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Simple Summary: Detection of new diagnostic markers would greatly influence the institution of preventive (control) protocols in bovine mastitis. Using the quantitative proteomics workflows and statistics for milk analysis, we identified that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphy- lococci and streptococci intramammary natural infection and inflammation, and subsequently gained a deeper understanding of the immunopathology of mastitis-related infection and inflam-mation for various etiological agents. Abstract: Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiol-ogy and host immune responses, independent of the mastitis level. After identification of Staphy-lococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantita-tion through the SEQUEST algorithm. Statistical analysis employing R was used to identify dif-ferentially abundant proteins between the groups. Protein classes, functions and function-al-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflam-mation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific- pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

The idea of dual sire insemination in dog breeding is to give equal chances to both males to fertilize the eggs due to the specific oestrus cycle of the bitch. Two sexually mature, privately owned females Petit Basset Griffon Vendeen (bitch A and B) aged 3 to 5 years were subjected to dual- sire inseminations. Prior to insemination, the semen of stud dogs (n=5) was collected and evaluated for volume, concentration, total sperm count, motility, membrane integrity (HOS test), percentage of live spermatozoa and sperm morphology (eosin nigrosin staining). The time of insemination was based on serum progesterone (P4) concentrations, where P4 of >5–10 ng/mL was considered ovulation. Bitch A was inseminated in two oestrus cycles with fresh mixed semen from two males using the endoscopic transcervical insemination technique (TCI). Bitch B was inseminated in one oestrus cycle by laparoscopic intrauterine deposition of frozen thawed semen. Semen evaluation showed minimum deviations in all tested parameters between the chosen males on the days of insemination. Depending on sperm concentration and quality, the volume of ejaculates was adjusted, resulting in an equal number of motile spermatozoa from two males in each insemination, providing them an equal chance for fertilisation. Confirmation of pregnancy was carried out 24 days after insemination by ultrasonography. The whelping outcome was obtained directly from the owner of the bitches. Puppy blood was taken immediately after whelping from the umbilical vein into EDTA tubes. Blood samples were also obtained from the dam and both sires for DNA profiling. Parentage was determined for each dual-sired pup by using Thermo Scientific Canine Genotypes Panel 1.1. All inseminations resulted in pregnancy and whelping. A total of 14 puppies were born in three litters. Mixed parentage was determined in 1 of the 3 resultant litters (bitch A). In conclusion, dual sire insemination is a useful breeding tool providing the opportunity to obtain puppies from multiple genetic backgrounds in a single litter. However, as this method usually produces offspring from single father, optimal insemination protocol should be established for producing a litter of mixed paternity.

BACKGROUND: Bacteria from Staphylococcus and Streptococcus genera are frequent causes of mastitis in dairy cows. We hypothesized that the quantitative changes in the milk proteome differed depending on presence of Staphylococci or Streptococci. METHODS: We analyzed 118 milk samples from cows with subclinical mastitis, clinical mastitis, and positive controls (group with normal somatic cell count and positive bacterial culture), classified into the groups with Staphylococci (SPH, N=51) or Streptococci (STP, N=67). Sample preparation included proteins՚ reduction, alkylation, precipitation using acetone, digestion with trypsin, and labeling with tandem mass tags. The liquid chromatography-tandem mass spectrometry analysis included separation using the linear gradient and scanning in the positive ion mode using the data-dependent analysis Top8 method, on a modular system consisting of Ultimate 3000 RSLCnano system (Dionex®) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific®). The proteome Discoverer® software was employed for proteins՚ identification and relative quantitation following the SEQUEST algorithm. Further, the data were analyzed with the R statistical package and MedCalc® software. RESULTS: Among the 386 identified proteins, 20 had different abundance in SPF and STP groups. Eleven proteins had a higher abundance in the SPF group. They included apolipoprotein E, a regulator of protein kinase C, regulators of the coagulation cascade, complement activity, antigen binding, inflammatory response, etc. Among nine proteins that were more abundant in the STP group, the most important were: apolipoprotein A1, coagulation cascade proteins, and proteins involved in cell proliferation, ion transport, homeostasis of thiol components, antigen processing, and presentation. Furthermore, three proteins showed the highest potential to differentiate between the SPF and STP groups with satisfactory accuracy. Those were protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I, which all had higher abundance in the SPF group. CONCLUSION: The milk proteome pattern differs depending on bacterial culture in milk. Further studies will additionally address pathophysiological changes and clinical utility of the observed differences.

Objective: The occurrence of bovine mastitis (inflammation of the mammary gland) is considered to be one of the biggest challenges on dairy farms, due to reduction of the animal welfare and milk production and, consequently, economic loss for the farm. Therefore, the diagnosis of mastitis at subclinical stage and early treatment is of the utmost importance. However, the lack of clinical symptoms and unambiguous biomarkers for the diagnosis in early disease stage, prevent further advancements. Thus, the objective of this study was to implement the quantitative tandem mass tag (TMT) approach in combination with statistical and bioinformatics data processing for determination of quantitative changes in milk proteomic profiles during development and progression of mastitis on dairy farms. Methods: Milk samples (N = 138) were aseptically collected on dairy farms after physical examination and before the milking. Based on the results obtained for somatic cell count (SCC) and bacterial culture (BC) examination, milk samples were classified into experimental groups: healthy positive control (group with normal SCC and positive BC, N = 20), healthy negative control (group with normal SCC and negative BC, N = 20), subclinical mastitis (moderately increased SCC and positive BC, N = 80), clinical mastitis (high SCC and positive BC, N = 18). Samples were processed using TMT label-based quantitative approach. High resolution LC-MS/MS analysis was carried out using the Ultimate 3000 RSLCnano system (Dionex, Germering, Germany) coupled to Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Protein identification and quantification were performed using Proteome Discoverer software (version 2.3., Thermo Fisher Scientific) with SEQUEST algorithm implemented and database search against Bos taurus FASTA files. Statistical and bioinformatic analysis were done using R and Cytoscape. Results: Altogether, 386 quantifiable proteins were identified. There were 145 proteins with statistically different abundance between the groups. Gene Ontology analysis of proteins with significantly altered abundances showed that milk proteins were involved in biological adhesion, immune system process, biological regulation, interspecies interaction between organism, response to stimulus, signalling and others. Based on the statistical and fold change difference between investigated groups complement component 3, cathelicidin-1, cathelicidin-7, lactoferrin, haptoglobin, and chitinase-3-like protein were identified as potential biomarkers for subclinical mastitis. Conclusion: Shotgun TMT-based high-resolution proteomic profiling allowed identification of potential milk biomarkers for differentiation of mastitis grade in dairy cows. After validation of selected proteins, these findings might provide a valuable contribution to understanding of mastitis pathophysiology and might be applicable to the development of a new cow-side diagnostic tool.

The objectives of this study were to investigate the effect of the dog’s age, semen quality, and the duration and season of semen transit on whelping rate and litter size after insemination with transported chilled extended semen. The sperm rich fraction was collected from 43 dogs of 18 breeds, which were presented at the Clinic for chilled semen transport, in the period from 2017 to 2021. Immediately after collection, the total sperm concentration and count, motility, membrane integrity (HOS test), the percentage of live spermatozoa and sperm morphology (eosin nigrosin staining) were evaluated. The sperm rich fraction was centrifuged and diluted with Tris – fructose - citrate extender with the addition of 20% (v/v) egg yolk, then chilled and prepared for shipping. A dose consisted of at least 200x106 live, motile, morphologically normal spermatozoa. The data on the dog’s age, chilled sperm transit time, the season of transit, and the whelping rate and litter size after insemination were recorded. The whelping rate was 55.8% with a mean (±SEM) litter size of 4.71±0.58 pups. The total number of spermatozoa was higher in artificial insemination (AI) that resulted in whelping compared to unsuccessful AI (P, Svrha rada bila je istražiti utjecaj dobi psa, kvalitete sjemena te trajanja i sezone transporta ohlađenog sjemena na postotak štenjenja i veličinu legla nakon osjemenjivanja kuja ohlađenim, transportiranim sjemenom. U razdoblju od 2017. do 2021., od 43 psa 18 različitih pasmina koji su došli na kliniku s ciljem transporta ohlađenog sjemena, prikupljena je spermom bogata frakcija ejakulata. Neposredno nakon uzimanja ejakulata ocjenjena je koncentracija, ukupan broj spermija, pokretljivost, integritet membrane (HOS test), postotak živih spermija i morfologija spermija (bojenje eozin nigrosinom). Spermom bogata frakcija je centrifugirana i razrijeđena s Tris – fruktoza – citratnim razrjeđivačem s dodatkom 20% žumanjaka, nakon čega je uzorak ohlađen i pripremljen za transport. Doza za osjemenjivanje sadržavala je najmanje 200x106 živih, pokretnih, morfološki normalnih spermija. Zabilježeni su podatci o dobi pasa, tranzitnom vremenu, sezoni transporta te postotku štenjenja i veličini legla nakon osjemenjivanja transportiranim ohlađenim sjemenom. Od ukupnog broja osjemenjenih kuja, 55,8% se oštenilo s prosjekom (±SEM) od 4,71±0,58 šteneta po leglu. Ukupan broj spermija bio je viši kod osjemenjivanja koja su rezultirala štenjenjem nego kod neuspješnih osjemenjivanja (P

Globalno zatopljenje predstavlja direktnu i indirektnu prijetnju preživljavanju mnogih živih bića na zemlji. Uz direktne prijetnje koje donose prirodne katastrofe koje prate klimatske promjene poput uragana, tsunamija, poplava i suša, tu je i vrlo značajan utjecaj toplinskog stresa. Konji čija je sezona razmnožavanja direktno povezana s toplijim dijelom godine i sezonom najduljih dana, nalaze se pred fiziološkim izazovom. Njihova zona komfora je do 25°C a dobro podnose temperature do 30°C. Donedavno su temperature iznad 25°C tijekom 24 i više sati u umjerenom pojasu bile iznimna a danas su česta pojava. Stoga je vrlo vjerojatno da će razmnožavanje konja kao vrste u uvjetima globalnog zatopljenja predstavljati sve veći izazov kojem nije moguće doskočiti niti metodama asistirane reprodukcije jer je utjecaj toplinskog stresa na rasplođivanje konja kompleksan i sveobuhvatan.

Mastitis je među najčešćim bolestima na farmama mliječnih krava za čije se liječenje i prevenciju koristi najveća količina antibiotika. Učestala, neispravna ili nepotrebna uporaba antimikrobnih sredstava predstavlja veliku prijetnju ubrzanom razvoju antimikrobne rezistencije. Prijenos antimikrobno rezistentnih patogena na ljude moguć je putem mlijeka i mliječnih proizvoda te su propisane smjernice za racionalnu primjenu antimikrobnih sredstava u veterinarskoj medicini. U mliječnoj se industriji najviše antibiotika koristi prilikom zasušivanja krava u svrhu prevencije nastanka mastitisa. U isto vrijeme antibiotici se nepotrebno primjenjuje i u životinja koje nisu inficirane. Iz tog razloga sve više zemalja prelazi na model selektivnog zasušivanja krava. Antibioticima se zasušuju samo inficirane četvrti ili vimena krava, odnosno krave s povećanim rizikom za nastanak infekcije. Jedan od većih problema prilikom uvođenja selektivnog zasušivanja krava je odabir kriterija prema kojem će se odabrati krave koje će biti zasušene antibiotikom. Protokoli za odabir krava trebaju biti dovoljno precizni, jednostavni za interpretaciju, jeftini, sigurni, certificirani i dostupni za uporabu u svim dijelovima zemlje. Kriterij za odabir krava može se temeljiti na mikrobiološkoj pretrazi mlijeka, određivanju BSS prilikom zasušivanja, incidenciji kliničkih mastitisa u laktaciji, paritetu i njihovim međusobnim kombinacijama. Bakteriološka je pretraga mlijeka najpouzdaniji pokazatelj, ali se broj somatskih stanica na posljednjoj mužnji ističe kao pouzdan pokazatelj na temelju kojeg se može provesti selektivno zasušenje bez negativnih učinaka na proizvodnju. Granična vrijednost broja somatskih stanica kao učinkovit i održiv model za donošenje odluka iznosi 100.000 st/mL mlijeka za primiparne i 200.000 st/mL mlijeka za multiparne krave. Uporaba antibiotika za zasušenje samo kod inficiranih četvrti krava omogućava dodatno smanjenje primjene antibiotika. Iako je tijekom godina postignut značajan napredak u razumjevaniju epidemiologije, imunologije, dijagnostike i patogeneze intramamarnih infekcija (IMI) u suhostaju u programima suzbijanja mastitisa naglašava se značenje smještaja krava u suhostaju i prijelaznom razdoblju. Važnost kontrole IMI u suhostaju u svakodnevnoj praksi se često podcjenjuje., Mastitis is the most common disease in dairy cows and most antibiotics are used for treatment and prevention. Frequent, inappropriate, or unnecessary use of antimicrobials poses a major risk for accelerated development of antimicrobial resistance. Transmission of antimicrobial-resistant pathogens to humans is possible via milk and dairy products, and guidelines for the rational use of antimicrobials in veterinary medicine are prescribed. In dairy farming, most antibiotics are used to prevent mastitis at drying off. At the same time, antibiotics are used unnecessarily even in uninfected animals. An alternative strategy that promotes lower antimicrobial usage is selective dry cow therapy, where only cows with mastitis receive antibiotic treatment. However, an issue in the implementation of selective dry cow treatment is the criteria applied for selecting the cow for drying off with antibiotics. Cow selection protocols should be sufficiently accurate, easy to perform and interpret, cost-effective, safe, certifiable, and applicable in different areas of a country. The criteria for cow selection can be based on bacteriological culture of milk, somatic cell count at drying off, incidence of clinical mastitis in previous lactation, parity, or any of their combinations. Bacteriological culture of milk is the gold standard, though somatic cell count at the last milking has proven to be a reliable indicator for cow selection to drying off with antibiotics without negative effects on milk production. The thresholds of

A 15-year-old intact female Pekingese was referred to the Clinic because of vaginal bleeding and polydipsia. The patient had a previous diagnosis of degenerative mitral valve disease ACVIM stage C, and was stable under standard therapy. An enlarged and painful abdomen was observed during examination. Haematology and biochemistry profile showed signs of inflammation, most probably due to infection, and increased BUN and creatinine levels. Abdominal ultrasound showed dilation of the uterus with anechoic area and right uterine horn filled with content. The patient was premedicated with midazolam (0.25 mg/kg IV) and methadone (0.15 mg/ kg IV). General anaesthesia was induced with propofol (3 mg/kg IV), followed by endotracheal intubation. The patient was connected to the anaesthetic machine with a rebreathing circuit and maintenance of the anaesthesia was provided by a combination of oxygen and isoflurane. Pain management was provided by the use of fentanyl boluses (2, 5 µg /kg IV). Patient monitoring included capnometry, non-invasive arterial blood pressure measurement, pulse oximetry, and ECG. Intraoperative findings were unilateral segmental aplasia of the left uterine horn and right horn filled with content and uterine body tumor. Ovariohisterectomy was performed with subsequent pathohistology. The pathohistology findings were: squamous metaplasia and cystic degeneration of the endometrial glands, uterine adenomyosis and squamous aplasia of the left uterine horn and left ovary. Postoperative care included pain management, antibiotic therapy, control of renal parameters and constant monitoring of breathing frequency with with attention to the occurrence of edema due to cardiac problems. The patient was discharged to home care two days after the procedure. In conclusion, this was a complex case of unilateral uterine horn aplasia, pyometra and uterine tumor complicated with a heart disease that in the end had positive outcome

Attempted preparation of a chelated Co\(^{IV}\) \(\beta\)-silylamide resulted in the unprecedented disproportionation to Co\(^{0}\) and a spirocyclic cobalt(IV) bis(\(\beta\)-silyldiamide): [Co[(N\(^{t}\)Bu)\(_2\)SiMe\(_2\)]\(_2\)] (1). Compound 1 exhibited a room-temperature magnetic moment of 1.8 B.M. and a solid-state axial EPR spectrum diagnostic of a rare S=1/2 configuration for tetrahedral Co\(^{IV}\). Ab initio semicanonical coupled-cluster calculations (DLPNO-CCSD(T)) revealed the doublet state was clearly preferred (−27 kcal mol\(^{−1}\)) over higher spin configurations only for the bulky tert-butyl-substituted analogue. Unlike other Co\(^{IV}\) complexes, 1 had remarkable thermal stability, and was demonstrated to form a stable self-limiting monolayer in preliminary atomic layer deposition (ALD) surface saturation experiments. The ease of synthesis and high stability make 1 an attractive starting point to investigate otherwise inaccessible Co\(^{IV}\) intermediates and for synthesizing new materials.