Effect of zinc oxide, potassium iodide and withdrawal diet as alternative moulting methods on performance of commercial laying hens

ABSTRACT

The aim of this study was to compare applicability of the non-feed removal method with the feed withdrawal method by using zinc Oxide and potassium iodide in moulting on productive performance. A total of 240 84-week-old Lohmann Selected Leghorn (LSL-Lite) laying hens were randomly distributed in 40 cages (ten treatments and four replicates per treatment). The results showed that crop pH and Ilium pH were similar among treatments. Moulting method affected body weight reduction and feed consumption (P < 0.05), maximum body weight reduction from 1 to 16 days during moulting was observed in T₃. Feed consumptions for hens fed ZnO diets (T₄) were lower, with hens fed the KI diet being intermediate (P < 0.05). During the second laying cycle no significant effect was found for egg weight and feed intake. Organ characteristics (weight organs and length oviduct) of hens fed a zinc-supplemented diet were lower than those non-moulted and hens fed a KI-supplemented diet. In conclusion, that each moulting method differently influenced various parameters studied for post moulting performance. Indicating that non-fasting moulting methods, which are better for animal welfare, are effective alternatives to fasting method.

KEYWORDS:

• Forced moulting
• laying hens
• potassium iodide
• productive performance
• zinc oxide

1. Introduction

It has been shown that the technique of induced moulting is an effective tool to improve the product performance and profitability of old laying hens (RolonA and Cunningham 1993). Moulting brings the flock into a second cycle of lay with improved eggshell quality and increased economic performance (Moghaddam et al. 2018). Various induced moult techniques that do not use fasting have been suggested. These alternative methods use dietary manipulations to create an imbalance of nutrients (Bozkurt et al. 2016). Exposing to repeated or chronic stress may disturb physiological homeostasis and immune function and ultimately lead to maladaptive stress responses in animals, such as a lack of adaption, a prolonged response, or even a low response to a new stressor (Verbeek E and BlacheD 2019). However, moulting remains a necessary management strategy to meet market needs as it is used as an effective intervention method to avoid interruptions in the egg market whenever pullet shortages occur (Flock D and AndersonK 2016).

Conventional induced moulting program usually involves a period of fasting for 10–15 days or more and the goal of this intervention is to synchronize the 25–30% body weight (BW) reduction in the flock (Ruszler 1998). The decision of using a force moulting program depends on several factors, such as price of replacement new flock, egg quality and price, feed cost, maximizes facility use and employed moulting method, (Garcia et al. 2001; Mazzuco and Hester 2005; Reddy et al. 2008). Last decades, induce moulting by temporary feed withdrawal (typically by withdrawing food for 7–14 days and sometimes also withdrawing water for an extended period) has become a concern to poultry industry due to welfare and food safety reasons (Hebert and Cerneglia 1978; Keshawarz and Quimby 2002; Bell 2003; Willis et al. 2009). Feed withdrawal results in severe stress on the body and followed that decline immunity and increase mortality rate in hens. Ideally, a good moulting method should get the flock out of production within 5–6 days, keep the flock out of production until it has rested, bring it back into production when needed, simple to implement, low in cost, result low mortality and lead to high subsequence performance (Lee 1982; Koelkebeck et al. 1992).

Some flocks experiencing high mortality rate during forced moulting (Park et al. 2004). It has been recommended that the flock must be managed so that mortality does not exceed 1.25% over the 1–2 weeks of (nearly complete) feed withdrawal, compared to a 0.5% to 1% monthly mortality in a well-managed flock under low stress condition (Bell and Kuney 1992).

Therefore, alternative methods of induce moulting which do not use total food withdrawal, e.g. creating a dietary mineral imbalance, generally result in lower mortality rates, these methods include feeding a diet with low-Na and high Aluminium (Berry and Brake 1985, 1987; Yousaf and Ahmad 2006)feeding a ration containing high levels of Zinc (Zn, 20,000 mg/kg) as Zn oxide (ZnO) (Reddy et al. 2008), feeding a diet containing 2500–5000 mg/kg I as potassium Iodide (Yousaf and Ahmad 2006), low-Ca diets (Breeding et al. 1992) or high-fiber and low-energy diets (Woodward et al. 2005) Other non-fasting moult methods include the feeding of wheat middling's (Biggs et al. 2003), a combination of wheat middling's and corn (Mazzuco et al. 2011), various ratios of alfalfa or layer ratio (Donalson et al. 2005; Petek and Alpay 2008), a whole grain barley diet (Onbasilar and Erol 2007), a combination of soy-hulls based diet and corn (Mazzuco et al. 2011) or feeding broken rice, rice bran or cassava meal for a short period (Gongruttananun et al. 2013), cottonseed meals (Davis et al. 2002), jojoba meal (Vermaut et al. 1997). The dietary supplementation of drugs or hormones, such as gonadotropin-releasing hormone (GnRH), melengestrol acetate, or thyroxin have also used for inducing moulting (Dickerman and Bahr 1989; Koch et al. 2007; Onbasilar and Erol 2007).

Hormones have major metabolic roles in reproductive function (e.g. egg production) and moulting progress of poultry. Kang et al. (2001) reported that high egg production is associated with higher weights of ovary and follicles and higher progesterone confirmed the positive relationship of progesterone expression with egg production. The elevated levels of prolactin in birds play a negative role in reproductive performance. This is further concurred when dopamine stimulates the prolactin secretion from anterior pituitary gland (Youngren et al. 1998). The egg production in bird’s declines with increasing the concentration of prolactin.

Maximal improvements in post moult egg production and egg quality could be achieved when the birds had a recovery period of 14–21 d. Hence, the objective of this study was to compare induced moulting methods and determining the best method.

2. Material and methods

2.1. Birds and diets

The birds were housed into galvanized wire cages with dimensions 25 *40 *45 cm and had free access to their assigned diet and water during the feeding period. Each cage was equipped with individual feeders and nipple drinker's environmental temperature and air relative humidity were maintained at 20–24°C and 60–70%, respectively. Hens were exposed to a light (L) to darkness (D) photoperiod of 16L: 8D daily before the start of the experiment. The experiment consisted of a 4-week moult period followed by 20-week post moult production period (84–104 week of age). All experimental protocols adhered to the guidelines of, and were approved by, the Animal Ethics Committee of RaziUniversity (Kermanshah, Iran) and were in accordance with the guidelines on animal welfare. Environmental conditions inside the house were not controlled and therefore varied with the natural conditions outside (July 2016).

Single comb White Leghorn hens (n=240, 84 week of age) were individually weighed and randomly allocated to treatments (Table 1). Ten moult treatments included: (T₁) withdrawal diet 2 days and received ZnO diet 2.5% 14 days then allocated layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) feeding ZnO diet 2.5% (Goodman et al., 1986) 16 days then allocated layer diet; (T₄) feeding ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days and received KI (Ruszler 1998) diet 0.5% 14 days then allocated layer diet; (T6) withdrawal diet 2 days and followed KI diet 0.5% 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₇) feeding KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet. Ingredients and nutrient composition of the diets are given in Table 2.

Table 1. Description of moulting technique.

Treatment	Description
T1	withdrawal diet 2 days and received ZnO diet 2.5% 14 days then allocated layer diet
T2	withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days;
T3	Feeding ZnO diet 2.5% 16 days then allocated layer diet
T4	ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days;
T5	withdrawal diet 2 days and received KI diet 0.5% 14 days then allocated layer diet;
T6	withdrawal diet 2 days and followed KI diet 0.5% 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days;
T7	Feeding KI diet 0.5% 16 days then allocated layer diet
T8	Feeding KI diet 0.5% 16 days then then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days;
T9	withdrawal diet 11 days followed by layer diet
T10	Layer diet

Table 2. Ingredients and chemical composition of experimental diets.

Ingredient,% of diet	ZnO diet (%)	KI diet (%)	Rest 50% Pr (%)	Rest 75% Pr (%)	Layer diet (%)
Corn	78.10	77.39	83.34	73.24	59.00
Soybean meal	10.40	9.10	2.27	11.63	23.17
Wheat bran	2.39	6.40	1.01	-	-
Alfa alfa meal	-	-	1.5	1.5	1.5
Soybean Oil	-	-	0.34	2.16	4.96
DCP	0.27	0.24	1.94	1.86	1.76
Oyster shell	4.98	4.99	8.74	8.75	8.72
Salt	0.35	0.35	0.35	0.35	0.35
Vitamin mixture	0.25	0.25	0.25	0.25	0.25
Mineral mixture	0.25	0.25	0.25	0.25	0.25
L-Lys	0.19	0.20	-	-	-
DL- Met	0.32	0.32	-	0.02	0.05
ZnO	2.5	-	-	-	-
KI	-	0.5	-	-	-
Calculated analysis
ME (kcal/kg)	2880	2880	2880	2880	2780
Crude protein (%)	10.8	10.73	7.8	11.6	15.42
Ca (%)	2.04	2.04	3.75	3.75	3.7

Notes: The vitamin and mineral premix provide the following quantities per kilogram of diet: Vitamin A, 10,000 IU (all-trans-retinal); cholecalciferol, 2000 IU; vitamin E, 20 IU (a-tocopheryl); vitamin K3, 3.0 mg; riboflavin, 18.0 mg; niacin, 50 mg; D-calcium pantothenic acid, 24 mg; choline chloride, 450 mg; vitamin B12, 0.02 mg; folic acid, 3.0 mg; manganese, 110 mg; zinc, 100 mg; iron, 60 mg; copper, 10 mg iodine, 100 mg; selenium, 0.2 mg; antioxidant, 250 mg. mg; iron, 60 mg; copper, 10 mg iodine, 100 mg; selenium, 0.2 mg; antioxidant, 250 mg.

2.2. Productive performance

Egg production and mortality were recorded daily at the beginning of the experiments and continued until 2 weeks after the peak of post moult production. These periods were used to determine the days of stopping and returning to egg production. Egg mass was calculated by multiplying egg weight by egg production. Feed intake (FI) per cage was recorded as the average FI/d/hen. Feed conversion ratio (FCR) was determined as feed consumed per egg mass produced.

2.3. Body weight and slaughter parameters

During the moulting period, hens were weighed individually at day zero (initial live BW weight), 2, 10 and at day 16 to estimate BW loss. At the 7-day moulting period, 30 birds (3 hens/group) slaughtered whereas digestive system parameters were studied, including abdominal fat, liver, intestine weights and intestine length. Also, the reproductive system weight and length were studied as for oviduct and ovary.

2.4. Egg quality traits

At six weeks of the experiment, eggs were taken from each treatment for three consecutive days, weighed and egg index (the height: length ratio) and shell weight, yolk and albumin height, width and length were recorded. The Haugh unit and yolk index were calculated as described by Stadleman (1977). Egg shell thickness was measured by averaging measurements at three separate locations (tip, flat end and equator) using an electronic digital caliper scale. Yolk colour was determined using a Roche yolk colour fan (DSM nutritional products Co.). Colour scales ranged from 1 (pale yellow) to 15 (intense orange).

2.5. Blood leukocyte

To study the effects of different treatments on blood leukocyte subpopulations, blood samples of two birds from each replicate were collected from the wing vein at the end of experiment, and EDTA-containing blood samples were stained according to the method described by Lucas and Jamroz (1961); subsequently, 200 leukocytes per samples were counted by an optical microscope. The ratio of heterophil (H) to lymphocyte (L) was also calculated.

2.6. Ph of the crop and Ilium

The pH of the crop and Ilium was determined by the insertion of a sterile glass pH electrode 2 through an incision in the crop wall, ensuring that the electrode remained in contact with the crop mucosal surface as described by (Ramirez et al.1997).

2.7. Statistical analysis

All data obtained from the trials were subjected to the analysis of variance procedure of statistical analysis system (SAS 2001) according to completely randomized design. All values are presented as mean and associated pooled SEM. Means were separated by Duncan new ‘multiple range test. The level of significance was determined at P <0.05. The model was Yij=μ+αi + eij, where Yij, μ, αi and eij represent measured characteristic, overall mean, effect of experimental diet, and random error term, respectively.

3. Results

3.1. Egg quality parameters

The values of egg quality traits are detailed in Table 3. There was no significant difference among dietary treatments for egg index, yolk index, albumen height, yolk colour, Shell weight and Shell thickness.

Table 3. Effect of moulting and non-moulting diets on eggshell quality during the post moult laying period.

Treatment	Egg index (%)	Yolk colour ( Roche )	Albumen height (HU)	Yolk index (%)	Shell weight (gr)	Shell thickness (mm / 100)
T1	73.6	7.11	89.3	42.2	7.1	34.1
T2	71.9	7.11	92.3	42.4	7.2	35.3
T3	73.0	7.00	98.0	43.3	7.3	33.1
T4	70.8	7.00	95.7	42.4	7.3	32.6
T5	70.1	7.00	97.5	43.7	7.6	34.2
T6	72.4	7.11	98.4	42.6	7.7	33.6
T7	73.4	7.33	91.5	43.1	7.7	34.5
T8	72.1	7.44	99.3	43.6	7.1	32.2
T9	71.2	7.00	97.3	43.1	7.2	35.4
T10	72.1	7.00	92.3	41.8	7.4	33.6
P value	0.21	0.05	0.08	0.07	0.07	0.35
SEM	0.34	0.03	0.90	0.23	0.37	0.88

Notes: Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄) ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

3.2. Organ weights

In this study, our results indicate that organ characteristics (weight organs and length oviduct) those hens fed ZnO and KI diets were significantly lower than hen's non-moulted hens (Table 4). The results of the effects of ZnO supplementation on organ characteristics of moulted hens showed significant (P<0.01) variations among treatments in the relative weights of ovary, oviduct, liver, pancreas, spleen and intestine (Table 3). The relative weights of the ovary of hens in T₇ (13.37 g) and T₈ (13.35 g) were higher, and differed (P<0.01) from those of moulted hens in T₁(2.36 g), T₂ (1.47 g), T₃ (1.69 g), T₄ (2.44 g), and T₅(2.53 g). Higher length of oviduct was observed in the non-moulted hensT₁₀(54.33 cm). Highly significant (P<0.01) variation in the relative weight of pancreas was found between the moulted and non-moulted hens. Non-moulted hens in T₁₀ had higher (P<0.01) pancreas weights than those in other treatments. Moulted hens in T₃ had lower (P<0.01) pancreas weights than those in other treatments. The relative weight of the liver was higher for non-moulted hens in T₁₀ (38.48 g) and differed (P<0.01) from those in all other treatments. Relative weight of liver of hens on T₂ (21.66 g), T₃ (22.64 g), T₄ (19.30 g), T₅ (22.36 g) and T₆ (23.03 g) were similar.

Table 4. Effect of moulting and non-moulting diets on reproductive, immune and intestinal weight.

Treatment	Ovary weight (g)	Oviduct length (cm)	Oviduct weight (g)	Liver weight (g)	Pancreas weight (g)	A.A.T.W (g)	Spleen weight (g)	Intestine weight (g)
T1	2.36^d	24^d	6.33^d	24.94^d	2.62^e	11.08^gh	1.32^c	52.13^d
T2	1.47^d	19.33^f	3.23^e	21.66^e	1.76^g	11.86^gh	1.32^c	49.11^d
T3	1.69^d	18^f	2.77^e	22.64^e	1.63^h	17.18^f	1.20^c	46.12^d
T4	2.44^d	21^def	4.26^de	19.30^e	2.19^f	16.17^gf	1.30^c	44.25^d
T5	2.53^d	23^de	4.62^de	22.36^e	1.76^g	51.92^e	1.29^c	51.15^d
T6	7.52^c	32.33^c	15.68^c	23.03^e	2.81^d	64.79^d	1.19^c	59.14^c
T7	13.37^b	41.33^b	22.75^b	32.50^b	3.23^c	77.84^c	1.50^b	68.24^b
T8	13.35^b	42.66^b	21.96^b	33.45^b	3.40^b	85.44^b	1.56^b	70.12^b
T9	1.67^d	17.33^f	2.49^e	17.78^e	1.77^g	9.78^h	1.27^c	41.11^e
T10	28.19^a	54.33^a	29.84^a	38.48^a	4.23^a	96.81^a	1.75^a	97.12^a
P value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
SEM	0.44	5	1.36	1.22	0.0001	8.24	0.008	8.70

Notes: Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄) ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

3.3. Body weight and mortality rate

There was a highly significant (P<0.05) effect of moulting method on BW reduction. Maximum BW reduction for 1–16 days during moulting was observed in treatment 3 (Table 5). Body weight lost with 28.5% from initial weight for the hens that were deprived of feed for 11-d. Percentage body weight loss (0.76%) of the non-moulted hens were very minimal and negligible. At the end of the moulting period (16-d), the hens fed a KI-supplemented diet had regained a substantial amount of weight and had final BW losses about 7–9%. The range of mortality in this experiment was between 2% and 7.93% among treatments. The higher mortality percentage was observed in the T₉ while the lowest mortality rates was shown in treatment 6.

Table 5. Effect of moulting and non-moulting diets on body weight loss in difference days and mortality rate.

Treatment	Body weight loss 2 d (%)	Body weight loss 10 d (%)	Body weight loss end of moult 16 d (%)	Mortality rate (% )
T1	6.5^a	23^c	34.1^a	6.30^c
T2	7^a	23.1^c	32^b	6.86^b
T3	5.1^ab	25.3^b	35^a	5.76^c
T4	4.5^b	23.9^bc	31.2^b	6.26^c
T5	6.8^a	8.6^d	9.1^c	3.70^d
T6	5.7^ab	8.4^de	8.6^c	3.13^e
T7	2.1^c	6.7^ef	7.8^c	3.63^de
T8	2.3 ^c	5.8^f	7.9^c	3.90^d
T9	6.1^ab	28.5^a	-	7.93^a
T10	0.60	0.83	0.76	2^f
P value	<0.001	<0.001	<0.001	<0.001
SEM	1	1	1	0.08

Note: Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄₎ ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

3.4. Egg quality factors

There were no differences in the broken egg, shell less, cracked shell and Ab normal egg among the treatment groups throughout the post moult period (P > 0.05, Table 6).

Table 6. Effect of moulting and non-moulting diets on broken egg, Shell less, cracked shell and Ab normal egg shell (%).

Treatment	Broken egg (%)	Shell less (%)	Cracked shell (%)	Ab normal egg (%)
T1	1.17	0.27	2.65	1.90
T2	1.78	0.28	2.03	1.86
T3	1.76	0.10	1.42	1.5
T4	1.16	0.09	1.69	1.56
T5	1.33	0.69	2.32	1.93
T6	1.61	0.78	2.68	1.86
T7	1.03	0.88	1.94	2.13
T8	1.54	0.92	2.02	1.75
T9	1.23	0.60	2.11	1.66
T10	2.09	1.03	3.23	2.18
P value	0.44	0.31	0.96	0.91
SEM	0.12	0.09	0.32	0.21

Notes: Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄) ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

3.5. Immunity response and the pH of crop and Ilium

It was no difference in crop and ilium pH between treatments (P > 0.05, Table 7). On the 6th of the experiment, the H: L ratio was significantly higher in T₉. The H: L ratio for deprived feed treatment was significantly higher than other treatments. This result in agreement with the findings of (Landers 2004), who stated that hens moulted by feed deprivation have a significantly higher H: L ratio when compared to birds that were not moulted.

Table 7. Effect of moulting and non-moulting diets on heterophil: lymphocyte ratio and Crop PH and Ilium PH.

Treatment	H/L (%)	Crop pH	Ilium pH
T1	0.64^bc	5.8	6.8
T2	0.60^cd	5.9	6.8
T3	0.67^b	5.5	6.2
T4	0.58^de	5.7	6.2
T5	0.52^ef	5.2	7.2
T6	0.56^de	5.1	6.7
T7	0.55^de	5.5	6.7
T8	0.48^f	5.4	6.5
T9	0.78^a	6.1	6.2
T10	0.40^g	4.8	7.1
P value	<0.001	0.6	0.3
SEM	0.0009	1	1

Notes: Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄) ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

3.6. Performance parameters

In the present study, egg production has dropped to zero on the 4th day of induced moult in each moulted treatment. Cessation of egg production within 7d following forced moult has been reported previously (Nordstrom 1980; Lee 1982; Gongruttananun et al. 2013). In all treatment groups egg production ceased during the moulting period (except T₁₀). Although, the non-moulted birds continued egg production throughout the duration of the experiment, highly significant (P<0.01) variations in the number of days to cessation of egg production was witnessed among the moulted groups of hens. Birds in T₃ and T₄ stopped egg production on day 6 and 7 after ZnO treatment, compared to those hens in T₇ and T₈ which ceased egg production on day 9 and day 8.33 after KI treatment respectively. Induced moulting showed significant (P<0.05) effect on egg production of caged as shown in Table 8. Maximum number of eggs produced by treatment T₃ (79.80%) in post-moult period followed by T₂ (77.45%), T₄ (76.66%) and T₁ (76.30± %) (Table 8).

Table 8. Effect of moulting and non-moulting diets on some performance parameters.

Treatment	Days to lay cessation( days)	Return to lay (days)	Time reach to 50% production (days )	Percentage peak production (%)	Time reach to peak production (days)
T1	5.33	34^e	66^e	76.30^ab	116^ab
T2	5.67	36^d	70^bc	77.45^ab	117^a
T3	6	38^cd	65^e	79.80^a	115^ab
T4	7	39^bc	68^cd	76.66^ab	114^b
T5	8	42^ab	78^a	71.34^cd	110^c
T6	7.67	45^a	75^b	70.17^de	114^b
T7	9	41^ab	72^b	67.56^ef	105^d
T8	8.33	45^a	73^b	65.18^f	106^d
T9	4	32^e	67^de	74.55^bc	108^d
T10	-	-	-	58.55	-
P value	<0.001	<0.001	<0.001	<0.001	<0.001
SEM	1	1.96	2.55	3.87	2.44

Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄) ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

3.7. Performance parameters during the moulting period

Moulting methods also showed significant (P<0.05) effect on feed consumption. Feed consumption of the non-moulted hens was higher, compared to the moulted birds. Feed consumptions for hens fed ZnO diets were lower, with hens fed the KI diet being intermediate. A greater reduction in feed intake of the moulted hens was observed for hens in T₄ (ZnO supplementation) 1–16 days. Feed intake of the hens fed the diet containing 2.5% ZnO was significantly lower compared to the other treatments (p < 0.05) (Table 9).

Table 9. Effect of moulting and non-moulting diets Average performance parameters obtained during the moulting period (1-16 days).

Treatment	Egg production 1–4 w k ( % )	Egg production 4–8 w k ( % )	Feed intake 1–16 days ( g/hen/ d)	Feed intake 16–24 days ( g/hen/ d)	Feed intake 24–32 days ( g/hen/ d)
T1	1.79^b	12.81^c	21.1^d	100.13^b	102.16^b
T2	2.42^a	12.54^c	16.93^d	65.46^d	90.43^cd
T3	1.92^ab	12.74^c	18.5^d	105.9^a	102.16^b
T4	1.82^b	13.49b^c	15.23^d	66.33^d	89.20^de
T5	2.22^ab	15.61^ab	45.16^c	90.43^c	91.96^cd
T6	1.98^ab	15.99^a	45.46^c	88.10^c	85.43^ef
T7	1.85^ab	15.59^ab	57^b	100.10^b	95.1^c
T8	1.66^b	15.15^ab	52.23^bc	88.56^c	83.23^f
T9	1.98^ab	12.45^c	-	98.23^b	93.9^cd
T10	-	-	97.5^a	109.3^a	113.13^a
P value	<0.001	<0.001	<0.001	<0.001	<0.001
SEM	0.093	1.43	21.3	9.15	6.96

Notes: Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄) ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

3.8. Performance parameters during the second laying cycle

Post-moult egg weight, egg mass, feed consumption, and feed conversion ratio (32d– end period) are depicted in Table 10. During the second laying cycle no significance on egg weight but There was a highly significant (P<0.05) effect of moulting method on egg mass and Feed conversion ratio. Statistical analysis of data on egg mass showed that egg mass value was lower (p< 0.05) in the birds fed T₁₀ compared other groups during experimental period; Moulting of laying hen using ZnO caused a significant (P<0.05) decline in post- moult feed conversion ratio. Feed conversion ratio of hen in T₁ (1.98 g/g) and T₂ (2.05 g/g) T₃(2.00 g/g) T₄(1.94 g/g) were significantly (P<0.05) lower than those of the non-moulted hen T₁₀ (2.91 g/g). No significant differences (P>0.05) in feed conversion ratio were observed for hens in T₆ (2.29 g/g) and T₇ (2.16 g/g) and T₈ (2.23 g/g) that feed with KI but were significantly (P<0.05) lower than those of the non-moulted hen T₁₀ (2.91 g/g). However, feed consumption of hens in total treatment were similar (P>0.05) during the post-moult period.

Table 10. Effect of moulting and non-moulting diets on Layer performance during the second laying cycle (32 days- end).

Treatment	Egg production 8–20 (%)	Egg weight (g)	Egg mass (g /d/ hen)	Feed intake (g / d /hen )	Feed conversion ratio
T1	69.06^b	71.5	49.38^ab	97.93	1.98^b
T2	71.08^ab	70.69	50.25^ab	103.16	2.05^b
T3	72.58^a	70.66	51.30^a	102.46	2.00^b
T4	71.12^ab	70.17	49.90^ab	97.03	1.94^b
T5	64.12^c	70.29	45.06^b	97.10	2.15^ab
T6	64.31^c	69.96	45^b	102.93	2.29^ab
T7	62.85^c	70.53	44.32^b	95.76	2.16^ab
T8	62.32^c	70.03	43.64^b	97.40	2.23^ab
T9	69.65^b	70.34	49^ab	100.06	2.04^b
T10	54.40^d	71.13	38.69^c	112.96	2.91^a
P value	<0.001	0.62	<0.001	0.48	<0.001
SEM	1.99	1.06	0.85	80.13	0.11

Notes: Means in a column with different superscripts are significant (P< 0.05).

(T₁) withdrawal diet 2 days and followed ZnO diet 2.5% 14 days then layer diet; (T₂) withdrawal diet 2 days and received ZnO (2.5%) diet for 14 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₃) ZnO diet 2.5% 16 days then layer diet; (T₄) ZnO diet 2.5% 16 days then allocated diet with 50% CP for 8 days followed by feeding diet that contained 75% CP 8 days; (T₅) withdrawal diet 2 days followed KI diet 0.5% 14 days then layer diet; (T₆) withdrawal diet 2 days followed KI diet 0.5% 14 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₇) KI diet 0.5% 16 days then layer diet; (T₈) KI diet 0.5% 16 days then feeding Pr 50% diet 8 days followed by feeding Pr 75% diet 8 days; (T₉) withdrawal diet 11 days followed by layer diet and (T₁₀) Layer diet.

4. Discussion

Moulting causes a remarkable regression in ovary and oviduct weight, which is associated with the loss of oestrogenic activity (Khajali et al. 2007). Adequate amounts of thyroid hormones are essential to induce moult. Specifically, elevated plasma T4 triggers old feather loss and stimulates new feather growth (Dawson 2015). Induced moulting leads to the involution of the reproductive tract, which is proportional to the loss of BW and that the rebuilding of the reproductive tract would result in the removal of fat accumulation and followed that increased tissue efficiency (Park et al. 2004; Hassanabadi and Kermanshah 2007). Kashmiri and Vatsalya (2011) reported that forced moulting resulted in body weight loss, regression of reproductive organs and decrease in the size of liver. It could be suggested that complete feed withdrawal immediately ceases egg production because fasting results in changes in the normal physiology in terms of reduced digestive enzyme, serum cholesterol triglycerides and very low density lipoproteins (VLDL), which are important for egg production (Peebles et al. 2004); however, there is individual variation among birds.

It has been suggested that inducing moulting in hens with using ZnO had a significant effect on the relative weights of the ovary and oviduct of the birds, therefore, suggests that higher consumption of dietary ZnO increases the rate of reproductive characteristics in moulted hens (Machebe et al. 2013). According, Zn is physiologically involved in production, storage and secretion of sex hormones, and also affects the receptor sites of these hormones (Reddy et al. 2008). The decrease in liver weight results from the removal of hepatic energy stores as glycogen and lipids that are metabolized in the liver (Berry and Brake 1985). The liver is the target organ of yolk phospholipoprotein synthesis, which is dependent on ovarian steroids, especially oestrogen (Sturkie 1976). Zinc interferes with insulin secretion therefore glycogen may not deplete in the liver (McCormick 1984), possible mechanism of Zn detoxification in the liver, including a high rate of bile production and synthesis of large quantities of the metallothionein (Zn storage protein), may have contributed to the maintenance of liver weight. A significant reduction in liver weight after force moulting may be due to the decrease in oestrogen secretion by the ovary and cessation of lay.

Losses of BW in range 25-30% have been reported in hens moulted using various amounts and types of Zn salt (Stadleman 1977; Khan et al. 2011). The decline in BW in the moulted hens is not unconnected with decreases in feed consumption of the hens during the moulting process, decrease in organ weights (especially the reproductive organs and liver), which occur simultaneously during with BW loss during moulting reported about 65–70% reduction in feed intake in hens moulted using Zn salt (Zn acetate or Zn propionate) (Park et al. 2004) that rely on this reduced palatability. In agreement with our results, according to them maximum post moult performance was in those birds which lost 27–32% BW during moulting (Baker et al. 1983). This reduction in BW may be for feed restriction, stress, decreased muscle mass, and mobilization of glycogen stores, adipose tissue catabolism, and utilization of adipose tissue, decreased liver weight and involution of the reproductive organs in addition to the loss of feather (Berry and Brake 1991)Ovarian and oviduct regression during moult is critical to achieving optimum post-moult egg laying performance (Gongruttananun et al. 2017). Body weight loss is directly related to the post-moult performance (Kazemi-Fard et al. 2013). Also, Machebe et al. (2013) found that, moulted birds that fed 2.0% Zn had greater (P<0.01) percentage loss in BW (15.41%), than those of moulted hens with 0.5% and 1.0% Zn (7.17%). Our results for BW are similar finding by Aygun (2013). The probable explanation of higher mortality rate in the control group may be due to higher levels of stress hormones such as cortisol.

It is reported that H: L ratio remained consistent with age until the moult induction with feed withdrawal and then was significantly increased (Davis et al. 2000). That is most likely related to the adaptation to changes in metabolic demands caused by physiological stress. Similar to our findings, other studies have found that heterophil cells increased during 10–14 d periods of feed withdrawal (Alodan and Mashaly 1999). An elevated H/L ratio has been used as a reliable indicator for chronic stress in different animals, including chicken (Davis and Mandy 2018). Higher H/L ratios control hens could be related to activation of HPA axis due to 3 heat exposures and induced moult, leading to an initial increase in synthesis and release of CORT, which subsequently stimulates an earlier release of heterophils from bone marrow into blood circulation and redistribution of lymphocytes from blood circulation to other organs (Caroprese et al. 2014). These changes cause a decrease in the number of lymphocytes and an increase in the number of heterophils, resulting in higher H/L ratio.

In agreement with the findings of the current study indicated that heterophi1: lymphocyte ratios were increased during induced moulting (Davis et al. 2000).

Previous studies, (Ricke 2001; Gutierrez et al. 2008) reported that moulting diets containing high Zn decreased S. enteriti discolonization in laying hens when compared with hens undergoing feed withdrawal. Reduced feed intake may be the main factor causing the decrease in the Lactobacillus population (Humphrey et al. 1993; Corrier et al. 1999; Durant et al. 1999), thereby allowing the pH to rise in hens that are deprived of feed.

In contrast, another author (Humphrey et al. 1993) reported that when chickens are undergoing malnutrition or starvation, the pH of crop can increase due to decreased Lactobacillus fermentation within the crop. Feed withdrawal for 9 d resulted in a decrease in lactic acid in the crop, accompanied by an increase in crop pH (Ramirez et al. 1997; Durant et al. 1999). However, the results of the present study indicate that the increases in crop pH by dietary moulting treatment for feed withdrawal, Zn acetate, or Zn propionate regimens may reduce the normal resident Lactobacillus population or lactic acid concentration in the crop. Zn acetate or Zn propionate feeding may be inhibitory to the Lactobacillus population due to the effect of Zn on microorganism growth. Dietary Zn may influence growth and infectivity of bacterial pathogens in animal.

The low feed intake, stress, and nutritional imbalance that results in the involution of the reproductive tract cause a cascade of neuroendocrine effects. The feed is fasting or the provision of feeds, which composition does not supply the nutrients required for body maintenance, causes stress and, consequently hypothalamus hyperactivity. The hypothalamus then releases corticotrophin (ACTHRF) and thyrotrophic (TSHRF) hormones, influencing the pituitary, which then starts secreting ACTH and TSH. The increase in the blood levels of these hormones leads to adrenal and thyroid hypertrophy and hyper function, causing gonadotropic hormone imbalance and egg production ceased (Garcia 2004). The fed-fasted hen compared with those fed the 30 ppm Zn diet required less days to cease egg laying, according to (Bell 2003), the hens cease laying eggs sooner, they will start producing eggs sooner. Declining of LH, estradiol and progesterone were coincident with the cessation of egg production.

Feed intake of layers fed 1% Zn as ZnO or Zn acetate were 22 and 16 g/ bird, respectively. During the second week of the induced moulting period, hens presented 0% egg production that could be due to reduced appetite or palatability (Fox 1989). It was also reported that feed intake may be reduced; because Zn cation (Zn²⁺) induces follicle atresia and interrupting egg production (Johnson and Brake 1992). Therefore, it is possible that the efficiency of the treatment with the diet with high Zn levels is directly related with feed intake. It is reported that a few days after ZnO exposure egg production ceased, in accordance with the quantity of the salts added to the diets (Machebe et al. 2013). This suggests that higher consumption of dietary ZnO increases the rate of reproductive quiescence in moulted hens. Layers submitted to induce moulting by feed fasting had higher relative egg production (Onbasilar and Erol 2007).

Several studies reported that 20,000 ppm dietary Zn is as effective as fasting for inducing moulting. Zn as oxide or acetate (10,000 mg/kg) causes production to decline from 60 to 0% in 6 d. Combining a low calcium diet with a relatively low level of Zn (2,800 ppm) induces reproductive involution and moulting similar to that of 20,000 ppm dietary Zn (Breeding et al. 1992). High dietary levels of I (iodine) were found to induce a pause in egg production with or without loss of feathers (Hebert and Cerneglia 1978).

5. Conclusion

The study reveals that each moulting method differently influenced various parameters studied for post moulting performance. Indicating that non-fasting moulting methods, which are better for animal welfare, are effective alternatives to fasting methods. Present study showed that in order to achieve good post moult performance of laying hens, farmers should practice the use of ZnO supplementation, as an effective method to achieve induced moulting.

Disclosure statement

No potential conflict of interest was reported by the author(s).