The spermatozoa of group 1 overall retained more mitochondria (MitoTrackerGreen) and mtDNA copies, irrespective of the fraction. Regardless of the initial ejaculate quality, the motile sperm contained significantly more mitochondria and mtDNA copies. The same trend was observed for mitochondrial membrane potential , where motile sperm displayed high ΔΨm. These results stay in agreement with transcript-level evaluation  of antioxidant enzymes , which protect cells from the reactive oxygen species. An overall higher level of glutathione synthetase (GSS) mRNA was noted in group 1 bulls, suggesting higher ability to counteract free radicals. No differences were noted between basal oxygen consumption rate (OCR) (Seahorse XF Agilent) and ATP-linked respiration for group 1 and 2 bulls. In conclusion, mitochondrial content and activity may be used as reliable markers for bovine sperm quality evaluation.Standard sperm evaluation parameters do not enable predicting their ability to survive cryopreservation. Mitochondria are highly prone to suffer injuries during freezing, and any abnormalities in their morphology or function are reflected by a decline of sperm quality. Our work focused on describing a link between the number and the activity of mitochondria, with an aim to validate its applicability as a biomarker of bovine sperm quality. Cryopreserved sperm collected from bulls with high (group 1) and low (group 2) semen quality was separated by  The main causes will oscillate within the genetic/epigenetic factors, sperm-carried RNA, sperm proteome, sperm ultrastructure, centrioles, and mitochondria (mt).Gamete quality (sperm and oocytes) is one of the critical factors directly determining reproductive success both in vivo and in vitro. The role of sperm in animal reproduction is not solely restricted to providing the haploid male genome to the oocyte. At the time of fertilisation, it delivers several components, including a versatile population of RNAs. Standard semen analysis  rely on sperm concentration count, morphology, and motility assessment. Yet, these parameters are not universal for predicting fertilisation success. It was shown that about 50% of couples undergoing an IVF (in vitro fertilisation) programme with failed fertilisation had normal pre-IVF semen analysis ,2. A simpower plants” of a cell, the role of mitochondria for male fertility is directly linked to motility, but these organelles are also crucial for sperm hyperactivation, capacitation, acrosin activity, acrosome reaction, and DNA integrity. Mitochondria produce physiological levels of reactive oxygen species (ROS), which address redox-related cell signalling pathways [idiopathic male infertility [Recent research in animal and human sperm physiology is placing an increasing interest on the mitochondrion as a biomarker of sperm health and fertility ,5,6,7. Apathways ,9,10. Elpathways . Mitochopathways ,13. Moreertility . Studiesertility . Exposinertility . Impaireertility . A high ertility .Agilent Seahorse XF analysers may be applied to evaluate sperm mt-respiration.Given that the potential for cell movement is dependent on metabolic energy resulting from mitochondrial functions, evaluation of mitochondrial membrane potential may be indispensable to predict the quality of sperm motility . ΔΨm is somatic cell type” mitochondria; late primary and secondary spermatocytes and early spermatids contain “condensed” and metabolically more efficient mitochondria. Late spermatids and mature spermatozoa have an intermediate-type organelles [Fundamentally, the sperm mitochondrial structure and function are similar to that of the somatic cells; however, these organelles do show some morphological and functional changes throughout spermatogenesis. Flow cytometry analysis revealed variations in DNA content, chromatin structure, and dramatic changes in the mitochondria of mouse germ cells during maturation from spermatogonia to elongated spermatids . Changesganelles . Studiesganelles .During spermatid differentiation, a subset of mitochondria line the sperm midpiece, while the rest undergo phagocytic degradation in the Sertoli cells . The mecConcluding from the available data, it is evident that standard methods of semen evaluation relying on concentration, movement, morphology, and sperm cell membrane integrity analyses do not allow drawing full conclusions on the ability of a sperm cell to fertilise an oocyte. Except for human-assisted reproduction, cattle is the only animal species for which insemination with cryopreserved semen, IVF, and embryo production are done on a large scale . TherefoWielkopolskie Centrum Hodowli i Rozrodu Zwierząt w Poznaniu, Poznańska 13, 63-004 Tulce, Poland (WCHiRZ). Semen samples were prepared according to the standard AI protocol for cryopreservation, packed in AI straws, frozen, and stored in liquid nitrogen. For each male, all of the straws were obtained from the same batch (production day) of collected semen. To secure comparable initial concentrations of sperm, one straw was used per single experimental procedure; i.e., one strew was thawed for the Mitotracker Green procedure, one was thawed for the JC-1 staining, one was used for RNA extraction, and one was used for mtDNA analysis. The experiment was performed on 2 groups of animals: (1) bulls of high AI performance (n = 7) and sperm quality , and (2) AI bulls (n = 10) that were temporarily disqualified from semen production, due to severely decreased parameters such as ejaculate volume, sperm concentration, and motility. These bulls were also subjected to veterinary examination, 5/10 animals revealed inflammation-related problems in testes or accessory glands. The remaining 5 animals did not have medical symptoms but had long intervals between semen collection and less physical activity. In all cases, the semen samples were additionally subjected to the standardised CASA analysis, which confirmed the results of standard semen analysis performed at the AI station. The CASA analysis was performed with Sperm Class Analyser® System  with a module set for frozen/thawed bull semen.Bull semen was obtained from the local centre for bull breeding and artificial insemination: TM  followed by 3 min centrifugation at 300× g. The supernatant was removed, and the remaining pellet (sperm) was used for further procedures.Prior to analysis, the AI straws were thawed , and the spermatozoa were cleared off the cryoprotectants by washing in 500 μL of BoviWashSwim up was done according to the standardised protocol [g). The obtained pellet was layered under 300 μL Sperm-TALP medium  in an Eppendorf vial, positioned at an angle of 45°, and placed for 60 min in an incubator . Finally, the upper fraction  was gently separated from the lower fraction.protocol  with somg). Then, the pellet was suspended in 200 μL MitoTracker Green dye solution (20 nM in Hepes) and incubated for 60 min at 37 °C.The staining was done according to the manufacturer’s protocol . Prior to the procedure, the sperm were fixed in 2% PFA in PBS , washed in phosphate buffered saline (PBS), and centrifuged .The staining was done according the manufacturer’s protocol . The sperm samples were incubated for 60 min at 37 °C in 300 μL Sperm-TALP medium containing 8 μM of the JC-1 dye. After incubation, the samples were briefly washed in PBS to remove the excess of the dye  Airyscan system Zeiss . Filter sets used included a 488 nm filter with band pass 491–553 nm (laser Argon2), 410–480 nm DAPI (laser Diode 405), and 543 nm with band pass 584–632 nm (laser HeNe1), and the Plan-Apochromat 63×/1.4 Oli DIC M27 objective was used.MitoTracker Green and the JC-1 dyes. MitoTracker Green dye (490/516 nm excitation/emission) was excited by the 488 nm laser, and the fluorescence signal was detected in channel 2 (532/555 nm). In case of JC-1 , the fluorochrome was also excited by the 488 nm laser. At high ΔΨm, JC-1 forms complexes known as J-aggregates, which emit an orange–red fluorescence with a maximum at 595 nm , at low ΔΨm, JC-1 remains in the monomeric form emitting green fluorescence with a maximum at 530 nm . The final ΔΨm of a cell is established as a ratio between the red and the green fluorescence.The analyses were performed on Amnis FlowSight , separately for the Flow cytometry acquisition speed was set up to low speed and the highest resolution. The acquisition was performed at 20× magnification using the INSPIRE software. Approximately 30,000 single cell events were acquired from each bull sperm sample suspended in 100 μL PBS. The obtained data were analysed using the IDEAS software. First, the cells (spermatozoa) were gated from debris using a plot of bright-field channel 1 “Area” versus channel 1 “Aspect Ratio” . Next, t2O) for 10 min on ice, followed by 1× wash in PBS (500 μL) and centrifugation at 1500× g, buffer based on Bianchi, et al. [6 sperm cells) and 8 to 21 ng/mL for fractions separated by the swim up procedure. Additionally (at the optimisation step), to verify the efficiency of our extraction protocol, RNA integrity (RIN) was measured with Agilent 2100 Bioanalyzer using Agilent RNA 6000 Pico chips. The obtained the RIN values were close to 3, which is indicative and characteristic for high-quality sperm [RNA was extracted with the RNeasy Micro Kit  according to the manufacturer’s protocol, with important modifications at the cell lysis step. Efficient lysis is crucial for successful RNA isolation from spermatozoa. Sperm cells  are particularly resistant to the standard lysis conditions of the commercially available kits . Moreover, in case of semen, there is always a danger that the sample might contain a certain amount of the somatic cells. After thawing and initial washing, the sperm samples were incubated in 500 μL of the Somatic Cell Lysis Buffer  according to the manufacturer’s protocol. cDNA was synthesised using both Random Hexameres and OligodT. Samples were stored at −20 °C.SOD1), peroxiredoxin (PRDX1), and glutathione synthetase (GSS) was evaluated for each of the experimental groups. The reactions were carried out on a Roche Light Cycler 96 instrument . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) served as the reference genes. TaqMan probes and primers  and frozen at −20 °C. DNA concentration was measured with NanoDrop c2000 .DNA was isolated with a High Pure PCR Template Preparation Kit  according to the manufacturer’s protocol. Sperm samples were lysed with Tissue Lysis Buffer  and incubated with Proteinase K and Binding Buffer . The following steps included serial washes on filter columns and elution of total DNA (with 150 μL of the Elution Buffer), according to the protocol provided. All centrifugation steps were done at 7500× CYTB) gene and nuclear β-actin gene (ACTB). Primer and TaqMan probe sequences  are listed in ACTB and CYTB gene copy numbers. The CYTB copy number per single sperm cell  was established as a ratio of CYTB expression level to ACTB gene expression.A real-time PCR and absolute quantification module was used to calculate the number of copies of mtDNA per single spermatozoa. Quantification was done based on the standard curve method. A series of 10-fold dilutions of known concentrations  were prSeahorse XF Cell Mito Stress Test was applied. The compounds used during the measurements enabled the assessment of the key parameters of mitochondrial function. As there in no commonly used protocol for OCR/ATP-linked respiration and overall mitochondrial function evaluation in spermatozoa, the extensive optimisation of the procedure was performed (data not shown). Among the optimised elements, cell concentration from 105 to 106 per well, a well coating matrix (fibronectin versus ConA), sperm capacitation (Sperm-TALP +1 μg/mL Heparin pre-incubation), energy source (glucose vs. fructose), and a broad range of carbonyl cyanide 4-trifluoromethoxy-phenylhydrazine (FCCP) concentrations (0.125–2.0 μM) were tested. The below optimised protocol was chosen as the one showing the minimal divergence, and the sperm cell response was the most reliable.For oxygen consumption rate and ATP-linked respiration evaluation, the g for 5 min. The pellet was re-suspended in Agilent Seahorse XF Base Medium  with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose, pH was adjusted to 7.4.Semen samples (group 1 and 2) recovered from the AI straws were thawed in a 37 °C water bath for 1 min and diluted in 1 mL of Sperm-TALP medium. The sample was centrifuged at 150× 6 sperm cells in 50 μL were seeded per well of 8-well XF Seahorse Cell Culture Microplates  coated with fibronectin . To ensure spermatozoa attachment, 70× g plates were centrifuged for 1 min, and subsequently, 150 μL of assay medium  per well was added. The prepared microplates were incubated for 20 min at 37 °C.First, 8 × 10Seahorse XF Cell Mito Stress Test kit . After the sensor cartridge calibration, the microplate was placed in the Seahorse XF device, and the basal respiration was detected as baseline OCR. During the next step of the analysis, the OCR was measured after sequentially automatic injections of (1) Oligomycin A , (2) FCCP , and (3) Rotenone .The metabolic analysis was performed according to the procedures described in the 4 cells were seeded per well the day before the analysis to ensure proper cell adhesion to the plastic cell-treated surface of the microplate.For proof-of-concept analysis (control of the assay), the A549 human eukaryotic cell line was analysed. The scheme of A549 analysis mirrored the protocol applied for sperm cells with one exception; 4 × 10p-values were adjusted for multiple comparisons using Benjamini–Hochberg correction method.Due to the lack of normal distribution, the significance of differences between group 1 and group 2 bulls was assessed using two-sample Wilcoxon test available in rstatix package . The p-vMitoTracker Green and JC-1 staining), for the studied groups of bulls of high (1) and low (2) sperm quality parameters  were subjected to statistical analyses. Each time, the values of fluorescence intensity detected for single spermatozoa  were used for calculations. Differences in fluorescence intensity between these groups were assessed using mixed models equations with the random bull effect, as analysing multiple sperm cells per bull resulted in repeated observations for each individual. The statistical analyses were performed in the lme4 package [MitoTracker Green and JC-1 stainings. Prior to the analyses, the fluorescence intensities were normalised to meet the assumptions of the applied statistical analyses. Fluorescence intensities obtained for the MitoTracker Green staining were transformed by taking the square root of the original values. In case of JC-1, the ratio of red (emission detection 610/630 nm) to green (emission detection 532/555 nm) fluorescence, which represents the state of the mitochondrial condition (ΔΨm) was log-transformed and used in further analyses. For analyses, the following general model was used:MitoTracker Green staining, or a natural logarithm of a ratio between the red and the green fluorescence detection channels for the JC-1 dye, recorded for the l single spermatozoa. The i was a fixed effect of the k-th bull’s sperm quality parameters that could take two values  or poor-quality (group 2)) and j was a fixed effect indicating whether the evaluated sperm cell originated from the upper or the lower swim up fraction. The k, and p ≤ 0.05 were considered statistically significant.The results obtained by flow cytometry  were subjected to testing for normal distribution using the Kolmogorov–Smirnov and Shapiro–Wilk tests. The differences in the mRNA levels were analysed using the nonparametric two-tailed Mann–Whitney U test. The differences in the mtDNA copy number between the experimental groups were tested using the Kruskal–Wallis and the two-tailed Mann–Whitney U tests. All data with p ≤ 0.05 and p ≤ 0.008.As described in the M&M section, the semen samples were collected from two groups pf bulls: (1) high AI performance and sperm quality and (2) temporarily disqualified from semen production, due to severely decreased semen/sperm parameters. The initial choice of animals was based on the standard semen evaluation protocol and the veterinary examination done at the WCHiRZ centre (data not shown). Since the initial laboratory evaluation was done on fresh semen, and the assessed parameters included only ejaculate volume, sperm concentration, movement assessment, and morphology, CASA analysis on frozen/thawed sperm was done to validate the observed differences between the experimental groups . CASA evMitoTracker Green), which allowed evaluating the overall mt number per single cell, based on the level of emitted fluorescence. The specificity of fluorescent labelling was validated by confocal imaging  mitochondria content  is needed for a sperm cell to secure its ability to fertilise the oocyte. For this reason, we have applied mt specific fluorescent staining ( imaging .p ≤ 0.01) than sperm cells of bulls assigned to group 2 (swim up. The results indicate that regardless of the studied bull group (high semen parameters versus low), the motile (upper) fraction contained significantly more mitochondria (p ≤ 0.01) than the lower, non-motile fraction (p ≤ 0.01) than bulls disqualified from AI semen collection (group 2), irrespective of the fraction  contained 3.102 times more mitochondria  displayed higher mtDNA copy number (p ≤ 0.05) than the lower fraction of group 2 bulls.The above results stay in agreement with the mtDNA copy number evaluation. On average, the upper fraction of group (1) bulls contained 27.75 copies of mtDNA, and 17.90 copies were calculated for the lower fraction. Similarly, in group (2) bulls, 21.01 and 10.54 copies of mtDNA were estimated for the upper and lower fraction, respectively. Generally, both the upper and the lower MitoTracker Green staining, JC-1 fluorescence detection revealed significant differences (p ≤ 0.01) between all of the analysed groups . Moreover, even the less motile, lower swim up fraction collected from group 1 bulls had significantly higher GSS mRNA content than the spermatozoa from the upper  fraction of group 2 bulls (p = 0.008). Significant differences were also noted when comparing the lower fractions of the two experimental groups (p = 0.045). An interesting observation was made of the SOD1 transcript abundance. No statistically significant differences were noted between the two swim up fractions obtained for high-performance bulls (group 1), but the spermatozoa of the upper (motile) fraction of group 2 contained more SOD1 transcript copies than the lower fraction (p = 0.035). Additionally, the higher SOD1 transcript level noted for group 2 (upper fraction) was also significantly higher than the SOD1 mRNA content in both of the fractions separated for group 1 .The level of oxidative stress is an important factor in determining male fertility. Due to the high rate of cell division and mitochondrial oxygen consumption, testicular tissue is particularly susceptible to the oxidative stress. It is critical to obtain a balance between the production of free radicals and its metabolism. Antioxidants prevent damage either by counteracting the free radicals or by preventing their formation in the testicular cells. The preventive antioxidant system incorporates enzymes such as SOD, GSS, and PRDX; thus, we have analysed and compared the level of the corresponding transcripts . The mRNSeahorse analyser) offer a real-time assessment of cell’s metabolic state and enable tracking potential impairment of the energetic pathways. We have used standard Seahorse protocol for Cell Mito Stress Test  and measurements of basal respiration. ATP-linked respiration and spare respiration capacity were done. Since we have aimed to pinpoint any potential differences in the OCR between high and low sperm quality bulls, the analyses focused on samples that were not subjected to the swim up separation. This approach was also taken due to a large number of spermatozoa required per each experimental well. Oligomycin treatment resulted in a drop of basal respiration within 20 min of administration, but the sperm cells did not show the standard response mitochondria to the FCCP injection. In standard conditions (in somatic cells), this should be reflected by a dynamic increase of the OCR due to the collapse of the mitochondrial membrane potential. Indeed, under the same conditions, this phenomenon was detected in our control assay . An example of such analysis is shown in It was suggested that bovine spermatozoa preferentially exploit OXPHOS as the main source of energy . The oxyswim up fractions [The mitochondrion is being recognised as an important biomarker of sperm quality, fertilisation potential and survival after cryopreservation ,7,41. Efractions ,42,43. WIn comparison to fresh ejaculate, the viability of frozen–thawed spermatozoa is still on average relatively low, and it differs significantly among the breeding bulls ,45,46. Tswim up fraction of bulls with high sperm quality parameters (group 1) contained significantly more mtDNA copies than the sperm of the lower (non-motile) fraction of group 2 bulls (27.75 versus 10.54 copies). Moreover, this trend was also conserved within both of the lower fractions, as non-motile sperm from group 1 had on average significantly more mtDNA copies (17.90) than the lower fraction of group 2 (10.54 copies). The differences between the two upper fractions were not statistically significant, but spermatozoa of the motile fraction of group 2 bulls had less copies of mtDNA than the upper fraction of group 1. Therefore, mtDNA copy number could become an indicator of defective/proper spermatogenesis, provided that species-specific, optimal mtDNA content will be established. The functional significance of limited mtDNA copy number in sperm still remains unclear. Plausibly, it could reduce the likelihood of ROS-mediated DNA damage.Depending on energetic demand, cells carry a different number of mitochondria, each containing a variable number of mtDNA copies . The typ6 white blood cells/mL in a semen sample [swim up and sperm washes prior to DNA extraction) eliminate this factor.The existing contradictions in the correlation of mtDNA copy number and male fertility status may arise from several factors such as species specific differences, the use of different methods to quantify the mitochondrial genome (Southern blot vs. PCR-based assays), or contamination with other cell types (immature sperm or leukocytes) that may be present in poor quality semen ,58. Leukn sample . In our MitoTracker Green accumulates in mitochondria regardless of their mitochondrial membrane activity. Significant differences were noted between all of the studied groups. On average, the motile fraction of group 1 sperm had the highest mitochondrial content, which was progressively reduced in its corresponding lower fraction and in both of the swim up fractions obtained from poor quality sperm , which eventually leads to necrosis or the release of mitochondrial pro-apoptotic factors [Alternations in mitochondrial membrane fluidity during cryopreservation have been suggested to raise the ΔΨm and induce the release of ROS, which in turn cause single/double-strand DNA breaks ,64,65. C factors ,68,69. T factors . Spermat factors ,72. Stud factors  have ind factors .swim up selected motile sperm of bulls with generally low sperm evaluation parameters (group 2) could be compared to the motile fraction of high performance bulls (group 1). Studies show that swim up allows successfully eliminating/separating sperm with DNA fragmentation from viable spermatozoa, significantly increasing the pregnancy success rate for sub-fertile male patients [swim up is one of the methods of choice for selecting the best quality spermatozoa. Recently, Magdanz et al. [swim up successfully selects motile spermatozoa with high ΔΨm, it may not level off the differences in sperm quality arising from “the male factor”. In our studies, the upper fraction from group 2 had significantly lower ΔΨm value than the upper fraction of group 1. The bottom fractions also differed, with group 2 having the lowest ΔΨm of all experimental setups. This observation may be reflecting alternations in mitochondrial dynamics caused by a disease or pathological processes affecting the testis. Studies in humans [swim up selection) might be reflecting the physiological status of an animal. All these males were disqualified from semen production due to severely decreased parameters such as: ejaculate volume, sperm concentration, motility, and morphology. On veterinary examination, some of these bulls revealed problems related to inflammation in testes or in accessory glands. Other males did not present any negative symptoms, but in the recent months had less physical activity or had long intervals between semen collection.Following the above observations, the question was raised of whether the patients . This mepatients . In cattz et al.  showed tn humans  and ratsn humans  showed tn humans  are asson humans . TherefoPathological processes affecting the testis may directly affect spermatogenesis and handicap mitochondrial morphology and function. Spermatogonia and early spermatocytes contain small mitochondria with low OXPHOS activity. Later in the process, higher oxygen consumption, elevated ATP production, and downregulation of glycolytic enzymes upregulates aerobic metabolism and OXPHOS. The respiratory chain complex assembly increases and cristae develop . ConsequDefective spermatozoa produce high levels of ROS that may also become toxic for the healthy sperm present in the semen . Antioxi2O2 and other ROS. PRDX1 transcripts were found in human spermatogonia and round spermatids, and the corresponding enzyme was located in sperm equatorial region, nucleus, and flagella [PRDX1 in all of the analysed samples. Although the differences were not statistically significant, the highest (close to significance) PRDX1 level was noted in the motile fraction of group 1 bulls. A decrease in the level of PRDXs is associated with an inability to maintain low levels of ROS. It was shown that human sperm motility parameters were significantly affected by PRDXs inhibition, the consequences of which included an increase of ROS and a decrease of ΔΨm and the intracellular level of ATP. Inhibition of PRDXs negatively affected sperm capacitation and ability to undergo the acrosome reaction. It also reduced the fertilisation rate, embryonic cleavage, and development rate in the IVF system [PRDX enzymes are recognised as vital factors in male fertility, and six members of the PRDX family were detected in testis and spermatozoa . They arflagella . The resF system ,92. PRDXSOD1 gene transcript in spermatozoa collected from the motile fractions of bulls with both high and low sperm quality parameters. Studies by [SOD1 level in the upper swim up fraction from bulls with poor semen quality (group 2). This may be reflecting the negative processes happening during spermatogenesis, where spermatogonia or spermatozoa need to compensate for the rising levels of ROS by increasing the transcription of the vital antioxidant agents.Activity of the SOD enzymes positively correlate with sperm concentration and overall motility, but a wide range of differences was noted among various mammalian species including humans, donkey, mouse, rat, rabbit, dog, and bull spermatozoa ,94,95,96GSS transcript level in non-motile sperm of both of the analysed groups compared to the spermatozoa of the upper swim up fraction obtained from high AI performance bulls (group 1). This may be supported by the observations that a significant decrease in intracellular GSH concentration was noted in seminal plasma and spermatozoa with severely impaired morphology in infertile men [GSS is the second enzyme in the glutathione (GSH) biosynthesis pathway, and similarly to PRDX1 and SOD1 is a potent antioxidant. We have noted a drastic decrease in the tile men .swim up selected), motile fraction of bovine spermatozoa has higher metabolic rates (OCR level) regardless of the bull breed. The sperm samples that were not subjected to swim up show almost no reaction to the administration of oligomycin, suggesting a low level of OXPHOS activity (little ATP expenditure). This could be possibly explained by the fact that hyperactivation and active movement requires higher oxygen consumption rates. In this respect, our data are consistent with these results, as we see a similar basal sperm metabolic rate over time, as presented by Magdanz et al. [Seahorse assay [Mitochondrial oxygen consumption is considered the central parameter of mitochondrial function. Studies on various species have shown that mitochondrial oxygen consumption is positively correlated with traditional measures of sperm function including motility and vitality ,31,100. The most important conclusion arising from this work is that the parameters that allow describing the activity and the number of mitochondria in each sperm cell may be used as reliable biomarkers for bovine sperm quality evaluation. For proper sperm function, it is necessary to create a balance between produced free radicals and cell metabolism. The sperm cells of proper mobility contain highly active mitochondria (with high ΔΨm), which corresponds to higher ability to produce energy. These results stay in agreement with transcript analysis of genes coding the antioxidant enzymes, which prevent the destructive action of ROS. The diagnostic methods used in this project have the potential to become applied in animal breeding centres and as a diagnostic tool in human medicine (fertility clinics).


Capripoxvirus, Orthopoxvirus, and Parapoxvirus genera can infect livestock, with the two former having zoonotic importance. In addition, they induce similar clinical symptoms in common host species, creating a challenge for diagnosis. Although endemic in the country, poxvirus infections of small ruminants and cattle have received little attention in Botswana, with no prior use of molecular tools to diagnose and characterize the pathogens.Poxviruses within the A high-resolution melting (HRM) assay was used to detect and differentiate poxviruses in skin biopsy and skin scab samples from four cattle, one sheep, and one goat. Molecular characterization of capripoxviruses and parapoxviruses was undertaken by sequence analysis of RPO30 and GPCR genes.The HRM assay revealed lumpy skin disease virus (LSDV) in three cattle samples, pseudocowpox virus (PCPV) in one cattle sample, and orf virus (ORFV) in one goat and one sheep sample. The phylogenetic analyses, based on the RPO30 and GPCR multiple sequence alignments showed that the LSDV sequences of Botswana were similar to common LSDV field isolates encountered in Africa, Asia, and Europe. The Botswana PCPV presented unique features and clustered between camel and cattle PCPV isolates. The Botswana ORFV sequence isolated from goat differed from the ORFV sequence isolated from sheep.This study is the first report on the genetic characterization of poxvirus diseases circulating in cattle, goats, and sheep in Botswana. It shows the importance of molecular methods to differentially diagnose poxvirus diseases of ruminants. Poxviruses exist throughout the world and are responsible for several economically significant zoonotic diseases affecting humans, wildlife, farming animals, and domestic animals , 2. GenePoxviridae family, which is divided into two subfamilies: Entomopoxvirinae, which infect invertebtates, and Chordopoxvirinae, which infect vertebrates [Chordopoxvirinae subfamily comprises 18 genera: Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, Cervidpoxvirus, Crocodylidpoxvirus, Yatapoxvirus, Centapoxvirus, Macropopoxvirus, Mustelpoxvirus, Oryzopoxvirus, Pteropopoxvirus, Salmonpoxvirus, Sciuripoxvirus and Vespertilionpoxvirus [Poxviruses are complex, linear, enveloped, double-stranded DNA viruses with large genomes of 130–360 kb in length , 6. Theytebrates . The ChoChordopoxvirus subfamily, are genera that affect livestock ruminants, some of which are capable of infecting the same animal species creating a challenge for clinical diagnosis. They include Capripoxvirus which is comprised of goatpox virus (GTPV), sheeppox virus (SPPV) and LSDV and Parapoxvirus, which includes ORFV, PCPV and bovine papular stomatitis virus (BPSV). Poxviruses can be transmitted through small abrasions of the skin , directly or indirectly from contaminated aerosols in the environment through the respiratory tract , and potential mechanical transmission by biting arthropods  [Among the nd SPPV) –12.In Botswana, poxvirus diseases in ruminants have received little attention, with no further characterization methods performed. The first publication on poxvirus diseases in Botswana was on ORFV in goats , withoutThis study describes the clinical presentation, molecular detection, and molecular characterization of poxvirus diseases from clinical samples. We report the first confirmed case of pseudocowpox in Botswana, and the first molecular characterization of LSDV, ORVF, and PCPV in the country.Six samples  from cattle, sheep and goat from Chobe, Central, Kweneng and Southern districts Table  were useSamples were submitted by field personnel to the Botswana National Veterinary Laboratory (BNVL) for routine testing. Initially, one sample from 2010, one from 2016 and two from 2017 were tested by histopathological examination and the findings were suggestive of poxvirus infections Table . In 2019Epidemiological information including clinical signs and attempted clinical diagnosis were collected from original sample submission forms that accompanied the samples submitted to BNVL.The samples were cut with a scalpel blade into small pieces and homogenized (10% w/v) in sterile phosphate buffered saline (PBS), then centrifuged at 2500 rpm for 10 min to collect the supernatant. DNA was extracted from 200 µl of the supernatant using DNeasy Blood and Tissue kit (Qiagen) following the manufacturer’s instructions. The DNA was eluted in 80 µl elution buffer, then kept at − 20 °C until further use.Orthopoxvirus, Capripoxvirus, and Parapoxvirus) and provides additional genotyping of the viruses within each of the three genera .The extracted DNA was tested using an HRM multiplex real-time assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance . The assThe PCR was set up in a 20 μl reaction volume containing 1 × SsoFast™ EvaGreen® Supermix (Bio-Rad), equal concentration (200 nM) of each of the forward and reverse primers (Table Capripoxviruses were amplified using primers [The G-protein-coupled chemokine receptor (GPCR) , and the primers  in TableParapoxviruses, the partial B2L gene was amplified by PCR in a reaction volume of 25 μl containing 500 nM of each of the primers [For the  primers  in TableAll PCR products were separated by electrophoresis on a 1.5% agarose gel at 100 V for 1 h. The positive PCR products were purified using the Wizard SV Gel and PCR clean-up system kit (Promega) according to the manufacturer’s instructions, then sequenced commercially by LGC Genomics (Germany). The sequences were edited and assembled using Vector NTI Advance™11.5 software . All sequences were submitted to GenBank.For phylogenetic reconstructions of the RPO30, GPCR, and B2L gene trees, multiple sequence alignments of the nucleotides sequences were performed separately for each gene using the muscle algorithm and the codon option implemented in MEGA7 . AdditioThe sequence alignment FASTA file was converted into a Nexus format file using Seaview programme version 4.7 . The BayIt appeared that most animals had lesions suggestive either of poxvirus infections, and sometimes, papillomavirus infections Table .Table 3SAs summarised in Table Two fragments for the RPO30 gene (554 bp and 520 bp) and three fragments for the GPCR gene  were successfully amplified and sequenced in all three LSDV positive samples . Similarly, an approximately 1210 bp fragment of the B2L gene was amplified and sequenced in the remaining three samples that were positive for PCPV (BOT_BOV/2010/6389) and ORFV . The complete RPO30 and GPCR genes and partial B2L gene sequences were submitted to GenBank database under accession numbers MW748471 to MW748479.In both GPCR and RPO30 gene phylogenetic trees, all three cattle samples from Botswana  clustered within the LSDV group  respectively.The RPO30 gene sequences were identical for all the Botswana LSDVs and identical to common field isolates, but different from the the vaccinal strains derived from KS1 and Neethling viruses. The Botswana isolates differed from LSDVs from Sudan, (GU119938) and (GU119944), by a single non synomymous nucleotide difference leading to amino acid substitutions at one position  to PCPV strains strain 3/07 (KF478804) isolated from cattle in Germany, and strain VR634 (GQ329670) collected from man infected by cattle in New Zealand.The B2L sequence of ORFV BOT_CAP/2019/74 was 100% identical to that of strain OV-SA00 (AY386264) isolated from a goat in the USA. The multiple sequence alignments of the B2L gene revealed 24 nucleotide changes between the B2L gene of BOT_CAP/2019/74 and BOT_OV/2017/158.This study presents the first confirmed case of PCPV in Botswana and the first molecular characterization of poxviruses affecting sheep, goats and cattle in the country from animals presenting clinical lesions consistent with poxvirus infections. The DNA of LSDV (3 samples), PCPV (1 sample), and ORFV (2 samples) were detected in clinical samples from 2010, 2016, 2017, and two recent samples from 2019 using an HRM assay for the differential diagnosis of poxvirus infections . It is wThe attempted clinical diagnosis of the poxvirus diseases were mostly consistent with the results of the HRM assay for LSDV and ORFV. The LSD suspected cattle had typical clinical signs that included skin nodules/bumps/lesions , 24 and For all samples, the sequencing data was in full agreement with the HRM data. The RPO30 and GPCR gene sequences of the Botswana LSDV samples were identical even through they were collected from three different years, 2016, 2017 and 2019, and from 3 different geographic locations . This suggests that the virus is well conserved and that the same strain is responsible for all cases.Moreover, the Botswana LSDV sequences presented similar features to common field isolates of LSDV encountered in Africa, Europe and Asia, including the presence of the 12-nucleotide deletion in their GPCR gene , 29–31.The existence of PCPV in cattle has been reported worldwide –35. In AThe path of introduction of the PCPV strain to Botswana is unknown and requires further investigation. Additional genetic analysis of the PCPV strain is also required to determine if it is genetically distinct enough to be classified as cattle,reindeer or camel variant of PCPV virus.The phylogenetic tree based on the B2L gene sequence, and the scrutiny of the multiple sequence alignment showed that the sample collected in sheep in 2017 in northern Botswana differed from that of the sample collected in goat in 2019 in the southern part of the country, suggesting that at least two different strains of ORFV are circulating in the country.The caprine ORFV B2L sequence was very similar to an ORFV OV−SA00 (AY386264) isolate collected from a goat in the USA in 2000. The ovine ORFV sequence differed from most ovine sequences, but displayed 99.6% amino acid similarity to isolate FJ-ZX (KC568400) collected from a goat in China in 2012.This paper reports the first molecular detection and characterization of poxvirus diseases circulating in sheep, goat and cattle in Botswana. It shows the importance of molecular methods for the differential diagnosis of poxvirus diseases in ruminants . Based on our findings, we recommend the HRM assay be used as a rapid screening and confirmatory tool for poxvirus infections during disease investigations and epidemiological studies. PCPV is a zoonotic disease that farmers in Botswana have limited information on. Publication of this information coupled with improved public health awareness campaigns to local farmers will provide the necessary information about pseudocowpox virus to educate future recognition of disease and timely reporting and treatment. Considering PCPV’s risk of dissemination, strict hygiene measures should be applied to prevent transmission to humans when detected. Country-wide surveillance is recommended to determine the prevalence of LSDV and PCPV infections in cattle, ORFV in sheep and goats in Botswana, and to identify infection risks for other animals and humans. The correct identification of the etiologic agent of pox-like lesions in ruminants is essential to allow for proper pox disease management and control, and to improve veterinary interventions, including vaccination of non-infected surrounding herds in the case of LSD.


The variables backfat thickness (BFT), Ca, Mn, Cu, C14:0, C15:0, and C20:0 showed the highest coefficients of variation. Three clusters were defined by the HCA. Out of all carcass traits, only BFT differed significantly (p < 0.001) among clusters. Clusters significantly (p < 0.001) differed for total lipids (TLIPIDS), moisture, dry matter (DM), fatty acid composition, cholesterol content, and mineral composition (except for Fe). The variables that define the canonical variate “CARCASS” were BFT and degree of marbling (MARBLING). TLIPIDS was the main variable for the “PROXIMATE” canonical variate, while C16:0 and C18:1c had the most relevant contribution to the “LIPIDS” canonical variate. BFT and MARBLING were highly cross-correlated with TLIPIDS which, in turn, was significantly affected by the IM lipid content. Carcass traits were poorly correlated with mineral content. These findings allow for the possibility to develop selection criteria based on BFT and/or marbling to sort carcasses, from grass-fed cattle fattened under tropical conditions, with differing nutritional values. Further analyses are needed to study the effects of sex condition on the associations among carcass traits and lipidic components.Hierarchical cluster (HCA) and canonical correlation (CCA) analyses were employed to explore the multivariate relationships among chemical components  of lean beef  Beef is known as one of the main sources of protein with high biological value, bioavailable minerals , vitamins of the B-complex  and other nutritional components  ,5. It isBos indicus influence. A couple of reports in Brazil [It is well known that intrinsic factors like species, breed, gender, age, and the structure of the type of muscle ,13,14,15n Brazil ,27 indicn Brazil . The comFor decades, the meat industry and scientists have used carcass characteristics to predict palatability-related attributes and/or consumer acceptability. Indeed, most of the carcass quality grading systems rely on the relationships between individual (or combined) carcass traits and sensory attributes of meat . Key chalongissimus thoracis) quality traits, and lean meat mineral composition (20 elements). In this case, the researchers employed a combination of univariate (simple correlation) and multivariate  techniques that allowed them to compare the relationship between minerals, not only individually but also in a factorial fashion (five factors) with the animal/carcass performance and the beef quality traits. This study [To our knowledge, there is limited information regarding the nutritional quality/value of meat specifically focused on a possible relationship between the anatomical or other physical characteristics of the intact beef carcass and its meat nutrient composition. This information gap needs to be addressed/closed particularly for beef produced under tropical, grass feeding conditions given the alleged health claims linked to its consumption . We propis study  only incis study ,33 indicis study ,35,36,37The underlying principle of CCA is to investigate the relationship between the variables by developing several independent canonical functions that maximize the correlation between the linear composites known as canonical variates . The CCAKnowing the degree of association of the multivariate relationships between the nutrient composition and the quality traits of dressed beef may allow identifying predictors of the meat nutrient composition that can be assessed on the hanging carcass and, eventually, the possibility to develop selection criteria for sorting carcasses with different nutritional values.longissimus dorsii lumborum (LDL) and selected carcass traits of cattle fattened on pasture under tropical conditions.This study aimed to explore the multivariate relationships among chemical components  of lean beef Carcass traits and nutrient composition data from a randomly selected group of 120 slaughtered cattle  were collected for this observational study. This sample was representative of slaughter male cattle derived from the prevailing production systems in the Venezuelan tropics where livestock is mostly fattened on pasture with little or no supplementation . Out of Postmortem Inspection of Cattle [postmortem, the chilled carcasses were subjected to evaluation. Skeletal and lean maturity  scores and subcutaneous backfat thickness (BFT) were determined following USDA guidelines [The animals were harvested at a commercial packing house following the procedures of the Venezuelan Standards of Bioethics and Biosecurity for Research with Living Organism , and thef Cattle . After bidelines . The subidelines . The degidelines .After evaluation, chilled carcasses were cut out following conventional butchering procedures according to regulation 792-82 of the Venezuelan Commission for Industrial Standard , trimmedDuplicate samples were analyzed for crude protein (CP) content following the Kjeldahl procedure; moisture (WATER) and dry matter (DM) were estimated by weight loss at 105 °C for 24 h, and ash at 550 °C during 6 h  with som−1 of fresh tissue.Duplicates of 10.0 g of ground meat were calcined in a furnace at 550 °C for 6 h. Sample handling and mineral analyses were conducted according to the methodology described by Giuffrida-Mendoza et al. [Cholesterol content of each steak sample was determined in triplicate, according to the procedure described by Rhee et al. .3/CH3OH [Fatty acids (FA) were determined by gas chromatography as described by Slover and Lanza . A dupli3/CH3OH  to yield3/CH3OH .The data analysis was performed using the IBM SPSS 23 statistical software . The oriTwo hierarchical cluster analyses (HCA) were performed. The first HCA was applied to explore the presence of any pattern or relationship between the 32 variables under study , using the linkage (between groups) method. To measure the degree of association between variables, Pearson’s correlation coefficient was applied with the measurement transformed into absolute values. The second HCA was applied to group all the samples using Ward’s method with the squared Euclidean distance measure and considering the sex condition to describe how the variables are presented within each cluster.To validate the clusters obtained, an ANOVA with two main factors (sex condition and cluster) was applied on each variable. The results from the two HCA were represented by dendrograms. To analyze the relationship among the subgroups of the variables proximate, mineral, and lipid components with respect to the subgroups of carcass traits, a canonical correlation analysis (CCA) was carried out. Wilk’s Lambda and Bartlett tests were used to determine the significance of canonical correlations.The acronyms of the variables studied in this research and their definitions are shown in N = 109) were 48 (44%), 24 (22%) and 37 (33.9%), respectively . Among the proximate components, TLIPIDS from separable lean only presented the greatest variation (between 0.93 and 6.67 g.100 g−1). Out of the 30 fatty acids under study, the most abundant were C16:0 (0.028–1.288), C18:0 (0.053–0.705), C18:1c (0.27–1.749) and C18:1t (0.117–0.981). Overall, MUFA constituted 57.65% of the total; PUFA represented less than 5% of the total and the rest (37.35%) corresponded to SFA.The descriptive statistics of the experimental data are presented in The first HCA allowed to determine how variables were grouped by degree of similarity as calculated by the squared Euclidean distance with a similarity index ranging from 0 (higher similarity) to 25 (lower similarity); a close distance between variables indicating high correlation. This first HCA also allowed to provide a simple representation of the total data composed of 32 variables and to explore how the variables correlated to each other. p < 0.001) among clusters (p < 0.0001). Clusters differed (p < 0.0001) in fatty acid composition and CHOLEST (p < 0.05).The HCA by samples resulted in a dendrogram with three clusters, sufficiently distant to expect relatively different values among the groups . ANOVA aclusters . In the −1); therefore, this subgroup also exhibited the highest values in most of the fatty acids evaluated (p < 0.0001). Cluster 2 is mostly composed of bulls with the lowest BFT (0.226 cm) and total lipids . On the other hand, cluster 3 was more balanced, comprised of 56.5% of bulls and 53.5% of steers, with similar values in BFT and TLIPIDS; this cluster represented the group of samples with similarities in fat-related traits and differences in mineral content. In general, cluster 3 showed the highest values in Ca, Mg, P, K, Zn, Cu and Mn (p < 0.0001). Cluster 1 is mainly represented by steers with the highest values in BFT (0.724 cm) and TLIPIDS (4.16 g.100 gp < 0.0001). Canonical redundancy analysis revealed that the first canonical correlation represents 75.96% of the explained variance, which indicates that there is a high degree of association between carcass traits and the proximate composition.The first canonical correlation between U (representing the proximate composition traits) and V (representing the subgroup of carcass traits) was significant  and V (named as “CARCASS”), respectively. Standardized canonical coefficient or canonical weights of TLIPIDS had the greatest contribution to the canonical variate “PROXIMATE” . The corr = −0.836 and r = −0.855, respectively). On the other hand, the variable CFINISH had a moderate contribution to the canonical variate “CARCASS” (r = 0.751). The very low standardized coefficients of SM, LM, and CW indicated an irrelevant contribution of these variables to the canonical variate “CARCASS”.Canonical cross correlation describes the correlation between variables and the opposite canonical variate. The variable TLIPIDS showed the highest canonical cross-loading with the canonical variate “CARCASS” . On the The variance of the canonical variate “PROXIMATE” associated with the variables of its own group represented 30.70% of the total data variation, which could be attributed to the high loading value of the variable TLIPIDS. The cross variance between “PROXIMATE” and “CARCASS” only accounted for 12.10% of the total variance; a low value that is also associated with the contribution of the TLIPIDS variable. The contribution of the canonical variate “CARCASS” was 34.10% of the total variance. The canonical correlation between PROXIMATE and CARCASS subgroups of variables aligns with the results obtained from the linear correlation between the variables related to fatness . The corp = 0.002), showing a not very strong correlation coefficient (r = 0.629), and represented 59.17% of the explained variability. The CCA between the subgroups of variables related to lipid composition traits and the subgroup of carcass traits revealed six canonical correlations. The first canonical correlation was highly significant (r = −0.912); and C18:1c  showed the highest correlation coefficients with the canonical variety “LIPIDS”. This indicates that Palmitic and Oleic acids were the fatty acids that mostly contributed to the conformation of the canonical variate “LIPIDS”. The variable cholesterol content presented a low canonical weight and, therefore, low correlation with its canonical variety. The C16:0  with the highest standardized canonical coefficient also exhibited the highest correlations with the carcass trait variables . Other fThe variance of the canonical variate “LIPIDS” accounted for 38.8% of the total data variation. The cross variance between “LIPIDS” and “CARCASS” only represented 19.10% of the total variance. This low value is potentially related to a low correlation between these two subgroups of variables, and/or the smaller number of CARCASS variables as compared to the lipidic components.Six possible canonical correlations were obtained between mineral content and carcass traits; however, none of them were statistically significant. Based on the available data and the statistical technique applied, there is insufficient evidence to demonstrate the existence of any relationship between mineral components and carcass traits.Longissimus lumborum taken from crossbred cattle varying in age, sex condition, and diet. Also, these values are similar to those reported for the fresh Longissimus muscle derived from Bangladeshi beef (zebu type) finished on pasture [The carcasses evaluated in this study are representative of South American tropical cattle fattened on pasture, which presents more variation in degrees of fatness, carcass finish, and conformation traits than their counterparts that are subjected to more standardized, intense feeding protocols. The results obtained in this study also concur with carcass characteristics ,57,58 an pasture  and othe pasture . The min pasture ,15,64,65The relatively low Ca content in these beef samples is potentially related to the quality of vegetation consumed/grazed by these animals. Pastures and forages constitute the main food sources for fattening cattle in Venezuela. The nutritional value of pastures depends on the amount of nutrients present in these plant species, which are absorbed from the soil; consequently, it will be the characteristics of the soil that defines the development of the plant with respect to the concentration and availability of the mineral elements present.According to Araujo , tropicaThe relationship between meat sensory traits and physicochemical characteristics has been studied using multivariate analysis techniques in beef ,69; howeThe first HCA allowed a simple representation of the total data with 32 variables and to explore how the variables correlated. The second HCA grouped all the observations by similarities in three clusters with a significant variation in BFT, TLIPIDS, mineral and fatty acid composition. It has been demonstrated that sex greatly influences protein and fat deposition in cattle and defines distinct differences in body composition ,70,71; nBos indicus) and regardless of sex, it is known that Bos indicus-influenced cattle individuals, exhibits lower levels of marbling when compared to Bos taurus biological types. The second factor could be the low energy content of the grass-based diet which did not facilitate a greater expression of the inherent differences between the sex conditions in terms of lipid content. Finally, it should also be considered that only separable lean was used for chemical analyses. Otherwise, if the meat sample had not been devoid of the surrounding subcutaneous and intermuscular fat depots, the differences between the sex conditions would have been more noticeable because the steers would give loins with a more significant amount of fat per 100 g of fresh tissue than bulls. Needless to say, a relatively greater fat content would bring a concomitant reduction in the concentration of other proximate components.It is noteworthy to highlight that this was not a controlled experiment designed to compare sex conditions. Therefore, genetics, management (slaughter weight and age), nutrition and other confounding factors  could afThere is a consensus that the use of a CCA as a multivariate approach is appropriate for evaluating the interrelations among meat quality and carcass traits ,73. For c had the largest contribution for the “LIPIDS” canonical variate. We analyzed canonical correlations by paired groups of variables: proximate composition, lipids, and mineral contents with carcass traits. The correlation coefficients between the original variables and the canonical variates allow establishing the weight of each original variable in the conformation of the canonical variate. The main variables that define the “CARCASS” canonical variate were BFT and MARBLING, meaning that these two variables had the highest contribution to the corresponding canonical variate. TLIPIDS was the main contributing variable for the canonical variate “PROXIMATE”, while C16:0 and C18:1As expected, carcass fatness-related traits  exhibited the highest cross-correlations with TLIPIDS , suggestc components presented the highest correlations with carcass traits. The palmitic acid (C16:0) represents the main product of the de novo fatty acids synthesized from carbohydrates and volatile fatty acids of the diet, and it can be elongated to stearic acid (C18:0), and then to arachidic (C20:0) [In our study, the C16:0 and C18:1 (C20:0) . Also, tp < 0.05) with all carcass traits but negatively correlated with hot carcass weight, while no significant correlation (p > 0.05) was detected between contents of Fe or Zn and carcass traits. Garmyn et al. [p > 0.05) with any carcass trait. Age-related carcass traits  were not correlated (p > 0.05) with the proximate, mineral or lipidic compositions.According to the available data and the CCA applied, there is not enough evidence to assume that there is an association between mineral component variables and carcass traits. These results validate the findings of the HCA which indicate that carcass traits have a weak correlation with the mineral composition of the meat. The results obtained in this work are comparable with those of Duan et al. , who repn et al.  reportedn et al.  reportedBFT represents between 10 to 13% of total carcass fat tissue and it is dependent on genetics, nutrition, and finishing systems of ruminants; also, these may be influenced by sex and age, considering that the nutrient dynamics in the ruminant’s body differs between sexes, and these differences become more evident with age . A meta-r > 0.5) related to TLIPIDS, despite exhibiting a very low range of scores [between 1  and 3 (Slight); Bos indicus-influenced, grass-fed cattle. In fact, BFT data (r = 0.62) than when the USDA scores were used (r = 0.56) while Kruk et al. [r = 0.60). In fact, Silva et al. [The process of IMF deposition depends on many factors such as sex condition, age, and nutrition ,79,80. MBFT data  show a hk et al.  had repok et al. ; howeverk et al.  reportedk et al.  found tha et al.  pointed a et al.  when mealongissimus muscle, and showed that only raw steaks with “Practically Devoid” MARBLING level contained significantly less cholesterol (on wet basis) than did raw steaks with any of the other seven MARBLING scores. In the present study, CHOLEST showed a low loading value, indicating a weak association with carcass traits. Catillo et al. [Rhee et al.  reportedo et al.  reportedo et al.  found thlongissimus muscle.Canonical correlation analysis is an optimized multivariate technique for evaluating the existence or non-existence of relationships between complex groups of variables. In this study it proves to be a powerful tool to study the relationship between the selected set of carcass traits and the proximate, lipid and mineral components, particularly when it is expected that certain degree of interaction exists among these three groups of chemical variables. This work demonstrates an important relationship between backfat thickness, marbling and the content of total lipids and fatty acids in beef LDL muscle from cattle fed on pastures under tropical conditions. Instead, carcass traits are poorly associated with beef mineral content. These findings allow for the possibility to develop selection criteria based on BFT and/or marbling to sort carcasses with differing nutritional values. For the experience gained in beef carcass grading in Venezuela, the evaluation of marbling levels requires more intense training and supervision of graders than the BFT measurement. Moreover, marbling is seldom used to grade beef carcasses in the developing, tropical countries. Therefore, it is more practical to use BFT in future regression analyses to explain the variation in lipid composition of beef Further analyses are needed to determine the potential influence of sex condition on the magnitude of the associations among carcass traits and beef fatty acid composition.


Oral antitumor therapy has significantly improved clinical outcomes in multiple tumor entities. However, following a standard dosing regime, strong interindividual variability in patients’ plasma concentrations can be observed for many oral antitumor drugs. This results in risks of reduced therapeutic effect and increased side effects. Monitoring these variable plasma concentrations is an important tool in evaluating multiple factors influencing drug exposure and, if necessary, adjusting therapeutic doses. Here, we developed a method for the simultaneous measurement of 57 oral antitumor drug plasma concentrations. Detection and quantification were achieved using liquid chromatography coupled to an Orbitrap mass spectrometer, which can be easily expanded to newly approved oral antitumor drugs in the future. Applicability of the method was proven by measuring 71 plasma samples from 39 patients undergoing oral antitumor therapy. In summary, the developed method provides an important tool for exposure measurements of oral antitumor drugs.Oral anticancer drugs have led to significant improvements in the treatment of multiple tumor entities. However, in patients undergoing oral antitumor therapy, plasma concentrations are highly variable, resulting in risks of reduced therapeutic effects or an increase in side effects. One important tool to reduce this variability is therapeutic drug monitoring. In this work we describe a method to simultaneously quantify the plasma concentrations of 57 oral antitumor agents. Quantification of these drugs was achieved using liquid chromatography coupled to an Orbitrap mass spectrometer. The method was fully validated according to the FDA guidelines and constitutes a simple and robust way for exposure monitoring of a wide variety of oral anticancer drugs. Applicability to clinical routine was demonstrated by the analysis of 71 plasma samples taken from 39 patients. In summary, this new multi-drug method allows simultaneous quantification of 57 oral antitumor drugs, which can be applied to exposure monitoring in clinical studies, taking into account the broad variety of oral antitumor drugs prescribed in clinical routine. Oral anticancer drugs play an important role in the treatment of multiple tumor entities  and haveMost of the oral anticancer drugs—especially protein kinase inhibitors—are prescribed in a one dose fits all approach ,9,10. HoSome studies demonstrated an association between low plasma concentrations and poorer treatment outcomes for a number of oral anticancer drugs such as alectinib, imatinib, and vemurafenib ,9. SimilA crucial measure to evaluate and minimize the above mentioned factors is the determination of a patient’s plasma concentration via therapeutic drug monitoring (TDM) ,14,25. CThe method presented in this manuscript addresses the need to cover a broad range of oral anticancer drugs as a prerequisite for systematic data on plasma concentrations of these drugs. We developed and validated a liquid chromatography-mass spectrometer (LC-MS) based method that enables the simultaneous quantification of 57 oral anticancer drugs in plasma samples, using a straightforward sample preparation. The presented method and technology (1) offer the possibility to conduct systematic studies on the impact of plasma concentrations of these drugs on clinical outcomes, (2) is suitable to implement routine drug exposure measurement for a wide variety of oral antitumor therapy prospectively, and (3) allows for the easy addition of further, newly approved compounds.2H4]-everolimus and [2H5]-lenvatinib were purchased from Alsachim . Next, [2H4]-olaparib was purchased from Biomol , and [2H3]-clopidogrel was bought from @rt molecule . Dimethyl sulfoxide (DMSO), water, acetonitrile (ACN), formic acid (FA), and methanol (MeOH)  were purchased from VWR chemicals . Pooled blank human plasma was used from the Klinische Pharmakologie Erlangen 37 (KPE37) study . An Acquity UPLC® BEH C18 column  equipped with an Acquity UPLC® BEH C18 VanGuardTM Pre-Column  was used for chromatographic separation. The Q-ExactiveTM Focus mass spectrometer (MS) was hyphenated with a heated electrospray ionization (HESI) source .The liquid chromatography-mass spectrometer system consisted of a Thermo Scientific Dionex UltiMatev/v) FA (eluent A) and MeOH with 0.1% (v/v) FA (eluent B). The injection volume was 5 µL. The gradient program is shown in Chromatographic separation was achieved using a gradient elution with water containing 0.5% . Eight individual working standards were prepared by stepwise dilution of this standard solution with concentrations from 10 to 200% cmin  depending on their respective solubility . Validation results were analyzed with Microsoft Excel 2016. min at steady state being the lower limit of validation (LLOV) and 200% cmin the upper limit of validation (ULOV).Validation of the MS assay and sample preparation was carried out according to the guidelines for Bioanalytical Method Validation of the US Food and Drug Administration (FDA) . Blank p2) was permanently greater than 0.995 (weighting 1/x). To meet FDA accuracy requirements, all calibration points with a concentration above the LLOV and six calibration levels overall had to be between 85 and 115% with a maximum coefficient of variation (CV) of 15%. Sensitivity was tested by determining the response of the lowest non-zero standard of the calibration standards compared to the response of a blank sample (prepared with IS). The acceptance criterion was fulfilled if analyte response in the blank sample was five times lower compared to the response in the LLOV.Eight-point calibration curves between LLOV and ULOV of every analyte were plotted, whereby four replicates of each point were used to assess if the coefficient of correlation , ULOV) that were stored at −80 °C and thawed three times at 4 °C. After thawing, samples were frozen for 24 h between cycles. Bench-top stability was evaluated at ambient conditions with or without light for 24 h. Stability at 4 °C was tested by storing the four validation standards in a fridge at 4 °C for 24 h and 6 weeks. Similarly, stability was analyzed after incubation at 56 °C for 1 h with an Eppendorf Thermomixer compact . Autosampler stability was assessed by measuring ready for injection samples after being kept in the autosampler at 4 °C for 24 and 72 h, respectively. Long-term stability was tested after storing three replicates of the four validation standards at −20 °C and −80 °C for 1, 2, and 3 months. Stability was accepted if measured values were within the FDA range, i.e., ≤15% for 2 × LLOV, MOV and ULOV, and ≤20% for LLOV, of their nominal concentration.For recovery investigation three replicates of five validation standards , MOV, and ULOV) were prepared by spiking working solutions before and after plasma protein precipitation into pooled human plasma. Recovery was defined as the ratio of pre-extraction to post-extraction addition signal intensity. For matrix effect investigation, working solutions were spiked into water (instead of human plasma) before the extraction was performed. Ratios between the analyte responses of post-extraction addition into pooled human plasma and into water informed about the extent of matrix effects.Selectivity was investigated by analyzing six blank plasma samples from healthy individuals and was accepted if the samples were free of interferences at the retention time(s) of the analyte(s) and IS. Acceptance criteria for any interference was a signal response ≤ 20% of that of the LLOV and ≤5% of that of the IS.Carryover was assessed by injecting two blank samples (without IS) directly after running the highest calibration level. To fulfill the acceptance criteria, signal intensities had to be below 20% of the peak area of an LLOV reference and less than 5% for IS.Dilution integrity was analyzed by preparing pooled human plasma samples spiked with analytes to a four-fold concentration than the ULOV, followed by an eight-fold dilution of this sample with pooled human plasma.2O) were tested for chromatographic separation. Optimal separation of all 57 oral anticancer drugs was achieved using a gradient of water containing 0.5% FA and MeOH containing 0.1% FA  of mobile phases  were determined for all analytes and were permanently ≥0.995. Carryover was observed in eight analytes with values from 0.03%  to 12.7% (midostaurin). Selectivity investigations showed that after injection of six blank plasma samples from healthy individuals, there was no interfering signal with a response of ≥20% of LLOV and ≥5% of IS detectable. Sensitivity results fulfilled the acceptance criteria, with the exception of the two lowest calibration levels of panobinostat . Thus, panobinostat fulfilled acceptance criteria from LOV to ULOV. All results are shown in All 57 oral antitumor drugs fulfilled the FDA acceptance criteria. Correlation coefficients  and panobinostat (CV 20.2%). Intra- and interday accuracy and precision at different concentration levels , ULOV) are shown in Results of recovery and matrix effect investigations are listed in Detailed stability test results are shown in For concentrations four times higher than ULOV, 49 of the oral anticancer drug dilutions showed accuracies (85.4–115.0%) within the acceptance criteria. Brigatinib, dacomitinib, encorafenib, osimertinib, pazopanib, regorafenib, rucaparib, and trifluridine did not fulfill the acceptance criteria. Detailed results of dilution integrity investigations are shown in min). The measured concentrations of these 41 samples are presented as % cmin in A total of 71 blood samples from 39 patients treated with 14 different oral anticancer drugs were collected (see The present work constitutes a new validated multi-drug MS method for simultaneous determination of 57 oral antitumor drugs in human plasma. Recently, various research groups aimed at the development of methods for the simultaneous quantification of oral antitumor drugs ,37,38,39In the present work we circumvented this limitation by employing an Orbitrap MS. This high mass resolution MS enables the analyte identification based on a highly resolved mass to charge ratio and fragmentation is not necessarily required. Using this analytical hardware, it was possible to develop a method for the simultaneous quantification of 57 oral antitumor drugs, which is to the best of our knowledge the highest number of drugs covered by a single method. In addition, our method offers the advantage of being easily extendable to include further oral antitumor drugs and drug metabolites of interest. This is possible due to the full scan mode in which the Orbitrap MS was operated in this study. Furthermore, acquisition in full scan mode allows for retrospective detection of metabolites that are not the focus of this research, but might be in the future. In previous literature, one other method applied an Orbitrap MS for the quantification of oral antitumor drugs, however, only six kinase inhibitors were quantified simultaneously .min after adequate dilution.Validation results indicated accordance with the requirements of the FDA guidelines . For conPrevious publications reported stability issues for some oral anticancer drugs. For instance, osimertinib and afatinib concentrations decreased under ambient conditions ,41. Thesmin for three samples, nine were within 30–150% cmin and two were >150% cmin. The average concentration of the plasma samples at ±4 h of trough level was 10.2 ng/mL (range 0.4–59.5 ng/mL, CV 140.8%, The applicability of the method to a real-life clinical setting was subsequently assessed in a pilot study, including patient plasma samples from the CCC Erlangen-EMN. 71 plasma samples were collected, of which 41 samples were taken ±4 h of trough level according to the patient’s statement. In line with the literature, the results demonstrate a significant interindividual variability of the plasma concentrations of the respective antitumor drugs . For exaThe high interindividual variability in plasma concentrations found in the present work and in previous publications ,9,10,14 A variety of factors influence the pharmacokinetics of oral antitumor drugs and consequently clinical outcome. Routine measurement of plasma concentrations via TDM has not been implemented for most oral antitumor drugs yet. This is most likely due to the requirement of applying multiple analytical methods to cover the broad range of approved drugs, which has deemed routine drug exposure measurement too labor-intensive and time-consuming to date. The new method presented here employs a high resolution Orbitrap MS in full scan mode in order to overcome this hurdle. The high mass resolution allows for the simultaneous measurement of 57 oral antitumor drugs without the need for a change of analytical hardware. It also provides an opportunity to easily expand the included analytes with newly approved oral antitumor drugs and metabolites of clinical relevance. Consequently, the presented method sets a foundation for future studies to investigate the putative benefits of routine drug exposure measurement for a wide range of oral antitumor drugs.


TSC1 or TSC2 result in dysregulated mTOR activation, leading to the occurrence of TSC.Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by facial angiofibromas, epilepsy, intellectual disability, and the development of hamartomas in several organs, including the heart, kidneys, brain, and lungs. Mutations in either A 44-year-old man was hospitalized for acute lumbago and hematuria.The patient presented with facial angiofibromas, epilepsy, fibrous plaques, periungual fibroma, renal angiomyolipomas (AML), pulmonary lymphangioleiomyomatosis (LAM), liver hamartomas, and osteosclerosis. A diagnosis of TSC was made based on clinical manifestations.Next-generation sequencing (NGS) was performed to screen for potential variants, which were verified using Sanger sequencing. The final variant was analyzed using a minigene assay.TSC2 variant  was identified using NGS and confirmed using Sanger sequencing. The in vitro minigene assay showed that the variant c.336_336 + 15delGGTAAGGCCCAGGGCG caused erroneous integration of a 74 bp sequence into intron 4. This novel variant was not found in his unaffected parents or 100 unrelated healthy controls.A potentially pathogenic novel TSC2 variant, c.336_336 + 15delGGTAAGGCCCAGGGCG, in a patient with classical TSC and demonstrated that this variant leads to aberrant splicing using a minigene assay. Our results extend the understanding of the mutational spectrum of TSC2.We identified a novel heterozygous  The morbidity of TSC is nearly 1:10,000 among newborns, and most patients are diagnosed during the first 15 months of life. TSC is characterized by facial angiofibromas, epilepsy, intellectual disability, and development of hamartomas throughout the body, particularly in the brain, skin, heart, and kidneys. Owing to the different locations of lesions, the spectrum of clinical manifestations is very wide among individuals. Some patients are severely affected at an early age and at multiple sites throughout the body. The major causes of mortality in patients with TSC are seizures and renal complications. TSC is believed to have a high spontaneous mutation rate, as suggested by the large number  of aperiodic cases without a family history.TSC1 and TSC2, which act as tumor growth suppressors and encode the proteins hamartin and tuberin, have been found to be responsible for mTOR overactivation, which may be the underlying mechanism of pathogenesis.Tuberous sclerosis complex  is an autosomal dominant genetic disease caused by mutations in  Owing to genetic heterogeneity, the clinical phenotype of the disease presents high variability, thus making the prediction of the phenotype on an individual basis challenging. Even monozygotic twins with the same mutation can exhibit different clinical expressions. The atypical clinical presentation indicates the potential shortcomings of the current diagnostic criteria for TSC. A pathogenic variant of either TSC1 or TSC2 detected by genetic analysis represents a separate diagnostic criterion, regardless of clinical findings. With advances in genomic DNA sequencing technology, it is now easy to analyze the entire sequence of TSC genes. However, molecular diagnosis of TSC remains challenging because of the difficulty in correctly assessing gene variants of unknown significance. A functional analysis is required to determine the pathogenicity of these variants.The diagnosis of TSC depends mainly on clinical diagnostic criteria.TSC2 gene variant, c.336_336 + 15del16, by Next Generation sequencing (NGS) and Sanger sequencing. An in vitro minigene assay was performed to investigate the effect of this variant on the splicing process.Here, we present the clinical manifestations of a patient with TSC and detected a novel 2TSC1 and TSC2 genes associated with TSC. The sequencing data were aligned to the human reference genome hg19 . Sanger sequencing was performed to verify the NGS results. Primers were designed using PRIMER 5 software for PCR amplification, according to the potential pathogenic variants obtained by NGS. After purification, the PCR products were sequenced using an ABI PRISM 3730 Genetic Analyzer. Finally, a minigene assay was performed to study the effects of the variants at the transcript level. Mutant and wild-type pMINI-TSC2 vectors were transfected into 293T cells. After transfection for 48 hours, the total RNA was extracted and reverse-transcribed into cDNA. Following, the cDNA was amplified by PCR, analyzed by agarose gel electrophoresis, and sequenced.Written informed consent was obtained from the individual and approval of the Institutional Medical Ethics Committee of Suining Central Hospital. Genomic DNA was extracted from the peripheral blood samples of the patient. After quality control of the extracted DNA samples, a targeted NGS panel  was used to analyze the exons and flanking intronic regions of the 3 Owing to the ruptured hemorrhage of his renal AMLs, the patient eventually underwent surgery. After year of follow-up, the patient remained stable.A 44-year-old man from the Han dynasty presented with a chief complaint of “acute lumbago and hematuresis” for three days and was admitted to the urology department of our hospital. He had a long history of seizures and was being treated with antiepileptic medication. Facial angiofibromas, fibrous plaques, and multiple periungual fibromas were discovered on physical examination Fig. . BiochemTSC2 variant c.336_336 + 15del16, resulting in the deletion of the last nucleotide G in exon 4 and the subsequent 15 bases in the intron region, is more likely to cause a complete loss of the splice donor site of intron 4. This variant was more likely to cause splice defects. Minigene assay electrophoresis showed that the c.336_336 + 15del16 variant led to a slightly larger TSC2 mRNA transcript than that in the wild-type clone. Sequencing showed that pMINI-TCS2-WT was considered to have normal splicing, whereas the mRNA sequence of pMINI-TCS2-MUT was changed, the original splicing site was lost, and erroneous insertion of a 74 bp sequence into intron 4 caused the splicing position moved back 90 bp (r.336delins336 + 16_336 + 90) . This variant was not found in his family members or in the 100 unrelated controls  and polycystic kidney disease (PKD). In such patients, PKD is frequently associated with TSC2/PKD1 contiguous gene syndrome. The central nervous system is almost invariably involved, with up to 85% of patients presenting with epilepsy, and at least half of the patients have intellectual disabilities or other neuropsychiatric disorders, including autism spectrum disorder. In this study, the patient was characterized by classical multisystematic symptoms, including severe renal AMLs, pulmonary LAM, multiple calcified subependymal nodules, liver hamartomas, skin impairment, and osteosclerosis, indicating a more severe grade of illness. The patient eventually underwent surgery because of rupture hemorrhage of the renal AMLs.TSC is an autosomal dominant neurocutaneous disorder affecting multiple organs. Two genes involved in TSC have been identified; the TSC2 variant c.336_336 + 15del16 of exon 4 was identified. This variant leads to deletion of the last nucleotide G in exon 4 and the subsequent 15 bases in the intron region. Notably, this position may play an important role as a splicing modulator that may alter the splicing of the TSC2 transcript. The minigene splicing assay confirmed that the variant c.336_336 + 15del16 led to the loss of the original splicing site and erroneous insertion of a 74 bp sequence into intron 4, causing the splicing position moved back 90 bp (r.336delins336 + 16_336 + 90). This variant may disturb normal splicing alterations during splicing. Therefore, this variant might lead to a defective tuberin and severe impairment of the normal hamartin–tuberin interaction, thus resulting in upregulation of the mTOR pathway, which in turn might cause the disease. Notably, splice mutations may play a more important role in human hereditary diseases. Classical splicing variants that affect nucleotides at the splice acceptor and donor sites lead to aberrant RNA splicing. However, all types of variants  can lead to splicing defects either by disrupting or creating signals. Zhang et al reported a missense mutation, c.3610G > A, in which the last nucleotide of exon 29 in TSC2 was replaced. This leads to the substitution of a single amino acid from glycine to arginine at amino acid position 1204 (p. Gly1204Arg), affecting normal splicing. Qiu reported a novel TSC1 frameshift mutation (TSC1 c.1550_1551del) that triggered aberrant splicing simultaneously, leading to TSC formation. Additionally, the in vitro minigene assay is an attractive alternative for assessing the impact of intronic variants on splicing.In this study, using NGS and Sanger sequencing of TSC genes, a novel heterozygous  the c.336_336 + 15del16 variant was considered as the LOF variant (loss of function), which conforms to very strong disease-causing evidence , strong pathogenicity evidence (the patient had a de novo mutation and no family history), and moderate pathogenicity evidence , namely PVS1 + PS2 + PM2, which was considered pathogenic.The c.336_336 + 15del16 variant was considered a novel variant because it was not found in the ExAC, ESP, 1000G, and HGMD databases and was absent in his parents and 100 healthy controls. According to the classification standards and guidelines of ACMG genetic variation,TSC2 gene is a pathogenic variant. This variant affects mRNA splicing, leading to loss of tuberin function, which may be the underlying cause of TSC. The identified variant extended the TSC2 mutational spectrum and, to some extent, increased our understanding of the molecular mechanism of TSC pathogenesis.In summary, we highlighted the clinical features of patients with classical TSC. DNA sequencing and minigene splicing assays showed that the novel splicing variant c.336_336 + 15del16 within exon 4 and intron 4 of the We thank the patient who agreed to use the images and clinical data.Data curation: Linli Liu, Gaowu Yan.Funding acquisition: Chunshui Yu.Writing – original draft: Linli Liu.