Amino acids

Amino acids (methionine, lysine, threonine and tryptophan) were obtained from Evonik México S.A. de C.V., México. Mesquite gum powder from Prosopis laevigata trees was provided by the Universidad Autónoma Metropolitana-Iztapalapa. Gum powder is a highly branched complex polyelectrolyte formed mainly by L-arabinose and D-galactose, and minor proportions of 4-O-methyl-D-glucuronate and L-rhamnose, in a 2:4:1:1 ratio, and a protein content of around 4.8% db (dry basis), which is responsible for its excellent emulsifying properties (Roman-Guerrero et al., 2009). Soybean oil was purchased from a local supermarket (Toluca, State of Mexico, Mexico). Tween 20™ and sodium alginate were acquired from Sigma Aldrich, S.A. de C.V. (Toluca, State of Mexico, Mexico). Distilled water was produced in the lab by reverse osmosis.

Experimental diet

An experimental diet was formulated to cover the nutritional requirements of cattle in the finishing stage. The composition of the diet was determined in the Bromatology laboratory of the Faculty of Veterinary and Zootechnics of the Universidad Autónoma del Estado de México (Table 1). Representative samples of the experimental diets were taken and dehydrated at 60 °C for 72 h. The dry residues were ground in a hammer mill (Thomas Wiley® Laboratory Mill model 4, Swedesboro, NJ, USA), with a 1 mm sieve. The percentages of moisture, dry matter, ash, nitrogen and ether extract were determined according to the respective AOAC methodologies (1997). The analysis of neutral and acid detergent fiber was performed in an ANKOM200 fiber analyzer (ANKOM Technology Corp, Macedon, NY, USA) with alpha-amylase and sodium sulfite according to Van Soest et al. (1991). The determination of organic matter was calculated by subtracting the ash content. The percentage of protein was determined by multiplying the nitrogen content by 6.25.

Table 1 Ingredients and composition of the experimental dietPreparation of nano encapsulation of the amino acidsPreparation of W1/O emulsion

The amino acids were dissolved in distilled water at a 1:1 (w/v) ratio. Tween 20 (6% by weight) was added to the soybean oil at 60 °C and stirred at 800 rpm for 15 min to ensure complete dissolution. Then, the W1/O emulsion (i.e. water, phase 1, is dispersed in oil) was prepared by slowly adding the amino acid solution to the oil phase in a 1:1 (w/w) ratio and mixing using an Ultra-Turrax T50 homogenizer (IKA®-WERKE Works Inc., Wilmington, NC, USA) at 8000 rpm for 6 min (Su et al. 2022)

Preparation of W1/O/W2 double emulsion

For the external aqueous phase (W2), mesquite gum (10% by weight) and sodium alginate (2% by weight) were dissolved in distilled water. This was then mixed with the freshly prepared W1/O at 25 °C. The W1/O/W2 emulsions were mixed in a (1:2, w/w) ratio with an Ultra-Turrax T50 homogenizer (IKA®-WERKE Works Inc., Wilmington, NC, USA) at 8000 rpm for 5 min. The emulsions were stored at 4 °C after preparation (Su et al. 2022).

Characterization of the emulsionDroplet size measurement

For the measurement of droplet size, the method of Liu et al. (2020) with some modifications was used. The evaluation of the size distribution and the volume-weighted mean diameter of the droplets in the double emulsion was determined using a static light scattering instrument (Mastersizer 3000, Malvern Instruments, Worcestershire, UK).

Zeta potential measurement

To measure the zeta potential of the droplets, a particle electrophoresis instrument (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK) was used (Mikulcova et al. 2018). The samples were diluted 1:100 (w/v) in deionized water under agitation.

Microstructure of the W1/O/W2 double emulsion

To observe the microstructure of the W1/O/W2 double emulsions, an optical microscope with a 100× oil immersion objective and a confocal laser scanning microscope were used. The W1/O/W2 double emulsions were stained using Nile Red (0.1 mg/mL) and Fluorescein-FITC (0.1 mg/mL), respectively. A 5 µL sample was spread on the microscope slide with a coverslip at 25 °C.

Encapsulation efficiency

The W1/O/W2 emulsion was mixed with distilled water in a 1:3 (w/w) ratio, then centrifuged at 6500 rpm for 15 min, and the supernatant was collected using a syringe and subsequently filtered using a drain membrane (pore size: 0.45 μm) and stored at 4 °C until analysis.

Amino acid content was determined by high-performance liquid chromatography (Agilent HPLC 1100, USA) using an Agilent Hypersil ODS column (5 μm, 4.0 mm × 250 mm) and a UV detector (λ = 338 nm). The mobile phases consisted of A: 27.6 mmol/L sodium acetate-triethylamine-tetrahydrofuran (500/0.11/2.5, v/v/v) and B: 80.9 mmol/L sodium acetate-methanol-acetonitrile (1/2/2, v/v/v). Elution was performed at a flow rate of 1.0 mL/min with a gradient of 8 to 100% of mobile phase B for 20 min, followed by 100 to 0% of mobile phase B for 4 min. The encapsulation efficiency of the double emulsion was calculated using the following equation:

\(}\;\left( \% \right)=\left( }0 - }} \right)/}0 \times }00\% \)

Where EE is the encapsulation efficiency (%), M0 is the weight (mg) of the amino acids initially dissolved in the internal aqueous phase (W1) of the W1/O/W2 emulsions, and M is the weight (mg) of the amino acids released to the external aqueous phase (W2) after centrifugation.

Storage stability of the W1/O/W2 double emulsion

The W1/O/W2 emulsions containing encapsulated lysine, methionine, threonine, and tryptophan were stored at 4 °C after preparation. The external phase was collected every 7 days for a period of 28 days to determine the encapsulation efficiency of the amino acids.

In vitro ruminal incubation

Ruminal contents were obtained from four slaughtered cattle (350–450 kg live weight) at slaughterhouse in the municipal of Toluca, Mexico, mixed and strained through 4 layers of cheesecloth into a flask with O2 free headspace. The rumen contents were transferred in an airtight thermos to the Bromatology laboratory of the Faculty of Medicine, Veterinary and Zootechnics of the Autonomous University of the State of Mexico, Toluca, Mexico. Samples of 500 mg of the diet as a substrate (previously ground and weighed) were weighed into 160 ml serum bottles. After that, 10 ml of particle-free ruminal fluid was added to each bottle, and 40 ml of the buffer solution of Goering and Van Soest (1970), with no trypticase added, was immediately added in a 1:4 (v/v) proportion. The different amino acids (free form, nano-encapsulated) were added to the glass vials in their respective concentrations (lysine: 0, 0.2, 0.4, 0.6 g/g diet DM; methionine: 0, 0.15, 0.3, 0.6 g/g diet DM; threonine: 0, 0.1, 0.15, 0.2 g/g diet DM; tryptophan: 0, 0.08, 0.1, 0.12 g/g diet DM).

A total of 288 bottles (3 bottles of each triplicate sample within each of the 4 amino acids with 4 different levels and in two forms (free and nano- encapsulated), in 3 runs on different weeks, with 3 bottles as blanks (i.e., rumen fluid only), were incubated for 48 h. Once all bottles were filled, they were immediately closed with rubber stoppers, shaken and placed in the incubator at 39 °C. The volume of gas produced, methane, carbon monoxide and hydrogen sulfide production were recorded at 2, 4, 6, 24, 28, 30 and 48 h of inoculation.

At the end of incubation (i.e., 48 h), bottles were uncapped, pH was measured immediately with a pH meter (GLP 22, Crison Instruments, Barcelona, Spain), and fermentation was stopped by swirling the bottles in ice. The contents of each bottle were transferred as filtered fermentation residue to determine the apparently degraded substrate.

Total gas, methane, carbon monoxide and hydrogen sulfide production

Total gas volume was measured in PSI (pounds per square inch) at 2, 4, 6, 24, 28, 30 and 48 h of incubation as described by Theodorou et al. (1994) using a digital manometer with an accuracy of 2% (Manometer model 407910, Extech® Instruments, Nashue, NH, USA). CH4, CO and H2S were quantified according to Acosta et al. (2022). At the end of each measurement, the gas accumulated at the top of the vials was released with a syringe without a plunger to avoid further gas accumulation and partial dissolution of the evaluated gases (Tagliapietra et al. 2010).

Rumen pH and dry matter degradability

After 48 h of incubation, the contents of the glass vials were filtered through 25 mm porosity bags (Filter bag F57, ANKOM Technology Corp., Macedon, NY, USA). The liquor (filtrate) was collected in a glass beaker and the pH was measured immediately using a glass electrode potentiometer (Hanna® Instruments model HALO® HI11102). The residues adhering to the walls of the glass vials were removed by rinsing with distilled water and collected in the same bags as used for the initial filtration. The residues were dried at 60 °C for 72 h. Dry matter digestibility (DMD) was calculated considering the initial weight of the diets and the weight of the obtained dried residues.

Calculations and statistical analysis

The kinetics of total gas, CH4, CO and H2S production were estimated by adjusting the gas volume with the NLIN procedure of SAS (2002) according to the model proposed by France et al. (2000):

\(}=} \times [}-}^}(} - })}}]\)

where y = volume (ml) of total gas, CH4, CO and H2S at time t (h). b = asymptotic production of total gas, CH4, CO and H2S (ml/g DM). c = production rate of total gas, CH4, CO and H2S (ml/h). Lag = initial lag time before total gas, CH4, CO and H2S production starts (h).

Metabolizable energy (ME) (MJ/kg DM) was estimated according to the equation proposed by Menke et al. (1979):

\(}=}.}0+\left( } \times }} \right)+\left( } \times }} \right)\)

where CP = crude protein (g/kg DM). GP = total gas production (ml/200 mg DM) at 24 h of incubation.

Total short-chain fatty acid concentrations (SCFA) (mmol/200 mg DM) were calculated according to Getachew et al. (2002) as:

\(}=\left( } \times }} \right) - 0.00425\)

where GP = total gas production (ml/200 mg MS at 24 h of incubation).

In addition, the ratio of CH4 to SCFA (CH4: SCFA; mmol mmol-1), ME (CH4: ME; g MJ-1) and OM (CH4: OM; mL/g) were calculated.

The experimental design was completely randomized with a 4 × 2 × 4 factorial arrangement (4 amino acids – methionine, lysine, threonine, and tryptophan), 2 forms (free and nano-encapsulated), and (4 doses). The data from the three replicates of each treatment were averaged in each run and the averages obtained, for each run, were used as the experimental unit. The analysis was performed using the GLM procedure of SAS (2002) with the following statistical model:

\(}_}}}=\mu +}_}}+}}_}}+}}_}}+}+}} \times } \times }} \right)_}}}+}}}.\)

where, Yijk is the response variable, µ is the general mean, Ai is the effect of the dietary type of amino acid, Fj is the effect of the form of amino acid (free and nano), Dk is the effect of extract doses, and (A × F × D)ijk is the effect of the interaction between the type of amino acid, forms (free and nano-encapsulated) and their doses used, and εijk is the experimental error. The comparison of means was performed using Tukey’s test, and they were considered significantly different when p ≤ 0.05.