Induced molting has been practiced by commercial farms in many countries to recover egg production of aged laying flocks for a long time. Successful molting can also prevent age-related decline in eggshell quality . The conventional feed and water withdrawal procedure has been generally used for laying flocks because it is simple and practicable  with a satisfactory post-molting laying performance . However, feed deprivation causes severe stress on the body followed by a decrease in immunity and an increase in mortality. It also makes laying flocks more susceptible to Salmonella infection . Thus, forced molting by temporary feed and water withdrawal has become a concern to the layer industry due to animal welfare and food safety issues.
An ideal molting method should proceed in a way that protects animal welfare and health without fasting or causing too much stress . European Union (EU) and many other countries have adopted non-fasting molting methods due to the incompatibility between animal welfare and fasting. Likewise, in the Republic of Korea, forced molting based on fasting may soon become a restriction in accordance with the Animal welfare comprehensive plan. Many studies have compared conventional fasting to other non-fasting procedures, including low nutrients diet deficient in protein, calcium, and sodium [5,6], high zinc diet [7,8], and feeding high levels of wheat middling  or alfalfa . However, non-fasting procedures have not always been proven to be consistent in ceasing egg production except for a high zinc diet . Feeding a high zinc diet is one alternative to non-fasting molting. It causes less severe stress and provides satisfactory results [8,11]. The objective of this study was to evaluate the effects of four different non-fasting molting methods on the time required to stop and return to egg production and egg quality during molting and post-molting periods.
MATERIALS AND METHODS
The experimental protocol was approved by the Institutional Animal Care and Use Committee at Konkuk University (KU20169).
A total of 400 60-week-old Lohmann Lite layers were used in this study. Prior to experiments, layers had an acclimation period of two weeks. Layers were randomly allotted to the following five dietary treatment groups (eight replicates of 10 for each group) so that egg production was similar for each group: 1) fasting group, layers received no diet from day 1 to day 10 as control; 2) group 2 layers received the molt-inducing diet recommended by the breeding company (MD); 3) group 3 layers received a wheat bran-based diet as a group with low levels of energy, crude protein (CP), amino acids (AA), and Ca (WB); 4) group 4 layers received a commercial layer diet with 8,000 ppm zinc (CZn); and 5) group 5 layers received the molt-inducing diet with 8,000 ppm zinc (MZn). The zinc was provided in the form of zinc oxide (800 g Zn/kg ZnO).
The formula and chemical compositions of experimental diets are shown in Table 1. Experimental diets and water were provided for ad libitum consumption except for the control group during the molting period. At the start of experiment, eight birds from each group were selected, weighed individually, and marked on their legs. A room temperature of 25 ± 3°C and a natural photoperiod of 14 h of light were maintained throughout the entire experimental period. Proximal compositions of formulated diets are shown in Table 1. Analyzed values of zinc were slightly higher than calculated values.
1) Vitamin and mineral mixtures provided the following nutrients per kg of diet: vitamin A, 40,000 IU; vitamin D3, 8,000 IU; vitamin E, 10 IU; vitamin K3, 4 mg; vitamin B1, 4 mg; vitamin B2, 12 mg; vitamin B6, 6 mg; vitamin B12, 0.02 mg; pantothenic acid, 20 mg; folic acid, 2 mg; nicotinic acid, 60 mg; Mn, 25 mg; Zn, 50 mg; Fe, 60 mg; Cu, 10 mg; Co, 0.15 mg; Se, 0.10 mg.
MD, group received induced molting diet; WB, group received wheat bran based diet; CZn, group fed commercial layer diet with 8,000 ppm zinc; MZn, group fed induced molting diet with 8,000 ppm zinc; DDGS, distiller’s dried grains with soluble; ZnO, zinc oxide; CP, crude protein; P, phosphorus; Lys, lysine; Met, methionine; TSAA, the total sulfur amino acids; MEn, nitrogen correted metabolizable energy; DM, dry matter.
Diets were freshly added every day and feed intake was recorded weekly by replicate. Eggs were collected at a fixed time every day. The number and weight of eggs laid were recorded during the molting period. Abnormal eggs were excluded from egg weight measurement. Egg mass was calculated as hen-day egg production multiplied by the average egg weight.
The number of days to the cessation of laying of each treatment of layers was also recorded. When egg production reached 0% and body weight (BW) loss reached 20% or more, molting was terminated. The molted layers were fed the commercial diet immediately. The molting experiment lasted up to 28 d. Even if the molting conditions were not met, layers were fed the commercial diet after 28 d of molting period.
At the end of the molting experiment, one bird per replicate was sacrificed by CO2 asphyxiation. Its ovary and growing oocytes samples were carefully removed and weighed. Relative weights of these samples were expressed as a percentage of live BW.
During the post-molting period, individual BWs of eight hens from each group were monitored at the start and the end of the experiment. All birds were fed the commercial layer diet containing 15% of CP and 2,700 kcal/kg of nitrogen corrected apparent metabolizable energy (AMEn) during the post-molting experiment. The diet and water were provided for ad libitum consumption. Diets were freshly added every day and feed intake was recorded weekly by replicate. Eggs were collected at a fixed time every day. The number and weight of eggs laid were recorded during the post-molting period. Abnormal eggs were excluded for egg weight measurement. Egg mass was calculated as hen-day egg production multiplied by average egg weight. Numbers of days to start laying, days to return to 50% egg production, and days to return to pre-molting egg production were also recorded.
Egg quality was determined bi-weekly during the post-molting experiment. Five eggs from each replicate were collected, weighed, and stored overnight at room temperature (20°C) for subsequent analyses. The breaking strength of sampled eggs was measured using a DET-6000 digital egg tester (Nabel, Kyoto, Japan). Eggshell thickness without shell membrane was measured using a micrometer (series 547-360 Digimatic micrometer, Mitutoyo, Japan). Egg yolk color was determined by comparison with Roche yolk color fan (Hoffman-La Roche, Basel, Switzerland). Albumen height was also measured using the DET-6000 digital egg tester (Nabel). Haugh unit calculation was then performed.
Data were analyzed by the GLM procedure of SAS  with cage lot (two adjacent cages) as experimental units for evaluating egg production and egg quality and individual birds as the unit for other criteria. Significant differences were determined using Duncan’s multiple range test at p < 0.05.
RESULTS AND DISCUSSION
The productivity and BW of layers before and during molting experiment are shown in Table 2. Before molting, the average egg production exceeded 80%, showing no significant difference among groups. Mean egg production in fasting control and groups fed diets with ZnO were significantly lower (p < 0.001) than those of MD and WB during 7 d of molting. Egg production in the latter two groups were reduced by 10 % or slightly more than those before molting. The mean egg weight was the lowest in the fasting control group but the highest in the WB group during 7 d of molting. Egg weights in groups fed diets with ZnO were significantly lower than that of the WB group, but higher than that of the fasting control group (p < 0.001).
Egg production in fasting control was rapidly reduced. It ceased at 5.9 d of molting. In both molt treatment with ZnO, egg production was similarly reduced and ceased after 6.9 d and 7.0 d of molting period, respectively, none of which differed significantly from that of the control. After egg production had stopped, layers kept fasting or fed diets with ZnO to obtain the weight loss goal until 10 d of the experiment. Layers fed MD and WB did not reach cessation of laying even on the 28 d of molting period. Therefore, it was judged that molting was incomplete in these two groups fed diets without ZnO.
Conventional feed withdrawal procedure seems to be the fastest way to stop egg laying, even if it goes against animal welfare. When feed deprivation is used in a molting procedure, egg laying is stopped on 6 d . Machebe et al.  have reported that layers fed diets with high levels of zinc (5,000 to 20,000 ppm as ZnO) show rapid reduction in egg production and cease laying within 9 d of molting experiment. Similarly, layers fed diets with varying levels of ZnO show dramatic reduction in egg production and stop laying within 6.8 d of experiment in a linear manner . On the other hand, diets with low levels of CP or nutrients and WB have not always been proven to be consistent in ceasing egg production . Hwangbo et al.  have found that layers fed diet containing 90% or 70% wheat bran show reduced egg production of 31.0% or 28.4%, respectively, and that laying does not stop completely until the end of a 4-wk experiment. When layers are fed a low CP diet, egg production slows down and stops after 4 weeks of feeding . In the present study, layers fed a MD with low nutrients and a WB did not reach the cessation of laying even on the 28 d of molting period. Both groups (MD and WB) did not reach the expected molting index either.
Average feed intake of CZn and MZn were significantly lower than those of MD and WB during 10 d of molting (p < 0.001). There were significant differences in BW and weight loss % among groups at different molting periods (10th d for control group and groups fed diets with ZnO; 28th d for groups fed diets without ZnO). Layers in the fasting group had the highest weight loss, followed by MZn and CZn at 10th d of molting period. Weight loss % in MD and WB groups were relatively low (−16.6 and −13.1, respectively)
Weight loss is directly associated with a decrease in feed intake. Layers fed diets with ZnO had lower feed intake (21.8 g, and 18.4 g/hen per day) than did layers fed MD and WB. The decreased feed intake could be due to low palatability of high levels of zinc  or ability of zinc cation to induce follicular atresia . In this study, observations of BW and weight loss regardless of molting period (10th d or 28th d) were in agreements with previous studies. Laying hens that are subjected to conventional feed withdrawal lose 20% of BW on 5 d of experiment . In contrast, BW loss % in MD and WB groups did not reach the weight loss goal even on the 28 d of molting period. The BW loss of layers subjected to fasting and fed diets with ZnO were within the ranges suggested by Baker et al.  for proper post-molting performance. Layers fed diets with ZnO had greater BW loss (at an average of 20.2% from initial BW) at the end of 10 d molting period than any other non-fasting treatments even on the 28 d.
Relative weights of ovary and growing oocytes in layers subjected to fasting and fed diets with ZnO were significantly lower than those of MD and WB groups at 10th d or 28th d of molting period each (p < 0.001). Layers subjected to conventional fasting had the lowest relative weight, followed by CZn and MZn, none of which differed significantly (Table 2).
The regression of ovary is directly related to the rejuvenation process . Weight loss of ovary and growing oocytes occurred simultaneously with BW loss in this study. There was no significant difference in relative ovary weight among fasting control and groups fed diets with ZnO. These observations are consistent with a previous report . It has been suggested that high levels of dietary zinc can induce follicular atresia, which can lead to subsequent cessation of egg production by interfering with the process of ovulation in laying hens .
Egg production, the number of days to start laying, days to return to 50% egg production, and days to return to pre-molting egg production in three groups with complete molting are shown in Table 3. During the first 2 wks of post-molting, MZn showed higher egg production than those in the other two groups (p < 0.01). Egg weight and daily egg mass in the MZn group were significantly higher than those of fasting control (p < 0.05). There was no significant difference in feed intake among three groups during this period. Days to start laying in two groups fed diets with ZnO were significantly reduced than that in the group subjected to fasting (7.9 d and 7.8 d vs. 9.5 d) (p < 0.05). There was no significant difference in days to return to 50% egg production or return to pre-molting levels among groups.
At the start of the post-molting experiment, the BW was the lowest in the fasting control group (p < 0.01). However, layers recovered the BW loss after 6 wks of feeding. The weight gain % ranged from 16.9 for WB group to 33.1 for fasting control (p < 0.001). From 3 to 6 wk of post-molting period, egg production in control group and groups fed diets with ZnO were numerically higher but not significantly higher than those of incomplete molting groups. Egg weight in the control group was significantly higher than the group fed MD without ZnO (p < 0.05). There was no significant difference in daily egg mass or feed intake among groups during this period (Table 4).
There was no significant difference in eggshell strength among groups at 2 wk of post-molting period (Table 5). The eggshell strength in the CZn group was significantly higher than those of incomplete molting groups at 6 wk of post-molting period (p < 0.05). The Haugh unit was the highest in the fasting control group than in other groups at 2 wk of post-molting period (p < 0.01), although it was not significantly different at 4 wk or 6 wk of post-molting period. Yolk color values in the control group and groups fed diets with ZnO were significantly higher than those of incomplete molting groups at 4 wk of post-molting period (p < 0.001).
It was shown that 0% egg production could not be obtained by MD or WB group in this study. Thus, these two groups were excluded from the recovery of egg production. The MZn group showed higher egg production and egg weight than those of the fasting control group during the first 2 wks of recovery (p < 0.05). Days to start of laying were about 1 d earlier in groups fed diets with ZnO, although there were no significant differences in days to return to egg production or to pre-molting levels among the three treatments (Table 3). Similarly, Goodman et al.  have found no significant differences in days to return to 50% egg production or subsequent laying performance during 22 wks of experiment.
From 3 to 6 wks of post-molting period, egg production in the control group and groups fed diets with ZnO were numerically but not significantly higher compared with those of incomplete molting groups (Table 4). During this period, egg production and egg weight results were shown to be similar between layers subjected to fasting and layers fed diets with ZnO during the post-molting period. As previously reported, egg production of layers fed diets with ZnO as well than layers molted by fasting performed during post-molting period . With similar methods of molting, Onbaşılar and Erol  have also found no difference in egg production between conventional fasting and a zinc-based diet. It has been suggested that properly induced molting is an effective way to improve eggshell quality in aged laying flocks [4,21]. The eggshell strength was higher in the CZn group than those of incomplete molting groups at 6 wk of post-molting period (p < 0.05). Difference in eggshell strength tended to lack significance between the group subjected to fasting and groups fed diets with ZnO. These results were consistent with results of Karimi et al.  that showed no significant difference in eggshell quality between post-molt eggs from layers molted by fasting and those by a high zinc diet. Based on egg production and egg quality, molting diets with ZnO are similarly effective in inducing molting and increasing post-molt performance than the conventional fasting method.
The present study provides available information on the time required to stop and return to egg production by application of non-feed withdrawal molting during molting and post-molting periods. Results of this study indicate that a non-feed withdrawal molting using ZnO can be used in the industry with success. In addition, these molting diets do not seem to disturb the normal physiology of layers based on performance indices. It is considered that more than 5,000 ppm of zinc is required for satisfactory molting . But the use of zinc in feed should be strictly regulated due to the environmental emission of zinc. Further studies are needed to clarify the minimum levels of dietary zinc while effective for non-feed withdrawal molting.