Milk Proteins

The mice were sacrificed 2 h after performing the final behavioral tasks. Thereafter, their hippocampal and colon tissues were lysed with ice-cold lysis RIPA buffer containing 50 mM Tris–HCl (pH 8.0), 1% phosphatase inhibitor cocktail and a protease inhibitor cocktail, 150 mM sodium chloride, 0.5% sodium deoxycholate, 1.0% Igepal CA-630 (NP-40), and 0.1% sodium dodecyl sulfate (SDS)^{38 (link)}. The lysates were centrifuged at 10,000 g and 4 °C for 10 min. The resulting supernatants were electrophoresed by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane using Western Blot Transfer Sysem (Bio Rad Laboratories, Inc, Hercules, CA), blocked with 5% non-fat dried-milk proteins, probed with the antibodies for p65, p-p65, BDNF, claudin-5, occludin, caludin-1, iNOS, COX-2, and β-actin, and washed twice with phosphate buffered saline with tween 20. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. The protein bands were detected by using enhanced chemiluminescence detection kit.

Buffalo mammary gland tissue was obtained from local slaughterhouse (New Delhi, India) for isolation of BuMEC. We followed essentially the same protocol used by Ahn et al. [21] (link) for isolation of BuMEC with minor modifications. Briefly, mammary parenchyma tissue was collected from a disease-free buffalo udder after slaughter and transported aseptically to the laboratory in ice in sterile HBSS (Sigma, USA) containing 100 U/ml penicillin, 5 µg/ml streptomycin and 50 ng/ml amphotericin (HBSS-PS). The tissue pieces were trimmed of connective tissue, including fat and washed three times with HBSS-PS. The tissue was minced with sterile blade and digested with 0.05% collagenase (Sigma, USA), 0.05% Hyaluronidase (Sigma, USA) for 3 h at 37°C. The digested tissue were further treated with 0.25% trypsin EDTA (Sigma, USA), 1% Dispase (Stem cell Technologies, USA) and DNaseI (Stem cell Technologies, USA) at a concentration of 1 mg/ml for 30 min at 37°C and filtered through 40 µ cell strainer (Stem cell Technologies, USA). The filtrate was centrifuged at 80×g for 1 minute. The pellet was washed three times with phenol red free DMEM-F12 (Sigma, USA) containing 10% FBS. The cells were seeded at a density of 2×10⁵ cells/35 mm dish (Nunc, Denmark) in growth medium, which was containing DMEM/F12 supplemented with 5 µg/ml bovine insulin (Sigma, USA), 1 µg/ml hydrocortisone (Sigma, USA), 1 µg/ml apotransferrin (Sigma, USA), 10 ng/ml EGF (Sigma, USA), 10% FBS, 100 U/ml penicillin, 5 µg/ml streptomycin and 50 ng/ml amphotericin. For induction of milk protein expression, BuMECs were grown in the growth medium supplemented with 5 µg/ml Prolactin (Sigma,USA). The cells were cultured in an incubator at 37°C under 5% CO₂. For cryopreservation, 10⁶ cells/ml were suspended in freezing medium constituting 70% DMEM/F 12, 20% FBS (Hyclone, USA) and 10% DMSO (Sigma, USA). Cell suspensions were distributed into 1 ml aliquots in cryovials and stored in liquid nitrogen. We used selective trypsinization steps to enrich the mammary epithelial cells (MECs) preferentially and remove the fibroblast cells from the primary culture. For selective trypsinization 0.25% trypsin-EDTA (Sigma, USA) was added to the confluent monolayer of heterogeneous population of cells and allowed to act for three min at 37°C. The trypsinization was stopped by adding fresh growth media, and the detached fibroblast cells were removed. The cells in monolayer which remained attached to the surface were allowed to grow by addition of fresh growth medium. The cells were subjected to 7 continuous passages for selection of homogeneous population of BuMECs. The BuMECs were routinely evaluated for sterility by growing them in antibiotic free media. The cells were also tested for incidence of mycoplasma contamination using Myco Alert Mycoplasma detection kit (Lonza, USA).

All the experimental procedures were approved by the University of Illinois (Urbana-Champaign) Institutional Animal Care and Use Committee (no. 17166). In total, 16 multiparous Holstein cows past peak lactation (>60 DIM) were used in a randomized complete block design conducted from February to April 2020. All cows were housed in a freestall system equipped with individual feeding gates (American Calan Inc., Northwood, NH) and were fed once daily at approximately 0800 hours. Milking occurred two times per day at 0400 and 1530 hours. Cows were blocked by DIM (97.1 ± 7.6 d) and parity (3.4 ± 0.62), MY, BCS, and SCC (Supplemental Table 1). After 1 wk of adaptation, cows were randomly assigned within block to one of two treatments (eight per treatment): 1) a control group receiving the basal TMR with no supplementation (CON) or 2) basal TMR with 19 g/d of a Saccharomyces cerevisiae fermentation product (NTK; NutriTek®, Diamon V, Cedar Rapids, IA), top-dressed. This is the commercially recommended dose for the type of animal used in this trial. The ration was formulated to meet NRC (2001) requirements and is presented in Table 1. The diet was formulated for cows at 180 DIM, BW of 703 kg, MY of 35.4 kg/d with a target of 3.50% milk fat and 3.12 milk protein and predicted DMI of 24.9 kg/d. The diet included corn silage, alfalfa hay, canola and corn gluten meal, ground shelled corn, rumen-protected lysine and methionine, rumen-inert fat, rumen-bypass protein, vitamin and mineral mix, and the ionophore Rumensin (Elanco, Greenfield, IN). The experimental period lasted 68 d and was divided into two phases: 1) Phase 1 (P1) from the beginning of the experimental period until d 63, and 2) Phase 2, from d 64 to 68, which represents the FR period. Cows were euthanized at the end of FR for a separate experiment. During Phase 2, cows were restricted to 40% of their ad libitum intake of the previous 5 d. This amount of restriction was chosen based off previous work in which 40% restriction was validated as a method to induce intestinal barrier dysfunction in dairy cattle (Kvidera et al., 2017b (link)).