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.

Curcuma longa rhizome is the source of turmeric. Curcumin exhibits some encouraging antiparasitic properties in helminths. The purpose of this investigation was to determine the ovicidal and larvicidal activities of curcumin in Toxocara canis eggs in vitro. Curcumin dilutions (36.8 mg/ml, 18.4 mg/ml and 3.6 mg/ml) were prepared by adding RPMI-1640. The eggs and hatched infective-stage larvae were incubated with curcumin dilutions for 6, 12 or 24 hours. The ovicidal activity was evaluated after 28 days. Larvicidal activity was assessed after completing each incubation time. In the present study, no changes in the eggshell structure were observed in all curcumin groups. The lowest embryogenesis rate (75%) was observed only at the highest curcumin dilution (36.8 mg/ml) at the 12- and 24-hour incubations, but the difference was not found statistically significant. No significant larvicidal effect of curcumin was detected. The percentage of moving larvae was 80% at 12 hours and 76% at 24 hours in the highest curcumin dilution (36.8 mg/ml). T. canis larvae survived in RPMI-1640 for four days after being incubated with 36.8 mg/ml curcumin for 24 hours. However, the untreated larvae were still active at this time. Further studies focusing on the migration of T. canis infective larvae in animal models may shed light on the effect of curcumin, which is rapidly metabolized in the body and absorbed at low levels from the intestine, on the migrating larva.

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.

An in vitro experiment was conducted to study the in vitro cellulose disappearance of the following seven experimental feeds such as 1) Control feed; 2) Experimental feed (75%) + Fresh brewery waste (25%) (EFFBW); 3) Experimental feed (75%) + Dried brewery waste (25%) (EFDBW); 4) Fresh brewery waste (FBW); 5) Dried brewery waste (DBW); 6) Paddy straw (PS) and 7) Brewery waste incorporated paddy straw (BWIPS) incubated for 2, 6, 12, 24, 48 and 72 hours in Rumen Simulation Technique (RUSITEC). During initial incubation periods (2, 6, 12 and 24 hours), control feed, EFFBW, EFDBW and FBW showed higher in vitro cellulose disappearance than other experimental feeds, whereas, during later incubation periods (48 and 72 hours), the DBW, paddy straw and BWIPS showed higher in vitro cellulose disappearance than other experimental feeds incubated in RUSITEC. Among the feeds experimented in RUSITEC, disappearance of cellulose was more in the control feed, EFFBW and EFDBW when compared to other experimental feeds. Similarly, the cellulose disappearance was more in BWIPS compared to paddy straw. The in vitro cellulose disappearance rates at all incubation periods were higher for FBW than those for DBW.

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.

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.

Toxoplasma gondii, an obligate intracellular parasite, is the aetiological agent of toxoplasmosis, a disease that affects approximately 25% – 30% of the world’s population. At present, no safe and effective vaccine exists for the prevention of toxoplasmosis. Current treatment options for toxoplasmosis are active only against tachyzoites and may also cause bone marrow toxicity. To contribute to the global search for novel agents for the treatment of toxoplasmosis, we herein report the in vitro activities of previously synthesised benzyltriazole derivatives. The effects of these compounds against T. gondii in vitro were evaluated by using a expressing green fluorescent protein (GFP) type I strain parasite (RH-GFP) and a type II cyst-forming strain of parasite (PruΔku80Δhxgprt). The frontline antitubercular drug isoniazid, designated as Frans J. Smit -isoniazid (FJS-INH), was also included in the screening as a preliminary test in view of future repurposing of this agent. Of the compounds screened, FJS-302, FJS-303, FJS-403 and FJS-INH demonstrated 80% parasite growth inhibition with IC50 values of 5.6 µg/mL, 6.8 µg/µL, 7.0 µg/mL and 19.8 µg/mL, respectively. FJS-302, FJS-303 and FJS-403 inhibited parasite invasion and replication, whereas, sulphadiazine (SFZ), the positive control, was only effective against parasite replication. In addition, SFZ induced bradyzoite differentiation in vitro, whilst FJS-302, FJS-303 and FJS-403 did not increase the bradyzoite number. These results indicate that FJS-302, FJS-303 and FJS-403 have the potential to act as a viable source of antiparasitic therapeutic agents.