According to data published by European Comission1, the total production of eggs (for hatching and direct consumption) in the European Union in 2016 was 7.5 million tonnes, being Spain the 4^th largest producer of eggs in the EU, currently with approximately 45 million hens based on egg production2. Thus, poultry farming is an important agricultural activity in Spain, with genetic improvement, sanitation, efficient management of production costs and nutritional programs, as well as, handling its main pillars of development.

Nowadays, the protein and amino acid sources used in birds’ feeding have a special importance given its high cost. In addition, under current production conditions, the threonine (Thr) is the third limiting amino acid in maize- and soya-based feeds, mainly because cereals have a low content of it, and represents the critical point in formulations of minimum cost, due to its influence in the crude protein level of diet.

Therefore, these diets may give rise to a deficiency of Thr, being advisable to supply it by its synthetic form3. When synthetic amino acids are supplied and diets are formulated in terms of digestible amino acids (instead of total amino acids), it is possible to substitute maize and soya for alternative ingredients, ensuring an equivalent amount of digestible amino acids4.

Besides that, Thr supplementation allows the reduction of crude protein level of diets, contributing to the reduction of N-excretion in the environment3. It is required by poultry to form body protein, collagen and elastin, as well as, the feathers. Also, being found in gastrointestinal epithelium (mucosal cells and digestive enzymes) and as a component of immunoglobulins4.

Considering the aforementioned, this work aimed at determining the digestible Thr requirement and the Thr/Lys ratio to optimize the egg’s productivity and quality in commercial Isa Brown laying hens from 55- to 58- and 59- to 61-week-old.

The experiment was carried out at the Technical School of Agricultural Engineering of the Polytechnic University of Madrid in accordance with Royal decree 53/2013, which establishes the basic rules applicable to protection of experimental animals and other scientific purposes5.

A total of 216 Isa Brown laying hens from 55 to 61 weeks of age were housed in groups of 4 birds in 54 cages of 508 x 450 x 450 mm (571.50 cm²/bird). The cage was the experimental unit and each cage had two drinkers, and an available feeder length of 10.16 cm/bird. The average temperature during trial was 20 ± 2°C, and the 16L:8D regimen (16 hours light : 8 hours dark). Animals received water and feed ad libitum throughout the experiment.

The birds consumed a basal diet containing 146.9 g/kg of crude protein, 3.7 g/kg of digestible Thr (dig. Thr) and 2,744 kcal/kg of metabolizable energy. The basal diet was supplemented with increasing levels of L-Thr so that six diets containing 3.7, 4.0, 4.3, 4.6, 4.9 and 5.2 g/kg of dig. Thr and a Thr/Lys ratio of 0.58, 0.63, 0.67, 0.72, 0.77 and 0.81, respectively. The diets were isoenergetic and isonutritic, except for dig. Thr levels and Thr/Lys ratio (Table 1). Diets did not contain antibiotics or growth promoters and were supplied for hens as flour. Each cage was randomly associated with one of the six experimental treatments with nine replicates each.

The mean daily feed intake (DFI) per replicate, initial live body weight (LBW), mean body weight gain (BWG), hatch index (HI) and mean egg weight were evaluated. The feed:gain ratio (F:G) and egg mass were calculated daily. To calculate BWG, all hens from each replicate of each treatment were weighed at the start of the trial (55 weeks), at week-58 and at the end of trial (61 weeks of age). Mortality was monitored daily. Also, the eggs were classified by commercial classes according to regulation n. 589/2008 that details the application rules of regulation n. 1234/2007 which stablishes the rules of egg’s commercialization.

External and internal egg quality parameters were analyzed, including Haugh unit (HU), albumen height, yolk color, eggshell strength at break and eggshell thickness. The HU was calculated using the formula described by Haugh6 according to which: HU = 100 log (h + 7.57 - 1.7 W^0.37); where: h = albumen height (mm) and W = egg weight (g). The albumen height (mm) was measured with a dial micrometer gauge movement by handle rotation (B. C. AMES, model S-6428, Waltham, MA, USA) for which the eggs (albumen + yolk) were placed on a level and flat glass surface. The yolk egg color was analyzed using the Roche® colorimetric fan method7.

For the egg texture testing, the equipment model TA-XT2i (Stable Micro Systems Ltd., Godalming, UK) was used connected to a computer and controlled by specific software. The thickness of eggshell (μm) was measured using a Mitutoyo America Corp.â (Aurora, IL, USA) digital micrometer (model 293 MDC-MX) at 3 points above the egg midline, followed by the calculation of arithmetic mean.

Data were analyzed by repeated measures analysis using the MIXED procedure of SAS8and significant effects were considered when P<0.05. Linear and quadratic polynomial contrasts were used to determine the dig. Thr optimal levels.

The interaction between Thr-level and the experimental period was not significant for any of the studied parameters, and because of that, only the results for the main effects are shown. Neither the Thr-level nor the experimental period had any effect on the studied variables (Table 2) from hens of 55- to 61-week-old. The average daily values of DFI, BWG, F:G, HI, egg weight and egg mass were 128 g/day, 16.32 g, 2.31 g/g, 84.2%, 66.8 g and 56.4 g, respectively. It should be mentioned that DFI and BWG were higher for the 55- to 58-wk-old hens than for those of 59- to 61-wk-old. However, the F:G remained constant (2.3 g/g).

Neither the Thr-level in diet nor the experimental period influenced the egg quality parameters (P>0.05) (Table 3). The obtained average values were 6.94 mm and 6.75 mm for the albumen height, and 89.7 and 88.7 for the HU of hens of 55 to 58 weeks of age and, 59 to 61 weeks of age, respectively. Also, an average value of 5.28 kg/cm² and 5.09 kg/cm² was obtained for the eggshell strength at break and, 342 μm and 339 μm for the eggshell thickness of 55- to 58- and 59- to 61-wk-old hens, respectively.

However, a quadratic effect (P<0.001) of the Thr level in diet was observed on the commercial classification of eggs above 63 g. Thus, the L-egg class was affected by Thr-levels according to the quadratic regression estimate (y = -12.897x² + 114.51x - 180.69, R² = 62%) with the maximum point of 4.44 g/kg L-Thr for egg production within L-class, a percentage of 73.63% of eggs within L-class.

The XL-egg class also had the effect (P<0.01) of the Thr-levels according to the quadratic regression estimate (y = 11.94x² - 105.1x + 242.09, R² = 59%), considering the minimum for XL-egg class production at 4.4 g/kg and 11.86% of XL-egg class estimated. The requirement of Thr for obtaining large eggs (L- and XL-egg classes) was 563.2 mg for the average feed intake of 128 g/bird/day.

Sá et al.3 unlike the results of our trial, verified an effect of dig. Thr levels on egg production of 34- to 50-wk-old semi-heavy hens and, Escobar Alvarez9 found effects of dig. Thr levels as responsible for greater egg production (91.72%) of laying hens from 33- to 41-wk-old. The egg weight in our study varied from 67.0 to 67.4 g for levels of dig. Thr of 3.7 to 5.2 g/kg, corresponding to a Thr intake of 473.6 to 665.6 mg and, corroborates the findings of Sá et al.3, where different levels of dig. Thr also did not affect the egg weight and of Leeson and Summers10 that reported the Thr supplementation (5.3 to 6.7 g/kg) in diets with high-protein content (220 g/kg crude protein) did not affect productivity or egg’s weight.

On the other hand, Faria et al.11observed an increase on egg weight from 55.18 g with diet containing 3.5 g/kg to 58.36 g with diet containing 5.3 g/kg dig. Thr, concluding that low levels of Thr in diet reduced egg weight of 31- to 38-wk-old light laying hens. Besides that, the same authors found that diets containing more than 5.3 g/kg dig. Thr increased the feed intake and egg production, as well as, the daily egg mass; however, they did not observe any effect on body weight of 45- to 52-wk-old hens when diets were supplemented with 4.5 or 5.3 g/kg of dig. Thr. Besides that, Teixeira et al.12, evaluating levels of dig. Thr for light laying hens noticed an improvement on egg weight of hens fed diets containing 6.1 g/kg of dig. Thr.

Likewise, the daily egg mass per bird was not improved neither by higher than 3.7 g/kg Thr levels (or 473.6 mg of dig. Thr with a feed intake of 128 g/bird/day) nor by the evaluated periods, showing higher values for periods 55 to 59-wk and 59 to 61-wk of age in comparison to the results reported by Valério et al.13, that used levels of 0.510 to 0.635 mg of dig. Thr for the daily egg mass per bird of 40.4 g; although the birds in our trial were older (55 to 61-wk-old) than those of Valério’s et al.13.

Escobar Alvarez9 obtained results of 57.09 and 56.51 g for the daily egg mass per bird after 744 mg and 775 mg of dig. Thr in diet. And, according to Rostagno et al.14, the nutritional requirement of dig. Thr is 596 mg for semi-heavy hens with daily egg mass of 55 g and body weight of 1.6 kg; what means 25% more than the recommended in our assay (473.6 mg). In the same way, the F:G ratio was not influenced by the different Thr levels in the diet or by the evaluated periods, being in agreement with Teixeira et al.12, who used levels of 3.7 to 7.9 g/kg of dig. Thr in diet provided to 44-wk-old laying hens and found no effect (P>0.05) on the F:G per dozen eggs. Therefore, a dig. Thr/dig. Lys ratio of 58% in diet of our trial did not present significant differences, so the addition of dig. Thr showed no improvements on F:G. Nunes et al.15 determined the nutritional requirements of Thr for semi-heavy hens from 50- to 66- wk-old using five levels of dig. Thr: 4.6, 4.9, 5.2, 5.5, 5.8 g/kg and reported no effects of the dig. Thr levels on internal egg quality parameters (yolk and albumen index, specific gravity, yolk percentage, thickness or shell weight per surface area). Besides that, the results of Schmidt et al.16 with semi-heavy hens of 79- to 95-wk-old agreed with our results, and did not present differences on variables of Haugh unit, hatch index, albumen index, as well as, on performance parameters (feed intake and egg weight). For these authors, the recommendation for semi-heavy hens from 79 to 95 weeks of age should be 509 mg/bird. For adult birds, amino acid deficiency results in the catabolism of body protein, especially those present in skeletal muscle. In the case of hens that have reached their physical maturity and are in production phase, the problem is aggravated by the great demand of protein for egg synthesis. Sá et al.3 evaluating 34- to 50-wk-old semi-heavy hens observed that HU presented a quadratic effect (P

The minimum dig. Thr levels found in our study (4.41 g/kg equivalent to the average intake of 564.48 mg/bird) for L-egg class production and (a dig. Thr of 4.44 g/kg equivalent to the average daily dig. Thr intake of 583 mg) for XL-egg class production are lower compared to the recommendations of Rostagno et al.14 and Rostagno et al.17 (diets with 6.96 g/kg of total Thr and 5.96 g/kg of dig. Thr for laying hens with average daily feed intake of 105 g, daily egg mass of 55 g and 1.60 kg of body weight). Although, for other evaluated characteristics, higher levels than 3.7 g/kg, equivalent to a dig. Thr intake of 473.6 mg (corresponding to a dig. Thr/dig. Lys ratio of 58% and an average feed intake of 128 g) did not improve the productivity and quality traits of laying hens.

The NRC18 recommends diets with dig. Thr levels of 5.2 g/kg for semi-heavy layers with an average daily feed intake of 110 g, and FEDNA19 recommends levels of 5.0 g/kg total Thr and of 4.3 g/kg dig. Thr for laying hens older than 45 weeks of age. In the study of Sá et al.3 with increasing levels of dig. Thr (4.10, 4.45, 4.80, 5.15 and 5.5 g/kg) in diet with 16% of crude protein for 34- to 50-wk-old light laying hens, the recommended dig. Thr level was 5.1 g/kg or a daily dig. Thr intake of 583 mg. Thus, considering the mentioned above, the differences found among authors could be due to lineages used, environment, management, and changes in production systems20.

The results suggest that 55- to 61-week-old Isa Brown laying hens fed diet containing an amount greater than 3.7 g/kg dig. Thr, equivalent to a dig. Thr intake of 473.6 mg did not improve their productivity and quality traits; however, for a higher percentage of L- and XL-egg classes, the level of dig. Thr in diet should be greater than the recommended level of 4.4 g/kg.

To Nutreco N. V. company, Poultry and Rabbit Research Centre for the financial support.