the groups. Mineral-supplemented groups (G2 and G3) showed a significant change (p < 0.05) in testosterone production during the 120th and the 180th day of the trial. Scrotal circumference and temperature gradient of the scrotal surface did not show any significant change. Supplementing growing bull calves with specific trace minerals above the basal level (70, 17.50, 65 and 1.75 ppm of Zn, Cu, Mn and Cr) has no direct beneficial effect on the growth parameters but can have positive effects on their antioxidant status, immunity and reproductive development as the related blood parameters were positively affected.

Examining the reproductive success of bull kelp (Nereocystis luetkeana, Phaeophyceae, Laminariales) in climate change conditions.

Climate change is affecting marine ecosystems in many ways, including raising temperatures and leading to ocean acidification. From 2014 to 2016, an extensive marine heat wave extended along the west coast of North America and had devastating effects on numerous species, including bull kelp (Nereocystis luetkeana). Bull kelp is an important foundation species in coastal ecosystems and can be affected by marine heat waves and ocean acidification; however, the impacts have not been investigated on sensitive early life stages. To determine the effects of changing temperatures and carbonate levels on Northern California's bull kelp populations, we collected sporophylls from mature bull kelp individuals in Point Arena, CA. At the Bodega Marine Laboratory, we released spores from field-collected bull kelp, and cultured microscopic gametophytes in a common garden experiment with a fully factorial design crossing modern conditions (11.63 ± 0.54°C and pH 7.93 ± 0.26) with observed extreme climate conditions (15.56 ± 0.83°C and 7.64 ± 0.32 pH). Our results indicated that both increased temperature and decreased pH influenced growth and egg production of bull kelp microscopic stages. Increased temperature resulted in decreased gametophyte survival and offspring production. In contrast, decreased pH had less of an effect but resulted in increased gametophyte survival and offspring production. Additionally, increased temperature significantly impacted reproductive timing by causing female gametophytes to produce offspring earlier than under ambient temperature conditions. Our findings can inform better predictions of the impacts of climate change on coastal ecosystems and provide key insights into environmental dynamics regulating the bull kelp lifecycle.