ABSTRACT Laboratory animals have frequently been used in scientific and preclinical pharmaceutical drug safety and efficacy research. Although the introduction of new in silico assays and computer modeling for drug discovery has shown promise in reducing laboratory animal trials, there is still a need to develop in vitro alternatives to in vivo animal models. The in vitro spheroid wound model is one of the best options for developing alternative techniques to animal research as it is the most widely used laboratory animal model. The aim of the study is to using 3D in vitro wound modeling as an alternative to in vivo wound healing assays. In the study, a three-dimensional cell culture (organoid culture) with cell/cell and cell/matrix junctions was generated using the most common Fibroblast and HaCaT cell lines hanging drop technique to replicate the healing stages in the injured skin area. After spheroid epidermal structures were formed, inhibitors and activators were added to the culture medium and their effects on the wound line and 3D cells produced were determined. It was noted that the number of spheroid structures increased significantly and cell-cell interactions became visible in the additional activator groups compared to the control groups. When the inhibitor-treated group was compared with the control groups, it was observed that the formed structures completely disappeared or decreased in amount and cell-cell interactions could not be established. In conclusion, this study offers an alternative to using laboratory animals to evaluate potential medicines and/or extracts in wound healing experiments.

Adipokines are a group of proteins that are synthesized from adipose tissue. Omentin is a type of adipokine which is detected in human serum and it is evaluated as a secreted factor from adipose tissue, placenta and ovarium. The knowledge about the effect of omentin on reproduction is limited. In the presented study, investigation of omentin effect on in vitro uterine contractions was evaluated. The uteruses of the rats which were collected from 20 virgin female rats, were isolated and suspended in the isolated organ bath. The experimental protocols were carried out in 3 groups. The effect of omentin on spontaneous uterine contractions in protocol 1, induced by oxytocin (2.5 mIU/mL) in protocol 2, and induced uterine contractions with PGF2α (10-6 M) in protocol 3 was tested. The effect of omentin on isolated uterine tissues was tested with the administration of 10, 100 and 300 ng/mL omentin. 300 µL of distilled water was applied to the control of each group. In this study, 10, 100 and 300 ng/mL of omentin applied for 10 minutes did not have an effect on uterine contractility in all study groups (p>0.05). As a result, further studies with higher doses and longer incubation times are recommended.

The purpose of this study was to investigate the effect of gamithromycin on rat uterus smooth muscles and to evaluate the possible mechanism of action. Forty-four uterine tissues isolated from 16 female Wistar rats weighing 200-250 grams were used in the experiments. In the preliminary experiments, gamithromycin was tested at concentrations of 10-7 M, 10-6 M and 10-5 M; where 10-5 M was selected for the experiments. In Group 1, the uterus segments were treated for 10 min with 10-5 M gamithromycin following 10 min control contractions. In Group 2, the effect of 10-5 M gamithromycin over 2.5 mIU / mL oxytocin contraction was evaluated. In Group 3, the effect of 10-5 M gamithromycin was evaluated over 10-8 M atropine incubation. In Group 4, 10-5 M gamithromycin was applied for 10 min followed by 0.0625 μg/ml of cloprostenol application. The same protocols were applied for dimethylsulfoxide (DMSO) as control. Frequency, average amplitude and peak amplitude values ​​of the contractions were assessed. In Group 1, gamithromycin in were shown to increase the contractility at 10-5 M significantly (P≤0.01); while no statistically significant difference was observed in comparison to DMSO (P> 0.05). For the other tested groups, no statistically important difference were observed (P> 0.05). The nonsignificant difference of the results of this study can be attributed to the chemical form of gamithromycin and the concentration used. In order to be able to fully assess the effects and possible mechanism of gamihtromycin on the uterine smooth muscle, higher gamithromycin concentrations should be studied and, if possible, further studies should be performed with different agonist and antagonist agents.

This study investigated oxyclozanide, a salicylanilide anthelmintic, as an alternative treatment for Methicillin-resistant Staphylococcus aureus (MRSA) in bovine mastitis. With conventional treatments facing challenges due to antibiotic resistance, oxyclozanide demonstrated promising in vitro antimicrobial effect against MRSA isolates. The minimal inhibitory concentration (MIC) ranged from 0.5 to 1.0 μg/ml, indicating its efficacy. Oxyclozanide’s mechanism involved disrupting the transmembrane structure of bacteria, presenting a potential strategy against MRSA. This suggested the repurposing of oxyclozanide as a viable option for combating MRSA in bovine mastitis, prompting further investigation into its precise mechanism and therapeutic potential.

The present study was conducted to study the effect of maize (Zea mays) cob on replacing paddy straw in the Total Mixed Ration (TMR). Twelve cross bred calves of about 5 to 8 months of age with body weight ranging from 41 to 79 kg were divided into two groups of six each in completely randomized design. Five complete diets were prepared (TMR1 to TMR5) using maize cobs at the level of 0%, 25%, 50%, 75% and 100% replacement of paddy straw in the diets containing 12 % CP and 60 % TDN of 50:50 concentrate to roughage ratio. There was significant (P<0.01) differences among the diets in OM, CF, NFE, TA, AIA, NDF, ADF, Lignin, Hemicellulose and Cellulose contents. In vitro rumen fermentation study showed significantly (P<0.01) higher total gas (ml/200 mg/48 h), in vitro dry matter and organic matter degradability in maize cob based ration than paddy straw contained ration (51.17 vs 36.00; 62.87 vs 57.25; 64.80 vs 59.93). Paddy straw (100%) based diet as control ration and maize cob (100 % replacement of paddy straw) based diet as treatment ration fed to growing calves for sixty days in growth trial. A seven day digestion trial was conducted in the middle of the experiment. The digestibility (%) of DM, OM, CP, EE, CF and NFE were significantly (P<0.01) higher in the maize cob based diet fed group than paddy straw based diet fed group. The average body weight gain (kg) and FCR (kg DMI/ kg gain) were significantly (P<0.05) higher in maize cob fed animals (19.47; 6.10) than paddy straw fed animals (16.02; 7.34). The feed cost per unit of weight gain in calves fed paddy straw based diet was numerically higher (30.78 %) than calves fed on maize cob based ration. It could be concluded that maize cob based ration could replace paddy straw at 100 % level in total mixed ration without affecting feed intake and nutrient digestibility and may also improve the body weight gain and reduce feed cost in growing calves.

Adhesion of equine spermatozoa to homologous oviduct epithelial cells (OEC) in vitro results in specific changes in spermatozoa and OEC function. To test the hypothesis that adhesion of spermatozoa affects protein synthesis and secretion by OEC, the following treatment groups were established in culture: OEC with culture medium only; control spermatozoa in culture medium only; OEC in coculture with spermatozoa; and OEC and spermatozoa in coculture, but physically separated by a microporous membrane. The experiment was replicated within each of 4 ejaculates from 3 stallions. De novo protein secretion by OEC was measured and compared by incorporation of [35S]methionine, and evaluated, using two-dimensional polyacrylamide gel electrophoresis and fluorography. Monolayers of OEC secreted a large number of proteins of molecular mass ranging from 14 to 205 kd. Adhesion of spermatozoa consistently caused reduced synthesis of 2 OEC secretory proteins and new or increased synthesis of 6 proteins. When spermatozoa and OEC were separated by a microporous membrane, some but not all of these changes were duplicated. Synthesis of 3 OEC secretory proteins, unaffected by binding of spermatozoa, was reduced when spermatozoa were prevented from contact with OEC by a microporous membrane. Adhesion of equine spermatozoa to homologous OEC monolayers and presence of equine spermatozoa resulted in qualitative and quantitative changes in synthesis and secretion of proteins by OEC. These changes have implications for storage, longevity, and maturation of spermatozoa.

Piroxicam and other nonsteroidal anti-inflammatory drugs (NSAID) have antitumor activity against naturally acquired cancer in dogs and human beings, and against experimentally induced tumors in rodents. We are investigating potential mechanisms of NSAID anti-tumor activity. The direct cytotoxicity of piroxicam, indomethacin, and aspirin against 4, canine tumor cell lines (transitional cell carcinoma, squamous cell carcinoma, melanoma, and soft tissue sarcoma) was determined in short-term growth rate assays and in clonogenic assays. Piroxicam was evaluated alone and in combination with the lipoxygenase inhibitor zileuton, and in combination with the chemotherapeutic agents cisplatin and carboplatin. The 50% inhibitory concentrations (IC50) against melanoma cells in short-term growth rate assays were: 530 micromolar piroxicam, 180 micromolar indomethacin, and greater than 1 mM aspirin. These IC50 values were over 10 times greater than serum concentrations of these drugs that could safely be achieved in vivo. The IC50 of zileuton combined with piroxicam (280 micromolar) was not different from the IC50 of zileuton alone (230 micromolar; ANOVA P = 0.47) in melanoma cells. Similarly, addition of piroxicam did not alter the IC50 of either cisplatin (1.6 micromolar) or carboplatin (6.1 micromolar). These results suggest that NSAID, at serum concentrations achievable in vivo, do not have direct cytotoxicity against canine tumor cells tested. It is unlikely that the in vivo antitumor activity of NSAID is attributable to a direct cytotoxic effect.

Systemic administration of dexamethasone led to a significant reduction in the size of the tuberculin reaction in response to intradermal injection of bovine purified protein derivative in 18 cattle experimentally sensitized to Mycobacterium bovis (P < 0.01) and 8 cattle naturally infected with M bovis (P < 0.001). The reaction in 6 of the 7 M bovis-infected cattle that received dexamethasone was classified as negative for the standard interpretation of the single intradermal comparative tuberculin test. Significantly fewer BoCD2+ (P < 0.05) and BoCD4+ T cells (P < 0.001) were present at the reaction site and in blood of dexamethasone-treated cattle, compared with untreated control cattle. Significantly fewer cells expressing the interleukin-2 receptor and WC1+ gamma delta T cells (P < 0.001), and a significantly greater number of cells expressing the ACT2 antigen (P < 0.05) were found at the reaction site in dexamethasone-treated cattle than in controls. The number of BoCD8+ T cells at the reaction site and in blood was not significantly affected by administration of dexamethasone. In vitro production of interferon-gamma by lymphocytes incubated with bovine purified protein derivative also was significantly lower (P < 0.01) in the dexamethasone-treated cattle.

Parenchymal cells were isolated from the liver of male calves, and monolayer cultures formed were treated with glucocorticoids to examine whether haptoglobin, appearance of which is associated with hepatic lipidosis (fatty liver) in cattle, is induced by steroid hormones. Without addition of dexamethasone, only trace amounts of haptoglobin were detected in culture medium. With addition of dexamethasone (10(-12) to 10(-4)M), considerable amounts of haptoglobin were released into the medium. Maximal release was observed at concentrations of 10(-8) to 10(-6)M dexamethasone. Haptoglobin release was similarly induced by cortisol, although the effect was less potent than that of dexamethasone. Actinomycin D (a known protein synthesis inhibitor) dose-dependently reduced amounts of haptoglobin released in response to 10(-8)M dexamethasone. Dexamethazone also induced annexin I, which is known to be synthesized in response to glucocorticoids. Dexamethasone treatment resulted in reduced protein kinase C activity in the cell cytosol, which has been shown to be an early event in dexamethasone-treated cells. Other than glucocorticoids, estradiol induced haptoglobin release, whereas progesterone was less effective. The association of haptoglobin with hepatic lipidosis can be reasonably explained by the fact that haptoglobin production by the liver is induced by glucocorticoids and estradiol, and these steroid hormones are triggers for development of hepatic lipidosis in cattle.

The in vitro activity of trimethoprim (TMP) and 9 sulfonamides and their combinations in 6 concentration ratios was tested against 62 Salmonella strains isolated from horses over a 3-year period in the Netherlands, using the agar-dilution method. Most of the isolates were S typhimurium strains (n = 52); the others were S heidelberg (n = 3), S hadar (n = 2), S thompson (n = 2), S enteritidis (n = 1), S infantis (n = 1), and S derby (n = 1). The minimal TMP concentration at which 50% of the Salmonella strains were inhibited (MIC50) was 0.12 micrograms/ml. Sulfachlorpyridazine (SCP; MIC50, 16 micrograms/ml), sulfamethoxazole (SMX; MIC50, 32 micrograms/ml), and sulfadiazine (SDZ; MIC50; 32 micrograms/ ml) were the most potent of the sulfonamides tested. The antimicrobial effect of the sulfonamides, in combination with TMP (additive, synergistic, or antagonistic), was expressed by the fractional inhibitory concentration (FIC) index. Concentrations of SDZ and SCP with TMP had marked synergism at all tested TMP-to-sulfonamide concentration ratios (1:1 to 1: 160; FIC index, 0.10 to 0.50); SMX had synergy with TMP at all ratios, except 1:1 (FIC index, 0.10 to 0.27). Sulfamethazine, sulfamerazine, sulfadoxine (SDX), sulfatroxazole, sulfadimethoxine, and sulfacetamide had MIC50 greater than their breakpoint MIC value and are, therefore, less potent drugs. However, synergy with TMP was found for these less potent sulfonamides at certain concentration ratios, depending on the sulfonamide used. Sixteen Salmonella strains were resistant to TMP, all sulfonamides, and TMP-sulfonamide combinations; 14 of these strains were S typhimurium phage type 200, 1 was S typhimurium phage type 61, and I was S typhimurium phage type 10. Four additional Salmonella strains were resistant to the sulfonamides alone (1 S typhimurium phage type 171 and 3 S typhimurium strains that could not be biotyped). Results of this study indicate that SDZ, SCP, and SMX are the best sulfonamides to combine with TMP for treatment of salmonellosis in equids, because they are the most potent sulfonamides and have strong synergism with TMP at a wide range of TMP-to-sulfonamide concentration ratios.