Sodium sulphite

Dulbecco’s Modified Eagle Medium (DMEM) powder with high glucose (without sodium bicarbonate) and DMEM liquid were procured from Sigma (USA). Fetal bovine serum (FBS), trypsin-ethylene diamine tetra acetate (Trypsin–EDTA) and penicillin–streptomycin were purchased from Gibco BRL (California, USA); sodium bicarbonate, sodium sulphite, calcium chloride dihydrate (CaCl₂.2H₂O), dimethyl sulphoxide (DMSO) and thiazolyl blue tetrazolium bromide (MTT) reagent were bought from Sigma-Aldrich (St Louis, MO, USA). The plasmids encoding BRCA1 (catalog no. 61586) and BRCA2 (catalog no. 16245) tumor suppressor genes were ordered from ‘Addgene’ (USA), where _pcBRCA1-385 was a gift from Lawrence Brody and _pCIN BRCA2 WT was a gift from Mien-Chie Hung^{41 ,42} . The antibiotic, ampicillin (Fisher Scientific), Luria Bertani (LB) agar from Merck (Germany) and LB broth were procured from Laboratorios CONDA (Spain). Two human breast cancer cell lines MCF-7, and MDA-MB-231, one mouse breast cancer cell line 4T1 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). For Western Blotting, Tween-20 and Pierce ECL Western blot detection reagents were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Dithiothreitol (DTT) (Bio-Rad), bovine serum albumin (BSA) (Merck, USA), protease inhibitor and phosphatase inhibitor cocktail were obtained from Sigma. Monoclonal IgG primary antibodies raised in rabbit for phospho-MAPK, total MAPK, GAPDH, and the horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit IgG antibody were from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Enzymatic assays were performed on enzymatic extracts to determine both cellulase and xylanase activities of every isolated fungal strain, using the DNS method [75 (link)]. Briefly, this method allows the measurement of reducing sugar production by enzymatic extract from a substrate of sodium carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) and wheat arabinoxylan (Megazyme, Wicklow, Ireland) for cellulase- and xylanase-assays, respectively. Both substrates, sodium carboxymethyl cellulose and arabinoxylan, were prepared at 1% in 0.05 M pH 5.3 sodium citrate buffer (citric acid 21 g/L, Acros Organics, Geel, Belgium, and sodium citrate tribasic dihydrate 29.6 g/L, Sigma-Aldrich, St. Louis, MO, USA). A solution of DNS 1% was prepared (DNS, Sigma-Aldrich, St. Louis, MO, USA, 1%; Phenol, Fluka, Honeywell, Charlotte, NC, USA, 0.2%; sodium sulphite, Sigma-Aldrich, 0.05%; NaOH, Fluka, 1%). Commercial enzymatic solutions were used as control, being solutions of 1% cellulase and 1% xylanase from T. longibrachiatum (Sigma-Aldrich, St. Louis, MO, USA). Reaction consisted of mixing 1.8 mL of substrate solution with 200 µL of enzymatic solution (fungal extract or control), heating this mix at 50 °C during 5 min, adding 3 mL of 1% DNS solution, heating the mix at 100 °C during 15 min, and reading the OD at 540 nm (Shimadzu, Kyoto, Japan) after a cooling step of tubes in cold water. One unit of enzyme activity was defined as the amount of enzyme allowing the release of 1 µmol of product (xylose for xylanase and glucose for cellulase) per minute in the assay condition.

Feed refusals were weighed daily to estimate feed intake. Representative samples of hay, concentrate and CBS used in the trial were collected at the beginning of the adaptation period. They were ground with a cutting mill to pass a 1 mm screen sieve (Pulverisette 15-Fritsch GmbH, Idar-Oberstein, Germany). AOAC International procedures (40 ) were used to determine DM (method no. 930.15), ash (method no. 942.05), crude protein (CP, method no. 984.13), acid detergent fiber and acid detergent lignin (ADFom and ADL, method no. 973.18). Ether extract (EE, method no. 2003.05) was analyzed according to AOAC International (41 ). Neutral detergent fiber (aNDFom) was analyzed according to Mertens (42 (link)); α-amylase (Merck, Darmstadt, Germany) and sodium sulphite (Merck, Darmstadt, Germany) were added, and results were corrected for residual ash content.
Rumen degradable protein (RDP) was analyzed according to Licitra et al. (43 (link)).
The energetic value of feeds was expressed as net energy for lactation (NE_L) and was estimated using National Research Council equations (39 ).
The FA composition of hay, concentrate and CBS was assessed using a combined direct transesterification and solid-phase extraction method as reported in Dabbou et al. (44 (link)). Separation, identification, and quantification of fatty acid methyl esters (FAME) were performed as described by Renna et al. (45 (link)). The results were expressed as g/kg DM. The daily intake (g/head) of each individual FA and groups of FA from the diet was estimated considering the daily intake and the analytically determined FA composition of each feedstuff.
The contents of total extractable phenols (TEP) and different polyphenol fractions (non-tannin phenols, NTP; condensed tannins, CT) in CBS, were assessed as detailed in Iussig et al. (46 (link)). Briefly, polyphenols were extracted twice with aqueous acetone (70:30 v/v) and subjected to ultrasonic treatment for 20 min at room temperature in an ultrasonic water bath (Bransonic-21, Branson Ultrasonics, Danbury, CT, USA). Polyvinyl-polypyrrolidone was used to separate NTP and total tannins (TT), according to a modified Folin-Ciocalteu method. The butanol-HCl-iron method was used to determine CT. The absorbance was recorded at 725 nm (TEP and NTP, expressed as gallic acid equivalents) and 550 nm (CT, expressed as leucocyanidin equivalents) using a UV–vis spectrophotometer (Shimadzu UVmini-1240, Shimadzu Corporation, Kyoto, Japan). Total tannins (TT) were computed as the difference between TEP and NTP. Hydrolysable tannins (HT) were estimated as the difference between TT and CT (47 (link)). The amounts of phenolic compounds daily ingested by the goats belonging to the CBS group was estimated based on the analyzed phenolic composition of CBS and the determined intake of concentrate.
All the analyses were performed in duplicate.