2.1. Process of Low-Temperature Crystallization of Fish Oil and Feed Additive Elaboration

Raw fish oil was obtained during the production of fish meal, as a result of boiling fish waste (mainly herring and sprat from the Baltic Sea) and then pressing the resulting pulp. The resulting post-production waters (temperature > 90 °C) were directed successively to a clarifying and separating centrifuge to separate the crude fish oil. In order to reduce the content of dioxins and dioxin-like compounds, fish oil was subjected to a purification process [23].

The concentration of n-3 FAs in fish oil, mainly EPA and DHA, was increased by the process of low-temperature crystallization (LTC) [24], according to a modified version of the method elaborated by Bodkowski et al. [25]. The process of FA transesterification was carried out using 2 M methanol solution of potassium hydroxide. First, 2500 mL of acetone (Avantor Performance Materials Poland Inc., Gliwice, Poland) was added to 1000 mL of fatty acid methyl esters (FAME) of fish oil, and it was mixed for 2 min (300 rpm) using a magnetic stirrer (Ikamag EDA 9, IKA^® Poland Ltd., Warsaw, Poland) and then was placed in the freezer (Thermo Electron Corporation, Waltham, MA, USA) at −70 °C for 18 h. After the freezing process, the crystallized fraction was separated from the liquid part by means of a laboratory kit for vacuum filtration using a Buchner funnel (Laboport, KNF Neuberger, Inc, Trenton, NJ, USA). In the last stage, acetone was evaporated (40 °C, vacuum 200 mbar) from the liquid fraction using a rotary vacuum evaporator (Laborota 4011, Heidolph Instruments GmbH & Co. KG Vertrieb Labortechnik, Schwabach, Germany) and it was used again in the next cycle.

To protect UFA in fish oil after the process of low-temperature crystallization (LTC-FO) against oxidation, α-tocopherol dissolved in methanol (all reagents from Avantor Performance Materials Poland Inc., Gliwice, Poland) was added at 200 mg/100 g [26]. Up to the time of application tests on cows (max. 2 weeks), LTC-FO was stored in refrigerated conditions (approx. 4 °C) in closed dark bottles.

Chromatographic analysis of FAME was performed using a gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and SP-2560 capillary GC column (100 m length × 0.25 mm inner diameter (i.d.), d_f = 0.20 μm; Supelco, Bellefonte, PA, USA). The temperatures of the injector and detector were 250 and 260 °C, respectively. Nitrogen was used as the makeup gas, while helium was the carrier gas. The flow rate of the carrier gas was 1.1 mL/min. The oven program was as follows: initial temperature 140 °C (2 min), increase 2 °C/min up to 225 °C, isotherm for 10 min, increase 4 °C/min up to 240 °C, isotherm for 10 min. Two microliters of the sample was injected in split mode (100:1). Particular FAs were identified by comparison with FAME standards (GLC #47885, #47571, #H4515, #20290-75-9, #17269, #10417-94-4, #D2659 Sigma-Aldrich, Chemie GmbH, Schnelldorf, Germany). The process of transesterification and chromatographic analyses were carried out according to the procedures ISO [27] and AOCS [28]. The process of low-temperature crystallization of fish oil (FO) and chromatographic analysis of FO and LTC-FO were conducted at the Department of Food and Environmental Chemistry, National Marine Fisheries Research Institute in Gdynia, Poland (6 analyses for FO and LTC-FO, from different batches of oil).