The effect of Apium graveolens L., Levisticum officinale and Calendula officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro.

Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5; 75; 150 and 300 µg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5-carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 µg/ml (109.0±4.3%) followed by a decline at 300 µg/ml (89.4±2.3%). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 µg/ml (88.8±11.66%; 87.4±6.0%) and 300 µg/ml (86.2±9.3%; 84.1±4.6%). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 µg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 µg/ml. Finally, the disturbance of GJIC was significantly affected at 300 µg/ml of Levisticum officinale (82.5±7.7%) and Calendula officinalis L. (79.8±7.0%). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.

Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.

Meat and fat quality of Krskopolje pigs reared in conventional and organic production systems.

Data on production traits of the only Slovenian autochthonous pig breed, the Krskopolje pig, is very scarce. Krskopolje pigs are reared in conventional and organic production systems, which were compared in the present study. After weaning, 24 barrows were assigned within litter to either conventional (CON) or organic (ECO) rearing system. Group CON (n=12) was housed indoors in two pens (7.5 m2) with partly slatted floor. Group ECO (n=12) was held in a sty with sheltered area (concrete floor, bedded with straw, 16 m2) and outdoor paddock area (100 m2). The trial started when pigs had 68±8 kg BW and 157±6 days of age. Two diets were formulated with equivalent ingredients and composition. For ECO diet the ingredients used were ecological. Group ECO received a diet with 12.4 MJ metabolisable energy (ME) per kilogram and 12.9% CP and group CON a diet with 12.7 MJ ME/kg and 13.6% CP. Feed distribution was limited to 3.5 kg per pig daily. In line with the rules for organic production, ECO pigs were additionally given alfalfa hay ad libitum. After 73 days on trial, the pigs were slaughtered and carcass, meat and fat quality was evaluated. Meat quality traits (pH, colour, water holding capacity), fatty acid composition, lipid and protein oxidation, collagen content and solubility were analysed in longissimus lumborum (LL) muscle. Fatty acid composition, lipid oxidation, and vitamins A and E concentrations were determined in backfat. There were no significant differences in growth rate and carcass traits between ECO and CON pigs, however, ECO pigs tended (P<0.10) to have higher daily gain and lower dressing percentage, higher (P<0.001) pH 45 min and lower (P<0.01) pH 24 h postmortem, affecting (P<0.10) also water holding capacity and objective colour parameters (P<0.05) of LL muscle. There were no differences in intramuscular fat (IMF) content of LL muscle, however, IMF of ECO pigs had lower (P<0.05) proportion of saturated and higher (P<0.01) proportion of monounsaturated fatty acids accompanied by higher (P<0.001) values of thiobarbituric reactive substances (TBARS). In backfat, ECO pigs showed lower (P<0.05) vitamin E content, higher (P<0.001) TBARS, higher (P<0.01) degree of unsaturation (percentage of polyunsaturated fatty acids), and also higher (P<0.05) vitamin A concentration than CON pigs, which can be related to alfalfa hay supplementation of ECO pigs. In brief, organic rearing of Krskopolje pigs did not affect performances but had an effect on meat and fat quality.