Induced molt using cassava meal. 2. Effects on eggshell quality, ultrastructure, and pore density in late-phase laying hens

Induced molt using cassava meal. 2. Effects on eggshell quality, ultrastructure, and pore density... Abstract This experiment was conducted to investigate the effect of a non-fasting induced molt using cassava meal on the eggshell quality, ultrastructure, and porosity in late-phase (74 wk old) H&N Brown laying hens. Hens were randomly assigned to 3 treatments of 90 birds each: 1) Controls with no induced molt (CONT); 2) molted by full feeding with cassava meal for 3 wk (FP3); and 3) molted by full feeding with cassava meal for 4 wk (FP4). Following the treatments, groups 2 and 3 were fed a pullet developer diet for 3 weeks. During the molt period, the birds were exposed to an 8L:16D photoperiod and had access to drinking water at all times. Thereafter, all hens were fed a layer diet (17% CP) and exposed to a 16L:8D photoperiod until the end of the study. Compared to the CONT treatment, significant reductions (P < 0.05) in shell weight, thickness, and breaking strength were identified on the sixth d of feeding the molt diet. Significant (P < 0.05) improvements in these parameters were observed for the FP3 and FP4 treatments during the post-molt period, with the greater degree in the FP4 treatment. In addition, scanning electron microscopy revealed a smaller size of mammillary knobs accompanied by a higher density of mammillae in eggs taken from the molted treatments. Evidence of type B mammillae was detected in an egg produced by the CONT hens, whereas confluent and cuffing mammillae were observed in an egg taken from the FP4 birds. Reduced pore densities were found in the molted treatments in some periods of the post-molt production as compared to the CONT treatment. It was concluded that feeding the cassava molt diet for 4 wk could be an effective non-fasting molt method for improving eggshell quality, ultrastructure, and porosity in post-molt laying hens. INTRODUCTION Eggshell quality is an important contributing factor in table and hatching eggs. The eggshell significance is related to its function to provide a container for the egg contents and protect them from bacterial contamination. Approximately 8 to 10% of the eggs laid for the table industry suffer shell damages during routine handling, causing economic losses (Hamilton et al., 1979). Therefore, the shell must be strong enough to prevent failure during packaging and transportation processes. Most eggshell quality measurements have particularly focused on the amount of eggshell mass, including its weight, thickness, and specific gravity. However, the strength of the eggshell depends not only on its weight and thickness but also on the quality of its microstructure. As reviewed by Roberts and Brackpool (1994), Wilhelm von Nathusius, the first person who studied the ultrastructure of the avian eggshell, described and named the mammillary layer of the eggshell. Reid (1984) reported that there was some evidence of a correlation between mammillary changes and poor shell quality. Solomon (1991) observed the greatest variation in eggshell ultrastructure occurred in the mammillary layer and described a number of abnormalities. Bain (1992) summarized the structural variations increasing the resistance of eggshells to unstable fracture, such as early fusion, cuffing, and confluent mammillae, and those decreasing the resistance of the shell, such as late fusion and type B mammillae. Eggshell pores are small holes that transfer gases and water vapor between the embryo and the outside environment. The pore system of the avian eggshell is located at a specific location between the eggshell cones to provide gas and humidity exchanges. In chicken eggs, typical pores have a funnel-shaped orifice opening at the outer surface of the shell at the level of the cuticle, and a single channel passing through the vertical crystal layer and the palisade region to open at the inner surface of the eggshell between adjacent mammillae (Chien et al., 2008). Pore formation begins at the level of the mammillary layer with the grouping of 4 to 5 mammillary bodies (Solomon, 2010). The distribution of pores in the eggshell surface of the domestic hens is estimated to be between 100 and 300 pores/cm2 (Gilbert, 1971). Klingensmith and Hester (1985) investigated the relationship between pore density and shell quality and reported that hard-shelled eggs exhibited a lower density of pores as compared to that of soft-shelled eggs. Molting in avian species is characterized by a period of rest whereby egg production ceases, reproductive tract regresses, and renewal of feathers occurs (Berry, 2003). Decreasing d length during the molt period initiates gonadal regression, prompting molting and rejuvenation (Follett and Sharp, 1969). Induced molt of laying hens is used by commercial egg producers to extend the reproductive period of their flock. Conventional induced molting programs usually involve a restricted or shorter photoperiod to natural light (Hambree et al., 1980), and the removal of feed (Christmas et al., 1985) and occasionally drinking water (Brake and Thaxton, 1982). Egg production resumes and increases rapidly to a profitable rate following the artificial molt (Hurwitz et al., 1975; Lee, 1982). Albumen quality (Lee, 1982; Tona et al., 2002) and eggshell quality (Hurwitz et al., 1975; Lee, 1982; Christmas et al., 1985) also are improved by induced molting in the subsequent laying cycle. However, the conventional molting method has drawn criticism due to concerns of animal welfare and food safety. Various non-fasting induced molting techniques have been suggested. These alternate methods use dietary manipulations to create an imbalance of particular nutrients (Biggs et al, 2003; Gongruttananun et al., 2013; Bozkurt et al., 2016). There is a deficiency of information concerning the effects of a non-fasting induced molt technique on eggshell ultrastructure and pore density. Therefore, the objective of the present study was to determine the effects of an induced molt technique using cassava meal on eggshell quality, microstructure, and pore density in late-phase (74 wk) laying hens. MATERIALS AND METHODS All animal care procedures were approved by the Animal Ethics Committee of Kasetsart University. The Ethics regulation code is ACKU 00159. Before the experiment began, 270 H&N Brown late-phase laying hens (72 wk old) of similar body weight (BW) (1875 ± 20 g) were housed in a caged layer shed with water and feed provided for ad libitum consumption and a 16-hour exposure to daily photoperiod. The mean temperature of the house was 27.6 ± 1.2 °C, and the average light intensity was 4.7 ± 0.8 lx. The feed was a commercial layer diet calculated to contain 17% CP, 2,800 kcal of ME/kg of feed, and 3.5% Ca. Five replicate groups of 18 hens each (6 adjacent cages containing 3 hens/cage, 40 × 45 cm) were allotted to 3 treatments in a completely randomized design. Birds were weighed and allocated to each replicate to achieve a similar mean BW for each treatment. Egg production, egg weight, and egg quality were measured for 2 wk (72 to 73 wk of age) before the treatments began, in an attempt to keep a similar distribution of production rate, egg weight, and egg quality among the treatments. The 3 treatments were designated as follows: non-molted control treatment (CONT), in which the layer diet was provided and hens were exposed to the 16L:8D photoperiod throughout the study; the other 2 treatments were induced to molt by full feeding with a cassava molt diet for 3 (FP3) or 4 (FP4) wk according to the following procedure. At 74 wk of age, the FP3 and FP4 treatments were carefully transferred to a windowless molting house equipped with mechanical ventilation. The mean temperature of the house was 28.03 ± 1.31°C, and the mean light intensity was 15.4 ± 1.8 lx. Birds in each replicate of the molted treatments were housed together in one of the 10 pens located in the molting house. Each pen was 2.9 × 3.0 × 2.9 m (width × length × height). The induced molt period was divided into a 3-, or 4-week “stress period” and a 3-week “recovery period.” Birds in the FP3 and FP4 treatments had the stress period of 3 and 4 wk, respectively, during which the cassava molt diet and drinking water were accessible at all times. At the end of each stress period, the FP3 and FP4 birds were weighed, and body weight loss was calculated (the data are given in the companion paper; Gongruttananun et al., 2017). Then, birds in all molted treatments had a 3-week recovery period, during which a pullet developer diet and drinking water were provided at all times. At the end of each recovery period, the birds were carefully transferred back to their original house, where the CONT birds were being kept and maintained under the same management regime until the end of the study (95 wk of age). During the molt period, the photoperiod was reduced from the usual 16 h per d (16L:8D) to 8 h per d (8L:16D). The molting protocol is shown in Table 1, and the ingredient composition of the experimental diets, and the preparation of the cassava molt diet are given in the companion paper (Gongruttananun et al., 2017) Table 1. Molting procedure used in the experiment. Treatment1  Treatment type  Drinking water  CONT  Control (hens not induced to molt, consuming ad libitum on a layer diet under a lighting program of 16L:8D throughout the experimental period)  Provided  FP3  Cassava meal offered for 3 wk (74 to 76 wk of age) and a pullet developer diet offered for 3 wk (77 to 79 wk of age) under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  FP4  Cassava meal offered for 4 wk (74 to 77 wk of age) and a pullet developer diet offered for 3 wk (78 to 80 wk of age), under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  Treatment1  Treatment type  Drinking water  CONT  Control (hens not induced to molt, consuming ad libitum on a layer diet under a lighting program of 16L:8D throughout the experimental period)  Provided  FP3  Cassava meal offered for 3 wk (74 to 76 wk of age) and a pullet developer diet offered for 3 wk (77 to 79 wk of age) under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  FP4  Cassava meal offered for 4 wk (74 to 77 wk of age) and a pullet developer diet offered for 3 wk (78 to 80 wk of age), under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  1CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Eggshell quality measurement At the termination of the acclimatization, at 73 wk of age, the eggshell quality of all eggs collected from each replicate was measured for the pretest data. The measurement was repeated on d 6 of the molt period. During the post-molt period, 5 eggshell quality measurements were taken and recorded at the ages of 81, 85, 87, 91, and 93 weeks. The eggshell strength was measured using an Eggshell Destruction Strength Meter (Model EFG-0503 Robotmation, Co., Ltd., Tokyo, Japan). Thereafter, the eggs were broken at the equatorial region, and the interior contents were allowed to drain out. The eggshell, with the membranes, was cleaned with tap water and dried at room temperature for one week. After drying, the eggshell was weighed on an electric balance (Model PB 1501, Metler, Toledo, OH), and its thickness was measured in millimeters with a digimatic micrometer (Mitutoyo Corporation, Kanagawa, Japan). Three measurements were taken on the equatorial region on each eggshell, and the mean of 3 measurements was calculated. Preparation of samples for eggshell ultrastructural examination Two eggs from each replicate laid on the same d at 73 wk of age (pretest), on d 6 of feeding the molt diet (during the molt period), and at 84 wk of age (during the post-molt period) were randomly collected for the examination of the microstructure of the eggshell. The selected eggs were broken, the interior contents were removed, and the shells were cleaned with tap water. The specimens were prepared by cutting a piece (1 cm2) of shell from the equatorial region of each egg. The shell membranes were carefully removed after soaking in tap water. The loosely adhering membranes were peeled from the edge of the specimens inward. The samples were then soaked in 1.0 N NaOH for 72 h to eliminate any protein materials incorporated in the shell before being processed and studied, according to the method of Kaplan and Siegesmund (1973). Thereafter, the samples were rinsed in distilled water and left to dry at room temperature. All samples were mounted with their uppermost inner sides on aluminum stubs and coated with gold using an ion coater (Eiko Engineer IB-2, Eiko Engineering Co. Ltd., Ibaraki, Japan) for direct observation by a scanning electron microscope. These specimens were examined using a JEOL JSM-5600LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) operated at 10 kV, at magnifications of 100×, 200×, and 500×. The incidence of ultrastructural variants at the level of the mammillary layer was assessed according to the method and terminology developed by the Poultry Research Unit, University of Glasgow (Solomon, 1991). Photographs of replica surfaces were made to facilitate counting the number of mammillary knobs per unit of the interior surface of the eggshell. The density of mammillae of each shell was expressed as the number of knobs per unit. The average diameter of the mammillary knobs was estimated from the measured mammillary knob density, assuming regular circular geometry, according to the method of Van Toledo et al. (1982). Measurements of eggshell pore density Pore density was examined before the treatments (at 73 wk of age), on d 6 of feeding the molt diet and during the post-molt period, at 83, 87, 91, and 95 wk of age. Five eggs from each replicate laid on the same d were randomly collected for the examination of pore density. Egg contents were removed, and the shells were cleaned with tap water. Four 1 cm2 eggshell chips were taken from each area of 3 regions of the collected eggs: the large end, equator, and small end for the measurements of pore density (Peebles and Brake, 1985), and shell membrane etching was processed by soaking in 1.0 N NaOH for 72 h, according to the method of Kaplan and Siegesmund (1973). Thereafter, the shell samples were put in a biopsy cassette and dipped in a concentrated nitric acid solution for 7 seconds. The reaction was stopped by rinsing in distilled water. The shells were again removed from the tissue cassettes and left to dry overnight, then painted on the inner side with trypan blue in a 2% acetic acid solution, using a fine paintbrush. Pores were counted using a stereomicroscope (Model Nikon 204,825, Nikon Instruments Inc., Tokyo, Japan) at a magnification of 40× and expressed in number per .25 cm2. Pore density of each replicate was computed for the mean of the pores per .25 cm2 of shell surface found in the 12 areas counted on each egg of the 5 selected eggs. Statistical analysis The experiment was conducted as a completely randomized design with 3 treatments. Data were analyzed using the statistical software package SAS, version 9.0 (SAS Institute Inc., 2002). The GLM procedure was used to analyze the effects of the treatment on shell weight, shell thickness, shell breaking strength, mammillary density, mammillary knob diameter, and pore density. An arcsine transformation was used for all percentage data. Duncan's multiple-range test was used to estimate significant differences among treatment means (Snedecor and Cochran, 1980). Significance was based on P < 0.05. The experimental unit was each group of 18 hens for all traits studied. For the determination of eggshell microstructure and pore density, 2 and 5 eggs per replicate were used, respectively. Results in the tables are presented as means and the pooled SEM. RESULTS At the end of each stress period, BW loss of birds in the FP4 treatment was approximately 30.13%, whereas the mean of the FP3 hens was 25.23% (data are given in the companion paper; Gongruttananun et al., 2017). Eggshell quality Table 2 demonstrates the effects of molt treatments on shell weight of the experimental birds in comparison to the CONT treatment. It was found that on d 6 of feeding the molt diet, averages of shell weight of the 2 molted treatments were significantly lower than those of the CONT treatment. During the post-molt period, shell weights of the 2 molted treatments were significantly higher than those of the CONT treatment observed at 87, 91, and 93 wk of age. The effects of molt treatments on shell thickness and shell breaking strength are presented in Tables 3 and 4, respectively. During the molt period, the 2 molted treatments had an inferior value of these parameters as compared to the CONT treatment. During the post-molt period, the FP4 treatment had a significantly higher level of shell thickness measured at 81, 85, 91, and 93 wk of age, whereas shell thickness of the FP3 treatment was significantly higher than that of the CONT treatment only at 85 wk of age. During the post-molt period, the means of shell breaking strength of the FP4 treatment were significantly higher than those of the CONT treatment in every period of the measurements. A significant improvement in shell breaking strength also was observed for the FP3 treatment, except for that measured at 81 wk of age. There was no significant difference in shell weight or shell breaking strength between the FP3 and FP4 treatments throughout the study. Table 2. Effect of non-molted and molted treatments on shell weight throughout the study.1   Shell weight (%)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  8.78  8.83a  8.98  8.79  8.65b  8.71b  8.84b  FP3  8.68  7.35b  9.13  9.10  8.98a  9.12a  9.28a  FP4  8.94  7.27b  9.40  9.23  9.13a  9.22a  9.35a  Pooled SEM  0.37  0.83  0.42  0.29  0.18  0.25  0.22  P-value  0.552  0.001  0.326  0.095  0.003  0.017  0.008    Shell weight (%)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  8.78  8.83a  8.98  8.79  8.65b  8.71b  8.84b  FP3  8.68  7.35b  9.13  9.10  8.98a  9.12a  9.28a  FP4  8.94  7.27b  9.40  9.23  9.13a  9.22a  9.35a  Pooled SEM  0.37  0.83  0.42  0.29  0.18  0.25  0.22  P-value  0.552  0.001  0.326  0.095  0.003  0.017  0.008  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1Data are means of 5 groups of 18 hens each. 2CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Table 3. Effect of non-molted and molted treatments on shell thickness throughout the study.1   Shell thickness (mm)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  0.351  0.359a  0.373b  0.356b  0.356  0.358b  0.362b  FP3  0.343  0.278b  0.374b  0.375a  0.358  0.369b  0.371a,b  FP4  0.351  0.269b  0.390a  0.368a  0.374  0.408a  0.388a  Pooled SEM  0.014  0.028  0.010  0.007  0.013  0.011  0.009  P-value  0.629  <0.001  0.046  0.007  0.104  <0.001  0.002    Shell thickness (mm)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  0.351  0.359a  0.373b  0.356b  0.356  0.358b  0.362b  FP3  0.343  0.278b  0.374b  0.375a  0.358  0.369b  0.371a,b  FP4  0.351  0.269b  0.390a  0.368a  0.374  0.408a  0.388a  Pooled SEM  0.014  0.028  0.010  0.007  0.013  0.011  0.009  P-value  0.629  <0.001  0.046  0.007  0.104  <0.001  0.002  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1Data are means of 5 groups of 18 hens each. 2CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Table 4. Effect of non-molted and molted treatments on shell breaking strength throughout the study.1   Shell breaking strength (N)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  29.79  32.35a  30.82b  30.29b  27.68b  28.88b  28.66b  FP3  29.44  18.47b  37.38a,b  34.93a  32.98a  33.43a  32.51a  FP4  29.04  19.90b  39.77a  36.50a  35.00a  33.59a  33.97a  Pooled SEM  3.44  5.41  4.04  2.08  1.21  2.13  1.54  P-value  0.943  <0.001  0.012  0.001  <0.001  0.007  <0.001    Shell breaking strength (N)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  29.79  32.35a  30.82b  30.29b  27.68b  28.88b  28.66b  FP3  29.44  18.47b  37.38a,b  34.93a  32.98a  33.43a  32.51a  FP4  29.04  19.90b  39.77a  36.50a  35.00a  33.59a  33.97a  Pooled SEM  3.44  5.41  4.04  2.08  1.21  2.13  1.54  P-value  0.943  <0.001  0.012  0.001  <0.001  0.007  <0.001  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1Data are means of 5 groups of 18 hens each. 2CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Eggshell ultrastructure Table 5 summarizes the density and diameter of the mammillary knobs of selected eggshells. There were no significant differences for the density of the mammillae and the diameter of the mammillary knobs among the experimental groups at pre-molt and on d 6 of feeding the molt diet. During the post-molt period, the density of the mammillae of the FP3 (229.9 knobs/mm2) and FP4 (231.7 knobs/mm2) treatments was significantly (P < 0.05) higher than that of the CONT treatment (182.4 knobs/mm2). Conversely, a significantly lower value of the diameter of the mammillary knobs was observed in the 2 molted treatments (0.074 to 0.075 mm) as compared to that of the CONT treatment (0.085 mm). Scanning electron microscopy inspection at a magnification of 100 × revealed normal formation of mammillae with rounded caps in all samples of the shells taken from the 3 experimental groups measured on d 6 of feeding the molt diet (Figure 1). However, a depression in the thickness of the shells was detected in the FP3 and FP4 treatments compared with the CONT treatment as revealed by a transverse view (200×) (Figure 2). Figure 3 shows a mammillary view of the shells produced from hens in the 3 treatment groups examined during the post-molt period (95 wk of age). The examination at a magnification of 500× showed evidence of type B mammillary bodies in a shell sample produced from the CONT hens (Figure 4). The photograph revealed evidence of confluence in one shell sample produced from the FP3 hens. In addition, the evidence of confluence, including of cuffing, was detected in one shell sample taken from the FP4 treatment. During the post-molt period, shell thickness of the FP3 and FP4 treatments was greater than that of the CONT treatment as illustrated by a transverse view (200×) (Figure 5). Figure 1. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 1. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 2. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that a thinner shell thickness is illustrated in the shell structure of the FP3 and FP4 treatments; 200× (scale bar = 200 μm). Figure 2. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that a thinner shell thickness is illustrated in the shell structure of the FP3 and FP4 treatments; 200× (scale bar = 200 μm). Figure 3. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 3. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 4. View largeDownload slide Enlargement of a portion of Figures 3A (A), 3B (B), and 3C (C and D) showing ultrastructure variations of the mammillary layer of eggs. Note that type B mammillary bodies (B) are illustrated in the CONT treatment (A), confluence is observed in the FP3 treatment (B), and cuffing (C) and confluence (D) are revealed in the FP4 treatment; 500× (scale bar = 100 μm). Figure 4. View largeDownload slide Enlargement of a portion of Figures 3A (A), 3B (B), and 3C (C and D) showing ultrastructure variations of the mammillary layer of eggs. Note that type B mammillary bodies (B) are illustrated in the CONT treatment (A), confluence is observed in the FP3 treatment (B), and cuffing (C) and confluence (D) are revealed in the FP4 treatment; 500× (scale bar = 100 μm). Figure 5. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that the thickness of shell of the FP3 and FP4 treatments is greater than that of the CONT treatment; 200× (scale bar = 200 μm). Figure 5. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that the thickness of shell of the FP3 and FP4 treatments is greater than that of the CONT treatment; 200× (scale bar = 200 μm). Table 5. Effect of non-molted and molted treatments on mammillary density and mammillary knob diameter of the eggshell produced by the experimental hens throughout the study.   Number  Mammillary density (knobs/mm2)  Mammillary knob diameter (mm)    of  Pre-molt  During molt  Post-molt  Pre-molt  During molt  Post-molt    samples  (73 wk)  (Day 6 of molt)  (95 wk)  (73 wk)  (Day 6 of molt)  (95 wk)  Treatment1                CONT  10  206.5  159.8  182.4b  0.076  0.090  0.085a  FP3  10  194.1  148.4  229.9a  0.088  0.094  0.075b  FP4  10  220.7  170.8  231.7a  0.071  0.086  0.074b  Pooled SEM    49.9  30.5  37.7  0.011  0.007  0.008  P-value    0.67  0.46  0.02  0.48  0.31  0.01    Number  Mammillary density (knobs/mm2)  Mammillary knob diameter (mm)    of  Pre-molt  During molt  Post-molt  Pre-molt  During molt  Post-molt    samples  (73 wk)  (Day 6 of molt)  (95 wk)  (73 wk)  (Day 6 of molt)  (95 wk)  Treatment1                CONT  10  206.5  159.8  182.4b  0.076  0.090  0.085a  FP3  10  194.1  148.4  229.9a  0.088  0.094  0.075b  FP4  10  220.7  170.8  231.7a  0.071  0.086  0.074b  Pooled SEM    49.9  30.5  37.7  0.011  0.007  0.008  P-value    0.67  0.46  0.02  0.48  0.31  0.01  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Eggshell porosity Table 6 depicts the effects of induced molt treatments on pore density of the experimental birds. Before the treatments and on d 6 of feeding the molt diet, there was no significant difference in means of pore density among the treatment groups. During the post-molt period, pore densities of the FP4 treatment were significantly higher than those of the CONT treatment observed at 83 and 95 wk of age. A significant reduction in pore density also was found at 83 wk of age in the FP3 treatment. Table 6. Effect of non-molted and molted treatments on pore density (pores/.25 cm2) of the eggshell produced by the experimental hens throughout the study.   Number of  Pre-molt  During molt  Post-molt (wk)    samples  (73 wk)  (Day 6 of molt)  83  87  91  95  Treatment1                CONT  25  30.6  31.7  31.7a  32.0  32.9  31.7a  FP3  25  28.2  33.1  28.5b  29.6  30.4  28.2a,b  FP4  25  30.6  35.0  28.4b  30.4  28.3  27.7b  Pooled SEM    2.2  8.0  1.7  2.4  2.8  2.0  P-value    0.215  0.806  0.018  0.305  0.076  0.022    Number of  Pre-molt  During molt  Post-molt (wk)    samples  (73 wk)  (Day 6 of molt)  83  87  91  95  Treatment1                CONT  25  30.6  31.7  31.7a  32.0  32.9  31.7a  FP3  25  28.2  33.1  28.5b  29.6  30.4  28.2a,b  FP4  25  30.6  35.0  28.4b  30.4  28.3  27.7b  Pooled SEM    2.2  8.0  1.7  2.4  2.8  2.0  P-value    0.215  0.806  0.018  0.305  0.076  0.022  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large DISCUSSION From the results of the current study, shell weight, shell thickness, and shell breaking strength of the FP3 and FP4 hens declined significantly during the molt period, and after the molt all these parameters were significantly improved. The findings of the current study demonstrating that induced molting has beneficial effects on improvements in shell quality during the post-molt period are consistent with those reported by several investigators (Roland and Bushong, 1978; Baker et al., 1983; Roberts and Brackpool, 1994; Biggs et al., 2003; Bozkurt et al., 2016). However, it is apparent that the degree of improvement in shell quality of the FP4 treatment was greater than that of the FP3 treatment. This suggests a result of BW loss up to 30.13% in the FP4 hens, whereas BW loss of the FP3 birds was only 25.23%. Baker et al. (1983) reported that improvements in post-molt performance were related to an up to 31% increase in BW loss of hens (of their original BW). The investigators also suggested that hens that lost the greatest amount of BW exhibited the greatest improvements in eggshell quality in the subsequent laying cycle. Eggshell strength is significantly important for egg producers in reducing the economic losses from broken eggs. Eggshell breaking strength decreases through aging (Tumova et al., 2014). The strength of the eggshell depends both on its weight and thickness and the quality of its ultrastructure (Roberts and Brackpool, 1994). In the present study, eggshells were collected at 3 phases: prior to, during, and following the molt, to examine the microstructure and pore density. It is apparent that the diameter of mammillary knobs of the FP3 and FP4 treatments significantly reduced during the post-molt period as compared with that of the CONT treatment. On the other hand, the density of mammillae of the molted treatments was significantly higher than that of the non-molted treatment. These results are in agreement with several studies (Robinson and King, 1970; Bunk and Balloun, 1978; Klingensmith and Hester, 1985) that reported that a higher density of mammillae resulted in a stronger shell. In addition, results from the present study revealed evidence of type B mammillae in a shell sample produced from the CONT treatment. The type B mammillary bodies are small spherical bodies located within the mammillary layer of the eggshell, which may or may not attach to the shell membrane layer (Reid, 1984). These structures have been observed in poor shell quality by some investigators (Bunk and Balloun, 1978). In a previous study (Gongruttananun et al., 2013), the author found type B mammillary bodies in the mammillary layer of shells produced by non-molted hens aged 101 weeks. It is possible to postulate that aging is an important factor causing the evidence of type B mammillae, suggesting that the efficiency of shell gland function reduces with the age of hens. Nascimento et al. (1992) reported previously that eggshell quality, including ultrastructural features, decreased with the age of the hen. Interestingly, the results from the present study found evidence of confluent mammillae in the shell samples taken from the FP3 and FP4 hens. Also, evidence of cuffing was observed in the shell sample from the FP4 treatment. Roberts and Brackpool (1994) pointed out that the confluence, the condition in which the mammillary caps combine to form large areas of fusion at the cap sites (Solomon, 1991), made the region itself stronger and influenced the formation of the palisade layer of the shell. Cuffing, the distribution of additional calcite crystals around and between the adjacent mammillary cores (Reid, 1984), may increase the strength of the shell by increasing its effective thickness (Roberts and Brackpool, 1994). In addition, it is of interest to note that the density of pores of the shells taken from the molted treatments was significantly lower than that of the CONT treatment at some periods of the post-molt production (Table 6). These suggest that reduced pore density could be a contributing factor in the strength of the shell. From the present study, beneficial effects of the improvements in shell quality, ultrastructure, and porosity of which were found in the molted treatments, especially the FP4 treatment, would be due to remodeling of shell gland tissues following oviducal regression (Heryanto et al., 1997), leading to shell gland function improvement. Remodeling of the shell gland tissues also may be responsible for removing substances that are obstacles to the function of the shell gland, such as lipid. Baker et al. (1980) reported that the lipid content of the shell gland increased as hens aged. In another study, Baker et al. (1981) demonstrated that induced molting removed lipid accumulation in the shell gland and reduced the incidence of shell-less eggs. It is well documented that removal of lipid accumulation in the shell gland can be achieved when BW loss during molting is greater than 25%, which coincides with maximum oviducal regression (Baker et al., 1980; Brake and McDaniel, 1981; Brake et al., 1981). In summary, the results obtained from the present study indicated that feeding cassava meal for 4 wk and allowing a 3-week recovery period under an 8L:16D photoperiod is an effective non-fasting molt method for improving eggshell quality, microstructure, and porosity in laying hens. The results suggest that improving an eggshell's ultrastructure and reducing pore density may increase the strength of shell. ACKNOWLEDGEMENTS The research project was funded by the Kasetsart University Research and Development Institute, Kasetsart University, Bangkok, Thailand. REFERENCES Bain M. M. 1992. Eggshell strength: A relationship between the mechanism of failure and the ultrastructural organisation of the mammillary layer. Br. Poult. Sci.  33: 303−319. Google Scholar CrossRef Search ADS   Baker M., Brake J., Krista L. M.. 1980. Histological study of uterine lipid distribution in the laying hen. Poult. Sci.  59: 1557. (Abstr.). Google Scholar CrossRef Search ADS   Baker M., Brake J., Krista L. M.. 1981. Total body lipid and uterine lipid changes during a forced molt of caged layers. Poult. Sci.  60: 1593. (Abstr.). Google Scholar CrossRef Search ADS   Baker M., Brake J., McDaniel G. R.. 1983. The relationship between body weight loss during an induced molt and postmolt egg production, egg weight, and shell quality in caged layers. Poult. Sci.  62: 409−413. Berry W. D. 2003. The physiology of induced molting. Poult. Sci.  82: 971−980. Google Scholar CrossRef Search ADS   Biggs P. E., Dauglas M. W., Koelkebeck K. W., Parsons C. M.. 2003. 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Weight loss characteristics of the body, liver, ovary, oviduct and uterine lipid during a forced molt and their relationship to postmolt performance. Poult. Sci.  60: 1628. (Abstr.). Bunk M. J., Balloun S. L.. 1978. Ultrastructure of the mammillary region of low puncture strength avian eggshells. Poult. Sci.  57: 639−647. Google Scholar CrossRef Search ADS   Chien Y. C., Hincke M. T., Vali H., Mckee M. D.. 2008. Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro. J. Struct. Biol.  163: 84−99. Google Scholar CrossRef Search ADS PubMed  Christmas R. B., Harms R. H., Junqueira O. M.. 1985. Performance of Single Comb White Leghorn hens subjected to 4 or 10-day feed withdrawal force rest procedures. Poult. Sci.  64: 2321−2324. Google Scholar CrossRef Search ADS   Follett B. K., Sharp P. J.. 1969. Circadian rhythmicity in photoperiodically induced gonadotropin release and gonadal growth in the quail. Nature  223: 968−971. 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Induced molt using cassava meal. 2. Effects on eggshell quality, ultrastructure, and pore density in late-phase laying hens

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Abstract

Abstract This experiment was conducted to investigate the effect of a non-fasting induced molt using cassava meal on the eggshell quality, ultrastructure, and porosity in late-phase (74 wk old) H&N Brown laying hens. Hens were randomly assigned to 3 treatments of 90 birds each: 1) Controls with no induced molt (CONT); 2) molted by full feeding with cassava meal for 3 wk (FP3); and 3) molted by full feeding with cassava meal for 4 wk (FP4). Following the treatments, groups 2 and 3 were fed a pullet developer diet for 3 weeks. During the molt period, the birds were exposed to an 8L:16D photoperiod and had access to drinking water at all times. Thereafter, all hens were fed a layer diet (17% CP) and exposed to a 16L:8D photoperiod until the end of the study. Compared to the CONT treatment, significant reductions (P < 0.05) in shell weight, thickness, and breaking strength were identified on the sixth d of feeding the molt diet. Significant (P < 0.05) improvements in these parameters were observed for the FP3 and FP4 treatments during the post-molt period, with the greater degree in the FP4 treatment. In addition, scanning electron microscopy revealed a smaller size of mammillary knobs accompanied by a higher density of mammillae in eggs taken from the molted treatments. Evidence of type B mammillae was detected in an egg produced by the CONT hens, whereas confluent and cuffing mammillae were observed in an egg taken from the FP4 birds. Reduced pore densities were found in the molted treatments in some periods of the post-molt production as compared to the CONT treatment. It was concluded that feeding the cassava molt diet for 4 wk could be an effective non-fasting molt method for improving eggshell quality, ultrastructure, and porosity in post-molt laying hens. INTRODUCTION Eggshell quality is an important contributing factor in table and hatching eggs. The eggshell significance is related to its function to provide a container for the egg contents and protect them from bacterial contamination. Approximately 8 to 10% of the eggs laid for the table industry suffer shell damages during routine handling, causing economic losses (Hamilton et al., 1979). Therefore, the shell must be strong enough to prevent failure during packaging and transportation processes. Most eggshell quality measurements have particularly focused on the amount of eggshell mass, including its weight, thickness, and specific gravity. However, the strength of the eggshell depends not only on its weight and thickness but also on the quality of its microstructure. As reviewed by Roberts and Brackpool (1994), Wilhelm von Nathusius, the first person who studied the ultrastructure of the avian eggshell, described and named the mammillary layer of the eggshell. Reid (1984) reported that there was some evidence of a correlation between mammillary changes and poor shell quality. Solomon (1991) observed the greatest variation in eggshell ultrastructure occurred in the mammillary layer and described a number of abnormalities. Bain (1992) summarized the structural variations increasing the resistance of eggshells to unstable fracture, such as early fusion, cuffing, and confluent mammillae, and those decreasing the resistance of the shell, such as late fusion and type B mammillae. Eggshell pores are small holes that transfer gases and water vapor between the embryo and the outside environment. The pore system of the avian eggshell is located at a specific location between the eggshell cones to provide gas and humidity exchanges. In chicken eggs, typical pores have a funnel-shaped orifice opening at the outer surface of the shell at the level of the cuticle, and a single channel passing through the vertical crystal layer and the palisade region to open at the inner surface of the eggshell between adjacent mammillae (Chien et al., 2008). Pore formation begins at the level of the mammillary layer with the grouping of 4 to 5 mammillary bodies (Solomon, 2010). The distribution of pores in the eggshell surface of the domestic hens is estimated to be between 100 and 300 pores/cm2 (Gilbert, 1971). Klingensmith and Hester (1985) investigated the relationship between pore density and shell quality and reported that hard-shelled eggs exhibited a lower density of pores as compared to that of soft-shelled eggs. Molting in avian species is characterized by a period of rest whereby egg production ceases, reproductive tract regresses, and renewal of feathers occurs (Berry, 2003). Decreasing d length during the molt period initiates gonadal regression, prompting molting and rejuvenation (Follett and Sharp, 1969). Induced molt of laying hens is used by commercial egg producers to extend the reproductive period of their flock. Conventional induced molting programs usually involve a restricted or shorter photoperiod to natural light (Hambree et al., 1980), and the removal of feed (Christmas et al., 1985) and occasionally drinking water (Brake and Thaxton, 1982). Egg production resumes and increases rapidly to a profitable rate following the artificial molt (Hurwitz et al., 1975; Lee, 1982). Albumen quality (Lee, 1982; Tona et al., 2002) and eggshell quality (Hurwitz et al., 1975; Lee, 1982; Christmas et al., 1985) also are improved by induced molting in the subsequent laying cycle. However, the conventional molting method has drawn criticism due to concerns of animal welfare and food safety. Various non-fasting induced molting techniques have been suggested. These alternate methods use dietary manipulations to create an imbalance of particular nutrients (Biggs et al, 2003; Gongruttananun et al., 2013; Bozkurt et al., 2016). There is a deficiency of information concerning the effects of a non-fasting induced molt technique on eggshell ultrastructure and pore density. Therefore, the objective of the present study was to determine the effects of an induced molt technique using cassava meal on eggshell quality, microstructure, and pore density in late-phase (74 wk) laying hens. MATERIALS AND METHODS All animal care procedures were approved by the Animal Ethics Committee of Kasetsart University. The Ethics regulation code is ACKU 00159. Before the experiment began, 270 H&N Brown late-phase laying hens (72 wk old) of similar body weight (BW) (1875 ± 20 g) were housed in a caged layer shed with water and feed provided for ad libitum consumption and a 16-hour exposure to daily photoperiod. The mean temperature of the house was 27.6 ± 1.2 °C, and the average light intensity was 4.7 ± 0.8 lx. The feed was a commercial layer diet calculated to contain 17% CP, 2,800 kcal of ME/kg of feed, and 3.5% Ca. Five replicate groups of 18 hens each (6 adjacent cages containing 3 hens/cage, 40 × 45 cm) were allotted to 3 treatments in a completely randomized design. Birds were weighed and allocated to each replicate to achieve a similar mean BW for each treatment. Egg production, egg weight, and egg quality were measured for 2 wk (72 to 73 wk of age) before the treatments began, in an attempt to keep a similar distribution of production rate, egg weight, and egg quality among the treatments. The 3 treatments were designated as follows: non-molted control treatment (CONT), in which the layer diet was provided and hens were exposed to the 16L:8D photoperiod throughout the study; the other 2 treatments were induced to molt by full feeding with a cassava molt diet for 3 (FP3) or 4 (FP4) wk according to the following procedure. At 74 wk of age, the FP3 and FP4 treatments were carefully transferred to a windowless molting house equipped with mechanical ventilation. The mean temperature of the house was 28.03 ± 1.31°C, and the mean light intensity was 15.4 ± 1.8 lx. Birds in each replicate of the molted treatments were housed together in one of the 10 pens located in the molting house. Each pen was 2.9 × 3.0 × 2.9 m (width × length × height). The induced molt period was divided into a 3-, or 4-week “stress period” and a 3-week “recovery period.” Birds in the FP3 and FP4 treatments had the stress period of 3 and 4 wk, respectively, during which the cassava molt diet and drinking water were accessible at all times. At the end of each stress period, the FP3 and FP4 birds were weighed, and body weight loss was calculated (the data are given in the companion paper; Gongruttananun et al., 2017). Then, birds in all molted treatments had a 3-week recovery period, during which a pullet developer diet and drinking water were provided at all times. At the end of each recovery period, the birds were carefully transferred back to their original house, where the CONT birds were being kept and maintained under the same management regime until the end of the study (95 wk of age). During the molt period, the photoperiod was reduced from the usual 16 h per d (16L:8D) to 8 h per d (8L:16D). The molting protocol is shown in Table 1, and the ingredient composition of the experimental diets, and the preparation of the cassava molt diet are given in the companion paper (Gongruttananun et al., 2017) Table 1. Molting procedure used in the experiment. Treatment1  Treatment type  Drinking water  CONT  Control (hens not induced to molt, consuming ad libitum on a layer diet under a lighting program of 16L:8D throughout the experimental period)  Provided  FP3  Cassava meal offered for 3 wk (74 to 76 wk of age) and a pullet developer diet offered for 3 wk (77 to 79 wk of age) under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  FP4  Cassava meal offered for 4 wk (74 to 77 wk of age) and a pullet developer diet offered for 3 wk (78 to 80 wk of age), under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  Treatment1  Treatment type  Drinking water  CONT  Control (hens not induced to molt, consuming ad libitum on a layer diet under a lighting program of 16L:8D throughout the experimental period)  Provided  FP3  Cassava meal offered for 3 wk (74 to 76 wk of age) and a pullet developer diet offered for 3 wk (77 to 79 wk of age) under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  FP4  Cassava meal offered for 4 wk (74 to 77 wk of age) and a pullet developer diet offered for 3 wk (78 to 80 wk of age), under a lighting program of 8L:16D, then returned to a layer diet and a lighting program of 16L:8D  Provided  1CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Eggshell quality measurement At the termination of the acclimatization, at 73 wk of age, the eggshell quality of all eggs collected from each replicate was measured for the pretest data. The measurement was repeated on d 6 of the molt period. During the post-molt period, 5 eggshell quality measurements were taken and recorded at the ages of 81, 85, 87, 91, and 93 weeks. The eggshell strength was measured using an Eggshell Destruction Strength Meter (Model EFG-0503 Robotmation, Co., Ltd., Tokyo, Japan). Thereafter, the eggs were broken at the equatorial region, and the interior contents were allowed to drain out. The eggshell, with the membranes, was cleaned with tap water and dried at room temperature for one week. After drying, the eggshell was weighed on an electric balance (Model PB 1501, Metler, Toledo, OH), and its thickness was measured in millimeters with a digimatic micrometer (Mitutoyo Corporation, Kanagawa, Japan). Three measurements were taken on the equatorial region on each eggshell, and the mean of 3 measurements was calculated. Preparation of samples for eggshell ultrastructural examination Two eggs from each replicate laid on the same d at 73 wk of age (pretest), on d 6 of feeding the molt diet (during the molt period), and at 84 wk of age (during the post-molt period) were randomly collected for the examination of the microstructure of the eggshell. The selected eggs were broken, the interior contents were removed, and the shells were cleaned with tap water. The specimens were prepared by cutting a piece (1 cm2) of shell from the equatorial region of each egg. The shell membranes were carefully removed after soaking in tap water. The loosely adhering membranes were peeled from the edge of the specimens inward. The samples were then soaked in 1.0 N NaOH for 72 h to eliminate any protein materials incorporated in the shell before being processed and studied, according to the method of Kaplan and Siegesmund (1973). Thereafter, the samples were rinsed in distilled water and left to dry at room temperature. All samples were mounted with their uppermost inner sides on aluminum stubs and coated with gold using an ion coater (Eiko Engineer IB-2, Eiko Engineering Co. Ltd., Ibaraki, Japan) for direct observation by a scanning electron microscope. These specimens were examined using a JEOL JSM-5600LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) operated at 10 kV, at magnifications of 100×, 200×, and 500×. The incidence of ultrastructural variants at the level of the mammillary layer was assessed according to the method and terminology developed by the Poultry Research Unit, University of Glasgow (Solomon, 1991). Photographs of replica surfaces were made to facilitate counting the number of mammillary knobs per unit of the interior surface of the eggshell. The density of mammillae of each shell was expressed as the number of knobs per unit. The average diameter of the mammillary knobs was estimated from the measured mammillary knob density, assuming regular circular geometry, according to the method of Van Toledo et al. (1982). Measurements of eggshell pore density Pore density was examined before the treatments (at 73 wk of age), on d 6 of feeding the molt diet and during the post-molt period, at 83, 87, 91, and 95 wk of age. Five eggs from each replicate laid on the same d were randomly collected for the examination of pore density. Egg contents were removed, and the shells were cleaned with tap water. Four 1 cm2 eggshell chips were taken from each area of 3 regions of the collected eggs: the large end, equator, and small end for the measurements of pore density (Peebles and Brake, 1985), and shell membrane etching was processed by soaking in 1.0 N NaOH for 72 h, according to the method of Kaplan and Siegesmund (1973). Thereafter, the shell samples were put in a biopsy cassette and dipped in a concentrated nitric acid solution for 7 seconds. The reaction was stopped by rinsing in distilled water. The shells were again removed from the tissue cassettes and left to dry overnight, then painted on the inner side with trypan blue in a 2% acetic acid solution, using a fine paintbrush. Pores were counted using a stereomicroscope (Model Nikon 204,825, Nikon Instruments Inc., Tokyo, Japan) at a magnification of 40× and expressed in number per .25 cm2. Pore density of each replicate was computed for the mean of the pores per .25 cm2 of shell surface found in the 12 areas counted on each egg of the 5 selected eggs. Statistical analysis The experiment was conducted as a completely randomized design with 3 treatments. Data were analyzed using the statistical software package SAS, version 9.0 (SAS Institute Inc., 2002). The GLM procedure was used to analyze the effects of the treatment on shell weight, shell thickness, shell breaking strength, mammillary density, mammillary knob diameter, and pore density. An arcsine transformation was used for all percentage data. Duncan's multiple-range test was used to estimate significant differences among treatment means (Snedecor and Cochran, 1980). Significance was based on P < 0.05. The experimental unit was each group of 18 hens for all traits studied. For the determination of eggshell microstructure and pore density, 2 and 5 eggs per replicate were used, respectively. Results in the tables are presented as means and the pooled SEM. RESULTS At the end of each stress period, BW loss of birds in the FP4 treatment was approximately 30.13%, whereas the mean of the FP3 hens was 25.23% (data are given in the companion paper; Gongruttananun et al., 2017). Eggshell quality Table 2 demonstrates the effects of molt treatments on shell weight of the experimental birds in comparison to the CONT treatment. It was found that on d 6 of feeding the molt diet, averages of shell weight of the 2 molted treatments were significantly lower than those of the CONT treatment. During the post-molt period, shell weights of the 2 molted treatments were significantly higher than those of the CONT treatment observed at 87, 91, and 93 wk of age. The effects of molt treatments on shell thickness and shell breaking strength are presented in Tables 3 and 4, respectively. During the molt period, the 2 molted treatments had an inferior value of these parameters as compared to the CONT treatment. During the post-molt period, the FP4 treatment had a significantly higher level of shell thickness measured at 81, 85, 91, and 93 wk of age, whereas shell thickness of the FP3 treatment was significantly higher than that of the CONT treatment only at 85 wk of age. During the post-molt period, the means of shell breaking strength of the FP4 treatment were significantly higher than those of the CONT treatment in every period of the measurements. A significant improvement in shell breaking strength also was observed for the FP3 treatment, except for that measured at 81 wk of age. There was no significant difference in shell weight or shell breaking strength between the FP3 and FP4 treatments throughout the study. Table 2. Effect of non-molted and molted treatments on shell weight throughout the study.1   Shell weight (%)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  8.78  8.83a  8.98  8.79  8.65b  8.71b  8.84b  FP3  8.68  7.35b  9.13  9.10  8.98a  9.12a  9.28a  FP4  8.94  7.27b  9.40  9.23  9.13a  9.22a  9.35a  Pooled SEM  0.37  0.83  0.42  0.29  0.18  0.25  0.22  P-value  0.552  0.001  0.326  0.095  0.003  0.017  0.008    Shell weight (%)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  8.78  8.83a  8.98  8.79  8.65b  8.71b  8.84b  FP3  8.68  7.35b  9.13  9.10  8.98a  9.12a  9.28a  FP4  8.94  7.27b  9.40  9.23  9.13a  9.22a  9.35a  Pooled SEM  0.37  0.83  0.42  0.29  0.18  0.25  0.22  P-value  0.552  0.001  0.326  0.095  0.003  0.017  0.008  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1Data are means of 5 groups of 18 hens each. 2CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Table 3. Effect of non-molted and molted treatments on shell thickness throughout the study.1   Shell thickness (mm)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  0.351  0.359a  0.373b  0.356b  0.356  0.358b  0.362b  FP3  0.343  0.278b  0.374b  0.375a  0.358  0.369b  0.371a,b  FP4  0.351  0.269b  0.390a  0.368a  0.374  0.408a  0.388a  Pooled SEM  0.014  0.028  0.010  0.007  0.013  0.011  0.009  P-value  0.629  <0.001  0.046  0.007  0.104  <0.001  0.002    Shell thickness (mm)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  0.351  0.359a  0.373b  0.356b  0.356  0.358b  0.362b  FP3  0.343  0.278b  0.374b  0.375a  0.358  0.369b  0.371a,b  FP4  0.351  0.269b  0.390a  0.368a  0.374  0.408a  0.388a  Pooled SEM  0.014  0.028  0.010  0.007  0.013  0.011  0.009  P-value  0.629  <0.001  0.046  0.007  0.104  <0.001  0.002  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1Data are means of 5 groups of 18 hens each. 2CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Table 4. Effect of non-molted and molted treatments on shell breaking strength throughout the study.1   Shell breaking strength (N)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  29.79  32.35a  30.82b  30.29b  27.68b  28.88b  28.66b  FP3  29.44  18.47b  37.38a,b  34.93a  32.98a  33.43a  32.51a  FP4  29.04  19.90b  39.77a  36.50a  35.00a  33.59a  33.97a  Pooled SEM  3.44  5.41  4.04  2.08  1.21  2.13  1.54  P-value  0.943  <0.001  0.012  0.001  <0.001  0.007  <0.001    Shell breaking strength (N)    Pre-molt  During molt  Post-molt (wk of age)  Item  (73 wk)  (Day 6 of molt)  81  85  87  91  93  Treatment2                CONT  29.79  32.35a  30.82b  30.29b  27.68b  28.88b  28.66b  FP3  29.44  18.47b  37.38a,b  34.93a  32.98a  33.43a  32.51a  FP4  29.04  19.90b  39.77a  36.50a  35.00a  33.59a  33.97a  Pooled SEM  3.44  5.41  4.04  2.08  1.21  2.13  1.54  P-value  0.943  <0.001  0.012  0.001  <0.001  0.007  <0.001  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1Data are means of 5 groups of 18 hens each. 2CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Eggshell ultrastructure Table 5 summarizes the density and diameter of the mammillary knobs of selected eggshells. There were no significant differences for the density of the mammillae and the diameter of the mammillary knobs among the experimental groups at pre-molt and on d 6 of feeding the molt diet. During the post-molt period, the density of the mammillae of the FP3 (229.9 knobs/mm2) and FP4 (231.7 knobs/mm2) treatments was significantly (P < 0.05) higher than that of the CONT treatment (182.4 knobs/mm2). Conversely, a significantly lower value of the diameter of the mammillary knobs was observed in the 2 molted treatments (0.074 to 0.075 mm) as compared to that of the CONT treatment (0.085 mm). Scanning electron microscopy inspection at a magnification of 100 × revealed normal formation of mammillae with rounded caps in all samples of the shells taken from the 3 experimental groups measured on d 6 of feeding the molt diet (Figure 1). However, a depression in the thickness of the shells was detected in the FP3 and FP4 treatments compared with the CONT treatment as revealed by a transverse view (200×) (Figure 2). Figure 3 shows a mammillary view of the shells produced from hens in the 3 treatment groups examined during the post-molt period (95 wk of age). The examination at a magnification of 500× showed evidence of type B mammillary bodies in a shell sample produced from the CONT hens (Figure 4). The photograph revealed evidence of confluence in one shell sample produced from the FP3 hens. In addition, the evidence of confluence, including of cuffing, was detected in one shell sample taken from the FP4 treatment. During the post-molt period, shell thickness of the FP3 and FP4 treatments was greater than that of the CONT treatment as illustrated by a transverse view (200×) (Figure 5). Figure 1. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 1. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 2. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that a thinner shell thickness is illustrated in the shell structure of the FP3 and FP4 treatments; 200× (scale bar = 200 μm). Figure 2. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined on d 6 of the molt period. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that a thinner shell thickness is illustrated in the shell structure of the FP3 and FP4 treatments; 200× (scale bar = 200 μm). Figure 3. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 3. View largeDownload slide Scanning electron micrographs showing a mammillary view (100×) of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. (Scale bar = 500 μm.) Figure 4. View largeDownload slide Enlargement of a portion of Figures 3A (A), 3B (B), and 3C (C and D) showing ultrastructure variations of the mammillary layer of eggs. Note that type B mammillary bodies (B) are illustrated in the CONT treatment (A), confluence is observed in the FP3 treatment (B), and cuffing (C) and confluence (D) are revealed in the FP4 treatment; 500× (scale bar = 100 μm). Figure 4. View largeDownload slide Enlargement of a portion of Figures 3A (A), 3B (B), and 3C (C and D) showing ultrastructure variations of the mammillary layer of eggs. Note that type B mammillary bodies (B) are illustrated in the CONT treatment (A), confluence is observed in the FP3 treatment (B), and cuffing (C) and confluence (D) are revealed in the FP4 treatment; 500× (scale bar = 100 μm). Figure 5. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that the thickness of shell of the FP3 and FP4 treatments is greater than that of the CONT treatment; 200× (scale bar = 200 μm). Figure 5. View largeDownload slide Scanning electron micrographs showing a transverse view of an egg produced from a hen in the CONT (A), FP3 (B), and FP4 (C) treatment groups examined at 95 wk of age. CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 wk. Note that the thickness of shell of the FP3 and FP4 treatments is greater than that of the CONT treatment; 200× (scale bar = 200 μm). Table 5. Effect of non-molted and molted treatments on mammillary density and mammillary knob diameter of the eggshell produced by the experimental hens throughout the study.   Number  Mammillary density (knobs/mm2)  Mammillary knob diameter (mm)    of  Pre-molt  During molt  Post-molt  Pre-molt  During molt  Post-molt    samples  (73 wk)  (Day 6 of molt)  (95 wk)  (73 wk)  (Day 6 of molt)  (95 wk)  Treatment1                CONT  10  206.5  159.8  182.4b  0.076  0.090  0.085a  FP3  10  194.1  148.4  229.9a  0.088  0.094  0.075b  FP4  10  220.7  170.8  231.7a  0.071  0.086  0.074b  Pooled SEM    49.9  30.5  37.7  0.011  0.007  0.008  P-value    0.67  0.46  0.02  0.48  0.31  0.01    Number  Mammillary density (knobs/mm2)  Mammillary knob diameter (mm)    of  Pre-molt  During molt  Post-molt  Pre-molt  During molt  Post-molt    samples  (73 wk)  (Day 6 of molt)  (95 wk)  (73 wk)  (Day 6 of molt)  (95 wk)  Treatment1                CONT  10  206.5  159.8  182.4b  0.076  0.090  0.085a  FP3  10  194.1  148.4  229.9a  0.088  0.094  0.075b  FP4  10  220.7  170.8  231.7a  0.071  0.086  0.074b  Pooled SEM    49.9  30.5  37.7  0.011  0.007  0.008  P-value    0.67  0.46  0.02  0.48  0.31  0.01  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large Eggshell porosity Table 6 depicts the effects of induced molt treatments on pore density of the experimental birds. Before the treatments and on d 6 of feeding the molt diet, there was no significant difference in means of pore density among the treatment groups. During the post-molt period, pore densities of the FP4 treatment were significantly higher than those of the CONT treatment observed at 83 and 95 wk of age. A significant reduction in pore density also was found at 83 wk of age in the FP3 treatment. Table 6. Effect of non-molted and molted treatments on pore density (pores/.25 cm2) of the eggshell produced by the experimental hens throughout the study.   Number of  Pre-molt  During molt  Post-molt (wk)    samples  (73 wk)  (Day 6 of molt)  83  87  91  95  Treatment1                CONT  25  30.6  31.7  31.7a  32.0  32.9  31.7a  FP3  25  28.2  33.1  28.5b  29.6  30.4  28.2a,b  FP4  25  30.6  35.0  28.4b  30.4  28.3  27.7b  Pooled SEM    2.2  8.0  1.7  2.4  2.8  2.0  P-value    0.215  0.806  0.018  0.305  0.076  0.022    Number of  Pre-molt  During molt  Post-molt (wk)    samples  (73 wk)  (Day 6 of molt)  83  87  91  95  Treatment1                CONT  25  30.6  31.7  31.7a  32.0  32.9  31.7a  FP3  25  28.2  33.1  28.5b  29.6  30.4  28.2a,b  FP4  25  30.6  35.0  28.4b  30.4  28.3  27.7b  Pooled SEM    2.2  8.0  1.7  2.4  2.8  2.0  P-value    0.215  0.806  0.018  0.305  0.076  0.022  a,bMeans within a column with no common superscripts differ significantly (P < 0.05). 1CONT = non-molted control; FP3 = induced molt with cassava meal for 3 wk and had a recovery period of 3 wk; FP4 = induced molt with cassava meal for 4 wk and had a recovery period of 3 weeks. View Large DISCUSSION From the results of the current study, shell weight, shell thickness, and shell breaking strength of the FP3 and FP4 hens declined significantly during the molt period, and after the molt all these parameters were significantly improved. The findings of the current study demonstrating that induced molting has beneficial effects on improvements in shell quality during the post-molt period are consistent with those reported by several investigators (Roland and Bushong, 1978; Baker et al., 1983; Roberts and Brackpool, 1994; Biggs et al., 2003; Bozkurt et al., 2016). However, it is apparent that the degree of improvement in shell quality of the FP4 treatment was greater than that of the FP3 treatment. This suggests a result of BW loss up to 30.13% in the FP4 hens, whereas BW loss of the FP3 birds was only 25.23%. Baker et al. (1983) reported that improvements in post-molt performance were related to an up to 31% increase in BW loss of hens (of their original BW). The investigators also suggested that hens that lost the greatest amount of BW exhibited the greatest improvements in eggshell quality in the subsequent laying cycle. Eggshell strength is significantly important for egg producers in reducing the economic losses from broken eggs. Eggshell breaking strength decreases through aging (Tumova et al., 2014). The strength of the eggshell depends both on its weight and thickness and the quality of its ultrastructure (Roberts and Brackpool, 1994). In the present study, eggshells were collected at 3 phases: prior to, during, and following the molt, to examine the microstructure and pore density. It is apparent that the diameter of mammillary knobs of the FP3 and FP4 treatments significantly reduced during the post-molt period as compared with that of the CONT treatment. On the other hand, the density of mammillae of the molted treatments was significantly higher than that of the non-molted treatment. These results are in agreement with several studies (Robinson and King, 1970; Bunk and Balloun, 1978; Klingensmith and Hester, 1985) that reported that a higher density of mammillae resulted in a stronger shell. In addition, results from the present study revealed evidence of type B mammillae in a shell sample produced from the CONT treatment. The type B mammillary bodies are small spherical bodies located within the mammillary layer of the eggshell, which may or may not attach to the shell membrane layer (Reid, 1984). These structures have been observed in poor shell quality by some investigators (Bunk and Balloun, 1978). In a previous study (Gongruttananun et al., 2013), the author found type B mammillary bodies in the mammillary layer of shells produced by non-molted hens aged 101 weeks. It is possible to postulate that aging is an important factor causing the evidence of type B mammillae, suggesting that the efficiency of shell gland function reduces with the age of hens. Nascimento et al. (1992) reported previously that eggshell quality, including ultrastructural features, decreased with the age of the hen. Interestingly, the results from the present study found evidence of confluent mammillae in the shell samples taken from the FP3 and FP4 hens. Also, evidence of cuffing was observed in the shell sample from the FP4 treatment. Roberts and Brackpool (1994) pointed out that the confluence, the condition in which the mammillary caps combine to form large areas of fusion at the cap sites (Solomon, 1991), made the region itself stronger and influenced the formation of the palisade layer of the shell. Cuffing, the distribution of additional calcite crystals around and between the adjacent mammillary cores (Reid, 1984), may increase the strength of the shell by increasing its effective thickness (Roberts and Brackpool, 1994). In addition, it is of interest to note that the density of pores of the shells taken from the molted treatments was significantly lower than that of the CONT treatment at some periods of the post-molt production (Table 6). These suggest that reduced pore density could be a contributing factor in the strength of the shell. From the present study, beneficial effects of the improvements in shell quality, ultrastructure, and porosity of which were found in the molted treatments, especially the FP4 treatment, would be due to remodeling of shell gland tissues following oviducal regression (Heryanto et al., 1997), leading to shell gland function improvement. Remodeling of the shell gland tissues also may be responsible for removing substances that are obstacles to the function of the shell gland, such as lipid. Baker et al. (1980) reported that the lipid content of the shell gland increased as hens aged. In another study, Baker et al. (1981) demonstrated that induced molting removed lipid accumulation in the shell gland and reduced the incidence of shell-less eggs. It is well documented that removal of lipid accumulation in the shell gland can be achieved when BW loss during molting is greater than 25%, which coincides with maximum oviducal regression (Baker et al., 1980; Brake and McDaniel, 1981; Brake et al., 1981). In summary, the results obtained from the present study indicated that feeding cassava meal for 4 wk and allowing a 3-week recovery period under an 8L:16D photoperiod is an effective non-fasting molt method for improving eggshell quality, microstructure, and porosity in laying hens. The results suggest that improving an eggshell's ultrastructure and reducing pore density may increase the strength of shell. ACKNOWLEDGEMENTS The research project was funded by the Kasetsart University Research and Development Institute, Kasetsart University, Bangkok, Thailand. REFERENCES Bain M. M. 1992. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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