Testo

Arabidopsis thaliana (L.) Heynh. accessions Col-0 and C24 (Meyer et al., 2004 (link)) were grown under controlled conditions at 20/18°C, 60/75% relative humidity, 130–150 μmol m⁻² s⁻¹ photosynthetically active radiation (PAR) from Whitelux Plus metal halide lamps (Venture Lighting Europe Ltd., Rickmansworth, Hertfordshire, England, see Figure S1A for spectral composition of the emitted light) and a 16/8 h day/night regime in a walk-in growth-chamber. After 2–3 days of stratification at 5°C in constant darkness, seeds were germinated and seedlings cultivated under a 16/8 h day/night regime with 16/14°C, 75% relative humidity, and 130–150 μmol m⁻² s⁻¹ light intensity until 3 days after appearance of both cotyledons [usually reached at 4 days after sowing (DAS)]. For each experiment, light intensity (PAR lite Meteon, Kipp&Zonen, Reichenbach, Germany, 400–700 nm), air temperature and relative humidity (Testo 175-H2 data logger, Testo AG, Lenzkirch, Germany) were measured manually at the plant level. Pots were filled with a mixture of 85% (v) red substrate 2 (Klasmann-Deilmann GmbH, Geeste, Germany) composed of a blend of white and frozen through black phagnum peat, pH 5.5, supplemented with lime and NPK fertilizer (280 mg/l N, 200 mg/l P₂O₅, 360 mg/l K₂O, 100 mg/l Mg, 180 mg/l S, with micronutrients including chelated Fe) and 15% (v) sand and soil moisture was re-adjusted daily to 70% field capacity.
Soil water content corresponding to 100% field capacity was determined by weighing soil-filled pots after full watering and after drying for 3 day at 80°C. The weight corresponding to 70% field capacity was calculated in an analogous manner as for maize pots (see below).
One day prior to the experiment start, pots were filled with the soil mixture and watered to reach 70% field capacity. A blue rubber mat was placed as a soil cover and the pots were inserted into the carriers of the LemnaTec system where the weight of each pot was measured to determine the target weight. In the course of the experiment, the changes of weight that occurred in the intervals from 1 day to the next were used as measures of the amount of water lost from the soil and the equivalent volume of water was added through a peristaltic pump. A layer of textile material covered with a perforated black foil (to improve the background surrounding the pots in the top view images) was used in the supporting containers of the carriers to improve the water distribution. Prior to sowing, each carrier received 50 ml water pumped into the bottom container to increase the moisture during germination. Seeds were imbibed on moist filter paper for 48 h in the dark at 5°C. Thereafter, they were transferred to the soil using tooth picks. The pots were covered with plastic caps to maintain high humidity conditions during germination. These were removed after germination and development of the second rosette leaf.

For the CT_max test, a heating tank (25 × 22 × 18 cm) was filled with 9 L of 28 °C water. A water pump (Eheim Universal 300, Deizisau, Germany) was attached to a custom-made cylindrical steel heating case consisting of an inflow nipple, a wide outflow and a 300 W coil heater (Fig. 5). The pump pushed water through the heating cylinder and into the fish arena creating stirring, and the heating system was separated from the fish arena by a mesh. This setup ensured a homogenous temperature in the entire arena (<0.1 °C), whilst minimising the water current within the tank. A recently factory calibrated high precision digital thermometer with a ±0.1 °C accuracy (testo-112, Testo, Lenzkirch, Germany) continuously measured the water temperature in the fish compartment.Figure 5

CT_max experimental setup, ensuring homogeneous water temperature and consistent heating rate for all trials. (A) Water pump; (B) custom-made cylindrical steel heating case; (C) 300 W coil heater; (D) mesh (preventing fish swimming into the heating compartment); (E) fish compartment with 9 L of 28 °C water; (F) Photograph of the CT_max box.

A group of randomly selected individuals (3–6) were caught from their holding tank and transferred to the heating tank. The water was then heated at a steady rate of 0.3 °C per minute^{11 (link)}, in accordance with^{17 (link)}. A pilot experiment with zebrafish instrumented with small thermocouples showed that this heating rate caused a lag of heating of less than 0.2 °C between the ambient water temperature and the deep dorsal muscle (see below). In addition, oxygen concentrations at or above 100% saturation were retained throughout the test. Loss of equilibrium (LOE), defined as uncontrolled and disorganised swimming for two seconds, was chosen as the CT_max endpoint^{56 (link)}. Once LOE occurred in an individual, the water temperature was recorded with an accuracy of 0.1 °C, and the fish was immediately transferred into an individual tank of 28 °C water for recovery. Once the fish had recovered, it was anaesthetised, identified, weighed and photographed before being returned to its holding tank. Recovery of equilibrium generally occurred within two minutes, and normal behaviour was restored after approximately five minutes. During pilot experiments fish commenced feeding within fifteen minutes of a completed CT_max test, indicating that the thermal challenge didn’t cause major trauma. All but one of the fish recovered after the CT_max tests in the experiment (99% survival). Additionally, two treatment fish do not have a fourth CT_max measurement as they jumped out of the heating tank during the test.
The sham CT_max test consisted of the fish being put into the heating tank for the same duration (~40 minutes) as the treatment fish but the water was kept at 28 °C throughout. At the end, the fish were individually removed, anaesthetised, weighed, photographed and returned to their holding tank.
A condition index (K, equation 1) was calculated for each fish using the total length and weight measurements. Specific growth rate (SGR, equation 2) was calculated for four growth intervals, the first one from the date of first tagging until the first CT_max test, and weekly thereafter. $$K=(Weight/Lengt{h}^3)\times 100$$ $$SGR=(ln\left(W_t\right)-ln\left(W_0\right)/timeinterval)\times 100$$

During the experimental period, automatic feeding equipment (made by Institute of Animal Science Chinese Academy of Agricultural Sciences, Beijing, China and NanShang Husbandry Science and Technology Ltd. Henan, China) was used to record dry matter intake. Milking facilities (90 Side-by-Side Parallel Stall Construction, Afimilk, Israel) were applied to record milk production of each cow.
On the last day of the experiment, a gastric rumen sampler was used to collect rumen fluid samples through the esophagus at 3 h after the morning feeding. Collected samples were strained through four layers of cheesecloth to obtain rumen fluid. Rumen fluid was then divided into two parts. One part was processed to analyze the pH value, rumen volatile fatty acid (VFAs) and ammonia-N (NH₃-N). The other part was put into the liquid nitrogen immediately after adding stabilizer and then stored at −80 °C for DNA extraction. Rumen contents were strained through four layers of cheesecloth with a mesh size of 250 μm. The pH of each rumen fluid sample was measured immediately using a portable pH meter (Testo 205, Testo AG, Lenzkirch, Germany). Individual and total VFAs (TVFA) in the aliquots of ruminal fluid were determined by gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan). Concentration of NH₃-N was determined by indophenol method and the absorbance value was measured through UV-2600 ultraviolet spectrophotometer (Tianmei Ltd., China).
Milk samples were collected from individual cows during the last four consecutive days in 100-mL vials in each milking period. Samples were preserved with 2-bromo-2-nitropropan-1,3-diol and stored at 4 °C, before sent to the Milk and Dairy Products Quality Supervision and Testing Center, Ministry of Agriculture (Beijing, China) for analyses of milk protein, fat, lactose, somatic cell count and milk iodine content by a mid-infrared spectroscopy (Fossomatic 4000, Foss Electric A/S, Hillerød, Denmark).

The measurement of the leaf surface temperature was performed using the testo 865-i thermal imager (Testo SE & Co., KGaA, Titisee-Neustadt, Germany). After watering, the potted plants were placed on a grid frame, and a thermal image was taken from above. The image was analyzed using testo IRSoft software (ver. 5.0.5628.36882, Testo).

The temperature (T °C) and relative humidity (RH %) of the Mammoth Park ambient were measured every 30 min during the 18-month period (from 21 June 2021 to 9 November 2022). For this purpose, a Testo 176P1 data logger (Testo SE & Co. KGaA, Titisee-Neustadt, Germany) was installed on the wooden beam at a height of 250 cm from the floor. Furthermore, the moisture content (%) of wooden artifacts was measured using a Testo 606-2 measuring instrument (Testo SE & Co. KGaA, Titisee-Neustadt, Germany), which was set to wood mode.

The rectal temperatures of the mice were measured weekly using a Testo 925 thermometer (Testo, Lenzkirch, Germany).