Daily data of ambient temperature (T, °C) and relative humidity (RH, %) through July and August 2021, were obtained from Fariman meteorological stations (35o 42´ 0" N, 59o 50´ 0", Khorasan Razavi, Iran), located within 3 km distance of the experimental site. Daily readings were then considered by retrieving maximum daily T (Tmax) and RH value. Daily maximum THI was calculated, using the equation reported by Kendall et al. and Ouellet et al. : THI= (1.8 × Tmax + 32) − [(0.55 − 0.0055 × RH) × (1.8 × Tmax − 26)].
This equation has been proposed for animal experiments conducting in a climate with significant day and night and season temperature.
Animals, feeding and management
The animal study was reviewed and approved by Institutional Animal Care Committee, Ferdowsi University of Mashhad (Mashhad, Iran; Protocol number 101984). All experimental protocols were carried out in accordance with relevant guidelines and regulations in order to minimize animal pain, suffering and distress. The study is reported following the ARRIVE guidelines (https://arriveguidelines.org).Sixty-two high genetic merit multiparous Holstein lactating cows [684±16 kg of bodyweight, 28±7 days in milk (DIM), parity 2.9±0.6, milk yield 51.8±2.2 kg/d, milk fat 35± 0.21 g/L, milk protein 3.23± 0.05 g/L and BCS 2.76±0.21] were enrolled in this experiment. Cows were balanced by 305-d previous mature-equivalent milk yield, milk yield during the 3rd and 4th weeks of lactation, DIM, and parity, then assigned to two dietary treatments (n=31 per group). Animals fed a same basal diet for 7 days (5th week of lactation), then, were randomly assigned to 1 of 2 dietary treatments as control (only basal diet) or basal diet plus a rumen protected Zn-Met complex (RPZM; Loprotin, Kaesler Nutrition GmbH, Cuxhaven, Germany, as 0.131% of diet DM) beginning at 6th week of lactation for the total period of 6 weeks. The Zn-Met complex source contains 30% DL-Met and 7% Zn.The ingredients and chemical composition of the experimental diets are presented in Supplementary Table S1. During the study, cows were housed in two separated free stall rows, in a barn of a commercial dairy farm, Moghofat Maleck-Fariman, with 1120 milking cows. The barn was equipped with 4 ceiling fans (4X3 m, fan/58.5 m2), which were working for 24 h/d. Animals had free access to feed and water; diets were fed as a total mixed ration (TMR) three times per day at 0730, 1630 and 2230 h in amounts that ensured ad libitum consumption and approximately 3 to 5% feed refusals. Dry matter intake and apparent DM digestibility were determined as proposed by Velásquez et al. . Marker technique was applied to estimate fecal output and DM digestibility. Dry matter intake was then calculated by dividing fecal output by the indigestibility of DM. Samples of the diet ingredients were collected weekly, dried in a forced-air oven for 72 h at 55°C, and ground using a Wiley mill to pass a 1-mm screen, then analyzed for DM and the chemical composition. Dry matter was determined after 24 h at 95 °C (ISO 6496). Ash was determined after 3 h at 550 °C (ISO 5984). Nitrogen was assessed using the Kjeldahl method (Kjeltec 2300 Autoanalyser, Foss Tecator AB, Hoganas, Sweden) with crude protein (CP) as N × 6.25. Starch content was evaluated by an anthrone-sulphuric acid method using glucose as standard and estimated as 0.9 × glucose content after liberating the starch by heating in a boiling water bath in the presence of 2 N HCl. For neutral detergent fiber (NDF) and acid detergent fiber(ADF), the method of Goering and Van Soest was used.
Cows were milked three times daily at approximately 0400 and 1200 and 2000 h. The incidence of health problems was recorded for each cow accordingly throughout the experiment and appropriate treatment was considered if necessary.
Sample collection and processing
Feed refusals of each group were measured daily and total DMI per each experimental group was monitored by difference assuming a similar DM content of feed offered and the ort. Bi-weekly (weeks 7, 9 and 11 of lactation) samples of rectal feces were provided from 20 cows per each group to evaluate fecal output, DM digestibility and feed intake. Milk yield was recorded daily, and weekly milk samples were obtained at 3 consecutive milking. Samples were preserved with 2-bromo-2-nitropropane-1,3-diol and analyzed for protein, fat, lactose, milk urea nitrogen (MUN), SCC, solids nonfat (SNF), and total solids content using Fourier-transform infrared spectroscopy (FT-IR; CombioScope FTIR 600 HP, Delta instruments Drachten, The Netherlands) in a commercial laboratory (Sazan Rojan Alvand CO., Iran).
Fat corrected milk (FCM) standardized to 4 % fat was calculated using the following equation: FCM = [0.4 × milk yield (kg)] + [15 × milk fat (kg)] and energy corrected milk(ECM) was calculated as presented by Muñoz et al.: ECM= [0.3246 × milk yield (kg)] + [12.86 × fat yield (kg)] + [7.04 × protein yield (kg)]. Milk energy content (NEL) was calculated using the following equation: NEL= [(0.0929 × % milk fat) + (0.0563 × % milk true protein/0.93) + (0.0395 × % milk lactose)] × milk yield.
Blood was sampled bi-weekly (weeks 5, 7, 9 and 11 of lactation) from 15 cows per each group at 0800 h via puncture of the coccygeal vessels as proposed previously. The samples were kept at room temperature and the serum was separated within 0.5 h, then stored frozen at –20 °C until analyses for Glucose (GOD-PAP, https://parsazmun.ir), triglycerides (GPO-POD, www.Bionik.web.com), NEFA (Colorimetric method, RANDOX, www. Randox. Com), BHB (kinetic Enzymatic method, RANDOX, www. Randox. Com), urea (Urease-GLDH method, https://parsazmun.ir), cholesterol (CHOD_POG, http://paadco.co), HDL (direct enzymatic colorimetric method, http://paadco.co), SGOT (https://parsazmun.ir), SGPT (https://parsazmun.ir), calcium (https://parsazmun.ir), Zn (Colorimetric method, RANDOX, www. Randox. Com), total protein (https://parsazmun.ir), albumin (https://parsazmun.ir), haptoglobin (ZellBio GmbH, Lonsee, Germany), TAS (Manual calouric method, RANDOX, www.Randox.com), MDA (thiobarbituric acid reactive substances assay) and IL-1B (ZellBio GmbH, Lonsee, Germany). BothLDL and very-low-density lipoprotein (VLDL) was calculated using following equation: LDL= Total cholesterol – (HDL + VLDL), VLDL=Triglycerides/5.
Bi-weekly animal behavior indices including the time spent ruminating were recorded every 10 minutes per 24 h and calculated by multiplying total number of observed activities in each duration.Body condition score, rumen fill score and manure score were assessed bi-weekly from 7th week of lactation. Body condition score was recorded by the same operator using a 1 to 5 scale with 0.1 intervals as proposed by Ferguson et al.. Both rumen fill score and manure score were assessed 6 times per day started 2 h before the morning feeding by the same operator using a 1 to 5 scale. The visual characteristics for scoring the manure and rumen fill of Holstein dairy cows were done as previously described[30, 31].
One cow in the RPZM group was removed from the experiment at the end of the 9th week of lactation due to a physical injury. Besides, two cows in the control group were culled at the 7th and 8th weeks of lactation due to unknown sudden drop in milk production.
Data of milk yield and its component as well as blood biochemistry from the 5thlactation week (i.e., all animals received the similar basal diet) were used as covariate in the statistical analysis. The main aim of the statistical analysis of the experimental data was to determine the productive and metabolic responses of the cows fed the experimental diets. Data obtained weekly was statistically analyzed using the Proc Mixed procedure of SAS 9.1 (SAS Institute, Cary, NC, USA) for a completely randomized design with repeated measures included by the corresponding covariate. The model included the effects of group, week of lactation and the interaction between group and week and covariate (if necessary). Week of lactation was used as a repeated measurement, with cow within experimental groups as the subject. Experimental group, week, the interaction between treatment and week, and corresponding covariate were the fixed effects, and error was the random effect. Data of DM digestibility were averaged by cow and week of lactation, then, analyzed using a completely randomized design. Differences were considered significant at P < 0.05, whereas tendency was determined at 0.05 < P< 0.1. Data are expressed as the mean ± standard error of means.