## 2 Materials and Methods
### Immunoglobulin Content and Lysozyme Activity Analyses  
IgG, IgM and IgA were measured in donkey colostrum using specific human ELISA kits (KOMA BIOTECH, Seul, Republic of Korea), according to the manufacturer's instructions (reference K3231067, K0231069 and K0231064, respectively). ELISA (or enzyme-linked immunosorbent assay) is an immunoassay technique involving the reaction of antigens and antibodies in vitro. The kits contained all of the required reagents and material for the specific Ig quantitation: a precoated 96-well ELISA microplate (with antigen-affinity purified goat anti-human Ig), plate sealers, detection antibody (horseradish peroxidase-conjugated antigen-affinity purified goat anti-human Ig), standard protein (human reference serum), assay diluent (1% bovine serum albumin), color development reagents (tetramethyllbenzidine and H\({}_{2}\)O\({}_{2}\) solutions), stop solution (2 M H\({}_{2}\)SO\({}_{4}\)) and washing solution (phosphate-buffered saline powder with 0.05% Tween-20, pH 7.4). To each well of the precoated microplate, 100 uL of blank, standard or donkey colostrum sample was added and incubated at room temperature for 1 h with the plate sealer provided. The standards were diluted following the manufacturer's recommended dilutions. Then, 100 uL of the diluted detection antibody (1:20,000 to 1:50,000) was added per well and incubated for 1 h at room temperature in the sealed plate. Before, between and after the above steps, the wells were aspirated to remove the liquid and washed five times with the washing solution to remove unbound molecules. For the color development reaction, 100 uL of color development solution was added to the wells and incubated for about 10 min. The reaction was stopped by adding 100 uL of the stop solution, and the absorbance was read at 450 nm in less than 20 min using a Multiskan GO microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Triplicate determinations were performed for each Ig, and total concentrations were determined using the standard curves constructed with the diluted standards.  
Lysozyme activity was determined using a _Microoccus lysodeikticus_-based turbidimetric procedure recommended by Sigma Chemical Co. (Catalog Number MA393, Sigma, St. Louis, MO, USA), with slight modifications, with one unit of enzyme being defined as the amount of enzyme that causes a decrement of 0.001 units of absorbency at 450 nm. The principle of this assay is the lytic activity of lysozyme towards _Microoccus lysodeikticus_ cell walls (antibacterial activity) by measuring the loss of light intensity in the direction of incident beam propagation, with reference to a standard solution. A 0.015% (_w_/_v_) _Microoccus lysodeikticus_ cell suspension (substrate) was prepared by suspending _Microoccus lysodeikticus_ ATCC 4698 lyophilized cells in sodium phosphate buffer (66 mM, pH 6.24). To assure the freshness of this suspension, its absorbance at 450 nm (A450 nm) was measured at the beginning of the analyses and was consistently between 0.6 and 0.7, as it should be. Prior to the enzymatic reaction, the substrate was heated at 30 \({}^{\circ}\)C in a thermostatic bath. The reaction was initiated by adding 0.10 mL of appropriately diluted donkey colostrum to 2.5 mL of substrate and, after mixing by inversion, the decrease at 450 nm was immediately recorded for 3 min at 10 s intervals using a PerkinElmer Lambda 35 UV-Vis spectrophotometer (PerkinElmer Instruments, Waltham, MA, USA). Measurements were carried out against a blank containing the substrate and 0.10 mL of buffer, and each sample was measured in triplicate.  
For Ig content and lysozyme activity, the results were expressed as percentages (%), considering 100% the value obtained for colostrum before any treatment (untreated colostrum).