Experimental animals were managed according to the guidelines recommended in the Guide for the Care and Use of Agricultural Animal in Agricultural Research and Teaching (Federation of Animal Science Societies, 2010). All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Illinois (IACUC # 18109).
Experiment design, animals, and diets
Ninety-six, fall-calving beef cows (body weight [BW] = 601 ± 76 kg) from Dixon Springs Agricultural Center, Simpson, IL were utilized for this experiment. Prior to supplementation (d -77), cows were stratified by BW, body condition score (BCS), age, and fetal sex (64 male and 32 undetermined), and allotted into 8 predominately endophyte-infected tall fescue pastures with 12 cows/pasture (n = 4 pastures/treatment) and pasture groups were randomly assigned to 1 of 2 treatments. Each group of cows were rotated between two pastures every 2 wk, with average stocking density of 3.0 cows per hectare. Forage availability was measured with rising plate meter at each rotation [21]. Each cow was supplemented 0.77 kg dry matter (DM)/d soybean hulls mixed with either 80 g DM/d Strata + 80 g DM/d Prequel (PUFA, rich in linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid) or 155 g DM/d EnerGII (CON, rich in palmitic and oleic acids) for 77 ± 6 d prepartum (Table 1). Calcium salts of fatty acids (Strata, Prequel, and EnerGII) were provided by Virtus Nutrition LLC, Corcoran, CA. The two diets were designed to be isocaloric and isonitrogenous. Nutritional and fatty acid profiles of ingredients fed to cows during late gestation are presented in Table 2. Cows were supplemented 3 times a week (Monday, Wednesday, and Friday) in 3 portable metal bunks (3.66 × 0.76 m, accessible from both sides) per group during supplementation period. Supplements were typically ingested by cows within 15 minutes. Cows were also weighed and assigned BCS at the middle of supplementation (d -42), the end of the gestation (d -18), within one week post-calving (5 ± 2.4 d post-calving), at artificial insemination (AI)-pregnancy determination (d 113), and overall-pregnancy determination (d 186, same day as weaning) to monitor animal performance. Once weekly, cows that had calved and their calves were removed from the treatment groups, comingled to a common pasture, and managed as a single contemporary group from that point forward. Cows were supplemented with 2.27 kg/d/cow of dried distillers grains with solubles (DDGS; DM 81.2%, crude protein [CP] 30.4%, crude fat 10.6%, neutral detergent fiber [NDF] 32.9%, acid detergent fiber [ADF] 11.3%) and soybean hulls (DM 81.7%, CP 10.2%, crude fat 0.8%, NDF 61.7%, ADF 43.8%) in ratio of 50:50 during post-calving grazing period. Cows were provided with ad libitum hay (DM 76.0%, CP 9.4%, NDF 62.9%, and ADF 35.7%) from d 39 to weaning as forage availability declined in the fall. Cows were synchronized using the 7-day Co-Synch + controlled internal drug-release (Pfizer Animal Health, New York, NY) procedure [22] and artificially inseminated (AI; 78 ± 6 d post-calving). Ten days after AI, cows were exposed to 2 clean-up bulls for a 79 d breeding season. Pregnancy diagnosis to AI and overall pregnancy were performed 35 d and 98 d after AI. Pregnancy diagnosis was conducted by a trained technician with ultrasonography (Aloka 500 instrument, Wallingford, CT).
Cows were vaccinated at the initiation of the supplementation (d -77) and the end of gestation (d - 18). On d -77, cows were administered with 2 mL Leptoferm-5 (Zoetis, Florham Park, NJ), 1 mL Anaplasmosis vaccine (University Products L.L.C., Baton Rouge, LA), and 2 mL Auto. M. Bovis. (Pinkeye vaccine customized by Newport Laboratories, Worthington, MN). In addition, 2 Patriot Insecticide Cattle Ear Tags (Bayer, Shawnee Mission, KS) and Ivermectin (Norbrook, UK) were applied to the cows. On d -18, 5 mL Bovishield Gold FP5 VL5 HB (Zoetis), 5 mL Covexin 8 (Merck Animal Health), 7mL MU-SE (Zoetis), and 2 mL ScourGuard 4KC (Zoetis) and Cylence (Bayer) were administered.
There were 12 cows from PUFA groups that were removed from the trial from late-supplementation to rebreed. One cow was removed at late-supplementation (d -18) because of abortion. One cow was removed because of extremely poor BCS at calving. One was removed because of having twins. Five cows were detected open at calving. Three cows were removed because of loss of calves prior to when weigh-suckle-weigh was conducted. One cow from PUFA group died on December 2018. There was one cow from CON group was removed because of a stillborn birth. There were 5 and 8 heifer calves born from CON and PUFA cows, respectfully. Data from dams of heifer calves was included for analysis of cow performance data, while heifer calves were not included for analysis of calf birth BW, weaning BW, or mRNA expression.
Within 24 h after calving, calf birth BW was recorded and bull calves were castrated. Calves were provided with 1 mL BO-SE (Merck Animal Health), 20 mL Bovi-Sera (Colorado Serum Company, Denver, CO), and 1 mL Vitamin A/D (VetONE, Biose, ID) at birth. Calves were vaccinated on d 60 and d 172 with following: 2 mL Auto. M. Bovis. (Newport Laboratories), 5 mL Covexin 8 (Merck Animal Health), 5 mL Bovishield Gold FP5 VL5 HB (Zoetis), 2 mL Pulmo-Guard PHM-1 (AgriLabs, St. Joseph, MO), and 1/100 dose of Synanthic (Boehringer Ingelheim Vetmedica, Duluth, GA). Two mL Myco-B One Dose (American Animal Health, Fort Worth, TX) and 2 mL Inforce 3 (Zoetis) were also administered on d 60. Calves were weighed and weaned at 186 ± 6 d of age. Pre-weaning growth was evaluated based on weaning BW and pre-weaning ADG.
Sampling and analytical procedures
Blood samples were collected from cows at pre- (d -77) and mid-supplementation (d -42), and from cow/steer pairs within one week after calving (5 2.4 d post-calving). Blood samples (10 mL) were collected from the jugular vein of cows and calves by using polypropylene tubes (BD Vacutainer) containing sodium heparin for plasma, and placed on ice. After centrifugation for 20 min at 2,000 g and 4 , plasma was stored at -80 until later analysis. For steer calves and their dams, individual plasma samples were thawed in 0-4 water bath [23]. A glass rod was used to stir the water in the tub for accelerating the thawing process. Thawed plasma samples that were from the same grazing group were individually added into a pooled centrifuge tube for each sampling time point. Within a pooled sample, each individual plasma sample accounted for the same percentage of the total 1.5 mL pooled unit.
Relative concentrations of fatty acids in pooled plasma samples were analyzed by Metabolomics Center at the Roy J. Carver Biotechnology Center (Urbana, IL, USA). Extraction was conducted twice on 100µL of sample with 300µL of methanol:chloroform (1:2) solution. Organic phase was collected, evaporated and hydrolyzed with 500 µL of 3N methanolic HCL contained 2 g/L of butylated hydroxytoluene for 1 h at 85 ℃. Samples then were cooled to room temperature and extracted twice with 500 µL of hexane. Organic phase was collected, evaporated under nitrogen and re-suspended in 100 µl. Samples were analyzed using a gas chromatography-mass spectrometry system (Agilent Inc., Palo Alto, CA, USA) consisting of an Agilent 7890B gas chromatograph and an Agilent 5977A MSD. Separation was performed on a HP-5MS (60m × 0.25mm I.D. and 320 µm film thickness) capillary column (Agilent J&W, Palo Alto, CA, USA). The inlet temperature was 220 ℃, MSD interface temperature was 230℃ and the ion source temperature was adjusted to 230 ℃. An aliquot of 1µL injected in a splitless mode (20 mL/min for 0.75 min). The helium carrier gas was kept at a constant flow rate of 2 mL/min. The temperature program was: 2 min at 150 ℃, followed by temperature increase of 5 ℃ per min to 300 ℃ for 3 min. The mass spectrometer operated in positive electron impact mode at 69.9 eV ionization energy at m/z 50-600 scan range. Target peaks were evaluated using AMDIS v2.71 and Mass Hunter Qualitative Analysis B.08.00 (Agilent Inc., Palo Alto, CA, USA) software.
Milk production was determined before breeding (64 ± 9 d postpartum) on a subset of 59 cow/ steer calf pairs (7-9 pairs per grazing group) via weigh-suckle-weigh (WSW) as described by Beal et al. [24]. Day postpartum, cow BW at calving and cow age were stratified across subset groups. Milk samples were hand stripped on a subset of cows (4 cows/pasture group; n = 32) during WSW for milk composition and fatty acid profile analysis. Milk samples for composition analysis were shipped in a cooler with ice packs underneath the samples. Milk composition was analyzed by Dairy Lab Service Inc. (Dubuque, IA). For the milk samples that were collected for fatty acid profile analysis, the top fat layer was collected after centrifugation at 20,000 × g for 30 min at 4 ℃ [3] and stored at -80 ℃ until shipping out to Cumberland Valley Analytical Service Inc. (CVAS; Waynesboro, PA, USA). The analysis was conducted as previously described [25,26] with minor modifications. Briefly, approximately 1.6 mg of sample was weighed and extracted using a mixture of isopropanol and hexane (2:3, vol/vol). Extracts were collected and evaporated under nitrogen at 45 ℃ for about 8 min to dryness. Hexane and methyl acetate were used to dissolve lipid extracts, then fatty acid methyl esters (FAME) was synthesized by using methanolic sodium methoxide. The mixture was neutralized by oxalic acid, centrifuged and dried with calcium chloride. The FAME were separated and quantitated using PerkinElmer Clarus 590 (PerkinElmer, Shelton, CT) equipped with a fused-silica capillary column (SP-2560, 100 m 0.25 mm i.d. with 0.2-µm film thickness; Supelco Inc., Bellefonte, PA) and a flame ionizationdetector (FID). Hydrogen was used as the carrier gas.
Feed samples including pasture and supplement samples were collected every 2 weeks during supplementation period for proximate and fatty acid profile analysis. Forage samples were hand-clipped at the beginning of each rotation. Samples of supplement during the comingled grazing period were collected every 2 weeks for proximate analysis. All feed samples were stored at -20 ℃ until further processing. The pasture forage samples were composited on a monthly basis, while supplement samples were composited for the supplementation period. The samples that were for fatty acid profile analysis were freeze dried and ground through 1 mm screen using a Wiley mill (Arthur, H. Thomas, Philadelphia, PA). The rest of the samples that were for proximate analysis were composited and oven dried under 55 ℃ for at least 3 days, then ground through 1 mm screen using a Wiley mill. Ground samples were analyzed for DM (105 ℃ oven), crude protein (Leco TruMac, LECO Corporation, St. Joseph, MI), crude fat by using Ankom XT10 Fat Extractor (Ankom Technology, Macedon, NY), NDF and ADF by using an Ankom 200 Fiber Analyzer (Ankom Technology, Macedon, NY).
Fatty acid profile analysis of the feed samples was conducted by CVAS using method of Sukhija and Palmquist [27], which was modified as follows. One mL of internal standard (C13:0) was added to approximately 0.5 g of sample, followed by adding 3.0 mL of 5% methanolic HCL to sample. After vortex, sample was incubated in 70 ℃ water bath for 2 h. Sample was removed from water bath and cooled for 15 min. After cooling, 5.0 mL of 6% Potassium Carbonate and 1 mL of hexane were added to the sample. Sample was centrifuged at 752 × g at room temperature for 10 min. The organic layer was transferred to gas chromatography autosampler vial. Fatty acid profile was analyzed on PerkinElmer Clarus 580, split/splitless capillary injector, and FID detector with helium as the carrier gas. The fused silica capillary column was Restek Rtx-2330, 30 m × 0.32 mm i.d. × 0.20 µm.
Muscle and adipose tissue biopsy samples for mRNA expression were collected from every steer calf at birth (5 ± 2 d of age) and from a subset of steers (n = 24, 3 from each grazing group) at 3 wk prior to weaning (165 ± 4 d of age). The 24 steers were selected based on their BW at 64 ± 9 d of age being representative to the group average. For muscle biopsies, an area over the longissimus dorsi muscle from the first lumbar vertebra region on the left side of the animal was clipped closely and scrubbed 3 times thoroughly with betadine surgical scrub, and rinsed with 70% alcohol. Lidocaine was administered subcutaneously and intramuscularly (5-10 mL) over the biopsy site. After 10 min of lidocaine administration, a biopsy core of muscle (100-200 mg) was removed by using a biopsy needle (Bard MAGNUM; 12 gauge × 16 cm). The initial Longissimus muscle biopsy was taken 5 cm cranial to the hook bone half way between the axis and transverse processes of the lumbar vertebrae. After completing the biopsy, pressure was applied with sterile gauze to stop any external bleeding, the surrounding area was cleansed with sterile saline to remove blood, the incision was closed with a synthetic absorbable tissue adhesive Vetbond (3M Animal Care Products, St. Paul, MN, USA), and a topical antibiotic ointment was applied. Each subsequent biopsy was taken 5 cm cranial to the previous biopsy site. For subcutaneous adipose tissue biopsies, an area of approximately 15 × 15 cm over one side of the tail-head area was cleaned the same as muscle biopsy procedure. Lidocaine was administered similarly as muscle biopsy. Thereafter, a 3 to 4-cm incision was made with a sterile surgical blade and the skin was pulled back using forceps, exposing the subcutaneous adipose tissue. Samples of adipose (1 to 3 grams) were taken using forceps and a single-use sterile scalpel blade. The closure of the incision was conducted similarly as in muscle biopsy. Each subsequent biopsy was collected from the opposite side of the tail-head region. Biopsy samples were immediately frozen in liquid nitrogen and stored at -80℃ for later mRNA expression analysis.
mRNA expression
Biopsy tissue was weighed (50 mg for muscle tissue, 200 mg for adipose tissue) and RNA extracted using Qiazol Lysis Reagent (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s instruction. Genomic DNA was removed by using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). Concentration of RNA was measured with NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Quality of RNA was assessed using a 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) to calculate a RNA integrity value (RIN) for each sample. Values for RIN range from 1 to 10 (low to high quality) based on the area of 18S and 28S rRNA area and the height of the 28S rRNA peak. Extracted RNA had mean RIN values of 8.8 ± 0.48 for muscle tissue and 7.4 ± 1.61 for adipose. A portion of RNA was diluted to 100 mg/L using DNase/RNase free water prior to reverse transcription.
Complementary DNA (cDNA) was synthesized using 600 ng RNA, 54 µL DNase/RNase free water and 6 µL Random Primers (11034731001, Roche). The mixture was incubated at 65 °C for 5 min and placed on ice for 3 min. Fifty-four μL of master mix contained 24 μL 5X First-Strand Buffer, 6 μL Oligo dT18, 12 μL 10mM dNTP mix (18427088, Thermo Fisher Scientific), 1.5 μL RevertAid Reverse Transcriptase (200 U/µL, EP0442, Thermo Fisher Scientific), and 0.75 μL RiboLock RNase Inhibitor (40 U/µL, EO0381, Thermo Fisher Scientific). The reaction was carried out in a SureCycler 8800 Thermal Cycler (Agilent Technologies Inc., Santa Clara, CA, USA) using the following temperature program: 25℃ for 5 min, 42℃ for 60 min and 70℃ for 5 min. Resulting cDNA was then diluted 1:4 with DNase/RNase free water.
The primers used for Quantitative PCR (qPCR) are listed in Supplemental Table 1. The design and evaluation of the primers that had no references were conducted according to the method reported by Bionaz and Loor [28]. Sequence results for the DNA products of the designed primers are presented in Supplemental Table 2. The sequencing product was confirmed by BLASTN at the National Center for Biotechnology Information database (NCBI).
Quantitative PCR was performed using 4 μL diluted cDNA combined with 6 μL of a mixture composed of 5 μL of Sybr Green Fast Mix ROX (QuantaBio Inc., Gaithersburg, MD, USA), 0.4 μL of forward primer, 0.4 μL of reverse primer, and 0.2 μL of DNase/RNase free water. All cDNA samples were analyzed in triplicate and 7-point relative standard curve plus the negative control (DNase/RNase free water instead of cDNA template) were used. The amplification protocol was as follow: 2 min at 50℃, 5 min at 95℃, 40 cycles at 95℃ for 5 s, 60℃ for 30 s. After amplification, a melting curve analysis was performed over a range of 60-95℃ to verify that a single PCR product was generated at the end of essay. The data were calculated with the QuantStudio Real-Time PCR Software (version 1.3, Thermo Fisher Scientific). The final data were normalized using the geometric mean of internal control genes: GAPDH, ACTB, and RPLP0 for muscle samples, ACTB, BRPS2, and SLC35BC for adipose tissue samples. Treatment effect was analyzed for the abundance of the internal control genes; there were no treatment effects on abundance of any of the five internal control genes. Information about qPCR performance of the genes are presented in Supplemental Table 3 and 4.
Statistical analysis
Cow grazing group was considered the experimental unit for all response variables. Data, except AI pregnancy rates, were analyzed using the MIXED procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC, USA). Outliers were checked by using Proc Reg procedure of SAS removing data with a studentized t 3.0 prior to analysis. A random statement of cow group nested within treatment was included in all of the analysis under MIXED procedure when individual animal observational data was used. Treatment means were separated by the least square means function of SAS. The model for cow BW and BCS included treatment as fixed effect and cow age as covariate. The model for milk yield included the fixed effect of treatment, random effect cow group nested within treatment, and included cow age, cow milk expected progeny differences (EPD) and days postpartum as covariates. The model for calf plasma fatty acid relative concentrations, cow milk composition and milk fatty acid profile included treatment as a fixed effect and day-postpartum as a covariate. The model for steer weaning BW and pre-weaning ADG included the fixed effects of treatment and the covariate of weaning BW EPD Data concerning relative mRNA abundance but not normally distributed were transformed with a logarithmic function (LOG) or a Box-Cox family of power transformation to improve normality. For the analysis of relative mRNA abundance, treatment, time (either at-birth or at-weaning), and the interaction between treatment and time were used as fixed effects, group nested within treatment and sire were included as random effects. Repeated measure analysis of SAS was used for the analysis of cow plasma fatty acid relative concentrations. The model includes treatment, time (mid-supplementation or at-calving), and the interaction between treatment and time as fixed effects, and the concentration of the corresponding fatty acid at pre-supplementation as a covariate. The repeated measure was also used for the analysis of forage availability, and the model included treatment, time, and the interaction between treatment and time as fixed effects. Heterogeneous compound symmetry was used as the covariance structure for cow plasma fatty acid relative concentration and forage availability based on Akaike information criterion. The GLIMMIX procedure of SAS was used for the analysis of AI pregnancy rates, with treatment as fixed effect and age as a covariate in the model. A random statement including cow group nested within treatment, sire, and AI technician were included for the analysis of AI pregnancy rates. Overall pregnancy rate was initially analyzed with the same model as AI pregnancy rate; however, the data did not converge as some of the groups had 100% pregnancy rate. Thus, the MIXED procedure of SAS was used for the analysis of overall pregnancy rate, with each group being the observational unit. Significance was declared at P 0.05, and tendencies were declared from 0.05 P 0.10.