ABSTRACT Standard procedure for most conventional bone assays rely on bones being free of attached muscle or integumentary tissue. Use of an autoclave for bone cleaning is advantageous, as parts may be cleaned afterward by peeling the muscle away as opposed to tediously scrapping muscle tissue from the bone by hand. However, autoclave use for tissue removal has not been validated and published studies typically do not specify the cleaning method. One concern is that autoclave usage could cause mineral leaching out of the bone. The objective was to determine any change in bone mineral content as a result of autoclaving bone samples to remove muscle tissue. Ten pairs of frozen chicken legs were randomly selected and thawed from 72-wk-old W36 hens. Right legs were autoclaved at 121°C for 25 min in individual trays. Left legs were thawed and cleaned by hand. The tibia, meat, and exudate were collected from each leg. Cleaned bones were placed in a soxhlet to extract the fat for 30 h and ashed at 600°C for 8 h. Bone and muscle samples underwent microwave digestion in 10 mL of 70% nitric acid. Digested samples were analyzed for calcium using a flame atomic absorption spectrophotometer. Phosphorus was determined by a colorimetric assay measuring phosphate ion complexes. Statistical analysis was completed by paired t-tests. We found no significant calcium (P = 0.6319) or phosphorus (P = 0.1698) loss from bones autoclaved as compared with bones that were hand cleaned. This study provides evidence that affirms that the use of the autoclave on bones is a suitable method for tissue removal from the leg bones of adult laying hens. INTRODUCTION Measurements such as breaking strength, bone ash, and bone density are commonly used to assess bone mineralization which is an important reflection of bone status. The most commonly used bone is the tibia. This is partially because in young birds the tibia is the one of the fastest growing bones in the body and in hens it develops into a medullary bone at the onset of egg production; meaning that the tibia is a highly sensitive bone to deficiencies and other abnormal conditions. Additionally, the tibia bone is specifically called for in the official AOAC bioassay for vitamin D sources (1955). Typically, the first step in the preparation of bone samples is to clean the bone of any attached muscle and skin. Most studies skip detailing the cleaning processes of the bone samples in the methods section. However, when a study reports the cleaning methodology it falls under 2 categories: tissue removed by hand or tissue removed following immersion into boiling water. Tissue removed by hand is reported either as “Directly Excised” where they imply that the bone is cleanly cut out away from the bird with no flesh attached or “Knife cleaned” where the bone is removed and then scraped clean (Orban et al., 1993, Park, et al., 2003). The AOAC method (1955) calls for bones to be immersed in boiling water only long enough to loosen attached flesh, then deflesh the bone after cooling. One way to expedite the cleaning process is by placing the samples in an autoclave which gelatinizes the tissue for easy removal. The autoclave employs steam and vacuum pumps to remove chamber air to attain a high temperature for a set period of time. The appeal of the autoclave is clear but the use has not been formally evaluated. The autoclave has a much greater potential to change the composition of the bone than boiling water immersion. This is because autoclave cycles achieve a higher temperature than boiling water and causes a greater amount of cell damage in the bone (Böhm and Stihler, 1995). The harsh environment created inside the autoclave kills all cells and causes severe damage to the mineral matrix of the bone (Vanaclocha et al., 1997). This raises the concern that mineral content may be altered during the autoclave procedure. This study was aimed at investigating whether the use of an autoclave to facilitate bone cleaning caused mineral leaching from the bone. MATERIALS AND METHODS The samples were obtained from 72-wk-old W-36 laying hens (Regmi et al., 2016) and all procedures were approved by the Michigan State University Institutional Animal Care and Use Committee. Treatment Design Whole legs were stored in a –20°C freezer prior to treatment. Ten pairs of legs were split between 2 treatments and placed in a refrigerator (4°C) to thaw overnight. The left legs were cleaned by hand, removing all attached skin and feathers along with the muscle tissue with a scalpel. The right legs were put into individual containers without removing any attached skin, feathers or muscle tissue, and placed into the autoclave (733HCMC; Gentige, Wayne, NJ). The autoclave was set to run on a gravity cycle for an exposure time of 19 min at 121°C. Immediately following the completion of the autoclave, the tibia bone was freed of attached muscle tissue and skin. For each leg, the tibia bone and muscle tissue were separated into individual containers and skin was discarded. Any exudate found at the bottom of the containers was collected. Sample Analysis All collected tibiae were split into 3 parts using an autopsy saw (Stryker, Kalamazoo, MI), wrapped in cheesecloth, numbered, and placed into a modified soxhlet apparatus (LG-6920-100; Wilmad-Labglass, Vineland, NJ) for 30 h to achieve maximum ether extraction. Following extraction, they were ashed at 600°C for 8 h in a muffle furnace (Type 30,400 Furnace; Thermolyne). Bone ash and muscle tissue samples were weighed out in 0.4 g amounts, separate of one another in duplicate digesting overnight in 10 mL of 70% nitric acid (OmniTrace; MiliporeSigma, Temecula, CA) as originally described by Shaw, et al. (2002). Samples then underwent microwave digestion (MARS-5; CEM Corp., Matthews, NC) for 30 min at 1200 W reaching a maximum PSI of 200 and a temperature of 180°C. Digested samples were analyzed for calcium concentration using an atomic flame spectrophotometer (AA-700; Shimadzu, Kyoto, Japan). A peach leaves standard (National Institute of Standards and Technology, Gaitherburg, MD) was digested and analyzed with the samples (atomic absorption standard: ACAIKN-100, Ricca Chemical Co., Arlington, TX). Phosphorus concentration was determined via a colorimetric assay measuring phosphate ions reaction with molybdate complexes in the presence of Elon (p-methylaminophenol sulfate) solution acting as reducing agent (Gomori, 1942). The 96-well plates were read on a spectrophotometer (Spectramax 340, Molecular Devices, Sunnyvale, CA) at 700 nm. Statistical Analysis This study was a completely randomized design with 10 replicates of 2 treatments where bird tibia was the experimental unit. A paired t-test was conducted using SAS version 9.3 (PROC TTEST) to separate the treatment means. Significance was considered to be at P ≤ 0.05. RESULTS AND DISCUSSION Calcium and phosphorus concentrations for bone and muscle samples are shown in Tables 1 and 2. There were no differences in bone or muscle concentrations of calcium or phosphorus regardless of the cleaning procedure (P > 0.05). Tibiae calcium and phosphorus concentrations were not different between autoclave and hand-cleaned treatments (P > 0.05; Table 1). The muscle calcium and phosphorus concentrations were not statistically different between treatments (Table 2). The muscle mineral concentrations were consistently lower than the tibiae samples, which was expected based on natural rigor mortis events that deplete calcium and phosphorus stores in the muscle following death. Table 1. Calcium and phosphorus concentrations of tibiae samples assigned to different cleaning procedures1. Hand-cleaned2 Autoclaved2 P-value Bone Calcium (μg/g) 373 ± 2.08 372 ± 2.08 0.63 Phosphorus (mg/g) 194.9 ± 1.59 197.2 ± 1.59 0.17 Hand-cleaned2 Autoclaved2 P-value Bone Calcium (μg/g) 373 ± 2.08 372 ± 2.08 0.63 Phosphorus (mg/g) 194.9 ± 1.59 197.2 ± 1.59 0.17 1n = 10 samples per cleaning method. 2Treatment means ± standard errors. View Large Table 1. Calcium and phosphorus concentrations of tibiae samples assigned to different cleaning procedures1. Hand-cleaned2 Autoclaved2 P-value Bone Calcium (μg/g) 373 ± 2.08 372 ± 2.08 0.63 Phosphorus (mg/g) 194.9 ± 1.59 197.2 ± 1.59 0.17 Hand-cleaned2 Autoclaved2 P-value Bone Calcium (μg/g) 373 ± 2.08 372 ± 2.08 0.63 Phosphorus (mg/g) 194.9 ± 1.59 197.2 ± 1.59 0.17 1n = 10 samples per cleaning method. 2Treatment means ± standard errors. View Large Table 2. Calcium and phosphorus concentrations of muscle samples from the different cleaning procedures1. Hand-cleaned2 Autoclaved2 P-value Muscle Calcium (μg/g) 15.5 ± 6.53 19.5 ± 6.53 0.55 Phosphorus (mg/g) 1.64 ± 0.07 1.71 ± 0.07 0.31 Hand-cleaned2 Autoclaved2 P-value Muscle Calcium (μg/g) 15.5 ± 6.53 19.5 ± 6.53 0.55 Phosphorus (mg/g) 1.64 ± 0.07 1.71 ± 0.07 0.31 1n = 10 samples per cleaning method. 2Treatment means ± standard errors. View Large Table 2. Calcium and phosphorus concentrations of muscle samples from the different cleaning procedures1. Hand-cleaned2 Autoclaved2 P-value Muscle Calcium (μg/g) 15.5 ± 6.53 19.5 ± 6.53 0.55 Phosphorus (mg/g) 1.64 ± 0.07 1.71 ± 0.07 0.31 Hand-cleaned2 Autoclaved2 P-value Muscle Calcium (μg/g) 15.5 ± 6.53 19.5 ± 6.53 0.55 Phosphorus (mg/g) 1.64 ± 0.07 1.71 ± 0.07 0.31 1n = 10 samples per cleaning method. 2Treatment means ± standard errors. View Large These results support similar findings published in the literature. Hall et al. (2003) compared the AOAC method of bone cleaning with an autoclave method on the resulting tibia bone ash. This study found that the correlation coefficient was 0.972, which led the authors to recommend that both methods were acceptable for use when estimating bone ash of broilers. A similar study conducted by Orban et al. (1993) compared 2 methods of bone cleaning, directly excised and boiling water immersion on bone mineral content. This study reported no difference between the 2 methodologies on the bone mineral content. In this study, the autoclave method used was unique compared to similar studies. The autoclave program had right legs undergo a 19-min gravity cycle at 121°C in individual containers. The gravity cycle utilized downward displacement with positive pulse conditioning to remove chamber air by displacement with steam without utilizing external pressure control by vacuum pumps (Gentige Group, 2017). The study by Hall et al. (2003) autoclaved legs under a 6.82-kg of pressure for 8 to 12 min. The basis for their pressure setting is unclear. The settings for the autoclave procedure in this study were based in part on standard surgical autoclave settings for bones at an exposure time of 20 min at 121°C (Köhler et al., 1986). It is worth noting that the autoclave procedure in Hall et al. (2003) differed from this study in a second way. Their autoclaved bones did not undergo fat extraction prior to the ashing oven. Yan et al. (2005) demonstrated that tibia bone ash content is consistent regardless of fat extraction status. However, they did caution that unextracted tibiae may provide a less precise measurement due to the large amount of organic matter remaining inside the bone. The choice to use hand cleaned bones as the comparison treatment in this study was rooted in the concern that assisted methods, such as boiling water immersion could cause cellular damage (Park et al., 2003). There are considerations to address regarding the adoption of the autoclave methodology to future research projects. First, all studies to date were conducted on healthy birds that had all of their nutritional requirements met. It is well known that deficiencies in some minerals can lead to abnormal development of bones which may influence the effects of the autoclave procedure on bone characteristics. Secondly, to the authors’ knowledge all studies to date have investigated differences in cleaning methods on long bones only. Finally, the interaction of storage method and autoclave usage has not been investigated. This interaction has been reported on in orthopedic research, where concerns have been raised about an increased susceptibility to bone damage when frozen bones are placed in the autoclave (Wui et al., 2016). To summarize, there was no difference found between hand-cleaned and autoclaved tibia bones for either calcium or phosphorus concentrations. This is in agreement with similar studies that found no difference between, hand-cleaned, boiling water immersed, or autoclaved tibia bone ash content. Based on this, the autoclave is determined to be an appropriate tool to aid in the cleaning of tissue from bones. REFERENCES Association of Official Agricultural Chemists . 1955 . Official Methods of Analysis . 8th ed . Association of Official Agricultural Chemists , Washington, DC . Böhm P. , Stihler J. . 1995 . Intraosseous temperature during autoclaving . J. Bone Joint Surg. Br. 77-B : 649 – 653 . Google Scholar CrossRef Search ADS Gentige Group . 2017 . 700HC-E series vacuum/gravity steam sterilizers for healthcare applications. Accessed May 2017. http://ic.getinge.com/files/us-hc/product-documents/sterilization/700hc-e-sterilizer/productspecifications/hc-e-733-en-us-productspecifications.pdf . Gomori G. 1942 . A modification of the colorimetric phosphorous determination for use with photoelectric colorimeter . J. Lab. Clin. Med. 27 , 955 – 960 . Hall L. E. , Shirley R. B. , Bakalli R. I. , Aggrey S. E. , Pesti G. M. , Edwards H. M. . 2003 . Power of two methods for the estimation of bone ash of broilers . Poult. Sci. 82 : 414 – 418 . Google Scholar CrossRef Search ADS PubMed Köhler P. , Krelcbergs A. , Strömberg L. . 1986 . 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Res. 14 : 492 – 498 . Google Scholar CrossRef Search ADS © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Poultry Science – Oxford University Press
Published: May 24, 2018
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