Varying isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens.

To ascertain an appropriate level of isoleucine for LSL-LITE layers (23- to 30-week-old), diets containing total isoleucine concentrations (levels) of 0.66 (Control), 0.69, 0.72, 0.75, 0.78, 0.81, and 0.84% were fed as 7 treatments (2730 kcal/kg metabolizable energy) x 7 replicates x 10 birds per replicate. Significance for performance, egg quality, serum biochemistry, and ileal digestibility of protein was determined at P ≤ 0.05. Level, week, and level*week (L*W) were significant for production, egg mass, and feed intake. Level and week were significant for FCR. Week was significant for weight gain. Level was significant for egg weight, specific gravity, and shell thickness; week was also significant for these external egg parameters as well as shape index and proportional shell thickness. L*W was significant for all except shape index. For internal egg measurements, level was significant for proportional yolk, proportional albumen, yolk index, and yolk:albumen. Week was significant for internal egg parameters while L*W significantly affected Haugh unit, proportional albumen weight, yolk index, albumen index, and yolk color. Level was significant for globulin and glucose in serum. Isoleucine at 0.72%, 0.81%, and 0.84% produced the lowest FCR, an important standard in the poultry industry. Considering the low FCR of 1.45 and cost for inclusion as a dietary ingredient, 0.72% isoleucine was chosen for further studies with varying quantities of other branched chain amino acids in diets for young laying hens.

Introduction

It has been acknowledged that earlier recommendations for dietary amino acids for poultry may not be adequate [1]. Since 1994, researchers have continued to recommend appropriate levels of amino acids in layer diets. For instance, recommended optimum levels of isoleucine have included 550 to 660 mg/hen/day [2-4].

Huyghebaert et al. determined that the daily isoleucine requirements of layers (26- to 36-week-old) did not decrease while egg output increased [5]. On the other hand, several investigators noted that age and genetic variety (breed) of layers prompted further study associated with amino acids [6-8]. Values from producers of five layer breeders ranged from 640 to 767 mg/bird/day for isoleucine (Table 1), starting at a higher level than suggested above. Values for ideal digestible isoleucine for layers ranged from 79 to 86 [8].

Table 1

Digestible amino acid intake for isoleucine from five layer producers.

Hy-line 36 (Hy-line International, 2020)1	Hy(Hy-line brown Hy-line International, 2020)2	Lohmann brown lite (Tierzucht 2017)2	Lohmann brown classic (Tierzucht 2017)2	Isa brown (Isa Brown, 2011)3,4
6405	6565	5405	5705	7675

1First egg to production drops 2% below peak egg production (approximately 18–37 wk).

250% egg production to maximum egg production (approximately 21–40 wk).

3From 2% eggs production to 28 wk of age.

4Based on a feed intake of 105 g/bird/day.

5mg/bird/day.

As branched-chained amino acids (BCAAs), isoleucine and valine are important for maintaining gut immunity, antioxidant capacity, and critical metabolic processes [7,9,10]. Isoleucine is essential for growth, optimum egg mass, and egg production [11,12]. BCAAs are thought to be important in egg production due to regulation of fatty acid metabolism in the liver where production of lipoprotein could be rate-limiting for egg yolk formation [13]. It is metabolized extra-hepatically in skeletal muscles by BCAA transaminase, competes for transfer across the blood-brain barrier, and is transferred through the cellular membrane via the same pathway as other BCCAs [14,15].

Investigating appropriate quantities of available amino acids such as isoleucine will likely continue due to the world-wide increase in poultry and egg production [16,17]. Shivazad et al. suggested determination of the appropriate quantity of available synthetic amino acids due to less output of nitrogen and positive outcomes for environmental health [11]. Bregendahl et al. suggested ideal amino acid ratios relative to lysine irrespective of source for young White Leghorn layers [6]. Liu and Selle noted that the digestive dynamics of available synthetic amino acids differ from that of protein-bound amino acids [18]. All of these observations should be considered as investigators include new low cost sources of protein with varying quantities of BCAA’s in diets.

During feed formulation, the quantity of amino acids in less expensive protein sources can be determined and supplemental synthetic amino acids such as isoleucine can be used to meet the requirement; however, as with protein-bound amino acids, the appropriate requirement for synthetic amino acids is not clear. Leucine requirements are met by various protein sources in the diet while valine and isoleucine are often most examined [19]. For BCAAs, requirements are important because antagonism can occur, depressing performance [20,21].

The availability of synthetic isoleucine provides an opportunity to investigate its requirement in diets containing inexpensive sources of protein bound amino acids [17,20-22]. In the present work, we explored a range of levels (0.66% to 0.84%) for total isoleucine (bound plus synthetic) in the diet to understand how various performance and egg quality parameters for 23- to 30-week-old Lohmann LSL LITE layers were affected generally and to ascertain a specific level of isoleucine for further study with other BCAA’s.

Results and discussion

Levels (% L- Isoluecine) represents the total quantity of the amino acid in each diet.

Performance (Table 2)

Performance values (feed intake, egg production, mass, and layer weight) for the Control (0.66% isoleucine) were similar to expected values for Lohmann LSL-Lite layers [23].

Our results showed that 0.75% isoleucine produced less eggs when compared to 0.66–0.72%, and 0.81% isoleucine, but similar quantities compared to 0.78% and 0.84%. Thus, level decreased production with a linear trend. Dong et al. observed no change in egg production [or any performance measurement (egg mass, feed intake, FCR)] when feeding 0.54% to 0.94% digestible isoleucine to 28-week-old Lohmann Brown Layers in 0.10 intervals for an additional 12 weeks (with a one week acclimation period) [7]. Da Rocha et al. reported a quadratic effect on production when feeding isoluecine:lysine ratios (0.73:1, 0.78:1, 0.83:1, 0.88:1, 0.93:1 and 0.98:1) to Hy-line W-36 layers (24- to 40-week-old) and suggested a isoleucine:lysine ratio of 0.84:1 [4].

With increasing age (week), egg production significantly increased; level by week (L*W) was also significant. As noted by Bell and Weaver (2002), as layers age (week), production decreased [24].

Egg mass was affected by level with linear and quadratic effects. The statistically greatest egg mass (52.068 ± 1.45 g) occurred at 0.72% isoleucine and produced 2.32% more mass than the Control (50.88 ± 1.45 g). Peganova and Elder reported that greater than 1.0% isoleucine reduced daily egg mass for 24- to 32-week-old and 46- to 54-week-old Lohmann Brown layers [20]. When feeding Hy-Line W-36 (26- to 34-week-old), Bregendahl et al. noted that the ideal amino acid ratio for maximum egg mass was lysine (100):isoleucine [6]). Da Rocha reported a gradratic effect of level on egg mass [4].

Feed intake by level was significant with linear (downward) and quadratic responses.

The significantly lowest feed intake was associated with 0.75% isoleucine. At concentrations of greater than 1.05%, isoleucine reduced egg production, egg mass, egg weight, and feed consumption, likely associated with decreased dopamine, noradrenaline, and serotonin [25]. Peganova and Eder also observed that greater than 1.05% isoleucine was associated with a reduction in feed intake for Lohmann Brown laying hens (24- to 32-week-old) [20]. It is not clear if serotonin, as an appetite suppressant, affected feeding behavior at 0.75% isoleucine in our work because greater quantities of isoleucine did not cause the effect. Da Rocha et al. noted a quadratic effect on feed intake by level of isoleucine [4]. While level affected feed intake, there was no effect on bird weight gain, exhibiting temporal fluctuation. Perganova and Eder noted that dietary isoleucine greater than 0.8% caused a reduction in body weight [20].

In the poultry industry, FCR is most often selected as a useful performance variable in determining the most appropriate diet [26,27]. In our work, FCR linearly decreased significantly with level; the response was also cubic. Isoleucine at 0.72% and 0.84% significantly reduced FCR compared to all other levels except 0.78%. Da Rocha et al. reported a quadratic effect of isoleucine level on FCR [4]. Clark et al. compared feeding behavior of selected ISA Brown layers at 25 to 30 weeks of age [27]. Hens with FCR < 1.8 ± 0.02 (high feed efficiency) consumed less feed (as in our work) and preferred a diet with greater ash content and lower gross energy when compared to hens having high FCR > 2.1 ± 0.02 (low feed efficiency). Week was linear for FCR while L*W was not.

External egg quality (Table 3)

Level, significant for egg weight, had a quadratic effect; the greatest egg weight was produced by 0.69%, 0.81%, and 0.84% isoleucine with shared significance at 0.78%. Profit may be made if producers can consistently obtain appropriate amino acid ratios and a lysine:isoleucine at 1:0.69% with low cost feed ingredients while producing eggs with greater weight for markets where this parameter is the most important factor. Da Rocha et al. observed that increasing isoleucine:lysine (0.59, 0.63, 0.67, 0.71, 0.75, and 0.79) did not affect egg weight for Hy-line W36 laying hens (24- to 40-week-old) [4].

A linear downward trend was produced by level for egg shape. Eggs with a shape index < 76 may not withstand processing, packaging, and transportation due to reduced shell strength [28]. Duman et al. observed no effect of shape index on breaking strength [29]. While shape index may be important for commercial producers, it is likely less important in production for local markets or backyard production.

For specific gravity, level caused a linear (upward) effect, greatest at 0.84% (1.0888 ± 0.0043) compared to the Control (1.0820 ± 0.0043); there were quadratic and cubic effects. As specific gravity increases, shell weight should increase [30]. However, proportional shell weight was not significantly affected by level in our work.

Our results indicated that shell thickness was significant for level with quadratic and cubic effects. Isoleucine at 0.72% produced the lowest egg shell thickness (0.379 mm + 0.015 SE) that was between the medium (0.33–0.36 mm) and thick (0.39–0.41 mm) category as reported by Ketta and Tůmová [31]. More research is needed to elucidate how isoleucine level affects eggshell thickness. It is known that after fermentation of eggshells to produce alkaline protease, the protein hydrolyzate contained several essential amino acids including arginine, isoleucine, leucine, lysine, methionine, phenylalanine, and valine along with smaller quantities of cysteine; thus, eggshells have been proposed as a viable protein source [32]. As well, changes in epimerization of isoleucine in eggshell have been used to date the range of age for archeological sites [33]. All other levels of isoleucine in the present work where layers were raised in a cage system with no enrichment produced eggshells in the thick range as classified by Ketta and Tůmová for their eggs in a caged system with enrichment or pens with litter [31].

In their investigation of worldwide eggshell uniformity, Sun et al. noted that eggs were thickest (0.367 + 0.023mm) at the sharp end and thinnest at the blunt end (0.341 + 0.025 mm) [34]. Mean thickness of the shells across seven locations with and without the membrane was 0.369 ± 0.021 and 0.356 ± 0.022 mm, respectively [34]. Thus, all of the shells in our study met or exceeded mean thickness. All external quality measurements were significantly affected by week. L*W also affected all measurements excluding shape index where there was a linear trend.

Internal egg quality (Table 4)

Level had no effect on Haugh unit whereas week and L*W were significant due to fluctuations. Proportional yolk weight had linear, quadratic, and cubic effects due to level; the highest level of isoleucine (0.84%) produced the greatest value (26.26 ± 0.91g) compared to all other levels; and the yolk:albumen ratio (41.53 ± 0.0205g, with significant liner, quadratic, and cubic effects) was also greatest at this quantity of isoleucine. Perhaps 0.84% isoleucine increased deposition of lipid in yolk [13].

Level affected proportional albumen weight, with fluctuations, causing significant quadratic and cubic responses. Proportional albumen results differed from those of Da Rocha et al. [4] who found insignificance for isoleucine levels (0.59 to 0.79%) when feeding Hy-Line W36 layers (24- to 40-week-old). Yolk index was significant for level with linear and cubic effects.

In the present work, yolk color was not affected by level. As we observed, Da Rocha et al. reported insignificance for isoleucine levels (0.59 to 0.79%) on yolk color [4]. Although fat soluble carotenoids may not have been the same for our diets and those of Da Rocha et al., insignificance of level on yolk color in both studies likely indicated constancy of carotenoids across the diets of each individual study [4]. Dong et al. found no effect of feeding 0.54% to 0.94% isoleucine on internal quality measurements (albumen height, Haugh units, and yolk color) [7].

Week was significant for all internal measurements. L*W affected proportional albumen weight, yolk index, albumen index, and yolk color.

Comparative performance and external/internal egg quality measurements

The effect of level on performance parameters and external/internal egg quality measurements reported in the present study did not always agree with findings of other investigators even when the same range of isoleucine was added to layer diets. Age of layers, length of feeding, genetics, environmental factors, or imbalances of amino acid may be causative factors. As well, as noted above, Liu and Selle reported that the digestive dynamics of available synthetic amino acids differ from that of protein-bound ones [35]. More work is needed to unravel the digestive dynamics because protein bound amino acids (from inexpensive sources) and available synthetic ones likely will be fed to layers more often as in the present work. Other investigators suggested that due to possible imbalances, all BCAA’s should be monitored when investigating any one of them in commercial avian diets [36]. Moreover, as we reported, L*W often affects many parameters. Perhaps large scale coordinated investigations across several laboratories with several types of layers over their complete laying cycle are needed to find the most appropriate level of isoleucine, valine, and leucine for layers.

Biochemical analyses and crude protein digestibility coefficient (Table 5)

A downward linear trend for serum protein was observed for level. This result did not agree with those of Tewe who reported correlation of both protein quantity and quality with total serum protein [37].

For globulin, there was a significant downward linear trend. Levels of isoleucine from 0.66% (0.81 ± 0.16 g/dL) to 0.75% (0.85 ± 0.16 g/dL) produced a statistically similar response for globulin that was different from that for 0.84% (0.35 ± 0.16 g/dL). The reduction in globulins at 0.84% isoleucine was 58% compared to the Control (0.81 g/dL + 0.16 SE). Harlap et al. studied blood proteins in laying hens (Lohmann Whites, 26- to 80-week-old) [38]. They reported that the quantity of globulins was not related to the productive period but rather depended on a reduction in defenses with the greatest decrease at 80 weeks of age. At 30 weeks old, our control layers had globulin levels at or above 0.81g/dL (0.62% isoleucine to 0.75% isoleucine) that were gradually reduced to 0.35 g/dL at 0.84% isoleucine. The reduction of globulins indicated that increasing the isoleucine level beyond 0.75 may be related to a reduction of immunological defenses of the layers. This finding may be important; however, it is not clear if findings for Lohmann Whites are similar to all other breeds of layers, including LSL-LITE layers. Clearly, more research on these findings is indicated.

For glucose, level produced a downward linear response with quadratic and cubic trends. For ileal CP digestibility, there was a significant cubic effect for level. At 0.81% isoleucine, the coefficient was 19.65% less than that for the Control (82.71% ± 6.26 SE).

Total serum albumin, digestibility, and HI titers were not affected by level. Contrary to our work, Dong et al. reported a quadratic effect for isoleucine levels on serum albumin [7]. The genetic variation or subtype, susceptibility of host, age, breed, and environment (such as environmentally controlled houses versus open houses) are factors likely affecting ND titers of layers in our work and that of others [39]. Additionally, no affect for ND titers associated with level of isoleucine in our work could be explained by presence of specific circulating antibodies produced during low levels of infections associated with low endemic levels of NDV in the poultry industry worldwide [40]. As well, H9N2 virus was detected in 2015 in a Pakistani poultry worker, suggesting that H9 could be transmitted by staff to layers [41]. To date, NDV and AI are prevalent in Pakistan and are included in the commercial vaccination schedule [42].

Conclusion

For the purpose of our work, it was important to determine a specific level of isoleucine as an important parameter to compare to other BCAA’s. Isoleucine levels at 0.72% and 0.84% produced low FCR of 1.45 and 1.44, respectively, but shared significance with 0.78% (1.49). Also, at 0.72% isoleucine, other egg production, egg quality, and biochemical measurements were in the medium to high range. Considering cost of synthetic isoleucine as a dietary ingredient along with the low FCR, 0.72% was chosen as the constant for future studies for comparison of valine and/or leucine levels in layer diets.

Materials and methods

LSL-LITE layers (n = 490, at 23 to 30 weeks) were allotted to 7 treatments × 7 replicates × 10 layers per replicate. Two adjoining cages (60 x 63.5 cm, five birds each) constituted a replicate. Hens were fed the Control (basal diet + 0.66% L-isoleucine, as calculated, Table 6) and diets containing calculated total percentages of L-isoleucine at 0.69, 0.72, 0.75, 0.78, 0.81, and 0.84%.

Table 6

Nutrient composition of control diet for layers at 23 to 30 weeks.

Ingredients	Control feed	Nutrients %	Calculated1	Analyzed2
Corn	54.00	Dry Matter	90.32	90.27
Rice Tips	6.00	ME (Kcal / Kg)	2728.00	--
Soybean Meal	12.00	ME (MJ / Kg)	11.41
Canola Meal	5.00	Crude Protein	16.77	17.28
Sunflower Seed Meal	5.00	Ether Extract	3.06	--
Corn Gluten Meal 60%	2.00	Ash	2.27	--
Guar Meal	2.00	Crude Fiber	4.33	--
Poultry By-product Meal	2.00	Calcium	3.56	--
Canola Oil	0.70	Dig phosphorus	0.42	--
CaCO3	8.00	Total phosphorus	0.67	--
Di-Calcium Phosphate	1.80	Sodium	0.17	--
L-Lysine SO4	0.40	Potassium	0.62	--
DL-Methionine	0.15	Chloride	0.16	--
L-Threonine	0.10	Lysine	0.83	0.91
L-Tryptophan	0.05	Methionine	0.42	0.39
L-Isoleucine 3	0.10	Threonine	0.62	0.70
NaHCO3	0.40	Tryptophan	0.18	0.19
Vitamins Minerals Premix4	0.30	Cysteine	0.26	0.31
		Met + Cys	0.67	0.71
		Arginine	0.95	1.08
		Valine	0.67	0.88
		Isoleucine	0.66	0.78
		Leucine	1.29	1.38
		Histidine	0.37	0.43
		Phenylalanine	0.69	0.74
		Linoleic Acid	1.66	--
		Na + K--Cl (mEq / Kg)	214.31	--

1Calculated column estimated as digestible during feed formulation. For diets, quantities of synthetic isoluecine were added to yield total L-isoluecine at 0.69, 0.72, 0.75, 0.78, 0.81, and 0.84%.

2Analyzed for total amino acids (Amino Lab® Evonik SEA Pte. Ltd. Singapore—Lab code: SG16-0000618-001) [17].

3Purity > 98.0%.

4 Met or exceeded the minimum NRC requirements [43].

Feed in mash form and fresh water were provided ad libitum. Hens were housed in a semi-controlled environment with a constant 16L:8D photoperiod (10–15 Lux) where day to night temperatures were a maximum of 28.71 ± 0.12°C and minimum of 26.05 ± 0.11°C during August and September. The study was conducted on a commercial layer farm (Lahore, Pakistan) with uniform production performance (72.14% ± 1.66) and body weight (1.4 Kg ± 0.006).

The experimental protocol (including handling, care, and humane end point by cervical dislocation for ileal digestibility) was approved by the Ethical Review Committee, University of Veterinary and Animal Sciences, Lahore, Pakistan. As required in the approved protocol, birds were monitored twice daily for signs of illness (lethargy, not frequently consuming food, injury, or distress). There was no mortality. Hens remaining at the end of the study were returned to the layer farm.

Daily egg production, egg weight, feed intake, body weight gain, and FCR were determined. External measurements included egg weight, egg mass, specific gravity, shape index (egg width/egg length × 100), egg shell weight, and egg shell thickness [44-46]. Internal measurements included egg yolk color (Roche Yolk Color Fan) egg yolk index (egg yolk height/egg yolk diameter × 100), albumen index (albumen height/average egg albumen width × 100), and yolk to albumen ratio (egg yolk weight/weight of albumen) [47-49]. Proportional albumen ratio (egg albumen weight/egg weight × 100) was also determined. Haugh unit was calculated as:

HU = 100 × log (albumen height—1.7+ egg weight ^ 0.37 +7.57 [49].

Celite (2% of diet) was added three days before the end of the study. On the last day of the study, blood (~ 4 ml) was taken from the jugular vein of two birds per replicate. Blood samples were collected in gel vacutainers (gel clot activators-ImuMed®) [50]. Serum concentrations of total protein and glucose were measured spectrophotometrically (Merck, USA, Human Kits, Eggenstein, Germany). Newcastle Disease Virus (NDV) and H9 (subtype of avian influenza) antibody titers were determined by procedures of Rubbani et al. [51].

After blood samples were obtained, cervical dislocation was performed to obtain ileal digesta and analyzed [7,52-58]. and analyzed. Total nitrogen in feed and digesta (oven dried, 55–60°C) were used to calculate ileal digestibility of protein by Kluth and Rodehutscord as:

Digestibility Coefficient (%) = 100–100 x [(Celite in Diet x Protein in Digesta)/Celite in

Digesta x Protein in Diet] [59].

Statistical analysis

Data were analyzed by a one way ANOVA under a Completely Randomized Design and evaluated using orthogonal polynomials for linear, quadratic, and cubic responses [60]. Feed intake, FCR, body weight change, and egg quality parameters were analyzed by repeated measures (SAS, version 9.1, 2021) using PROC GLM [61]. Significance at P ≤ 0.05 was determined; P ≤ 0.10 was noted [62].

15 Jan 2021

PONE-D-20-35024

Varying digestible isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens

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Reviewer #1: Reviewer’s comments on Manuscript no PONE-D-20-35024.

The topic is very interesting and does allow knowledge transfer in the field of avian nutrition. However, the manuscript lacks certain details, which must be added. Major revision is required before it can be accepted for publication. The comments below will allow the authors to improve their manuscript:

Abstract:

Line 1 & 2: In control diet, 0.10 isoleucine was added to achieve 0.66% isoleucine levels. Mention the supplemental levels of isoleucine for all diets to achieve 0.66 to 0.84% isoleucine in complete feed.

Line 3 & 4: The authors must mention the control diet's crude protein value along with the ME values.

Line 6 &7: What is meant by weight change? Do you mean weight gain? Rephrase the term to clarify.

Line 12-14: Authors need to do a regression analysis to confirm the optimum levels of isoleucine for commercially important parameters. There may be different optimum levels for different parameters.

Introduction:

The introduction is not clear and needs to be elaborated and rewritten to justify higher isoleucine levels? What has changed, which demands higher levels?

Line 7-8: “Researchers have proposed optimum isoleucine levels to be from 550 to 660mg/hen /day”: But it is not clear how much of the supplemental isoleucine is needed in the diet to fulfil this requirement? This recommendation is for which breeds and what ages??

Line 8-9: Sentence needs revision. What do authors mean by genetic variation and age groups?

Results:

Table 1: was it for 20 to 30 weeks? Or 23 to 30 weeks?

Table 1: Units for Egg production and Feed intake is missing?

Table1: Change in bird weight? Not clear? I think authors means initial at minus final wt. If that is the case, then it should be mentioned as weight gain.

Table 1: Egg mass data is missing? Must add.

Page 6: line 2: Bodyweight change was significant for the week? Did it increase or decrease with age?

Page 6: Line 3: numerical differences mean nothing? The level of egg production was statistically lower for all treatments fed 0.75 to 0.84% supplemental isoleucine? Revise sentence to clarify what authors are trying to highlight?

Line 4: Highest numerical FCR? This is not true. Numerical differences should not be highlighted. Revise the sentence.

Table 2: Line 5-12: Needs revision: The authors mention that levels and weeks were significantly affected for external egg quality but did not explain the major differences, were they good or bad differences? The optimum level of isoleucine was different for each parameter. For example, For level as a factor, significantly higher egg weight was achieved at 0.69%, higher specific gravity was achieved at 0.84% isoleucine, and shell thickness was highest at 0.78% isoleucine.

Page 7: Internal egg quality (Supplement, Table 1):

It is not clear from the table what does propositional yolk and albumen weight means? Propositional to what? Please clarify?

For proportional yolk weigh, the optimum level of isoleucine was 0.84%. For proportional albumen weigh, the optimum level was 0.78%. The optimum level was 0.81% for yolk index, Y: A the optimum level was 0.84%. Can they all be mentioned in the text?

Regression analysis is required to determine the optimum level.

Table3: Were these levels within the expected physiological range? Not clear. Is an increase or decrease of these significant levels a good or a bad indicator? The whole section needs to be rewritten to clarify the differences between levels? Again, which level of isoleucine resulted in desired effects?

Table 3: A word is missing under quadratic (See the last row of the table?

Page 8: Discussion:

Line 11-12: Was Dong referring to egg production? Please mention.

Line 13: greater quantities? Be specific, mention what were the optimum levels for the egg production. Revise sentences.

Line 15-16: Excessive BCAAS? not clear, what is categorised as excessive levels? Revise the sentence. Which level of isoleucine can cause excessive BCAAs??

Line 16-17: Sentence needs revision: The authors need to clarify that Serotonin is an appetite suppressant. Therefore, speculation can be made that reduced feed intake at 0.75% can be associated with isoleucine level. Not clear what happened at 0.81? Please check and amend the sentence.

Page 9:

Line 3 to 4: "when less expensive feeds are used" what are less expensive feeds? Do you mean low CP feeds? Clarify the sentence.

Line 10- "Lowering isoleucine levels" What were the isoleucine levels used in Shim et al? Was the effect due to lowering of CP levels in general or was the effect specific to isoleucine levels? Not clear.

Line 13-14: what were the lower levels of isoleucine used by Dong? Add the information.

Page 10:

Line 2-3: effect of protein sources and levels of isoleucine are two different things. Clarify the sentence.

Line 3-5: Are you comparing isoleucine with Valine? Do you expect both AA's to affect the immunoglobulins in a similar way? or is it a typo? Please check and clarify.

Line 8-10: The CP levels of this study is 16%. It may be that increase in CP level can affect ND titer. There are many other factors that can affect the ND titre? These other factors can be explored to describe your results. The sentence needs clarification.

Line 11: what are "protein deprived chicks"? Not clear.

Line 15-16: Line 14 to 17: needs revision: recommendation for further studies is very vague. Can they be specific?

Material and methods:

• Throughout the text, it should be made clear that isoleucine levels are total and not SID.

• Methods should also specify L- isoleucine quantity added in each diet to meet the required % isoleucine levels.

Page 11:

Line 6: Methods should also specify the type of cage system that was used in this study.

Line 12: the formula used to determine ileal CP digestibility should be provided in the manuscript.

Line 15: Hempe et al: year of publication is missing

Line 16: Victor and Carver, 1936: Reference is too old. Please replace it with any recent reference if possible.

Line 16: Haugh 1938: Reference is missing in the reference list. Again reference too old.

How internal and external egg parameters were analysed? The information must be added in the methods section.

Page 12:

Line 3: Was digesta samples freeze or oven-dried. Add the information.

Statistical analysis:

• For egg production parameters it was factorial design (L*W), how can that be analysed as one way? It has to be a factorial design?

• Data must be analysed as regression analysis to confirm optimum levels of isoleucine for different parameters.

Reviewer #2: This is the review for the manuscript PONE-D-20-35024, entitled “Varying digestible isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens” for PLOS ONE Journal.

The present manuscript presents original data, which has been collected using appropriate standard methods. However, Minor concerns still need to be addressed or clarified.

Page 3 (Line 2): Abbreviations should be clarified when first mentioned e.g., AA = Amino acids. Clarify the three limited amino acids (including tryptophan).

Page 9 (Line 12): It is not clear why shell thickness changed with isoleucine supplementation. More explanation is required

Page 10 (Line 13): No discussions for the alteration in serum glucose content.

Page 10 (Line 19): what is 490?

Page 10 (Line 20): "and the Control + 0.69". Control or basal diet?

Page 12 (Lines 2 & 3): Many citations. Use one or two.

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Submitted filename: Reviewer comment PoneD 20 35024.docx

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5 Sep 2021

We have added information on data storage and support for research. Information about animals used has been added in Materials and Methods. As well, we have answered all questions/comments of reviewers.

Submitted filename: Isoluecine author comments and replies.docx

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11 Oct 2021

10 Nov 2021

Reviewer #1: The manuscript has been much improved. However, there are some minor corrections which are still required. Please see the comments which needs addressing:

Page 1:

Title: remove the word “digestible” as digestible levels of Isoleucine are not investigated in this study? It’s the L-Isoleucine levels that are studied in this experiment.

Digestible has been removed from the title.

Varying isoleucine level to determine effects on performance, egg quality, serum

biochemistry, and ileal protein digestibility in diets of young laying hens

Page 2:

Line 16: Further studies to investigate what? Not clear.

Considering the low FCR of 1.45 and cost for inclusion as a dietary ingredients, 0.72% isoleucine was chosen for further studies with varying quantities of other branched chain amino acids in diets for young laying hens.

Page 3:

Line 10: Table 1: Units are missing?

Added 5mg/bird/day.

Page 6:

Line 1: Replace the word “numerically” with “statistically” because the differences were statistically different.

Egg mass was affected by level with linear and quadratic effects. The numerically statistically greatest egg mass (52.068 + 1.45 g) occurred at 0.72% isoleucine and produced 2.32% more mass than the Control (50.88 + 1.45 g).

Page 7:

Table 1: wrong numbering: Table on page 3 is also Table 1?

Table 2. Isoleucine level and performance@ of layers for 23-30 weeks.

This table number was changed and then all other numbers for tables were changed in the entire manuscript.

Table 1. Symbol ### is similar for FCR and Weight gain: check and correct. Footnote for how FCR was calculated is missing.

Page 9:

Line 12: Fermentation? After which fermentation? Clarify?

Line 17: Reference is missing.

It is known that after fermentation of eggshells to produce alkaline protease, the protein hydrolyzate of egg shells contains several essential amino acids including arginine, isoleucine, leucine, lysine, methionine, phenylalanine, and valine along with smaller quantities of cysteine; thus, eggshells have been proposed as a viable protein source (Nagamalli et al., 2017).

Line 19: Incomplete reference. Year is missing in reference?

As well, changes in epimerization of isoleucine in eggshell have been used to date the range of age for archeological sites (Brooks et al., 1990).

Page 14:

Line 12: Were differences within an acceptable range? What is a reduction in levels indicating? Good or bad?

Harlap et al. (2021) studied blood proteins in laying hens (Lohmann Whites at 26- to 80-weeks-old). They reported that the quantity of globulins was not related to the productive period but rather depended on a reduction in defenses with the greatest decrease at 80 weeks of age. At 30 weeks old, our control layers had globulin levels at or above 0.81g/dL (0.62% isoleucine to 0.75% isoleucine) that were gradually reduced to 0.35 g/dL at 0.84 % isoleucine. The reduction of globulins indicated that increasing the isoleucine level beyond 0.75 may be related to a reduction of immunological defenses of the layers. This finding may be important; however, it is not clear if findings for Lohmann Whites are similar to all other breeds of layers, including LSL-LITE layers. Clearly, more research on these findings is indicated.

Line19: which viral strain?

The genetic variation or subtype, susceptibility of host, age, breed, and environment (such as environmentally controlled houses versus open houses) are factors likely affecting ND titers of layers in our work and that of others (Oberlander et al., 2020).

Page 15:

Rewrite conclusion section:

Conclusion

Data from this study is useful because researchers and producers can conduct regression analyses to determine the optimum level of isoleucine for various parameters. For the purpose of our work, it was important to determine a specific level of isoleucine as an important parameter to compare to other BCAA's. In the poultry industry, FCR is most often selected as a important useful performance variable in determining the most appropriate diet (Best, 2011; Clark et al. 2019). In our work, isoleucine levels at 0.72% and 0.84% produced low FCR of 1.45 and 1.44, respectively, but shared significance of FCR (1.49) with 0.78%. Also, at 0.72% isoleucine, other egg production, egg quality, and biochemical measurements were in the medium to high range. Considering cost of synthetic isoleucine as a dietary ingredient along with the low FCR, 0.72% was chosen as the constant for future studies for comparison of valine and/or leucine levels in layer diets. for further study to .

Line 4 and 5: Delete the sentence. This is not your conclusion. Line 8: Remove reference from the conclusion section. The authors can use this reference in the discussion section.

Line 11? Further studies to test what? Would you please clarify which “further” studies are required? To investigate what?

See above in Conclusion.

Page 15 vs 16: Material and Methods: Line 14 on page 15 says you had 10 layers per replicate, but in Line 13 on Page 16, authors say there is five birds/cage? Please check and confirm if you had 5 or 10 laying hens/replicate?

Two adjoining cages (60 x 63.5 cm, five birds each) constituted a replicate.

Page 17:

Line 1: Delete the line “ except for layers …..below.” Culls are not counted as Mortality.

There was no mortality. except for layers used to determine ileal digestibility as described below.

Line 13: what was the level of celite used?

Celite (2 % of diet) was added three days before the end of the study.

Line 20: Revise sentence.

Remove the word “above” and mention that it was ileal digesta.

After blood samples were obtained (above), cervical dislocation was performed to obtain ileal digesta (González Alvarado et al., 2007; Walk et al., 2012; Xu et al., 2015; and Cunniff, 2016).

Reviewer #2: (No Response)

26 Nov 2021

Varying isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens

PONE-D-20-35024R2

Dear Dr. Annie King ,

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Reviewers' comments:

19 Dec 2021

PONE-D-20-35024R2

Varying isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens

Dear Dr. King:

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Table 2

Isoleucine level and performance of layers for 23–30 weeks.

Isoleucine (%)	Lys:Ile#	Egg Production (%)	Egg Mass## (g)	Feed Intake (grams/day)	FCR###	Weight Gain#### (g)
0.66*	79.5	91.66ab	50.88bc	111.57ab	1.51a	16.33
0.69	83.1	92.17ab	51.35ab	111.90a	1.51a	13.29
0.72	86.7	92.37a	52.068a	110.67bc	1.45b	9.45
0.75	90.4	90.30c	51.18b	108.47d	1.52a	11.16
0.78	93.9	91.05bc	50.19c	109.63c	1.49ab	13.18
0.81	97.6	91.66ab	50.84bc	111.59ab	1.50a	13.35
0.84	98.8	91.08bc	51.38ab	110.14c	1.44b	20.14
SE		2.34	1.45	2.06	0.08	28.43
Week
23		79.96c	43.51g	106.81c	1.68a	9.79bc
24		87.07b	47.25f	106.87c	1.54b	21.98b
25		93.66a	50.13e	107.51c	1.48c	3.83cd
26		93.94a	51.43d	110.45b	1.49c	51.73a
27		93.79a	52.87c	115.43a	1.41d	3.54d
28		94.52a	54.23b	116.48a	1.49bc	3.95cd
29		94.77a	55.08a	110.14b	1.39d	7.48bcd
30		94.03a	54.51ab	110.85b	1.42d	5.50bcd
SE		1.77	1.095	1.56	0.06	21.49
Level		0.02	0.0002	<0.01	< 0.01	0.863
Week		< 0.01	< 0.01	< 0.01	<0.01	< 0.01
L*W**		< 0.01	< 0.01	< 0.01	0.64	0.4627
Linear		0.07	0.0311	0.004	0.04	0.5911
Quadratic		0.58	0.0818	0.001	0.41	0.1573
Cubic		0.25	0.4022	0.94	0.014	0.999

Mean (7 diets x 7 replicates x 10 birds per replicate) with different superscripts within a column differ significantly (P ≤ 0.05).

#Lysine: Isoleucine.

## Hen week production (%) multiplied by egg weight (g) and divided by 100.

FCR, kg feed consumed per 12 eggs produced.

####Weight gain over period of study.

*Control.

**Interaction of level × week.

Table 3

Isoleucine level and external egg quality of layers for 23–30 weeks.

Isoleucine (%)	Lys:Ile#	Egg Weight (g)	Egg Shape Index	Specific Gravity	Shell Thickness (mm)	Proportional Egg Shell Weight## (%)
0.66*	79.5	55.96b	74.69	1.0820bc	0.399a	10.386
0.69	83.1	56.44a	75.17	1.0817c	0.398ab	10.537
0.72	86.7	55.35d	75.18	1.0838bc	0.379c	10.452
0.75	90.4	55.47cd	74.61	1.0838bc	0.391b	10.540
0.78	93.9	55.77bc	74.75	1.0838bc	0.400a	10.304
0.81	97.6	55.99b	74.70	1.0841b	0.393ab	10.466
0.84	98.8	55.82b	74.95	1.0888a	0.398ab	10.530
SE		0.57	1.034	0.0043	0.015	0.4780
Week
23		54.42de	75.35ab	1.0669e	0.387d	10.949ab
24		54.27e	75.52a	1.0686e	0.424a	10.257cd
25		53.52f	74.99abc	1.0886bc	0.388d	9.8538e
26		54.73d	74.55cd	1.0965a	0.411b	11.156a
27		56.25c	74.82bcd	1.0950a	0.400c	10.45c
28		57.37b	74.55cd	1.0898b	0.417ab	10.87b
29		58.12a	74.37d	1.0874c	0.401c	10.12d
30		57.97a	74.25d	1.0788d	0.326e	10.019de
SE		0.43	0.78	0.0032	0.011	0.361
Level		<0.01	0.379	<0.01	<0.01	0.436
Week		<0.01	<0.01	<0.01	<0.01	<0.01
L*W **		<0.01	0.0892	<0.01	<0.01	<0.01
Linear		0.12	0.059	<0.01	0.6235	0.7596
Quadratic		0.002	0.15	0.0277	0.0006	0.861
Cubic		0.72	0.44	0.0302	0.0246	0.1000

@Means (7 diets x 7 replicates x 3 eggs per replicate) with different superscripts within a column differ significantly (P ≤ 0.05).

#Lysine: Isoleucine.

##Dried shell weight as percent of total egg weight.

*Control.

**Interaction of level × week.

Table 4

Isoleucine level and internal egg quality of layers for 23 to 30 weeks.

Isoleucine (%)	Lys: Ile#	Haugh Unit	Proportional Yolk Weight (%)##	Proportional Albumen Weight (%)##	Yolk Index	Albumen Index	Y:A###	Yolk Color
0.66*	79.5	94.26	25.36bc	64.258ab	43.246ab	12.09	39.65bc	3.464
0.69	83.1	94.12	25.24bc	64.221abc	42.834bcd	12.26	39.5bc	3.381
0.72	86.7	94.53	25.54b	64.035bc	43.061abc	12.05	40.18b	3.351
0.75	90.4	93.74	25.17bc	64.256ab	42.450de	12.23	39.21bc	3.357
0.78	93.9	94.15	24.92c	64.782a	42.622cd	12.40	38.71c	3.434
0.81	97.6	94.65	25.59b	63.950bc	43.341a	12.26	40.16b	3.304
0.84	98.8	94.09	26.26a	63.662c	41.954e	12.06	41.53a	3.488
SE		2.12	0.91	1.11	0.95	0.67	0.0205	0.37
Week
23		92.07e	25.69ab	63.89bc	43.232b	11.59e	40.53ab	2.939c
24		87.46f	25.25bc	64.491ab	40.703d	10.19f	39.39bcd	3.048c
25		92.09e	25.07c	65.073a	42.087c	11.58e	38.71d	3.578b
26		94.97cd	25.09c	63.745c	43.321b	12.51c	39.36dc	3.769ab
27		97.07b	24.94c	64.607a	44.795a	12.95b	38.77d	3.728b
28		99.78a	25.38bc	63.758c	43.184b	14.09a	40.08abc	3.973a
29		94.33d	25.95a	63.919bc	42.506c	12.02d	40.77a	3.143c
30		95.99bc	26.13a	63.847c	42.469c	12.59bc	41.17a	3.0c
SE		1.6	0.69	0.842	0.718	0.507	0.0155	0.280
Level		0.764	<0.01	0.013	<0.01	0.397	<0.01	0.483
Week		<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
L*W**		<0.01	0.4070	0.017	<0.01	<0.01	0.2456	0.0002
Linear		0.934	0.0024	0.17	0.0005	0.6936	0.0076	1.0
Quadratic		0.834	<0.01	0.04	0.6048	0.1931	0.0005	0.1285
Cubic		0.740	0.0056	0.02	0.0020	0.2247	0.0047	0.9171

@Means (7 diets x 7 replicates x 3 eggs per replicate) with different superscripts within a column differ significantly (P ≤ 0.05). % isoluecine as formulated.

#Lys/Ile for lysine: Isoleucine.

Egg albumen or yolk weight/egg weight × 100.

Yolk: Albumen.

*Control.

**Interaction of level × week.

Table 5

Isoleucine level, serum biochemistry, and ileal digestibility of crude protein values in layers for 23 to 30 weeks.

Isoleucine (%)	Lys: Ile#	Total Serum Protein	Serum Albumin	Globulin	Glucose	CP Digestibility Coefficient##	HI Titer
		-------------------g / dL----------			mg / dL	(%)	ND	H9
0.66 *	79.5	3.68	2.87	0.81a	170.14ab	82.713a	8.57	8.71
0.69	83.1	3.65	3.08	0.84a	208.29a	77.743ab	8.28	8.42
0.72	86.7	3.30	2.85	0.82a	220.71a	79.983ab	7.85	8.14
0.75	90.4	3.35	3.00	0.85a	194.71a	79.143ab	7.42	9.00
0.78	93.9	3.20	2.72	0.70ab	111.43bc	68.700ab	8.00	8.42
0.81	97.6	3.11	2.81	0.54ab	112.86bc	66.457b	8.57	8.57
0.84	98.8	3.00	2.81	0.35b	104.43c	77.563ab	8.71	7.71
SE		0.25	0.23	0.16	20.61	6.26	0.80	0.64
P-value
Level		0.07	0.77	0.03	< 0.01	0.1554	0.67	0.55
Linear		0.01	0.39	0.09	< 0.01	0.1266	0.74	0.85
Quadratic		0.22	0.79	0.73	0.06	0.9585	0.34	0.32
Cubic		0.69	0.69	0.57	0.07	0.0456	0.61	0.37

@Means (7 diets x 7 replicate x 2 birds per replicate) with different superscripts within a column differ significantly (P ≤ 0.05).

#Lysine: Isoleucine.

CP Digestibility Coefficient as ileal digestibility for crude protein.

*Control.