Gas chromatography

The pH values of the rumen fluids were analyzed using a pH meter (INESA, Shanghai, China). Volatile fatty acids (VFA) in the rumen fluid were determined by gas chromatography (Agilent Technologies, Santa Clara, CA, USA) referring to Stewart and Duncan [18 (link)]. The ruminal microbial protein (MCP) concentration was determined by the method of Makkar et al. [19 (link)], and ammonia nitrogen concentrations were determined by alkaline sodium hypochlorite–phenol spectrophotometry [19 (link)].

The volatile compounds of secondary metabolites from the crude ethyl acetate extract of fungi were determined using a gas chromatography (Agilent technologies). About 1 µL sample was injected in split mode and the flow of the carrier gas was adjusted to 1 mL/min. The injector temperature was maintained as 250 °C, whereas the detector temperature was 280 °C. The oven temperature was initially adjusted at 50 °C for 5 min and increased to 260 °C and held for 5 min. NIST database was used to find the compound of mass spectrum separated during analyses.

The approximate components of the whole fish and diets were measured through the assay method described in our previous study [14 (link)]. Briefly, moisture was assayed by drying samples to a constant weight at 105 °C. Ash content was measured by incinerating samples in a muffle furnace at 550 °C for 6 h. Crude protein was evaluated by the Kjeldahl method, and the crude protein content was calculated as % N × 6.25. Crude lipid content was assayed by the Soxhlet method. Each sample was analyzed 3 times.
According to the common method, as previously reported [13 (link)], the lipid of diets and muscle was separated through a mixture of chloroform and methanol with a volume ratio of 2:1., and then transesterification was performed with boron trifluoride etherate to prepare fatty acid methyl esters (FAME). Finally, the FAME was dissolved in n-hexane and separated by gas chromatography (Agilent Technologies Inc., Palo Alto, CA, USA), as previously described [13 (link)]. Briefly, FAME samples were applied using an on-column (HP-88, 100 m × 0.25 mm × 0.2 μm, Agilent Technologies Inc., Palo Alto, CA, USA) injection, and the oven temperature was programmed to go from 80 to 250 °C, at a rate of 40 °C per minute. The fatty acid composition was identified according to the commercial FAME standards (Sigma-Aldrich, Saint Louis, MO, USA), and calculated by the area normalization method.

To test whether selection for BAL after binge-like drinking during lights off (active cycle for mice) generalized to increase intake during lights on, we measured limited-access drinking in the light (DIL) and dark (DID) in separate groups of male and female Hs/Npt, HDID-1, and HDID-2 mice. As in Rhodes et al., mice were habituated to individual housing and a new sipper bottle for one week prior to testing [18 (link)]. HDID-1 (S30.G32-S31.G33, where “S” is the number of selected generations, and “G” is the number of total generations), HDID-2 (S25), and Hs/Npt (G81.V13, where V is the number of generations of this non-selected, genetically segregating stock in our VA facility) mice were tested simultaneously in the same experimental room on the same reverse light cycle day (n = 8–9/sex/time of day/genotype). Mice were offered one bottle of 20% ethanol (v/v in tap water) for 2 h (from ZT3-5 for the DIL mice and ZT15-17 for DID mice) on days 1–3 and for 4 h (from ZT3-7 for DIL mice and from ZT15-19 for DID mice) on day 4. A 20 uL periorbital blood sample was collected immediately after ethanol access on day 4 and BALs were determined using gas chromatography (Agilent, Santa Clara, CA, USA) as previously described [33 (link)].

Jugular blood samples (10 mL) of all yaks were collected on day 120 before the morning feeding. The serums were separated after centrifuge at 3500 rpm at 4 °C for 15 min and stored at −20 °C for analysis of serum biochemical and hormonal parameters. Then, six yaks in each group, which were close to the group average weight, were slaughtered humanely by captive bolt stunning and exsanguination according to the National Standard Operating Procedures of Cattle Slaughtering (GB/T 19477-2004). Digesta collected from dorsal, ventral, and caudal areas of the rumen were mixed and filtered through four-layer nylon cloth. Rumen fluid was collected in three 10-mL centrifuge tubes; one centrifuge tube of rumen fluid was immediately used to detect pH value using a pH meter (INESA, Shanghai, China), and the others were stored immediately at −80 °C for ruminal fermentation parameters and bacterial community composition analysis. The longissimus dorsi muscle samples were collected between the 12th and 13th ribs of the left side of carcass, and the intramuscular fatty acid profile was detected by using gas chromatography (Agilent Technologies, Santa Clara, CA, USA) as described in our previous study [12 (link)].

The Yellow River carp samples (n = 96) were collected from the Breeding Station of Henan Academy of Fishery Sciences, Zhengzhou, China. The cultured fish were approximately two years old and had been reared under the same conditions and fed with the same commercial diet (Liaoyang Yida Feed Co., Ltd., Liaoyang, China) before experiment. All the fishes were euthanized in MS222 solution before sampling. According to the experimental design, the muscle and liver tissues of each individual were collected and placed in RNALater (Qiagen, Hilden, Germany), which was kept at 4 °C for 3~6 h to immerse the reagent into the tissue samples, and then immediately frozen in dry ice. Then, 20 g of muscle tissue from each Yellow River carp was collected and stored at −80 °C, until sent to Qingdao Kechuang Testing Co., Ltd. (Qingdao, China) for determination of 35 types of fatty acids content. According to GB/T 22223-2008 standard [22 ], the samples were processed by gas chromatography with analytical instrument (Agilent, Santa Clara, CA, USA).