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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
* Department of Animal Sciences, Department of Family and Consumer Sciences, and Department of Natural Resources and Environmental Design, North Carolina Agricultural and Technical State University, Greensboro 27411
2 Corresponding author: willisw{at}ncat.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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0.05) in treatment NFW (3.48), NFM (3.22), and FFAW (3.33), whereas the greatest reduction was observed in treatment FFAM (2.72). The number of Salmonella bacteria recovered from the crop was significantly greater in the NFW treatment (3.43) and lowest in treatment FFAM (2.62). Treatment FFAM (30.0%) had the lowest BW loss and differed significantly from treatment NFW (42.3%), NFM (39.7%), and NFP (41.5%) but not from FFAW (38.0%) and FFAP (34.0%). Ovary weights for treatments NFW, NFP, FFAM, and FFAP did not differ significantly from each other but did so for NFM and FFAW (38.0%), which had the lowest weight. Return to egg production at 2 mo lagged behind in treatments FFAM, NFP, and FFM more than in any other treatments. At 3 mo, treatments FFAAM and NFW differed from the other treatments. Findings indicate that a greater decrease in the natural Salmonella population in the ceca and crop can be obtained with the combination of full-fed alfalfa plus mushroom extract in molting hens and induce a comparable molt with feed withdrawal.
Key Words: molting layer • mushroom • pokeweed • Salmonella • weight loss
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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These actions have fostered efforts by researchers to replace the stressful regimens of feed withdrawal to induce molting and thereby extend the laying cycle of hens. For example, using zinc to induce molting, Park et al. (2004) reported comparable results with conventional feed withdrawal methods utilizing 1% zinc acetate and 1% zinc propionate in the feed. In another study conducted by K. E. Anderson (North Carolina State University, Raleigh; personal communication), results showed that using a low-protein and low-energy molt diet without fasting provided comparable post-molt results.
The concern regarding Salmonella proliferation among molting chickens also has received considerable attention. Biggs et al. (2003), for example, demonstrated that hens fed on a wheat middling treatment diet went out of egg production within 8 d and that postmolt egg production in hens fed the wheat middling diets was comparable to hens fed on the 10-d feed removal treatment. Moreover, these investigators reported no Salmonella proliferation. Murase et al. (2006) also reported that feeding wheat bran could be successfully used as an alternative to complete feed withdrawal to force-rest aging hens and not increase Salmonella concentrations. In another alternative method to the non-feed-removal study, McReynolds et al. (2006) tested alfalfa and a combination of alfalfa and corn soy diet as a means of decreasing Salmonella during an induced molt. Although they reported a reduction in Salmonella with these alfalfa diets, they indicated a need for additional combination usage of products to enact a greater reduction in Salmonella. Although these previous studies show promise in terms of addressing the induction of molting and prevention of Salmonella duality, effective feeding regimens to achieve both are still in need of development and refinement.
Mushrooms are nutritionally functional foods containing compounds with antimicrobial activity. Moreover, they are rich sources of natural antibiotics. In a recent review of medicinal mushrooms by Rowan et al. (2003), these investigators reported that numerous bioactive polysaccharides from medicinal mushrooms appear to enhance innate and cell-mediated immune responses and exhibit antitumor or antivirus responses in animals and humans. The bioactive polysaccharides and polysaccharide-bound proteins from mushrooms are able to modulate many important immune cells because of their structural diversity and variability of the macromolecules (Rowan et al., 2003). In addition, they suggest that mushroom polymers (β-glucans) may trigger the stimulation of different immune cells in animals and humans by binding to a specific cellular receptor known as complement receptor type 3. Guo et al. (2004) investigated several mushroom and herb polysaccharides, as alternatives for an antibiotic, on growth performance of broilers, and found Lentinula edodes to be a significant growth promoter in broilers. Similarly, Willis et al. (2007) noted enhanced beneficial bifidobacteria production from mushroom extract (L. edodes) given to broiler chickens.
Another plant that has been found to exhibit antimicrobial effects is pokeweed. In research conducted by Edwards (2006), Salmonella Typhimurium was found to be sensitive to the crude water extract of pokeweed root, showing significant inhibition at as low as 20 mg/ mL in vitro tests. It is possible that active compounds, such as the proteins in pokeweed root extracts, could interact with the phospholipids of cell membranes, resulting in inhibition of bacterial growth. Many wild birds consume the fruits of the pokeweed plant and disperse the seeds in many places (McDonnell et al., 1984), yet, little is known about how the fruit positively or negatively affects birds. Barnett (1975) conducted a study with turkey poults and observed high mortalities (43%), enlarged hocks, and ataxia, after being fed a standard turkey starter mixed with liquefied berries at a rate of 0, 2.5, 5, and 10%. The addition of the pokeberry mixture was at the expense of the total diet. These young poults were given 10% pokeberries via the feed. This was the only scientific study found in the literature concerning poultry. The objective of this study was to evaluate the effectiveness of medicinal mushroom and pokeweed extract combined with alfalfa on the induction of a molt, weight loss, Salmonella population, and postmolt egg production in older hens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Fifty-four Single Comb White Leghorn hens approximately 77 wk of age that previously tested positive for Salmonella were used in this experiment without Salmonella challenge. Hens were removed from a commercial tier layer cage (64.8 x 68.6 cm) system, relocated to a cage unit in the same building, and allowed a 1-wk acclimation period with ad libitum feed and water with a 16L:8D photoperiod. The feed was a balanced unmediated corn-soybean pelleted ration that met or exceeded the NRC (1994) recommendations for nutrients. The ration was calculated to provide 2,818 kcal of ME per kilogram, 16.5% CP, 3.0% calcium, and 0.48% phosphorus. Hens were fed a 100% alfalfa meal diet without formulation or supplements, which according to the NRC (1994) has a ME value of 1,200 kcal/kg with about 17.5% protein. After acclimation, the hens were weighed, randomly placed in 3 replicate cage groups of 2 hens each, and assigned to 1 of the 9 treatment groups: 1) full-fed + H20 (FFW), 2) full-fed + mushroom (FFM), 3) full-fed + pokeweed (FFP), 4) nonfed + H20 (NFW), 5) nonfed + mushroom (NFM), 6) non-fed + pokeweed (NFP), 7) full-fed alfalfa meal + H20 (FFAW), 8) full-fed alfalfa meal + mushroom (FFAM), and 9) full-fed alfalfa meal + pokeweed (FFAP). Six hens per treatment were assigned, enabling the test of 1 hen per replicate cage group with 3 relocates per treatment group for egg production assessment. The 9 experimental feeding conditions were followed for 10 d. Nonfed hens received no form of feed or supplements for the 10-d period.
After assignment to experimental conditions, the photoperiod was adjusted to 8L:16D throughout the molting and resting period. At 10 d of molt, all hens were weighed and 1 hen from each of the 3 replicate groups for each condition (n = 27) was killed by cervical dislocation. The remaining hens in each treatment replicate (3) were placed back into the commercial tier layer system (58.4 x 53.3 cm) with the molt treatment given cracked corn until d 28 postmolt and then returned to a full-fed commercial layer ration. The light program was changed to 16L:8D to stimulate egg production. The total egg production reported for mo 1 was based on 20 d of collection after the 10-d molt.
Mushroom and Pokeweed Extracts
Shiitake mushrooms (L. edodes) were obtained from the indoor cultivation facility maintained by the Mushroom Biology and Fungal Biotechnology Laboratory at North Carolina Agricultural and Technical State University Farm (Greensboro). Lentinus edodes mycelium extract is a preparation of the water-soluble material from powdered mycelia extract harvested before the mushroom fruiting bodies develop. The major active constituent of this extract is reported to be heteroglycan protein conjugate polysaccharide. It contains 24.6% protein, 44% sugars, vitamins, and nucleic acid derivatives (Breene, 1990; Iizuka, 1997). A fine-dried shiitake powder (l-mm sieve, sample 100 g) was extracted by stirring with 1,000 mL of sterile deionized water at 25°C, 1,073 x g for 16 h. The mixture obtained was centrifuged for 30 min at 2,147 x g, and the supernatant was concentrated to 150 mL in a rotary evaporator at 55°C. The mixture was kept at 4°C before use as an additive in the water that was given to the hens starting at d 1 and continuing until d 10.
The pokeweed (Phytolacca americana) roots were harvested from a farm in Julian, North Carolina. The plant parts were separated and transported in a cooler to North Carolina Agricultural and Technical State University Food Microbiology Laboratory, where they were cut and kept frozen at –80°C. The samples were freeze-dried using a Labconco free-zone freeze dryer (Kansas City, MO) for 72 h. After freeze-drying, the samples were powdered using a grinder (Retsch 1640, Haan, Germany). The powdered materials were individually soaked in 100% deionized water or 80% ethanol (1:5 wt/vol) overnight with continuous stirring. The resultant liquid material was removed and centrifuged at 7,870.72 x g for 20 min at 4°C. The liquid supernatant was collected in a flask and the final residue was discarded. The water or ethanol, or both, in the supernatant was evaporated under reduced pressure using a Rotovapor (Buchi, Essen, Germany). The remaining water or ethanol was dried under vacuum using an Isotemp Vacuum Oven (Suwannee, GA). The dried materials were collected as crude water or ethanol extract of pokeweed roots. This mixture was made and replaced daily in the drinkers.
Salmonella Isolation
At the end of the molt, the hens were killed by cervical dislocation. The crops and ceca were excised aseptically and cultured for Salmonella. Spleen, liver, and ovary weights were recorded. Each crop and ceca was blended in a Stomacher 400 Lab System 4 (Seward, Norfolk, UK) for serial dilution examination. They were serially diluted (1:10) in 0.1% peptone (Becton, Dickinson and Company, Sparks, MD). One hundred microliters from each dilution tube was placed onto an XLD plate (Becton, Dickinson and Company) and spread evenly on the agar, and all plates were incubated for 24 h at 37°C. The number of colony-forming units of Salmonella was expressed exponentially as base-10 logarithm of Salmonella per gram of cecal or crop contents. Suspect colonies were confirmed biochemically (triple sugar iron and lysine iron slants) and by microscopic observations.
Statistical Analysis
Differences in the base-10 logarithm colony-forming units of Salmonella counts among different treatment groups were determined by ANOVA using the GLM procedures (SAS Institute, 2001). Significant differences were further separated using Duncan’s multiple range tests.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Means of Salmonella growth by experimental condition are shown in Table 1
. There were significant (P 0.05) differences in Salmonella concentrations between treatment groups in both crop and ceca samples. The chickens assigned to treatment 8 (FFAM) exhibited the lowest average of 2.72 log10 cfu of Salmonella that differed significantly from the NFW but not the FFM in treatment 2 of the ceca. Alfalfa has a high fiber content and has been shown to increase the transit time in the gastrointestinal tract of chickens. Because many bacteria utilize different substrates for growth, the increase in transit time favors bacterial degradation of fiber into fermentable substrates such as fructooligosaccharides to short-chain fatty acids. Significantly increased Salmonella populations were observed in treatments NFW, NFM, and FFAW.
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Several researchers have reported that feed withdrawal induces stress that manifests an increase in the susceptibility of hens to Salmonella Enteritidis, thereby increasing intestinal shedding and organ invasion (Holt and Porter, 1992; Thiagarajan et al., 1994; Holt et al., 1995). Recently, McReynolds et al. (2006) reported that alfalfa can be combined with layer rations to limit Salmonella Enteritidis infection and induce a molt comparable to feed withdrawal. Feed withdrawal has been linked to an increase in the number of broilers with crops colonized by Salmonella (Ramirez et al., 1997). Controlling the growth of Salmonella spp. during this stressful period should help to decrease organ invasion in laying hens. There are few reports in the literature utilizing extracts from these 2 sources; therefore, it is very difficult to provide informed discussion from the results of this study. Few if any conclusive studies regarding the efficacy of these extracts in poultry have been reported in the scientific literature. Wang et al. (1998) reported that natural medicinal products originating from fungi and herbs have been utilized as feed additives for farm animals in China for many years and have shown antimicrobial activities, immune enhancement, and stress reduction. In our study, no assessment of stress was measured. There were reports indicating that certain plant polysaccharides have a prebiotic effect in host animals (Cummings and Macfarlane, 2002). In a study conducted by Guo et al. (2004), mushroom extract (L. edodes) stimulated the number of potential beneficial bacteria such as bifidobacteria and lactobacilli while decreasing the number of harmful bacteria such as Bacteriodes spp. and Escherichia coli. Similarly, Willis et al. (2007) reported significantly greater bifidobacteria concentrations in the fecal droppings of young broiler chickens given mushroom extract for 3 consecutive weeks. It is not clear whether older chickens will respond in the same manner to this treatment regimen containing mushroom extract. The mechanism is not clear regarding the combined effect of alfalfa and mushroom extract to decrease the Salmonella populations. A possible explanation could come from the work of Hinton et al. (2000). They indicated that feed withdrawal purges the crop of fermentable carbohydrates that lactic acid-producing bacteria require for growth and lactic acid production. This action generally allows an increase in the numbers of salmonellae and other Enterobacteriaceae. The polysaccharides provided by the mushroom extract could have optimized this process. There was also a significant reduction of Salmonella in the crop with the combination of alfalfa meal plus pokeweed. Certainly, the use of alfalfa to decrease Salmonella is in agreement with the findings of McReynolds et al. (2006). They indicated a need for an additional combination additive to enact a greater reduction in Salmonella, and these 2 additives offer some potential. Many recent non-feed-withdrawal feeding programs are conducive to the commercial laying hen industry; however, implementation of these programs would be dependent on various factors. Some of the new experimental molting programs are still seeking improvement.
Means for BW loss by experimental conditions are shown in Table 2. Significant (P < 0.05) weight loss reduction differences were found with all treatment groups. When compared with the NFW treatment, weight losses were significantly lower in the FFAM treatment. There were no significant differences between other treatments with additives when compared with the NFW treatment. Hens in the NFW group has a reduction of 42.3% in BW and 39.7, 41.5, 38.0, 30.0, and 34.0% reductions in the NFM, NFP, FFAW, FFAM, and FFAP treatments groups, respectively, compared with the FFW treatment group that had a 4.2% reduction. The weight loss of the FFAM and FFAP treatment groups is comparable with the recommended BW losses, which are between 25 to 35% (UEP, 2002; Bell, 2003; Webster, 2003). Further evaluations are needed to understand completely the metabolic and physiological effects of these extracts used in molting.
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). The liver weight of the birds subjected to FFAP was significantly greater than the NFW-treated hens but did not differ from the NFP. The greater weight is most likely due to the alfalfa meal rather than any toxicity from the pokeweed. The decrease in liver weight in treatment NFW most likely resulted from the removal of hepatic energy stores as glycogen and lipids that are metabolized in the liver (Berry and Brake, 1985). The spleen weights varied between treatment. The NFW treatment had the lowest spleen weight of any of the treatments. Most spleen weights in the experimental treatments did not differ from the full-fed treatments. There were no consistant trends over treatments that were observed in the absolute weights of the spleen from birds going through a forced molt. The weights of the ovaries were significantly different from the full-fed treatments. The greatest degree of ovary regression was observed in birds treated with NFM and FFAW. Good regression was observed in treatments NFW, NFP, FFAM, and FFAP, which did not differ significantly from each other. Major regression during the molt is needed to obtain good long-term egg production and egg shell quality during the second production cycle. There appeared to be no health-related issues with the mature hens in any of the experimental treatments. Barnett (1975) observed that the weight gains of poults were decreased as the concentration of pokeberry increased using a 0, 2.5, 5, and 10% pokeberry inclusion into the feed. The Phytolacca plant is recognized for its high numbers of toxic chemicals. Numerous toxins have been isolated from the fruits (Steinmetz, 1960; Dwayne Ogzewalla et al., 1963; Barnett, 1975). Birds in general do not get affected as a result of ingesting berries, because their digestive system does not break down the seed in the berries that contains the toxin. In an unpublished research conducted by the authors, some weight depression was noted in young broiler chickens with increased concentrations of pokeweed root extracts. These observations indicated that young birds may be adversely affected by ingestion of pokeberries and other parts of this plant.
Means for egg production by experimental condition are shown in Figure 1. Return to egg production at 2 mo lagged behind in treatments FFAM, NFP, and FFM, more than in any other treatments. At 3 mo, treaments FFAM and NFW differed from the other treatments. This seems to correlate with a reduction of ovary weights in these treatments. The combined action of FFAP with regard to return of egg production was seen as comparable to FFAW. Moreover, there was a greater egg production trend for birds assigned to treatments FFP and FFAP that were given poke-weed extract. In conclusion, the use of mushroom extract combined with alfalfa proved to be effective in lowering Salmonella population during molt induction. Furthermore, pokeweed extract showed some potential to slow the growth of Salmonella. Body weight loss, ovary regression, and return to egg production were not adversely affected by the 2 extracts utilized in this experiment. Therefore, using these combinations of alfalfa and extracts would help decrease the stress from feed deprivation and bacterial pathogens in laying hens. Molt regimens with these extracts will need to be further investigated to determine the best dosage through experimental Salmonella challenges for molt induction performance and long-term health effects on hens.
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FOOTNOTES |
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Received for publication January 3, 2008. Accepted for publication August 7, 2008.
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REFERENCES |
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represents full-fed + H20 (FFW), broken line with - represents full-fed with mushroom (FFM), solid line with 
represents full-fed with pokeweed (FFP), solid line with 
represents nonfed + H20 (NFW), broken line with • represents nonfed + mushroom (NFM), solid line with • represents nonfed + pokeweed (NFP), broken line with -•- represents full-fed alfalfa meal + H20 (FFAW), broken line with x represents full-fed alfalfa + mushroom (FFAM), and thick solid line with 
represents full-fed alfalfa + pokeweed (FFAP).