*2.2. Diet Composition*

We used three specific diets with different EAA/NEAA ratios ranging from 100%/0% to 0%/100%, as previously described [27]. A summary of diet composition and EAA/NEAA ratios is shown in Table 1.


The Casein-Prot and Casein-AA diets were used as special test diets, since no composition of AAs in StD proteins was available even for the producer, and we needed to test the safety of life-long nutrition by free AAs.

All special diets provided quantitatively equal amounts of lipids, carbohydrate and micronutrients. All diets were thus iso-caloric and provided the same amounts of nitrogen, although nitrogen content was provided by different formulations of AAs or proteins, according to those presented in Table 1. All special diets were prepared for Nutriresearch s.r.l. (Milan, Italy) by Dottori Piccioni (Milan, Italy) in accordance with AIN76-A/NIH-7 rules [29].

Animals were randomly assigned to one of the six groups. Each group was fed exclusively with a specific diet [i.e., EAA-100% diet (*n* = 30), Casein-AA diet (*n* = 30), Casein-Prot (*n* = 30), StD (*n* = 40), EAA-30% diet (*n* = 30), or NEAA-100% diet (*n* = 30)]. All animals had free access to food and water.


**Table 1.** Diet composition.

\* Nitrogen (%) from free amino acids (AAs) only. ◦ Nitrogen (%) from vegetable and animal proteins and added AAs. ˆ whole casein protein. EAA-100% = free essential amino acid-exclusive diet; Casein-AA = Casein-like free AAs diet; Casein-Prot = Casein whole protein diet; StD = Standard diet; EAA-30% = free essential amino acid-poor diet; NEAA-100% = non-essential free amino acid-exclusive diet. The black line represents the limit between EAA (above) and NEAA (below). *bcaa* = branched-chain amino acids.

#### *2.3. Data and Sample Collections*

Body weight (BW), mean food and water consumption (g/days and mL/day, respectively) were calculated weekly in all groups. Mortality was monitored daily.

Animals from the two groups fed with NEAA-100% and EAA-30% had a mortality > 70% at the 7th week, and all those still surviving were euthanized for ethical reasons linked to a drop in weight, as discussed in results. Five animals from each of the other groups were euthanized after 12 and 18 months to check their morphometric and clinical parameters. Specifically, BW and nose–tail length (body length, BL) were measured. Blood samples from the hearts and urine from the bladders were immediately collected for further analysis. Glycaemia was also measured by a glucometer in venous blood samples collected from tail veins. Subsequently, the heart, kidneys, liver, spleen, *triceps surae*, retroperitoneal white adipose tissue (rpWAT) pad and brown adipose tissue (BAT) were quickly removed and weighed [30].

#### *2.4. Blood and Urine Analysis*

Blood samples were collected either in tubes containing the K3-EDTA anti-coagulant for cell count analysis, or in tubes without anti-coagulants for serum separation. The blood cell count was performed with a Cell-Dyn 3700 laser-impedance cell counter (Abbott Diagnostics Division, Abbott Laboratories, IL, USA). Serum and urine levels of albumin and creatinine were assessed with an ILab Aries (Instrumentation Laboratory, Lexington, MA, USA) automatic analyzer. Serum levels of haptoglobin (Hpg) [31] and the neutrophils to lymphocytes ratio (NLR) [32,33] were also assessed as inflammatory markers. These analyses were carried out by personnel of the "Division of Laboratory Animals" of IZSLER (Brescia, Italy).
