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Theoretical and empirical evidence indicates that low external validity due to rigorous standardization of study populations is a cause of poor replicability in animal research. Here, we report a multi-laboratory study to test whether heterogenization of study populations by using animals from different breeders increases the replicability of results from single-laboratory studies. We found that heterogenization by breeder was not potent enough to increase external validity sufficiently to improve replicability.

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions 1–4. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment 5. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between rearing facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multi-center study, where inbred mice from the same breeding stock were reared in five different facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that common variation among rearing facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organization. We detected changes in chromatin organization in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity and regulation of behavior. Our findings demonstrate that common environmental differences between rearing facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. We expect our findings to stimulate further research into the mechanisms and drivers of these epigenetic changes mediated by the laboratory environment. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and a need to account for the animals’ environmental background in study design to produce robust and replicable findings.

Bakterijska okužba z M.tuberculosis lahko povzroči tuberkulozo, bolezen, ki v večini primerov prizadane pljuča, lahko pa tudi ostale organe. V nekaterih primerih je lahko tudi smrtna. Standardno zdravljenje poteka s protibakterijskimi učinkovinami, natančneje z izoniazidom ali več učinkovinami hkrati (rifampicin, etambutol). Za učinkovito zdravljenje tuberkuloze je potrebno sočasno jemanje več zdravil. Čeprav se je pojavnost in smrtnost bolezni v zadnjih letih močno zmanjšala, je ta napredek pogosto spregledan zaradi naraščanja razširjenosti okužb z večkrat odpornimi bakterijami tuberkuloze ali še hujše oblike rezistence. Zaradi odpornosti določenih sevov na obstoječe metode zdravljenja se razvijajo nove oblike zdravilnih učinkovin za zdravljenje tuberkuloze, gre za t.i. direktne zaviralce encima InhA: tetrahidropiranski derivati, triklosan, piridomicin, 4-hidroksi-2-piridoni, tiadiazoli. Pri eksperimentalnem delu smo se osredotočili na bioizosterno zamenjavo osrednje fenilne skupine na tetrahidropiranskem tipu InhA zaviralcev. S pomočjo obročnih ekvivalentov smo fenilni obroč zamenjali s tiofenom in tiazolom. Med eksperimentalnim delom smo se srečali z določenimi tipi reakcij, kot npr.: radikalno halogeniranje – bromiranje redukcijo nitrilov do aminov sklopitvenimi reakcijami, kjer se je tvoril karboksamid. Uspeli smo sinetizirati dve končni spojini: spojino 8: 5-((3,5-dimetil-1H-pirazol-1-il)metil)-N-((4-(4-feniltiazol-2-il)tetrahidro-2H-piran-4-il)metil)tiofen-2-karboksamid in spojino 16: 2-((3,5-dimetil-1H-pirazol-1-il)metil)-N-((4-(4-feniltiazol-2-il)tetrahidro-2H-piran-4-il)metil)tiazol-4-karboksamid. Po končani sintezi smo analizirali končne spojine z jedrsko magnetno resonanco (1H,13C) in masno spektrometrijo visoke ločljivosti. Preverili smo tudi čistost dobljenih spojin s tekočinsko kromatografijo visoke ločljivosti. Končnim spojinam so na encimu InhA določili vrednost zaviralne koncentracije (IC50) na encimu. Glede na rezultate analiz se je tiofen izkazal za ustreznejšo zamenjavo fenila. Uspelo nam je pripraviti bolj čist tetrahidropiranski deriavat s tiofenom kot s tiazolom, poleg tega je izkazal tudi nižjo IC50 vrednost od tiazola. The bacterial infection with M.tuberculosis can cause tuberculosis, a disease that in most cases affects the lungs, but it can also affect other organs. Sometimes, it can also be deadly. The standard treatment uses antibacterial substances, specifically isoniazid or more substances at the same time (rifampicin, etambutol). For an effective treatment, it is necessary to take several medications simultaneously. Although the incidence and mortality of the disease lowered in the last years, this achievement is often overlooked because of increasing infections with multiple drug-resistant M. tuberculosis or an even worse form of resistance. Due to the resistance of strains to the existing methods of treatment, new forms of active substances for treatment of tuberculosis are being developed, these are direct inhibitors of enzyme InhA: tetrahydropyran derivatives, triclosan derivatives, pyridomycin, 4-hydroxy-2-pyridones, thiadiazoles. In the experimental part of our work, we focused on the effect of bioisosteric replacement of central phenyl group on the inhibitory activity of the tetrahydropiran type InhA inhibitors. With the help of ring equivalents, we replaced phenyl group with thiophene and thiazole. During this part of our work, we encountered a certain type of reactions, e.g. radical halogenation – bromination, reduction of nitriles to amines and coupling reactions. We synthesized two final compounds: compound 8: 5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-N((4-(4-phenylthiazol-2-yl)tetrahydro-2Hpyran-4-yl)methyl)thiophene-2-carboxamide, and compound 16: 2-((3,5-dimethyl-1H-pyrazol-1yl)methyl)-N-((4-(4-phenylthiazol-2yl)tetrahydro-2H-pyran-4yl)methyl)thiazole-4-carboxamide. After the synthesis was completed, we analyzed the final compounds with nuclear magnetic resonance and high resolution mass spectrometry. We also checked the purity of the compounds with high performance liquid chomatography. The final compounds were tested on encyme InhA for the values of IC50. Based on the results of the analysis, tiophene proved to be the better replacement of phenyl. We also managed to prepare a purer tetrahydropyran derivative with thiophene than with thiazole and it showed a lower IC50 value than tiazol.

Poor reproducibility is considered a serious problem in laboratory animal research, with important scientific, economic, and ethical implications. One possible source of conflicting findings in laboratory animal research are environmental differences between animal facilities combined with rigorous environmental standardization within studies. Due to phenotypic plasticity, study-specific differences in environmental conditions during development can induce differences in the animals’ responsiveness to experimental treatments, thereby contributing to poor reproducibility of experimental results. Here, we studied how variation in weaning age (14–30 days) and housing conditions (single versus group housing) affects the phenotype of SWISS mice as measured by a range of behavioral and physiological outcome variables. Weaning age, housing conditions, and their interaction had little effect on the development of stereotypies, as well as on body weight, glucocorticoid metabolite concentrations, and behavior in the elevated plus-maze and open field test. These results are surprising and partly in conflict with previously published findings, especially with respect to the effects of early weaning. Our results thus question the external validity of previous findings and call for further research to identify the sources of variation between replicate studies and study designs that produce robust and reproducible experimental results.

Salata kristalka sorte “Exquise” uzgojena je na otvorenom i u zatvorenom prostoru OPG-a iz Murskog Središća s ciljem određivanja kvalitete i održivosti. Kvaliteta salate utvrđena je odmah nakon berbe te nakon 5 i 13 dana skladištenja na 6 °C i 86 % vlage zraka u rashladnom prostoru. Utvrđene su sljedeće vrijednosti kemijskog sastava: sadržaj suhe tvari 6, 39-8, 86 %, sadržaj ukupne topljive suhe tvari 2, 00-7, 33 %, sadržaj ukupnih kiselina 0, 06-0, 18 %, vrijednost pH 5, 47-5, 92, sadržaj vitamina C 11, 38-19, 54 mg/100 g svježe tvari, sadržaj ukupnih fenola 37, 79- 165, 84 mg GAE/100 g svježe tvari te antioksidacijski kapacitet 1, 04-2, 24 mmol TE/L. Rezultati bioaktivnih spojeva pokazali su značajne statističke razlike između načina uzgoja salate i kod vremena skladištenja.

We studied how space allowance affects measures of animal welfare in mice by systematically varying group size and cage type across three levels each in both males and females of two strains of mice (C57BL/6ByJ and BALB/cByJ; n = 216 cages, a total of 1152 mice). This allowed us to disentangle the effects of total floor area, group size, stocking density, and individual space allocation on a broad range of measures of welfare, including growth (food and water intake, body mass); stress physiology (glucocorticoid metabolites in faecal boli); emotionality (open field behaviour); brain function (recurrent perseveration in a two-choice guessing task); and home-cage behaviour (activity, stereotypic behaviour). While increasing group size was associated with a decrease in food and water intake in general, and more specifically with increased attrition due to escalated aggression in male BALB mice, no other consistent effects of any aspect of space allowance were found with respect to the measures studied here. Our results indicate that within the range of conditions commonly found in laboratory mouse housing, space allowance as such has little impact on measures of welfare, except for group size which may be a risk factor for escalating aggression in males of some strains.

Prehrambena vrijednost salate značajna je zbog bogatog nutritivnog sastava, radi kojeg zauzima i značajno mjesto u svakodnevnoj prehrani. Cilj ovog rada bio je utvrditi razlike u količini nutrijenata u salati iz zaštićenog prostora i uzgoja na otvorenom, te mogućnosti skladištenja u rashladnom prostoru. Prema dobivenim rezultatima osnovnog kemijskog sastava najveći sadržaj svih istraživanih parametara utvrđen je kod svježe salate iz plastenika. S obzirom na vrijeme čuvanja salate (5 i 13 dana)vrijednosti osnovnog kemijskog sastava su značajno varirale, odnosno tijekom skladištenja sve navedene vrijednosti značajno su se reducirala uz izuzetak sadržaja ukupnih kiselina.

V diplomski nalogi sem z metodo analize izbljuvkov analizirala prehrano pegaste in lesne sove na Dravskem polju. Ugotovila sem, da je pegasta sova uplenila 13 različnih vrst malih sesalcev in ptice. Najpogosteje je pegasta sova plenila poljsko voluharico, sledi ji hišna miš. Plenila je še navadno belonogo miš, vrtno rovko, poljsko rovko, ptiče, dimasto miš, rumenogrlo miš, travniško voluharico, gozdno rovko, povodno rovko, črno podgano, velikega voluharja in vrtno voluharico. Medtem, ko je lesna sova na področju Štatenberga uplenila 6 različnih vrst malih sesalcev, ptiče, rake, žabe in hrošče. Lesna sova na področju Drevenika pa 8 različnih vrst sesalcev, ptiče, hrošče, rake in žabe. Pri lesnih sovah je bil najpogosteje uplenjen mali sesalec belonoga miš. Na podlagi najdenih čeljusti v izbljuvkih sem ugotovila razširjenost malih sesalcev na raziskovalnem področju. In my graduation thesis I have analysed a diet of barn owl and tawny owl on Dravsko polje. I realized that the barn owl fed 13 different kinds of small mamals and birds also. A barn owl was looting a bicolored shrew (Crocidura leucodon) and a mouse (Mus musculus domesticus) most often. A tamny owl was looting mood mouse (Apodemus sylvaticus) and yellow – necked mouse (Apodemus flavicollis) most often. I also found birds, crustaceans, frogs and beetles. On the basic of the founden jaws of small mammals I realized how extended are small mammals on Dravsko polje.

Zbog opće zabrinutosti javnosti zbog stanja okoliša u posljednjih 50 godina došlo je do rasta zanimanja za očuvanjem okoliša te ekološkom proizvodnjom hrane. Ideja da planet prolazi kroz ekološku krizu koja može ugroziti i samo postojanje života kakav poznajemo vrlo je raširena. U rješavanju tog problema od na određeni način može pomoći i međimurski konj. Međimurski konj je radni konj, najviše rabljen za obavljanje poljoprivrednih radova i transport roba. S prestankom potrebe za radnim konjima, pasmina izumire i održavaju je tek rijetki uzgajivači iz čiste ljubavi spram konja. Manjim dijelom rabe se za potrebe turističkih zaprega, terapijskog jahanja, za rad na polju i u šumi. Brz razvoj željeznice i pojava motornih transportnih sredstava u prvoj polovici 20. stoljeća smanjuje potražnju za radnim konjima, što dovodi do drastičnog smanjenja populacije međimurskog konja. Obzirom da ova pasmina ima svoju genetsku, kulturološku i određenu minornu gospodarsku vrijednost, u ovom radu istraženi su trendovi u okruženju, te mogućnost uključivanja međimurskog konja u ekološku poljoprivredu, ruralni turizam, terapijsko jahanje, te proizvodnju kobiljeg mlijeka. Ovaj rad prikazuje kratku povijest pasmine, njene karakteristike, brojno stanje, probleme te daljine uzgojne i uporabne mogućnosti.

Cognitive processes are influenced by underlying affective states, and tests of cognitive bias have recently been developed to assess the valence of affective states in animals. These tests are based on the fact that individuals in a negative affective state interpret ambiguous stimuli more pessimistically than individuals in a more positive state. Using two strains of mice we explored whether unpredictable chronic mild stress (UCMS) can induce a negative judgement bias and whether variation in the expression of stereotypic behaviour is associated with variation in judgement bias. Sixteen female CD-1 and 16 female C57BL/6 mice were trained on a tactile conditional discrimination test with grade of sandpaper as a cue for differential food rewards. Once they had learned the discrimination, half of the mice were subjected to UCMS for three weeks to induce a negative affective state. Although UCMS induced a reduced preference for the higher value reward in the judgement bias test, it did not affect saccharine preference or hypothalamic–pituitary–adrenal (HPA) activity. However, UCMS affected responses to ambiguous (intermediate) cues in the judgement bias test. While control mice showed a graded response to ambiguous cues, UCMS mice of both strains did not discriminate between ambiguous cues and tended to show shorter latencies to the ambiguous cues and the negative reference cue. UCMS also increased bar-mouthing in CD-1, but not in C57BL/6 mice. Furthermore, mice with higher levels of stereotypic behaviour made more optimistic choices in the judgement bias test. However, no such relationship was found for stereotypic bar-mouthing, highlighting the importance of investigating different types of stereotypic behaviour separately.