Effects of L-valine in layer diets containing 0.72% isoleucine.

In a previous study with LSL-LITE layers (-23 to 30-week-old), isoleucine at 0.72% and 0.84% produced values for FCR at 1.45 and 1.44, respectively and shared significance with 0.78% isoleucine (1.49). Considering that FCR is an important standard in the poultry industry due to the cost for adding feed ingredients such as synthetic amino acids and the low FCR of 1.45, 0.72% isoleucine was chosen for further study with LSL-LITE layers (n = 490 at 33- to 40-week-old) to determine effects on production and egg quality. The study included 7 diets (2730 Kcal kg metabolizable energy and constant isoleucine at 0.72%) containing varying quantities of valine [0.72 (Control), 0.75, 0.78, 0.81, 0.84, 0.87 or 0.90%] x 7 replicates x 10 hens/replicate. Significance at P ≤ 0.05 and P < 0.10 was determined. Level and week were significant for feed intake, egg production, and FCR; the interaction of level x week (L*W) was significant for feed intake and FCR. An isoleucine:valine of 1.233 corresponding to 0.72% isoleucine and 0.87% valine produced the lowest FCR of 1.30 (a 2.26% decrease compared to the Control at 1.33 ± 0.04). All measurements for external egg quality, except shape index and eggshell thickness, were significant for level. Week was significant for all parameters except shell thickness; L*W was significant for external quality measurements except shape index and shell thickness. Level, week, and L*W were significant for internal egg quality measurements. Serum protein and H1 titer were significant for level. Various production, egg quality, and biochemical measurements were significantly different from the control (0.72% isoleucine and 0.72% valine) at 0.81 to 0.87% valine. Findings of this study will aid researchers and commercial producers in narrowing the range of isoleucine, valine, and leucine needed for effects on particular parameters. Knowledge gained from this and others studies will eventually lead to an understanding of synergistic and antagonistic effects of branched chain amino acids in feed for various genetic types of layers throughout their productive lifetime.

Introduction

Branched chain amino acids (BCAAs) are precursors of other amino acids and proteins. Metabolized extra-hepatically, they are energy precursors and aid in repair of muscle proteins (Brosnan and Margaret, 2006) [1]. It has been assumed that leucine requirements can generally be met by various sources of protein in diets. Thus, research has focused on appropriate amounts of isoleucine and valine in the diet to maintain antioxidant capacity, gut immunity, and critical metabolic processes such as fatty acid metabolism (Azzam et al., 2015; Dong et al., 2016; Wen et al., 2019; Bai et al., 2021) [2-5].

In layers, isoleucine is said to be the fourth limiting amino acid after tryptophan (Harms and Ivey, 1993) [6]. Several studies have focused on isoleucine requirements for young hens. Harms and Russell (2001) provided 0.49 to 0.61% isoleucine to Hy-Line W36 (36- to 44-week-old) diets containing supplemental amino acids to ensure that isoleucine was first limiting [7]. Egg production, weight, and contents were significantly increased by all isoleucine additions. Results revealed daily isoleucine requirement of 589.2, 601.2, and 601.4 mg/day for egg production, weight, and content, respectively, indicating 12.6 mg isoleucine/g of egg content. A follow-up experiment for Hy-Line layer (35 to 43 week-old) diets with 0.39 to 0.61% isoleucine indicated significantly elevated egg mass, egg production, and egg weight for levels above 0.51% (Shivazad et al., 2002) [8]. Egg mass, egg weight, and egg production decreased with decreasing isoleucine. Feed consumption, energy intake, and body weight were significantly decreased at 0.45% isoleucine. Broken-line regressions showed requirements of 449.8, 497.0, and 469.0 mg isoleucine/day for egg production, weight, and mass, respectively, corresponding to 9.30 mg isoleucine/g egg mass. Parenteau et al. (2020) determined responses to isoleucine supplementation in low crude protein diets of laying hens (20- to 27 and 28- to 46- week-old) [9]. Findings showed that a reduction of crude protein by 2% was possible when diets were fortified with synthetic amino acids (methionine, lysine, threonine, and tryptophan) + isoleucine. Optimum responses occurred at 82 and 88% standardized ileal digestible isoleucine:lysine [9].

Results of earlier studies reported dietary valine requirements between 0.54 and 0.72% for maximum layer performance (Johnson and Fisher, 1958; Hurwitz and Bornstein, 1978) [10, 11]. Other results suggested from 610 to 786 mg valine/hen/day (McDonald and Morris, 1985; Jensen and Colnago, 1991) [12, 13]. NRC (1977) [14] recommended 550 mg/hen/day [15]. Later the recommendation was 600 mg/hen/day, followed by a final recommendation (to date) of 700 mg/hen/day (NRC,1994) [15]. As with isoleucine, some research has focused on valine requirements for layers of young to medium age (Harms and Russell, 2001) [16]. Hy-Line W-36 layers (39- to 47-week-old) were fed diets containing 0.525 to 0.700% valine. Egg production and egg weight were increased by 0.630% and 0.655% valine, respectively. Daily valine requirement of 592.5, 677.7, and 619.0 mg/hen/day were reported for improved egg production, weight, and contents respectively. A value of 13.1 mg valine/g of egg content was indicated by broken-line regression [16]. In work by Bregendahl et al. (2008) with Hy-Line W-36 hens (26- to 34-week-old), the digestible amino acid requirement used to calculate the ideal amino acid ratio for maximum egg mass was 501 mg/day of valine. The ideal amino acid ratio for maximum egg mass was 93% valine relative to lysine (100%) [17].

A critical review of amino acids requirements for laying hens was recently provided by Macelline et al. (2021) [18]. Values from producers of five layer breeders ranged from 640 to 760 and 700 to 819 mg/bird/day for isoleucine and valine, respectively (Macelline et al., 2021) [18]. Values for ideal digestible amino acid ratios for layers ranged from 79 to 86 and 88 to 102 mg/bird/day for isoleucine and valine, respectively (Macelline et al., 2021) [18].

As antagonists, isoleucine and valine, branched chain amino acids (BCAA’s) share the same pathway to cross the cellular membrane, are degraded by the same enzymes, and cross the blood-brain barrier at the same time (Benton et al., 1956; Rogers et al., 1993; Harper, 1984) [19-21]. As more synthetic amino acids are used in poultry diets, research will continue to ensure that complementary quantities of BCAA’s are maintained. The ideal amino acid ratio as determined by the Goettingen approach was lysine, 100; isoleucine, 75; and valine, 90. As determined by the Louvain approach, it was lysine, 100; isoleucine, 80; and valine, 91 (Soares et al., 2018) [22].

In a previous study with LSL-LITE layers (23- to 30-week-old), 0.72% and 0.84% total isoleucine in diets produced low FCR of 1.45 and 1.44, respectively, and shared significance of FCR (1.49) with isoleucine at 0.78% (Ullah et al., 2021) [23]. The corresponding ratios of lysine: isoleucine for these percentages were 0.72% = 1:0.867; 0.78% = 1:0.939, and 0.84% = 1:0.988. Due to the low FCR and cost consideration when adding synthetic isoleucine, 0.72% was designated for further study with various concentrations of valine to determine effects for layer performance as well as external and internal egg quality in LSL-LITE layers (33- to 40-week-old).

Results and discussion

The standard error of the mean (SE) is pooled for each individual measurement for production; external egg quality; internal egg quality; and serum biochemisty, digestibility, and titers. SE is included with the value for the Control of each parameter discussed below.

Production (Table 1)

Increasing valine had linear (p < 0.01), quadratic (p = 0.01), and cubic (p < 0.01) effects on feed intake. Except for 0.87%, all levels of valine significantly increased feed intake. At 0.78% valine, feed intake (111.52 kg) was 6.33% greater than the Control (104.88 kg ± 3.18). Lelis et al. (2014) also reported a linear increase in feed intake when feeding brown layers (42- to 54-wk-old) valine to lysine ratios of 84 to 100%, compared to our ratio of ~94 to 100% [24]. They suggested an ideal digestible valine to lysine ratio of 92% (0.607% digestible valine or 567 mg/hen/day of digestible valine) [24]. Differences in age and genetics of layers may explain differences in findings. Week (fluctuating values) and L*W were significant (p < 0.01) for feed intake in the present study.

Level produced a quadratic (p = 0.07) trend and a cubic (p < 0.01) effect for production of isoleucine:valine at 1.048 and 1.192, respectively.

The highest production (96.85% for 0.87% valine) produced an increase in weight of 2.77% compared to the Control (94.24% ± 2.01). Lelis et al. (2014) also noted that increasing ratios of valine:lysine had a quadratic effect on egg production of brown layers [24]. When Harms and Russell (2001) fed HyLine W-36 layers (39- to 47-week-old) diets containing 0.525 to 0.700% valine, egg production was increased at 0.630% [16]. Azzam (2015) found no effect on production when feeding 0.1 to 0.4% to Hyline Brown hens (40- to 47- week old) [2]. Results of Azzam (2015) are likely different from others due to the lower quantities (0.1 to 0.4%) of valine provided [2]. Age and breed of layers are likely important factors for differences in results as well.

For FCR, level was significant (p < 0.01); there were linear (p < 0.03) and cubic (p < 0.01) effects. A low feed intake and the highest egg production at 0.87% valine produced the lowest FCR (1.30) compared to the Control (1.33 ± 0.04). Lelis et al. (2014) reported a quadratic effect for FCR [24]. Azzam (2015) reported no effect on FCR with an increase of valine, likely due to the lower quantities fed [2]. Week was significant (p = 0.01) as values fluctuated over time. A trend (p = 0.10) was observed for L*W.

External egg quality (Table 2)

External egg quality parameters are shown in Table 2. Level was significant for all external egg quality measurements except shape index and shell thickness where there were trends at p = 0.08 and p = 0.10, respectively. Egg weight trended linearly (p = 0.09) with quadratic (p = 0.04) and cubic (p = 0.008) responses. Compared to the control (60.73 g ± 0.42), a significant peak (61.61g) for weight occurred at 0.84% valine.

Table 2

Valine level and external egg quality of 33- to 40-week-old hens.

Level (%)	Lys: val#	Iso: val##	Egg weight (g)	Specific gravity	Shape index	Shell weight (g)	Shell thickness (mm)	Prop shell weight$
Control*	0.867	0.986	60.73b	1.0739d	74.17b	6.61a	0.33 b	10.81ab
0.75	0.900	1.027	60.72b	1.0772bc	74.29ab	6.45ab	0.36a	10.60b
0.78	0.939	1.068	60.44c	1.0779b	74.60ab	6.59a	0.34b	10.84ab
0.81	0.975	1.109	60.17d	1.0802a	74.77a	6.56ab	0.34 b	10.95 a
0.84	1.010	1.151	61.61a	1.0760c	74.06b	6.39 b	0.33 b	10.31 c
0.87	1.048	1.192	60.58bc	1.0779b	74.20ab	6.46ab	0.34 b	10.61 b
0.90	1.084	1.233	60.55bc	1.0765bc	74.47ab	6.56ab	0.34 b	10.86ab
Pooled SE			0.42	0.002	0.98	0.29	0.039	0.46
Week (age)
33			59.05e	1.08b	74.71ab	6.00e	0.34abc	10.20d
34			60.06d	1.09a	74.39abc	6.25 d	0.34abc	10.32cd
35			60.62c	1.07c	73.92c	6.25 d	0.35ab	10.27d
36			61.00b	1.07d	73.95c	6.48 c	0.33c	10.59c
37			61.08b	1.10e	74.87a	6.91ab	0.33bc	11.29a
38			61.18ab	1.07e	74.14bc	7.01 a	0.33bc	11.53a
39			61.40a	1.07f	74.43bac	6.47c	0.34abc	10.58c
40			61.09b	1.08b	74.53abc	6.76 b	0.36 a	10.90b
Pooled SE			0.31	0.001	0.74	0.21	0.03	0.35
Level			< 0.01	< 0.01	0.08	0.04	0.10	< 0.01
Week			< 0.01	< 0.01	< 0.01	< 0.01	0.08	< 0.01
L*W**			0.01	< 0.01	0.06	< 0.01	0.21	0.03
Linear			0.09	< 0.01	0.22	0.29	0.02	0.43
Quadratic			0.04	< 0.01	0.60	0.18	0.15	0.15
Cubic			0.008	0.70	0.20	0.03	0.16	0.01

@ Means (7 treatments × 7 replicates × 10 layers per replicate) with the same superscript within a column do not differ significantly at P ≤ 0.05.

# Lysine:valine.

##Isoleucine:valine.

$Proportional shell weight, %

*Control as isoleucine and valine, both at 0.72% of the diet.

**Interaction of level by week.

Harms and Russell (2001) noted that for Hy-Line W-36 layers (39- to 47-week-old), egg weight started to increase at 0.655% valine when layers received 0.525 to 0.700% valine.16 Lelis et al. (2014) fed Dekalb Brown layers (42- to 54-week-old) while Azzam (2015) fed Hyline Brown layers (40- to 47-week-old) and found no effect of valine for egg weight or specific gravity.2, 24 Information for our study with SL LITE layers, those of Lelis et al. (2014) and Azzam (2015) with brown layers suggest a thorough investigation of the literature for effects of valine on egg quality traits form various genetic types of layers in the same age range 2, 24.

Level was linear and quadratic (both at p < 0.001) for specific gravity while egg shell weight was cubic (p = 0.03). Specific gravity peaked (1.0802) at 0.81% valine compared to the Control (1.0739 ± 0.002). Specific gravity and shell weight are closely related because specific gravity is the relationship between the shell weight and other egg components (Butcher and Miles, 2017) [25]. However, shell weight was not linear but cubic (p = 0.03). Shell weight was different from the Control (6.61 g + 0.29) at 0.84% valine (6.39 g), producing 3.33% less shell.

Increasing valine produced a trend for shell thickness at p = 0.10 and a linear effect (p = 0.02). Shell thickness increased at 0.75% valine (0.36 mm), producing 9.09% greater thickness compared to the Control (0.33 mm ± 0.039 SE). L*W was not significant for shell thickness (p = 0.21) and week produced a linear trend at p = 0.08 for shell thickness. The trend for shell thickness by level may have been confounded by a trend for week associated with deposition of minerals. This supposition was supported by findings of Park and Sohn (2018) who conducted a study to examine the histological change of the eggshell endometrium for 30-, 60-, and 72-wk-old commercial layers [26]. As well, they analyzed eggshell ultrastructure and ionic composition. These investigators noted that eggshell weight significantly increased between 30 (6.72 g ± 0.026) and 60 weeks (7.08 g ± 0.40). Na+, K+, and V2+ increased with age while CA2+, Co2+, and S2- significantly decreased. Ultimately, egg shell structure was affected causing increased weakness of the shell while thickness and density did not change.

Level of valine was significant (p < 0.01) for proportional shell weight and there was a cubic effect at p = 0.01. The lowest proportional shell weight occurred at 0.84% valine (10.31%), compared to the Control (10.82% + 0.42). The reduction was 4.63%. When eggs have similar weights, the amount of shell can cause a difference in specific gravity (Butcher and Miles, 2017) [25]. At 0.84% valine, egg weight was highest (61.61 g), proportional shell weight was lowest (10.81%); however specific gravity, while at the numerically lowest point (10.61), shared significance with other values. For proportional shell weight, week was significant at p < 0.01 and L*W was significant at p = 0.03.

Shape index for level (p = 0.08) and L*W (p = 0.06) were not highly significant. The importance of shape index as associated with shell durability is controversial. While some investigators noted that an index >76 is needed for shell durability during marketing, others show no correlation for these measurements (Sarica and Erensayin, 2004; Duman et al., 2016) [27, 28]. Unless eggs shells are extremely fragile due to feed source, disease, or handling, shape index may not affect sale of eggs in local farmers’ markets or for consumers’ use from backyard production.

Internal egg measurements (Table 3)

Level was significant for all internal measurements (Table 3). Albumen and yolk indices were linear, quadratic and, cubic at p = <0.01. The Control for the albumen index was 7.57 ± 0.38. It increased by 8.59% and 10.30% for 0.84% valine (8.22) and 0.90% valine (8.35), respectively. The yolk index for the Control (42.61 + 0.96) increased by 5.09% with valine (44.78) at both 0.75% and 0.78%. Haugh unit for the Control (93.32 HU ± 1.87) had the greatst increased of 2.90% at 0.84% valine (96.02 HU) and 3.24% at 0.90% valine (96.33 HU).

Table 3

Valine level and internal egg quality of hens at 33–40 weeks.

Levels (%)	Lys: val#	Iso: val##	Albumen index	Yolk index	Yolk color	Yolk: albumen	Haugh unit	Prop. yolk weight$
Control*	0.867	0.986	7.57c	42.61d	5.22ab	0.43a	93.31c	26.90a
0.75	0.900	1.027	7.55c	44.78a	5.13b	0.42ab	93.94c	26.54abc
0.78	0.939	1.068	7.43c	44.78a	5.16b	0.42ab	93.34c	26.40bc
0.81	0.975	1.109	7.49c	42.94c	5.34 a	0.43a	93.75c	26.80ab
0.84	1.010	1.151	8.22a	42.98c	5.27ab	0.40c	96.02a	25.70d
0.87	1.048	1.192	8.01b	42.78dc	5.25ab	0.41bc	94.97b	26.21c
0.90	1.084	1.233	8.35a	42.61dc	5.35 a	0.41bc	96.33a	26.60abc
Pooled SE			0.38	0.96	0.300	0.03	1.87	0.80
Weeks (age)
33			8.32a	43.00b	3.32g	0.41c	96.58a	26.04c
34			8.02bc	42.70bc	4.1 f	0.41bc	95.86a	26.19c
35			8.05b	43.54a	5.27e	0.40c	95.67a	25.99c
36			7.54d	42.95b	5.90b	0.42abc	93.65bc	26.42bc
37			7.87bc	44.09a	6.45a	0.43ab	94.43b	26.67ab
38			7.79c	43.90a	5.44d	0.42abc	94.15b	26.71ab
39			7.44d	42.8 1b	5.80bc	0.42abc	93.00c	26.69ab
40			7.39d	42.20c	5.65 c	0.43a	92.84c	26.95a
Pooled SE			0.29	0.72	0.22	0.02	1.41	0.61
Level			<0.01	< 0.01	0.04	< 0.01	< 0.01	< 0.01
Weeks			<0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01
L*W**			< 0.01	< 0.01	< 0.01	0.04	< 0.01	0.01
Linear			< 0.01	< 0.01	0.07	0.94	0.004	0.23
Quadratic			< 0.01	< 0.01	0.02	0.013	< 0.01	< 0.01
Cubic			< 0.01	0.003	0.93	0.01	< 0.01	0.03

@ Means (7 treatments × 7 replicates × 10 layers per replicate) with the same superscript within a column do not differ significantly at P ≤ 0.05.

# Lysine:valine.

##Isoleucine:valine.

$Feed consumed/kg egg.

* Control as isoleucine and valine, both at 0.72% of the diet.

**Interaction of level by week.

The yolk:albumen had quadratic (p = 0.013) and cubic effects (p = 0.01) for level. The Control value of 0.43 ± 0.03 for yolk:albumen decreased for hens fed 0.84% (0.40) and the reduction was 6.98%. The albumin index and Haugh unit exhibited a general downward trend as hens aged, r2 for these two variables was 0.95. These temporal effects likely confounded findings for level.

Proportional yolk weight had quadratic (p < 0.01) and cubic (p = 0.03) effects. The Control value was 26.90% ± 0.80 and there was a reduction of 4.46% for valine levels at 0.84% (25.70%). Level produced a linear trend for yolk color at p = 0.07 and a quadratic effect (p = 0.02).

For Lelis et al. (2014), egg weight and egg internal quality were not influenced by the different dietary digestible valine-to-lysine ratios [24]. Internal quality measurements associated with yolk at various isoleucine:valine used in the present study may be associated with synergistic or atagonistic effects on deposition of lipids influenced by branched chain amino acids and need further study (Bai et al., 2017) [5]. Yolk color may be affected by deposition of fat soluble carotenoids assoicated with effects of BCAA’s.

As noted by Macelline et al. (2021), the higher values for amino acid ratios for layers used in our study and that for Lelis et al. (2014) were warranted [18, 24]. These levels may be especially important for layers to produce egg content (protein and lipids) during the early to middle range of egg production, maintain antioxidant capacity, gut immunity, and critical metabolic processes (Azzam et al., 2015; Dong et al., 2016; Wen et al., 2019; Bai et al., 2021) [2-5].

Serum biochemistry and crude protein digestibility (Table 4)

No influence of level was noted for serum glucose. For serum albumin, there was a trend (p = 0.06) for level. Valine level was also significant (p = 0.02) for total protein due to fluctuation; there was no linear response. Valine at 0.81%, while not different from other values, produced a significantly lower value (3.22 g/dl) than that of the Control (4.30 g/dl ± 0.03) for total protein. The H9 titer did not respond to level of valine. The H1 titer for NDV was significant (p = 0.02) with a linear (p < 0.01) effect and a trend for a cubic response (p = 0.07). For level, there was a cubic (p = 0.07) trend and a linear effect (p = 0.02) for ileal protein digestibility.

Contrary to our results, Azzam et al. (2015) reported a significant quadratic effect on serum glucose for increasing valine that peaked at 0.2% but; there was no effect of valine level on total serum protein (Azzam, 2014) [2, 29]. The general downward response for total protein seemed to support findings of Bunchasak et al. (2005) [30] who found a significant response for decreased serum protein with increasing dietary valine (0.66, 0.76, and 0.85) in broilers [29]. However, Thornton et al. (2006) found insignificant effects of digestible valine on innate or adaptive immunity in broilers [31].

Our significance for level and a linear response for NDV titer agreed with early results of Bhardava et al. (1969) who found a positivie effect for increasing high levels of valine (0.9 to 1.5%) on NDV titers in diets of 4-day-old chicks [32]. These findings are likely related to the requirement of valine for antibody production (killer-cell activity and lymphocyte proliferation) as noted by Calder (2006) for mammals [33]. Results from Jian et al. (2021) revealed a significant quadratic decrease in serum IgA and IgM with increasing valine (0.59, 0.64, 0.69, 0.74, and 0.79%) in diets of 33- to 40- week-old Fengda No.1 laying hens. Serum IgM levels did not change among diets [34]. Investigators (Azzam et al., 2015) reported that high levels of valine did not negatively affect the immune function in layers [2]. As noted by Kidd et al. (2021), the quantity of leucine in the diet must be considered when examining the effects of valine on responses in broilers [35]. Perhaps future work on immune responses in layers should always include responses across levels of the BCAA’s simultaneously.

The supplemental amino acids, threonine, also boosted the immune function in laying hens as reported by Azzam et al. (2011a), Azzam et al. (2011b), and Dong et al. (2016) [3, 36, 37]. According to Dong et al. (2017), mucosal immunity was enhanced with increasing threonine [38]. These investigators suggested that when limiting amio acids in a low-crude protein diet of layers during peak production, threonine may be a limitig amino acid.

Conclusion

In our work, production, egg quality, biochemical measurements, and ileal digestibility were most significantly affected by varying level of valine from 0.81 to 0.87%, when isoleucine was constant at 0.72%. Information from the present work is valuable for both researchers and producers. Results of our previous work, indudicated that addition of isoleucine produced a low FCR at 0.72%. When investigating levels of isoluecne and valine in the present work with older lyers, FCR was reduced with 0.72% isolecine and 0.87% valine in the diet. More research will narrow the range for appropriate quantities of valine when isoleucine is held at 0.72% in diets for hens in early stages of egg production. Work on comparison of all BCAA’s should be performed on the same group of layers over their lifetime. In this way, use of BCAAs could be optimized for various genetic types throughout each period of production. Presently, producers can choose the amount of isolecucine (72%) and valine to maximalize their most important commecial parameter(s).

Materials and methods

Diets, housing, and care

Layers (490, 33-weeks-old LSL-LITE starting weight of 1.581 ± 0.065 Kg) were randomly assigned to 7 dietary treatments × 7 replicates × 10 layers per replicate for 8 weeks. Mash diets with 0.72% isoleucine + 0.72, 0.75, 0.78, 0.81, 0.84, 0.87 and 0.90% total valine were formulated and prepared. The diet containing 0.72% of both isoleucine and valine was considered the Control. Several low cost ingredients were used to replace more expensive soy (protein source) in the diet (Table 5) [39-42]. Diet and fresh water were provided ad libitum.

Table 5

Protein content in 100g of various components in layer diets.

Component	Grams/100grams
Conventional canola meal1	40.73 to 43.01
De hulled sunflower seed meal2	42.00
Soybean meal3	52.00
Poultry byproduct meal4	61.20

1Chen et al., 2015 [39].

2González-Pérez, 2015 [40].

3Banaszkiewicz, 2007 [41].

4Pesti, 1986 [42].

The total quantity of protein from all sources and amino acids are shown in Table 6 (Amino LAB®) [43].

Table 6

Feed and nutrient composition of the control diet.

Dietary Ingredients	%	Nutrients Composition	Calculated@	Analyzed*
			---(%)-----
Corn	54	Dry Matter	90.36	90.25
Rice Tips	6.1	Metabolizable Energy	2730	-------
Canola Meal	5.0	Crude Protein	16.70	17.21
Sunflower seed meal	5.0	Ether Extract	3.06	-------
Corn Gluten 60	2.0	Crude Ash	2.27	-------
Soybean Meal	12	Crude Fiber	4.33	-------
Guar Meal	2.0	Calcium	3.56	-------
Poultry By-Product Meal	2.0	Available phosphorus	0.42	-------
Oil	0.7	Total phosphorus	0.67	-------
CaCO3	8.0	Sodium	0.17	-------
DCP	1.8	Potassium	0.62	-------
Lysine-SO4	0.4	Chloride	0.16	-------
DL-methionine	0.15	Dig4 Lysine	0.83	0.909
L-Threonine	0.1	Dig Methionine	0.42	0.303
L-Tryptophan	0.05	Dig Threonine	0.62	0.609
L-Valine	0.05	Dig Tryptophan	0.18	0.186
L-Isoleucine#	0.1	Dig Cystine	0.26	0.294
NaHCO3	0.4	Dig M+C	0.67	0.597
Vitamin/Mineral Premix##	0.3	Dig Arginine	0.95	1.11
Total	100	Dig Valine	0.72	0.864
		Dig Isoleucine	0.73	0.764
		Dig Leucine	1.29	1.464
		Dig Histidine	0.37	0.427
		Dig Phenylalanine	0.69	0.825
		Linoleic acid	1.66	-------
		Na+K-Cl (mEq / Kg)	214.35	-------

@Digestible amino acids values were calculated during feed formulation.

*Values are total digestible amino acids (Amino Lab® Evonik SEA Pte. Ltd. Singapore, lab code: SG16-0000618-001) for all treatments).

#Purity of L-isoleucine was > 98.0.

##Vitamin/mineral premix/kg of feed: Vitamin A, 10000 IU: Vitamin D3, 2500 IU: Vitamin E, 15–30 mg; Vitamin K3, 3 mg; Vitamin B1, 1 mg; Vitamin B2, 4 mg; Vitamin B6, 3mg; Vitamin B12, 15 mcg; Pantothenic Acid, 8 mg; Nicotinic Acid, 30 mg; Folic Acid, 1 mg; Biotin, 50 mcg; Choline, 300 mg; Manganese, 100 mg; Zinc, 60 mg; Iron, 25 mg; Copper, 5 mg; Cobalt, 0.1 mg; Iodine, 0.1 mg; Selenium, 0.2 mg.

Bird care, egg quality, and measurements

The Ethical Review Committee, University of Veterinary and Animal Sciences (Lahore, Pakistan) approved the protocol housing, feeding, and care of layers. Housing conditions included a caged system (60 x 63.5 cm, five birds each) on a commercial layer farm with uniform egg production and body weight, constant 16L:8D photoperiod (10–15 Lux), a maximum (26.68 ± 0.21 SE °C), and a minimum (21.75 ± 0.24 SE °C) temperature during October and November. All procedures and measurements were those noted for Ullah et al., 2021 [23].

Statistical analysis

Data were subjected to a one way ANOVA under a completely randomized design (Steel et al., 1997) [44]. Weekly data for feed intake, FCR, body weight change, and egg quality parameters were analyzed by repeated measures SAS (version 9.1; SAS Inst. Inc. Cary, NC) using PROC GLM [45]. The model was Yijk = μ + τi + Pj + (τ × P)ij + εijk. where: Yijk = Y is observation of ith treatment on jth period, μ = the overall mean, τi = the effect of ith treatment (i = 1, 2,3…..7), Pj = the effect of jth period (P = 1, 2, 3…8), (τ ×P)ik = the effect of interaction between ith treatment and jth period, εijk = random error associated with kth observation on jth period on ith treatment NID ~ mean 0 and variance σ2. Data were also evaluated using orthogonal polynomials for linear, quadratic, and cubic responses. Statistical significance at P ≤ 0.05 and trends (P ≤ 0.10) were determined using Duncan’s Multiple Range Test (Duncan, 1955) [46].

12 Jan 2021

PONE-D-20-36625

Effect of L-valine in layer diets containing 0.72% isoleucine

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Authors

Regarding the manuscript title Effect of L-valine in layer diets containing 0.72% isoleucine

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, some observation in the present paper should be corrected and add to improve the quality of the paper.

Introduction

1- Need more information about the effects of isoleucine in layer hens.

2- Need recently references for using Valine in layer diets

Discussion

3- 3- In this part you focused on the studies which agree or disagree with your results without a)ny explanation why these findings were agree with disagree with you.

4- You must write the mode of action of L-valine and isoleucine on layer performance and egg quality

5- I advise to use some recently reference for using amino acids in poultry and the additives affect the egg quality and immunity

I recommend reading and using the following references:

Saleh A. A. (2016) Effect of Low-Protein in Iso-Energetic Diets on Performance, Carcass Characteristics, Digestibilities and Plasma Lipids of Broiler Chickens. Egyptian Poultry Science Journal. Vol (36) (I): (251-262).

Saleh A. A, Mohammed S. Eltantawy , Esraa M. Gawish , Hassan H. Younis , Khairy A. Amber , Abd El-Moneim E. Abd El-Moneim & Tarek A. Ebeid (2020) Impact of Dietary Organic Mineral Supplementation on Reproductive Performance, Egg Quality Characteristics, Lipid Oxidation, Ovarian Follicular Development, and Immune Response in Laying Hens Under High Ambient Temperature. Biological Trace Element Research. 195:506–514.

Saleh, AA., Ashia Zaki, Ahmed El- Awady, Khairy Amber, Neamat Badwi, Yahya Eid, Tarek A. Ebeid (2020) Effect of substituting wheat bran with cumin seed meal on laying performance, egg quality characteristics and fatty acid profile in laying hens. Veterinarski Arhiv 90 (1), 47-56,

Saleh A. A., Abeer A. Kirrella, Mahmoud A. O. Dawood, Tarek A. Ebeid (2019) Effect of dietary inclusion of cumin seed oil on the performance, egg quality, immune response and ovarian development in laying hens under high ambient temperature. Animal Physiology and Animal Nutrition. 103(6):1810-1817.

6- Material and Methods

Why didn’t you prepare your diets according the layer strain catalog (LSL-LITE)?

P13 L1 Mash and fresh water were provided ad libitum, what is mean of mash are you mean feed form?

Table 5. Feed and nutrient composition of diets.

You must provide the crude protein levels of (Canola Meal, Sunflower seed meal, Soybean Meal and Poultry By-Product Meal

Reviewer #2: This is the review for the manuscript PONE-D-20-35025, entitled “L-valine in diets with 0.72% isoleucine: effect on production, egg quality, serum biochemistry, and ileal digestibility of protein” for PLOS ONE Journal.

The manuscript aims at investigating the effect of dietary supplementation of L-valine on laying hen performance in terms of egg production and quality, serum biochemistry, and protein ileal digestibility.

I have the following more detailed comments that can help authors in the revision of the manuscript:

Page 4 (Table 1): Kindly check body weight values for laying hens. It seems not correct. These values more appropriate for egg weight not hen body weight.

Page 8 (Line 5): delete this sentence

Page 10 (Line 16): The effect of valine: lysine on egg production is not discussed. The authors only compared with the findings in the previous studies and nothing mentioned why this alteration has occurred.

Page 12 (Material and methods)

Page 12 (Line 19): 490 refers to what? Kindly write (N=490). Kindly provide the average initial hen weight.

Nothing mentioned about blood or serum collection or analysis, and ileal digestibility, egg quality assessment.

Kindly check the superscripts according to the pooled SE and presented values of mean through the whole manuscript

**********

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Reviewer #1: No

Reviewer #2: No

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10 Sep 2021

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

More information was provided throughout the manuscript to improve it.

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer 2 did not provide information on where the analysis was weak.

We conducted regression analysis on each measurement to determine all responses as discussed in the manuscript.

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Data will be deposited at the UC Davis Library, University of California, Davis, CA 95616.

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

No specific errors were noted.

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Authors

Regarding the manuscript title Effect of L-valine in layer diets containing 0.72% isoleucine

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, some observation in the present paper should be corrected and add to improve the quality of the paper.

More information has been added to all parts of the manuscript. In many places, it was totally rewritten.

Introduction

1- Need more information about the effects of isoleucine in layer hens.

2- Need recently references for using Valine in layer diets

More information was added to the Introduction.

Discussion

3- 3- In this part you focused on the studies which agree or disagree with your results without a)ny explanation why these findings were agree with disagree with you.

More discussion was added as advised.

4- You must write the mode of action of L-valine and isoleucine on layer performance and egg quality.

More discussion was added in the Introduction and Results and Discussion.

5- I advise to use some recently reference for using amino acids in poultry and the additives affect the egg quality and immunity

I recommend reading and using the following references:

Saleh A. A. (2016) Effect of Low-Protein in Iso-Energetic Diets on Performance, Carcass Characteristics, Digestibilities and Plasma Lipids of Broiler Chickens. Egyptian Poultry Science Journal. Vol (36) (I): (251-262).

Saleh A. A, Mohammed S. Eltantawy , Esraa M. Gawish , Hassan H. Younis , Khairy A. Amber , Abd El-Moneim E. Abd El-Moneim & Tarek A. Ebeid (2020) Impact of Dietary Organic Mineral Supplementation on Reproductive Performance, Egg Quality Characteristics, Lipid Oxidation, Ovarian Follicular Development, and Immune Response in Laying Hens Under High Ambient Temperature. Biological Trace Element Research. 195:506–514.

Saleh, AA., Ashia Zaki, Ahmed El- Awady, Khairy Amber, Neamat Badwi, Yahya Eid, Tarek A. Ebeid (2020) Effect of substituting wheat bran with cumin seed meal on laying performance, egg quality characteristics and fatty acid profile in laying hens. Veterinarski Arhiv 90 (1), 47-56,

Saleh A. A., Abeer A. Kirrella, Mahmoud A. O. Dawood, Tarek A. Ebeid (2019) Effect of dietary inclusion of cumin seed oil on the performance, egg quality, immune response and ovarian development in laying hens under high ambient temperature. Animal Physiology and Animal Nutrition. 103(6):1810-1817.

Many of these papers had to be requested from the authors or there was a monetary fee attached to them. The authors thank the Reviewer for the comment and found other useful articles.

6- Material and Methods

Why didn’t you prepare your diets according the layer strain catalog (LSL-LITE)?

In Material and Methods, we explained that other available and less expensive sources of protein were used to replace soy. We provided an evaluation of protein (and references) for the sources. It is not reasonable to assume that all researchers throughout the world can use the exact same diets used by the producers of the layers.

P13 L1 Mash and fresh water were provided ad libitum, what is mean of mash are you mean feed form?

We have explained that "mash" was the form of the diet. This has not been an issue with other manuscripts.

Table 5. Feed and nutrient composition of diets.

You must provide the crude protein levels of (Canola Meal, Sunflower seed meal, Soybean Meal and Poultry By-Product Meal

These were provided in Table 5 (Materials and Methods).

Reviewer #2: This is the review for the manuscript PONE-D-20-35025, entitled “L-valine in diets with 0.72% isoleucine: effect on production, egg quality, serum biochemistry, and ileal digestibility of protein” for PLOS ONE Journal.

The manuscript aims at investigating the effect of dietary supplementation of L-valine on laying hen performance in terms of egg production and quality, serum biochemistry, and protein ileal digestibility.

I have the following more detailed comments that can help authors in the revision of the manuscript:

We thank Reviewer # 2 for the comments.

Page 4 (Table 1): Kindly check body weight values for laying hens. It seems not correct. These values more appropriate for egg weight not hen body weight.

The information was removed.

Page 8 (Line 5): delete this sentence

It was deleted.

Page 10 (Line 16): The effect of valine: lysine on egg production is not discussed. The authors only compared with the findings in the previous studies and nothing mentioned why this alteration has occurred.

More information was added.

Page 12 (Material and methods)

Page 12 (Line 19): 490 refers to what? Kindly write (N=490). Kindly provide the average initial hen weight.

Please see Material and Methods - Diets, Housing and Care.

Nothing mentioned about blood or serum collection or analysis, and ileal digestibility, egg quality assessment.

This information is included in the first paper on isoleucine. We are also submitting revisions for this work, “Varying digestible isoleucine level to determine effects on performance, egg quality, serum biochemistry, and ileal protein digestibility in diets of young laying hens” and hope to have it be approved for publication and published before this present manuscript. Revisions for the manuscript on work with isoleucine should have been completed first; however, communication between colleagues in Pakistan and the US was delayed for some time.

_________

Kindly check the superscripts according to the pooled SE and presented values of mean through the whole manuscript.

These have been added throughout the Results and Discussion.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Yes.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Submitted filename: Valin review and comments 8 18 21.docx

Click here for additional data file.

23 Sep 2021

Effects of L-valine in layer diets containing 0.72% isoleucine

PONE-D-20-36625R1

Dear Dr. King,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Mahmoud A.O. Dawood, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

9 Dec 2021

PONE-D-20-36625R1

Effects of L-valine in layer diets containing 0.72% isoleucine

Dear Dr. King:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Mahmoud A.O. Dawood

Academic Editor

PLOS ONE

Table 1

Valine level and production parameters of 33- to 40-week-old hens.

Level (%)	Lys:val#	Iso:val##	Feed intake (kg)	Egg production (%)	FCR$
Control$ $	0.867	0.986	104.88d	94.24c	1.33b
0.75	0.900	1.027	108.37bc	95.41cb	1.33b
0.78	0.939	1.068	111.52a	95.41cb	1.37a
0.81	0.975	1.109	109.59b	95.55b	1.35ab
0.84	1.010	1.151	109.64b	94.31c	1.36a
0.87	1.048	1.192	104.32d	96.85a	1.30c
1.048	11.084	11.233
0.90	1.084	1.233	107.27c	95.39cb	1.35ab
Pooled SE			3.18	2.01	0.04
Week (age)
33			116.74a	93.85b	1.38a
34			107.97c	94.89ab	1.36ab
35			106.22cd	95.58a	1.34b
36			104.29e	95.91a	1.31c
37			100.45f	95.60a	1.28c
38			111.17b	95.39a	1.31c
39			110.84b	95.82a	1.38a
40			105.82de	95.43a	1.36ab
Pooled SE			2.41	1.52	0.03
Level			< 0.01	< 0.01	< 0.01
Week			< 0.01	0.01	< 0.01
L*W+			<0.01	*	0.10
Linear 00Linear			< 0.01	0.26	0.03
Quadratic			0.01	0.07	0.35
Cubic			< 0.01	< 0.01	< 0.01

@ Means (treatments × 7 replicates × 10 layers per replicate) with the same superscript within a column do not differ significantly at P ≤ 0.05.

# Lysine:valine.

##Isoleucine:valine.

$Feed consumed per kg eggs.

$ $ Control containing isoleucine and valine, both at 0.72% of the diet.

+Interaction of level by week.

* Data unavailable.

Table 4

Effect of valine level on serum biochemistry and ileal digestibility of crude protein in laying hens between 33–40 weeks.

Level (%)	Lys: val#	Ile: val##	Glucose (mg/dl)	Serum albumin (g/dl)	Total protein (g/dl)	H9 titer$	H1 titer for NDV$$	Digestability+
0.72*	0.867	0.986	148.29	2.90	4.30a	8.57	8.42ab	60.92ab
0.75	0.900	1.027	151.43	3.47	3.75ab	8.28	8.71ab	72.88a
0.78	0.939	1.068	146.86	2.88	3.42ab	8.14	9.14a	72.89a
0.81	0.975	1.109	149.29	3.21	3.22b	7.28	8.57ab	74.04a
0.84	1.010	1.151	142.71	3.28	3.42ab	8.42	8.00ab	67.88ab
0.87	1.048	1.192	117.71	2.77	3.57ab	7.57	7.71b	52.84b
0.90	1.084	1.233	146.14	2.64	3.37ab	8.57	8.00ab	66.11ab
Pooled SE			13.33	0.28	0.30	0.53	0.41	6.90
P value			0.20	0.06	0.02	0.13	0.02	0.07
Linear			0.11	0.15	0.55	0.67	< 0.01	0.02
Quadratic			0.82	0.77	0.43	0.41	0.957	0.57
Cubic			0.19	0.11	0.17	0.76	0.07	0.12

@ Means (7 treatments × 7 replicates × 2 layers per replicate) with the same superscript within a column do not differ significantly at P ≤ 0.05.

# Lysine:valine.

##Isoleucine:valine.

$Detection for subtype of avian influenza.

$$Detection for Newcastle Disease.

+Ileal protein digestibility

*Control as isoleucine and valine, both at 0.72% of the diet.