Target in Sight: A Comprehensive Review of Hydroxychloroquine-Induced Bull's Eye Maculopathy.

Purpose of Review: We review the latest screening and diagnostic techniques, and the most recent recommendations on the management of hydroxychloroquine retinopathy. Recent Findings: Hydroxychloroquine (HCQ) has been shown to cause retinal toxicity in a dose-dependent fashion. Early diagnosis is critical as the resultant retinopathy is not reversible. New imaging modalities, such as adaptive optics (AO), microperimetry, and retro-mode imaging, may show promise in the timely diagnosis of HCQ retinopathy. Summary: Automated visual fields and spectral-domain optical coherence tomography (SD-OCT) are the primary tests used in routine screening for HCQ retinopathy, but fundus autofluorescence (FAF) and multifocal electroretinogram (mfERG) have also been shown to be useful. A baseline ophthalmologic examination is recommended in all patients beginning long-term hydroxychloroquine therapy within the first year of starting therapy. Automated visual fields and SD-OCT should be included during this baseline exam in patients with pre-existing macular conditions. Afterwards, annual screening can be deferred for the first 5 years of HCQ treatment unless the patient has a major risk factor.

Analytical and clinical validation of diagnostic tests for the detection of leucospermia in beef bulls.

The objectives of this study were to validate diagnostic tests to detect polymorphonuclear cells (PMNs) in bull semen, and to determine the prevalence of leucospermia in beef bulls with varying semen quality. We hypothesized that all tests have comparable diagnostic value, and that leucospermia is more prevalent in unsatisfactory breeders in association with poor semen quality. For the analytical validation, one ejaculate was obtained from five bulls. Aliquots of 50 × 106 purified sperm were incubated in triplicate with six concentrations of purified bovine PMNs: 1) no PMNs, 2) 0.25 × 106 PMN/ml, 3) 0.5 × 106 PMN/ml, 4) 2.5 × 106 PMN/ml, 5) 5 × 106 PMN/ml, 6) 10 × 106 PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD45 immunolabeling. The number of leucocytes detected with the LEDT differed among treatments (P < 0.0001). The quantitative tests detected differences with the control treatment at a PMN concentration of ≥2.5 × 106 PMN/ml (P < 0.0001). Sperm motion parameters after 4 h of incubation at 38 °C were lower in samples with ≥5 × 106 PMN/ml (P < 0.05). For the clinical validation, semen samples from 305 beef bulls were evaluated. Unsatisfactory breeders (n = 83) had more CD45-positive cells (P = 0.016) and positive LEDT results (P = 0.008) than satisfactory breeders (n = 222). With CD45 immunostaining as the gold standard, the hemacytometer count had the highest clinical sensitivity (64.3 %) but the lowest specificity (73.3 %). A higher specificity was obtained with the peroxidase test (95.1 %) or semen cytology (98.8 %). In conclusion, the presence of ≥5 × 106 PMN/ml was associated with decreased semen quality in beef bulls. The hemacytometer count was the most sensitive bull-side test. But due to the low specificity, positive hemacytometer counts should be confirmed with the identification of peroxidase-positive cells or morphological identification of leucocytes on semen cytology. The CD45 immunostaining is the gold standard for the diagnosis of leucospermia in bulls.

Red Bull Energy Drink Impact on Salivary Glands in Wistar Rats: Can Blueberry Extract Reverse the Damage?

Energy drink (ED) consumption has become increasingly popular. Due to a lack of evidence, it was crucial to assess the effects of Red Bull (RB) consumption on the rat submandibular salivary gland and the potential therapeutic impact of blueberry (BB). Thirty rats were randomly assigned to five groups. Group 1 (Control) received distilled water. Group 2 (RB) received RB (10 mL/100 g/day) for 8 weeks. Group 3 (BB) rats were administered BB (500 mg/day for 8 weeks). Group 4 (RB + BB (L)) received RB for 8 weeks, and from the 5th week, were concurrently given BB (250 mg/day) for 4 weeks. Group 5 (RB + BB (H)) received RB for 8 weeks, and from the 5th week, were concurrently given BB (500 mg/day) for 4 weeks. At the end of the experiment, blood samples were collected, the animals were euthanized, and their submandibular salivary glands were harvested. Oxidative stress markers (MDA, GPx, CAT, and SOD) were assessed in both serum and tissue. Inflammatory markers (TNF-α, IL-6, and IL-10) were quantified in tissue. Submandibular gland specimens were prepared for light microscopy, and immunohistochemical staining was performed using anti-α-SMA. RB consumption resulted in a significant increase in MDA, TNF-α, IL-6, and IL-10, while GPx, CAT, and SOD levels decreased significantly. Degenerative changes in the gland's structure were observed in the RB group. A significant increase in α-SMA immunoreaction was detected in myoepithelial cells. Administration of BB, particularly at a high dose, ameliorated the aforementioned findings. In conclusion, blueberry administration exhibited therapeutic effects due to its antioxidative and anti-inflammatory properties.

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.

Higher abundance of DLD protein in buffalo bull spermatozoa causes elevated ROS production leading to early sperm capacitation and reduction in fertilizing ability.

BACKGROUD: Before fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm's ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction. RESULTS: In this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa. CONCLUSIONS: The present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize.

Quality and kinematic characteristics of Bali bulls frozen semen with purified green tea extract epigallocatechin-3-gallate antioxidant addition in diluent.

Background: The preservation of semen quality and kinematic characteristics during cryopreservation is crucial for the reproductive success and genetic management of livestock, particularly in Bali bulls. Aim: This study aimed to investigate the effect of adding purified green tea extract antioxidant Epigallocatechin-3-gallate (EGCG) in tris egg yolk diluent on the quality and kinematic characteristics of frozen semen from Bali bulls. Methods: Fresh and frozen semen samples were obtained from Bali bull and divided into four different treatment groups. P0 contained semen samples + diluent, while P1 to P3 consisted of semen samples + diluent supplemented with EGCG levels of 0.1, 0.15, and 0.2 mg/100 ml, respectively. Data were analyzed using One-way ANOVA and followed by Duncan's test if significant differences were found (p<0.05). Parameters observed included the assessment of fresh semen quality, kinematic analysis, post-thawing sperm viability, and abnormality. Results: The results indicated that the assessment of fresh semen quality showed macroscopic and microscopic semen quality according to SNI 4869-1:2021. Kinematic analysis revealed significant differences in DSL and STR parameters between P0 and P3 (p<0.05). EGCG supplementation also caused significant differences in motility between P0 and P3 (p<0.05). Viability and spermatozoa abnormality with EGCG supplementation did not show significant differences (p>0.05). Conclusion: The best results for motility, kinematics, and sperm morphology variables were found in P1 as it did not exhibit a decrease in motility, kinematics, and sperm morphology. Viability did not show significant differences between P1, P2, and P3, but the best results were found in P2 as it did not exhibit a decrease in viability with mean and standard deviation (66.84 ± 7.88). Abnormality variables also did not show significant differences between P1, P2, and P3, but the best results were found in P2 as it did not exhibit a decrease in abnormality with mean and standard deviation (23.80 ± 7.36).

Effect of oregano essential oil on intestinal immunoglobulin G in Holstein dairy bulls.

Introduction: Immunoglobulin G (IgG) is important in mediating humoral immunity and in the maintenance of immune homeostasis in the intestinal mucosa. Oregano essential oil (OEO) is a natural herbal extract that possesses antimicrobial, antioxidant, anti-inflammatory, and immunomodulatory properties. As the effects of OEO on intestinal mucosal immunity in Holstein dairy bulls remained unclear, we investigated the effect of dietary supplementation of OEO on IgG levels and IgG+ cells residing in the intestinal tract in Holstein dairy bulls. Methods: Twelve Holstein bulls in good health of approximately 10 months of age were selected for the experiment and randomly equally divided into two groups. The control (CK) group was fed a basal ration, and in the OEO group, the basal ration was supplemented with OEO (20 g/head/day). After 300 days of feeding, tissue samples of the jejunum, ileum, and colon of the bulls in each group were collected for histopathological analysis, immunohistochemistry, and enzyme-linked immunosorbent assays, respectively. Results: The jejunum, ileum, and colon of bulls in the CK group had obvious pathological damage, whereas the structure of each intestinal segment was clear and intact. In the OEO group, pathological damage was significantly reduced. IgG+ plasma cells were diffusely distributed in the lamina propria of the jejunum, ileum, and colon in the CK and OEO groups, with no significant difference between the groups. OEO supplementation significantly reduced the number of IgG+ plasma cells in each intestinal segment, with the highest decrease rate being noted for the ileum (22.87%), followed by the colon (19.45%) and jejunum (8.52%). ELISA test results and immunohistochemical results were mutually verified. The change in IgG content was consistent with the trend of change in the number of IgG+ plasma cells. Discussion: Our findings suggest that OEO supplementation does not alter the diffuse spatial distribution of IgG+ plasma cells in the intestines of Holstein dairy bulls, but lowers immunoglobulin levels to normal levels, significantly reduces intestinal damage, and may enhance mucosal immune defence barrier function by inhibiting inflammatory reactions.

Effect of cryopreservation and semen extender on extracellular vesicles isolated from bull semen.

Introduction: Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility. Methods: EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition. Results: Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender. Conclusion: This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.

Effect of exogenous cellulase pretreatment of straw-type fermented total mixed ration on digestibility and ruminal fermentation in growing beef cattle.

Objective: This study aimed to assess the impact of corn straw-based unfermented and fermented total mixed rations (TMR) supplemented with exogenous cellulase on the in vitro fermentation characteristics, growth performance, feeding behavior, apparent digestibility, rumen fermentation and digestive enzyme activities of Chinese Simmental bulls. Methods: Unfermented (direct spraying of exogenous cellulase onto TMR, TMR) and fermented (exogenous cellulase fermentation for more than 7 d, fermented total mixed rations [FTMR]) TMR were collected, dried, powdered and used as fermentation substrates. The fermentation liquid was ruminal fluid collected from Chinese Simmental bulls. The artificial rumen culture fluid were continuously cultured in vitro for 48 h. Based on the diets they were fed, 24 healthy Chinese Simmental bulls (average weight of 495.93 ±10.89 kg) were randomly divided into two groups, with 12 bulls in each group, which were fed TMR or FTMR. The study lasted 56 d. Results: In in vitro experiments, the neutral detergent fiber degradability and total volatile fatty acid, propionate, iso-butyrate, iso-valerate and valerate concentrations were greater in the FTMR group (p<0.05) than in the TMR group. However, the methane production, pH and A/P of the FTMR group tended to be lower (p<0.05) than those of the TMR group. In the in vivo experiments, the average daily gain, eating rate, and feed efficiency of the FTMR groups were greater (p<0.05) than those of the TMR group. Similarly, the NDF degradability of the FTMR group was greater (p<0.05) than that of the TMR group. Compared to those in the TMR group, the concentrations of total volatile fatty acids, iso-butyrate, propionate and butyrate were greater in the FTMR group (p<0.05), and the A/P ratio was lower (p<0.05). Similarly, cellulase, xylanase, and ß-glucosidase activities were greater (p<0.05) in the FTMR group than in the TMR group. Conclusion: Corn straw-based fermented total mixed rations supplemented with exogenous cellulase play a vital role in decreasing the structural carbohydrate content of TMR and ruminal methane production in vitro, improving nutrient digestion and absorption, optimizing rumen fermentation, and improving the growth performance of beef cattle.

Age-dependent variations in proteomic characteristics of spermatozoa in Simmental bull.

Increasing the age of bulls results in a decrease in reproductive function, including a reduction in sperm quality, which plays a vital role in determining the fertility of bulls. Through a proteomic approach, this research aims to analyze the influence of age factors on various proteomes contained in bull sperm. Frozen semen samples from Simmental Bulls were categorized into three age groups: two, four, and ≥10 years old. Subsequently, the post-thaw sperm cells obtained were separated based on molecular weight using 1D-SDS-PAGE. Peptides extracted from the bands produced in each age group were subjected to LC-MS/MS analysis. A total of 72 protein types were identified, with 45 being detected in the 4-year-old group and 41 expressed in both the 2 and ≥10-year-old groups. The results provided insights into proteins' role in sperm metabolism across all age groups. Specifically, the 2-year-old group exhibited the expression of proteins associated with acrosome assembly and spermatid development (SPACA1). In contrast, those in the 4-year-old group were linked to motility (PEBP4) and sperm decapacitation factor (PEBP1). Proteins expressed in the 2 and -year-old groups were discovered to be involved in fertilization processes (TEX101). In contrast, the ≥10-year-old age group was associated with hyperactive movement related to capacitation (Tubulin). In conclusion, age influenced the differences observed in the proteomic profile of post-thaw Simmental bull sperm using the 1D-SDS-PAGE tandem LC-MS/MS approach.

Use of presence vs absence of symphyseal teeth in jaws for the forensic analysis of bites by large traumatogenic shark species.

Identifying the species of shark responsible for a bite on humans is both complex and important for understanding and managing the shark risk. Depending on the species, tiny teeth may or may not be present in the symphyseal space at the junction of the upper and lower half-jaws. In the case of bites, these tiny teeth (if present) often leave specific marks that may enable species to be quickly and reliably distinguished. We first present the anatomo-morphological characteristics of the jaws of the three most traumatogenic species for humans which are the white, tiger, and bull sharks. The white shark has no symphyseal teeth, while the tiger and bull sharks do. On the basis of three confirmed real case studies involving those species, we then show that for the white shark, the wide symphyseal space between the first two teeth of each jaw usually leads to wounds including the presence of (quite) large flesh flaps, without any tooth imprint. Conversely, wounds following bites made by the tiger and bull sharks will generally leave characteristic small imprints of symphyseal teeth, especially in the case of incomplete or superficial bites. Although not systematic, this diagnostic approach provides fast, reliable, and clean results. The discrimination between two species with symphyseal teeth can then be made on the basis of complementary anatomic information such as jaw curvature and details linked to the anatomy of the teeth themselves, as well as the ecological context.

Theileria orientalis Ikeda infection does not negatively impact growth performance or breeding soundness exam results in young beef bulls at bull test stations.

Introduction: Theileria orientalis Ikeda genotype is an emerging cattle disease in the US. Since 2017, when T. orientalis Ikeda was discovered in beef cattle in two counties in Virginia, cattle infections have risen to include ~67% of Virginia counties and 14 states. Consistent with New Zealand studies, many infected herds in Virginia were >90% positive upon initial testing without overt evidence of infection. Central bull tests present a unique opportunity to study the effects of T. orientalis Ikeda infections, as bulls from multiple source herds are consolidated. The objective of this study was to determine if infection with T. orientalis Ikeda affected the average daily gain (ADG), adjusted yearling weight (AYW) and breeding soundness of bulls at two test stations in Virginia over a period of years. Materials and methods: The bulls were fed and housed similarly to compare their growth performance and breeding soundness. For T. orientalis Ikeda testing, DNA was extracted from whole blood for quantitative polymerase chain reaction. Results: The number of bulls infected with T. orientalis Ikeda at initial delivery to the stations increased significantly over the years studied. Multivariable linear regression models, using Angus bulls from Virginia test stations, indicated no significant effect on ADG or AYW in bulls that became test positive during the test or were positive for the duration, compared to Angus bulls that were negative for the duration. At LOC A, the odds of passing a breeding soundness exam (BSE) were not significantly different for bulls that turned positive during the test or were positive for the duration, compared to bulls that were negative for the duration of the test. At LOC B, bulls that became positive during the test were 2.4 times more likely (95% CI: 1.165-4.995, p = 0.016) to pass their BSE compared to bulls that remained negative throughout the test. Discussion: We do not suppose that an obscured infection of T. orientalis Ikeda is protective for bulls to pass a BSE. However, this study demonstrates an obscured infection of T. orientalis Ikeda does not negatively affect weight gain or achievement of a satisfactory BSE rating at the central bull test stations in Virginia.

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.

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.

Metagenomic identification of bull semen microbiota in different seasons.

A seasonal effect on sperm quality parameters was observed previously. Although identification of the bull semen microbiota by 16S rRNA sequencing was performed previously, it has not been carried out in commercial semen samples from different seasons, and its connection with sperm quality parameters has not been evaluated yet. The objectives in this study were; (i) to evaluate diversity of bull semen microbiota and sperm quality parameters in different seasons, and (ii) to find if specific bacteria were associated with seasonal differences in specific sperm quality parameters. Bull semen microbiota was identified in 54 commercial bull semen samples from 3 seasons (winter, spring, summer). Sperm quality was analysed by Computer Assisted Sperm Analyses (CASA) and Flow Cytometry (FC). From 28 phyla in all samples, six phyla were identified in samples from all seasons, with observed seasonal differences in their distribution. At genus level, 388 genera were identified, of which 22 genera had a relative abundance over 1 % and showed seasonal differences in bacterial diversity, and 9 bacteria genera were present in all seasons. Differences between spring and summer (P < 0.05) were observed for live hydrogen peroxide positive sperm cells. A trend towards significance (0.10 > P > 0.05) was observed for some CASA kinematics (VCL and LIN) and FC parameters (High respiratory activity, and live hydrogen peroxide positive sperm cells) between seasons. Nevertheless, associations between sperm quality parameters and specific bacteria were observed in spring.

First report of a neurofibrosarcoma in the penis of a bull.

Reproductive problems in cattle are frequent and have an important impact on production. In addition, inflammatory, traumatic and other diseases may be followed by the development of tumours, which are a cause of culling of breeding males. The main types of tumours diagnosed in the bull penis are fibropapilloma and squamous cell carcinoma. The aim of this study was to characterize a case of a tumour in the glans penis of a bull from a dairy farm in Santa Fe, Argentina. The neoplastic tissue was stained with haematoxylin and eosin and then analysed by immunohistochemistry to reveal its characteristic phenotype. Results showed positivity to vimentin, neuron specific enolase, proliferating cell nuclear antigen, S100 and glial fibrillary acidic protein. This suggested that the tumour was a neoplasm of neural origin, classified as neurofibrosarcoma, a peripheral nerve sheath tumour, here reported in the penis of a bull for the first time.

A systematic review of predictive, diagnostic, and prognostic biomarkers for detecting reproductive diseases in cattle using traditional and omics approaches.

Reproductive diseases and illnesses pose significant challenges in cattle farming, affecting fertility, milk production, and overall herd health. In recent years, the integration of various omics approaches, including transcriptomics, proteomics, metagenomics, miRNAomics, and metabolomics, has revolutionized the study of these conditions. This systematic review summarised the findings from studies that investigated reproductive disease biomarkers in both male and female cattle. After extracting 6137 studies according to exclusion and inclusion criteria, a total of 60 studies were included in this review. All studies identified were associated with female cattle and none were related to reproductive diseases in bulls. The analysis highlights specific biomarkers, metabolic pathways, and microbial compositions associated with bovine reproductive disease conditions, providing valuable insights into the underlying molecular mechanisms of disease. Pro-inflammatory cytokines such as IL-1ß, IL-8, IL-4, IL-6, TNFα and acute-phase response proteins such as SAA and HP have been identified as promising biomarkers for bovine reproductive diseases. However, further research is needed to validate these markers clinically and to explore potential strategies for improving cow reproductive health. The role of bulls as carriers of venereal diseases has been underestimated in the current literature and therefore needs more attention to understand their impact on infectious reproductive diseases of female cattle.

Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls.

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7 nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity.

Tritrichomonas foetus testing in bulls.

OBJECTIVE: To provide a video tutorial on how to perform preputial scraping for the testing of Tritrichomonas foetus in bulls. ANIMALS: Postpubertal bulls. METHODS: A preputial scraping device is inserted in the prepuce, and back-and-forth scraping movements are made to collect smegma and T foetus organisms. The sample is placed in an appropriate media and prepared for shipment to the diagnostic laboratory. RESULTS: Preputial sampling is an effective method for diagnosing T foetus in infected bulls. CLINICAL RELEVANCE: Bulls are the primary carriers of T foetus, a protozoan parasite responsible for bovine trichomoniasis. By sampling and testing bulls, veterinarians can identify infected animals and implement control measures to prevent the spread of the disease within herds. Trichomoniasis can lead to significant reproductive problems in cattle, including infertility, embryonic death, and abortions. Sampling bulls allows for the detection of infection, enabling prompt intervention to protect the reproductive health of the entire herd. Trichomoniasis outbreaks can result in substantial economic losses for cattle producers due to reduced conception rates, increased calving intervals, and decreased calf crops. Sampling bulls helps to mitigate these losses by identifying and removing infected animals from the breeding pool, thereby minimizing the spread of the disease and its associated reproductive inefficiencies. In many regions, T foetus testing in bulls is a regulatory requirement for cattle movement and trade. Sampling bulls and obtaining negative test results are often necessary for obtaining health certificates and complying with interstate or international movement regulations.

Selection based on the Breeding Soundness Evaluation is associated with the improvement of the reproductive quality of young Nellore bulls.

Breeding soundness evaluation (BSE) is the best methodology to estimate the fertility potential of future bulls and performing indirect selection for their fertility. However, the outcome of the BSE is influenced by several factors, including genetics, environment, and BSE guidelines. Herein, in this retrospective study, our first aim was to characterize the reasons for failure in 46,566 BSE from 2-year-old beef Bos indicus bulls (Nellore) born from 1997 to 2018. Our second aim was to determine whether or not BSE was associated with reproductive potential improvement of the bulls over the years. Due to changes in the BSE criteria, we used the same dataset, but only bulls born from 2002 to 2018 were included resulting in 35,856 BSE. For the second aim, the effect of the year and farm were included in the model of the multivariate logistic regression. We also determined if the main reasons for BSE failure decreased over time. Bulls were classified as approved (satisfactory potential breeders and qualified for natural breeding service) and not approved (deferred and unsatisfactory potential breeders). The reasons for BSE failure in Nellore bulls were poor semen quality (53.1 %) and physical defects (46.9 %), with the main physical defect being testis abnormalities (19.7 %). The overall percentage of bulls approved each year was 87.1 %, with no improvement over the years of study. However, the percentage of approved bulls at the first BSE increased over the years (P < 0.05). This increase was evident by a reduction in the difference between the overall percentage of the bulls approved vs the percentage of bulls approved at the first BSE. Furthermore, there was an increase in the percentage of bulls classified as satisfactory potential breeders in the BSE and an evident decrease in the percentage of bulls qualified only for natural breeding service (P < 0.05). In addition, an increase of the scrotal circumference (SC) of the herd was found (P < 0.05). These results indicate the overall quality of the bulls improved over the years. To associate and identify the main sperm abnormalities, 3461 not approved bulls were clustered. The most frequent defects were strongly coiled tail spermatozoa, proximal droplets, and acrosomal defects. Overall, there was no change in the frequency of bulls not approved by the sperm morphology nor the frequency of the main sperm abnormalities over the years. Nevertheless, the frequency of the defects remained very low, implying they were controlled. Additionally, abnormalities in the testis decreased over the years and was associated with the increase in the SC of the herd and a decrease of culled bulls due to low SC. In conclusion, this study demonstrates that there is an association between implementation and use of BSE with improvements in the reproductive quality of future generation bulls.