https://orcid.org/0000-0003-3630-0120
ABSTRACT
1. This study tested the hypothesis that the methyl-donor properties of betaine could reduce homocysteine concentrations, which has been recognised in a previous genetics study to be linked to bone quality. This was combined with phytase treatment, as phosphorus is critical for bone mineralisation.
2. Using a 2 × 2 factorial arrangement, a total of 1920 Lohmann LSL-lite chickens housed as 24 replicates of 20 chickens were fed one of four diets containing dietary betaine (0 or 1000 mg/kg) and phytase (300 or 1000 FTU/kg) from one day old until end-of-lay. Blood and bone samples were collected at 45 and 70 weeks of age.
3. Hens fed betaine had lower plasma homocysteine level (P < 0.05), higher tibia breaking strength (P < 0.05) and higher tibia bone density (P < 0.05).
4. Egg production and quality was excellent throughout the study and were not affected by the dietary treatments.
5. The addition of dietary betaine was successful at reducing plasma homocysteine concentrations and improving bone strength in laying hens, which could be used as an intervention to alleviate welfare concerns.
Introduction
The shell glands of laying hens utilise 10% of their total body calcium (Ca) daily to support the production of a shelled egg (Fleming Citation2008). During eggshell formation, 50–70% of Ca is derived from intestinal absorption (Bar Citation2009; Kerschnitzki et al. Citation2014) and the remainder derives from the unique avian medullary bone (MB) (Bar Citation2009). Driven by the influence of endogenous oestrogen at puberty, osteoblasts start producing mostly MB instead of compact bone (CB) (Dacke et al. Citation1993; Beck and Hansen Citation2004). MB is a type of woven bone that is structurally weaker than CB, only providing structural strength when in large volume (Fleming et al. Citation1998b). During egg-laying, osteoclasts that resorb bone will mobilise MB and CB. This can lead to an osteoporotic effect over time (Whitehead and Fleming Citation2000), defined as the reduction of structural mineralised bone which leads to increased fragility and susceptibility to fractures (Whitehead Citation2004). It has been reported that between 49 and 78% of hens will have had a bone fracture by the end of lay (Freire et al. Citation2003; Wilkins et al. Citation2004; Nicol et al. Citation2006; Wilkins et al. Citation2011; Petrik et al. Citation2015). This exceptionally high incidence of damage is a cause for concern.
While nutrition and environment contribute to bone quality, genetics has been shown to play an important role in the variation of susceptibility to osteoporosis and bone fracture (Bishop et al. Citation2000; Fleming et al. Citation2006). In previous work, next generation sequencing of the tibial transcriptome of hens carrying alternative alleles for a quantitative trait locus (QTL) for bone strength (Dunn et al. Citation2007) identified four differentially expressed genes at the QTL position (De Koning et al. Citation2020). The cystathionine beta synthase (CBS) gene was by far the strongest candidate with the largest difference in expression between hens with low and high bone strength genotypes. Alongside the difference in expression of the CBS enzyme, poorer bone quality was associated with higher concentrations of homocysteine (Hcy), the substrate for the CBS enzyme (Dunn et al. Citation2007; De Koning et al. Citation2020).
CBS plays a role in the trans-sulphuration pathway (Jhee and Kruger Citation2005). With the help of pyridoxine, CBS converts Hcy into cystathionine. Homocysteine is synthesised in the trans-methylation cycle, in which S-adenosylmethionine acts as a methyl donor to S-adenosylhomosyteine, which is then hydrolysed into Hcy and adenosine.
As CBS and Hcy are involved in the trans-methylation cycle, there are potential interventions which may be able to influence the level of Hcy, as it can be re-methylated into methionine through the remethylation pathway, where betaine acts as a methyl donor (Finkelstein Citation1998; Craig Citation2004). Betaine is a widely-used feed additive in swine, poultry and fish production (Eklund et al. Citation2005; Simon Citation1999). It has various functions including reducing the effects of heat stress in broiler chickens and the effects of osmotic stress in fish (Petronini et al. Citation1992; Simon Citation1999; Haldar et al. Citation2015).
Another widely-used feed additive in poultry production is phytase. Its function is to break down phytate found in plant seed and increase availability of phosphorus (P) which is bound to phytate (Chesson Citation1993; Selle and Ravindran Citation2007). P is one of the major components of bone mineralisation (Dimeglio and Imel Citation2019). The addition of higher concentrations of phytase, known as ‘superdosing’, can result in more complete phytate degradation (Cowieson and Adeola Citation2011; Cowieson et al. Citation2011). Phytase superdosing has been shown to improve egg production (Taylor et al. Citation2018) and increase bone strength (Rojas et al. Citation2018). The increase of P availability could help to restore any discrepancy in the Ca/P ratio for optimum bone mineralisation (Cowieson and Adeola Citation2011), and may have a synergistic effect with betaine to improve bone quality.
The aim of this study was to evaluate the effects of dietary betaine and superdosed phytase on bone quality, plasma Hcy, and egg production in laying hens. Ultimately these interventions could be incorporated as one component of programmes to improve bone health that are likely to include genetics, housing and nutritional approaches (Toscano et al. Citation2020) across the laying period.
Methods and materials
Animals
This study was approved by the Animal Welfare and Ethical Review Body at The Roslin Institute, UK and the Instituitional Animal Ethics Committee (IAEC), India.
Lohmann LSL-lite day old chicks (n = 1920) were reared following the Lohmann production and management guide (Lohmann Tierzucht GmbH Citation2018). In a 2 × 2 factorial arrangement, 1000 mg/kg betaine (Vistabet liquid, AB Vista, UK) was provided both alone and in combination with phytase (Quantum Blue, AB Vista, UK). The phytase control level was 300 phytase units (FTU)/kg, and the superdosed level was 1000 FTU/kg. These were mixed on-farm into pelleted feed (). The trial used a randomised design with 24 blocks where each contained 80 chicks in 20 cages. Each diet was randomly allocated to five cages of four chicks/hens per cage within the block. This ensured that, throughout the experiment, the chicks or hens were not single-housed after each sampling point. Samples were collected at two ages, 45 and 70 weeks of age. Eggs were collected daily. Feed intake was measured weekly, and body weights were measured every four weeks.
Table 1. The principal ingredients and nutrients of the basal laying hen diet fed at different stages. Betaine was added at two levels (0 or 1000 mg/kg) and phytase was added at two levels (300 or 1000 FTU/kg) according to the dietary groups
Blood and bone sampling
Blood samples with EDTA anticoagulant were taken at two ages; from 360 hens at 45 weeks, and 400 hens at 70 weeks. Blood samples were kept chilled on ice and plasma was prepared immediately by centrifugation. After blood sampling, the birds were humanely killed following Schedule 1 of the UK Animals (Scientific Procedures) Act 1986, and bone samples (keel, humerus and tibia) were collected. All samples were immediately frozen at −20°C until analysed. The whole bones were thawed prior to the removal of adhering soft tissues and radiographs of the bones were taken for radiodensity measurement in a Faxitron 43855D soft x-ray apparatus fitted with NTB EZ240 digital x-ray scanner (NTB GmbH, Germany). Exposure was at a voltage suitable for the bone type and age of hen. Each exposure included a 16-step aluminium (Al) step wedge with 0.25 mm increments for calibration purposes. Images were acquired using the IX-Pect acquisition and imaging software supplied with the scanner. Image processing and analysis were done on ImageJ 1.52n (https://imagej.nih.gov/ij/). After radiography, each humerus and tibia were subjected to a three-point bending test using a materials testing machine (JJ Lloyd LRX50, Sussex, UK) fitted with a 2500 N load cell. The bending jig span was 30 mm, and the loading speed was 30 mm/min. Breaking strength, defined as the maximum load achieved during the test, was recorded using the software Nexygen 2.0 supplied. Plasma Hcy concentrations were measured using Hcy enzymatic assay kit (HY4036, Randox Laboratories, County Antrim, UK), which has been validated for use in chickens (De Koning et al. Citation2020).
Statistical analysis
Statistical analysis was carried out using linear mixed models (LMM) with the main effects of betaine, phytase and age as fixed effects, and replicate as a random effect. Interactions with age were examined where appropriate. All models were fitted using Genstat 18th edition. Where appropriate, post hoc comparisons were made using the least significant difference (LSD) test. Linear regression (LG) analysis was performed on Hcy and bone strengths and densities. The level of attaining significance was P < 0.05. Results are presented as the mean ± standard error of the mean.
Results
Betaine had a significant effect on plasma Hcy concentrations (P < 0.05) (). Hens that were fed betaine had lower concentrations of Hcy than hens not receiving betaine and there was an interaction (P < 0.05) between betaine and phytase. In the hens fed the control level of phytase, those that were fed betaine had lower concentrations of Hcy than those not fed betaine. Age had an effect on Hcy concentrations (P < 0.01), whereby Hcy was higher in hens at 70 than in hens at 45 weeks of age. There was no effect of phytase on Hcy concentrations. Due to the large difference in numerical values of Hcy at the different ages, analysis was performed separately for 45 and 70 weeks of age. Betaine had a significant effect on plasma Hcy concentrations (P < 0.05) at 45 weeks of age, where hens that were fed betaine had lower concentrations of Hcy than those not fed betaine. There was no effect of betaine at 75 weeks of age.
Table 2. The effect of dietary betaine and phytase on plasma Hcy, cull body mass and bone quality traits measured at 45 and 70 weeks of age
Betaine had a statistically significant effect on tibia breaking strength (P < 0.05). Hens that were fed betaine had higher tibia strength than those not fed betaine. The effect of age on tibia breaking strength was verging on significance (P = 0.06). Tibial strength was higher in the hens at 45 than at 70 weeks of age. There was no effect of phytase on tibia strength.
Similar to the tibia breaking strength, betaine had a statistically significant effect on tibia density (P < 0.05). Hens fed betaine had higher tibia density than those not fed betaine. There was no effect of phytase or age on tibia density. There was no effect of betaine or phytase on humerus breaking strength, but there was an effect of age (P < 0.01). Humerus strength was higher in the hens at 45 than at 70 weeks of age.
Betaine and phytase did not have a significant effect on humerus density, but age did (P < 0.05). Humerus density was higher in hens at 45 than in hens at 70 weeks of age. There was an interaction between age and betaine (P < 0.05). In hens that were not fed betaine, there was a significant difference in humerus density at 45 than at 70 weeks of age. In the hens fed betaine, there was no significant difference in humerus density due to age. Betaine and phytase had no effect on keel density, but keel density was higher at 70 than at 45 weeks of age (P < 0.01).
There was a significant effect of phytase (P < 0.05) on feed intake per hen per day. Hens fed 1000 FTU/kg of phytase had higher feed intake than those fed the control amount. Otherwise, there was no effect of betaine or phytase on other zootechnical traits such as body weight, egg production, feed intake/egg mass. Similarly, there was no significant effect on egg quality parameters including egg mass, shell density, shell strength, shell mass, shell thickness, shell percentage or Haugh unit, as detailed in .
Table 3. The effect of dietary betaine and phytase on performance and egg quality traits
There was a significant negative correlation between plasma Hcy level and humerus breaking strength (P < 0.01, R2 = 2.6). The predicted humerus breaking strength was (166.7–0.70[Hcy]) N. Humerus breaking strength decreased by 0.70 N for every µM/l of Hcy. There was a significant positive correlation between plasma Hcy level and keel density (P < 0.01, R2 = 16.1). The predicted keel density was (0.55 + 0.003[Hcy]) mm Al. Keel density increased by 0.003 mm Al for every µM/l of Hcy. There was no correlation with Hcy for any of the other measured bone traits.
Discussion
This study tested the hypothesis that the addition of betaine to the diet of laying hens from hatch would have an effect on the level of plasma Hcy, which in turn would have an effect on bone strength traits. This hypothesis stemmed from the observation that the enzyme CBS, responsible for the conversion of Hcy to cystathionine and the removal of Hcy from the one carbon cycle, was identified as an expression QTL for osteoporosis in laying hens, and higher Hcy has been associated with poorer bone quality (Dunn et al. Citation2007; De Koning et al. Citation2020). The results observed in the current trial supported the hypothesis that a reduction in Hcy led to an increase in bone quality measurements, especially in the tibia. The results were supportive of the findings in human studies where higher plasma Hcy was associated with poor bone quality (Van Meurs et al. Citation2004; Blouin et al. Citation2009; Holstein et al. Citation2011).
High concentrations of Hcy have been shown to affect the enzyme lysyl oxidase which disrupts stable collagen cross-link formation (Herrmann et al. Citation2009; Thaler et al. Citation2011), which was shown in a study using chicks as an animal model (Masse et al. Citation1996, Citation2003; Bourckhardt et al. Citation2019). Hcy is known to increase bone resorption by increasing the number and activity of osteoclasts (Herrmann et al. Citation2005; Koh et al. Citation2006). In previous human studies, high concentrations of Hcy was associated with deteriorating cancellous bone (Holstein et al. Citation2011). In rats, bone and plasma Hcy concentrations were higher in animals with poorer bone quality (Herrmann et al. Citation2009). Similarly, this study found that laying hens with higher concentrations of plasma Hcy had poorer bone strength. This was particularly evident in the tibia breaking strength and density data, which was higher in the birds fed betaine.
When compared by age, the effect of betaine on Hcy was greater at 45 than at 70 weeks of age. The effect of Hcy may be greater during higher concentrations of bone remodelling, which occurs at a younger age during development (Berendsen and Olsen Citation2015), in addition to the remodelling that occurs during egg-laying.
The tibia breaking strength in chickens has been shown in previous studies to be a reliable measurement of bone strength (Fleming et al. Citation1998a, Citation2006). This is a bone strength trait with high genetic heritability (Bishop et al. Citation2000; Raymond et al. Citation2018). In this study, there was a significant and promising finding that the tibia was stronger in the birds fed betaine. Tibia breaking strength has good correlation with other bone traits (Bishop et al. Citation2000). While not significant, the humerus breaking strength for hens fed betaine was higher than in those not fed betaine. Thus, the results suggested that dietary betaine contributed to some increase in bone strength and supported the hypothesis that it can have a beneficial impact on overall bone strength.
There was a large difference in the plasma Hcy concentrations between ages. It was lower at 45 than at 70 weeks of age in chickens. However, the normal range of Hcy values is unknown in the chicken. In humans, the normal range is 5–15 µM/l but can reach >100 µM/l in severe cases of hyperhomocysteinemia (Kang et al. Citation1992). Mouse models (Zhou et al. Citation2001) showed Hcy concentrations ranging from 7.9 ± 1.3 µM/l in the control group to 146.1 ± 16.7 µM/l in the group fed very high concentrations of Hcy. In another study (Masse et al. Citation2003) Hcy concentrations in 12 week old chicks ranged from 43.8 ± 5.0 µM/l in the control group, and 358 ± 58 µM/l in the group fed very high concentrations of Hcy. More recently, De Koning et al. (Citation2020) showed that Hcy concentrations ranged from 16.7 to 18.6 µM/l in laying hens at 48 weeks of age, and 17.2 to 19.1 µM/l at 70 weeks of age. This showed that there are differences between studies which may reflect species and age differences, or methodological effects. In the current study, the samples were taken from all treatment groups, including the control group, and measured at each age, so the within-treatment effects on Hcy were comparable. However, differences between ages could be due to environmental as well as age effects.
The difference at the two ages in tibia breaking strengths was significant, and was verging on significance for humerus breaking strength. The bones were stronger at 45 than at 70 weeks of age. By 45 weeks of age chicken bones are fully developed and the decline in bone strength observed could have been due to an osteoporotic effect over time. Other studies have shown a similar effect of higher bone strength at around 45 weeks of age and lower bone strength at the end of lay (Fleming et al. Citation1998a). As discussed earlier, Hcy was higher at 70 weeks of age, which may affect the bone strength at this time.
In contrast to the humerus and tibia, density of the keel bone was higher at 70 than at 45 weeks of age. Bone density fluctuates during fracture healing and the higher keel density could have been due to the formation of radio-dense calluses that form around bone fractures (Clark et al. Citation2005; Li et al. Citation2019). The keel is one of the bones in the laying hen most susceptible to injury or fractures (Wilkins et al. Citation2004), and studies have shown that bone fracture incidence increases towards the end of lay (Clark et al. Citation2008).
With these effects, a correlation between bone quality traits and Hcy could be expected, and there was a negative correlation between Hcy and humerus breaking strength. The higher the concentration of plasma Hcy, the lower the humerus breaking strength. This supported the hypothesis that Hcy affects bone quality. However, there was a positive correlation between Hcy and keel density. While this was counterintuitive, calluses that arise from keel injuries would increase the bone density so that weaker keel bones would contrarily have higher density.
In the current study, hens that were fed high concentrations of phytase had a higher feed intake than those fed the control amount. While analysis indicated a significant effect on feed intake, the absolute difference was small, with the mean difference being only 0.52 g with a large error. This effect was not apparent in the feed intake/egg mass ratio, which is a more relevant feed conversion ratio in laying hen production. Superdosing phytase did not have an effect on any of the other traits measured. This could have been because the amount of P available in the feed with the control amount of phytase was sufficient for normal growth, bone development and maintaining bone homoeostasis. Production performance was good throughout the study and was not limited by any nutrient that superdosing phytase at 1000 FTU/kg presumably released. However, the phytase concentration used in this study was lower compared to the other studies (Kim et al. Citation2017; Rojas et al. Citation2018).
All treatment groups had good production levels and there was no difference between treatments. The addition of betaine and phytase to the diet in this study began from when the chicks were one day old to the end of the trial. The birds consumed similar amounts of feed per day, suggesting that supplementation did not affect palatability. Measurements of egg quality were taken, and none of the parameters showed differences between the treatment groups. The results suggested that the addition of betaine and phytase in the diet did not affect the development of the birds and their reproductive system – birds from all treatment groups had high production and produced similar quality eggs.
In summary, previous genetic analysis showed a strong candidate gene for bone strength in laying hens which, in turn, led to identifying betaine as a potential nutritional strategy to intervene in the trans-methylation cycle. The results of this study supported the hypothesis that the addition of dietary betaine can lower plasma Hcy and improve bone strength.
Acknowledgments
The authors express gratitude to Dr S. V. Rama Rao and the staff at Sri Ramadhoota Poultry Research Farm, Telangana, India.
Disclosure statement
The authors declare that they have no competing interests, with the exception of NW who is an employee of AB Vista which markets betaine and phytase products.
Additional information
Funding
References
- Bar, A. 2009. “Calcium Transport in Strongly Calcifying Laying Birds: Mechanisms and Regulation.” Comparative Biochemistry and Physiology A: Molecular & Integrative Physiology 152: 447–469. doi:10.1016/j.cbpa.2008.11.020.
- Beck, M. M., and K. K. Hansen. 2004. “Role of Estrogen in Avian Osteoporosis.” Poultry Science 83: 200–206. doi:10.1093/ps/83.2.200.
- Berendsen, A. D., and B. R. Olsen. 2015. “Bone Development.” Bone 80: 14–18. doi:10.1016/j.bone.2015.04.035.
- Bishop, S. C., R. H. Fleming, H. A. Mccormack, D. K. Flock, and C. C. Whitehead. 2000. “Inheritance of Bone Characteristics Affecting Osteoporosis in Laying Hens.” British Poultry Science 41: 33–40. doi:10.1080/00071660086376.
- Blouin, S., H. W. Thaler, C. Korninger, R. Schmid, J. G. Hofstaetter, R. Zoehrer, R. Phipps, K. Klaushofer, P. Roschger, and E. P. Paschalis. 2009. “Bone Matrix Quality and Plasma Homocysteine Levels.” Bone 44: 959–964. doi:10.1016/j.bone.2008.12.023.
- Bourckhardt, G. F., M. S. Cecchini, M. L. D. Hahmeyer, A. P. Remor, A. Latini, D. Ammar, Y. M. R. Muller, and E. M. Nazari. 2019. “Impact of Homocysteine on Vasculogenic Factors and Bone Formation in Chicken Embryos.” Cell Biology and Toxicology 35: 49–58. doi:10.1007/s10565-018-9436-y.
- Chesson, A. 1993. “Feed Enzymes.” Animal Feed Science and Technology 45: 65–79. doi:10.1016/0377-8401(93)90072-r.
- Clark, W. D., W. R. Cox, and F. G. Silversides. 2008. “Bone Fracture Incidence in End-of-lay High-producing, Noncommercial Laying Hens Identified Using Radiographs.” Poultry Science 87: 1964–1970. doi:10.3382/ps.2008-00115.
- Clark, W. D., E. L. Smith, K. A. Linn, J. R. Paul-Murphy, and M. E. Cook. 2005. “Use of Peripheral Quantitative Computed Tomography to Monitor Bone Healing after Radial Osteotomy in Three-week-old Chickens (Gallus Domesticus).” Journal of Avian Medicine and Surgery 19: 198–207. doi:10.1647/2003-037.1.
- Cowieson, A. J., and O. Adeola. 2011. “Board-invited Review: Opportunities and Challenges in Using Exogenous Enzymes to Improve Nonruminant Animal Production.” Journal of Animal Science 89: 3189–3218. doi:10.2527/jas.2010-3715.
- Cowieson, A. J., P. Wilcock, and M. R. Bedford. 2011. “Super-dosing Effects of Phytase in Poultry and Other Monogastrics.” Worlds Poultry Science Journal 67: 225–235. doi:10.1017/s0043933911000250.
- Craig, S. A. S. 2004. “Betaine in Human Nutrition.” American Journal of Clinical Nutrition 80: 539–549. doi:10.1093/ajcn/80.3.539.
- Dacke, C. G., S. Arkle, D. J. Cook, I. M. Wormstone, S. Jones, M. Zaidi, and Z. A. Bascal. 1993. “Medullary Bone and Avian Calcium Regulation.” Journal of Experimental Biology 184: 63–88.
- De Koning, D. J., N. Dominguez-Gasca, R. H. Fleming, A. Gill, D. Kurian, A. Law, H. A. Mccormack, et al. 2020. “An eQTL in the Cystathionine Beta Synthase Gene Is Linked to Osteoporosis in Laying Hens.” Genetics Selection Evolution 52. doi:10.1186/s12711-020-00532-y.
- Dimeglio, L. A., and E. A. Imel. 2019. “Calcium and Phosphate: Hormonal Regulation and Metabolism.” In Basic and Applied Bone Biology, edited by M. R. ALLEN and D. B. BURR, 257–282. London: Waltham, MA, Academic Press.
- Dunn, I. C., R. H. Fleming, H. A. Mccormack, D. Morrice, D. W. Burt, R. Preisinger, and C. C. Whitehead. 2007. “A QTL for Osteoporosis Detected in an F-2 Population Derived from White Leghorn Chicken Lines Divergently Selected for Bone Index.” Animal Genetics 38: 45–49. doi:10.1111/j.1365-2052.2006.01547.x.
- Eklund, M., E. Bauer, J. Wamatu, and R. Mosenthin. 2005. “Potential Nutritional and Physiological Functions of Betaine in Livestock.” Nutrition Research Reviews 18: 31–48. doi:10.1079/Nrr200493.
- Finkelstein, J. D. 1998. “The Metabolism of Homocysteine: Pathways and Regulation.” European Journal of Pediatrics 157: S40–S44. doi:10.1007/pl00014300.
- Fleming, R. H. 2008. “Nutritional Factors Affecting Poultry Bone Health.” Proceedings of the Nutrition Society 67: 177–183. doi: 10.1017/s0029665108007015.
- Fleming, R. H., H. A. Mccormack, L. Mcteir, and C. C. Whitehead. 1998b. “Medullary Bone and Humeral Breaking Strength in Laying Hens.” Research in Veterinary Science 64: 63–67. doi:10.1016/S0034-5288(98)90117-5.
- Fleming, R. H., H. A. Mccormack, L. Mcteir, and C. C. Whitehead. 2006. “Relationships between Genetic, Environmental and Nutritional Factors Influencing Osteoporosis in Laying Hens.” British Poultry Science 47: 742–755. doi:10.1080/00071660601077949.
- Fleming, R. H., H. A. Mccormack, and C. C. Whitehead. 1998a. “Bone Structure and Strength at Different Ages in Laying Hens and Effects of Dietary Particulate Limestone, Vitamin K and Ascorbic Acid.” British Poultry Science 39: 434–440. doi:10.1080/00071669889024.
- Freire, R., L. J. Wilkins, F. Short, and C. J. Nicol. 2003. “Behaviour and Welfare of Individual Laying Hens in a Non-cage System.” British Poultry Science 44: 22–29. doi:10.1080/0007166031000085391.
- Haldar, S., A. Singh, T. Ghosh, and D. Creswell. 2015. “Effects of Supplementation of Betaine Hydrochloride on Physiological Performances of Broilers Exposed to Thermal Stress.” Open Access Animal Physiology 111. doi:10.2147/oaap.s83190.
- Herrmann, M., A. Tami, B. Wildemann, M. Wolny, A. Wagner, H. Schorr, O. Taban-Shomal, et al. 2009. “Hyperhomocysteinemia Induces a Tissue Specific Accumulation of Homocysteine in Bone by Collagen Binding and Adversely Affects Bone.” Bone 44: 467–475. doi:10.1016/j.bone.2008.10.051.
- Herrmann, M., T. Widmann, G. Colaianni, S. Colucci, A. Zallone, and W. Herrmann. 2005. “Increased Osteoclast Activity in the Presence of Increased Homocysteine Concentrations.” Clinical Chemistry 51: 2348–2353. doi:10.1373/clinchem.2005.053363.
- Holstein, J. H., M. Herrmann, C. Splett, W. Herrmann, P. Garcia, T. Histing, M. Klein, et al. 2011. “High Bone Concentrations of Homocysteine are Associated with Altered Bone Morphology in Humans.” British Journal of Nutrition 106: 378–382. doi:10.1017/S0007114511000304.
- Jhee, K. H., and W. D. Kruger. 2005. “The Role of Cystathionine Beta-synthase in Homocysteine Metabolism.” Antioxidants & Redox Signaling 7: 813–822. doi:10.1089/ars.2005.7.813.
- Kang, S. S., P. W. K. Wong, and M. R. Malinow. 1992. “Hyperhomocyst(E)Inemia AS A Risk Factor for Occlusive Vascular-Disease.” Annual Review of Nutrition 12: 279–298. doi:10.1146/annurev.nutr.12.1.279.
- Kerschnitzki, M., T. Zander, P. Zaslansky, P. Fratzl, R. Shahar, and W. Wagermaier. 2014. “Rapid Alterations of Avian Medullary Bone Material during the Daily Egg-laying Cycle.” Bone 69: 109–117. doi:10.1016/j.bone.2014.08.019.
- Kim, J. H., F. M. Pitargue, H. Jung, G. P. Han, H. S. Choi, and D. Y. Kil. 2017. “Effect of Superdosing Phytase on Productive Performance and Egg Quality in Laying Hens.” Asian-Australasian Journal of Animal Sciences 30: 994–998. doi:10.5713/ajas.17.0149.
- Koh, J. M., Y. S. Lee, Y. S. Kim, D. J. Kim, H. H. Kim, J. Y. Park, K. U. Lee, and G. S. Kim. 2006. “Homocysteine Enhances Bone Resorption by Stimulation of Osteoclast Formation and Activity through Increased Intracellular ROS Generation.” Journal of Bone and Mineral Research 21: 1003–1011. doi:10.1359/jbmr.060406.
- Li, J., M. A. Kacena, and D. L. Stocum. 2019. “Fracture Healing.” In Basic and Applied Bone Biology, edited by M. R. Allen and D. B. Burr, 235–253. London: Waltham, MA, Academic Press.
- Lohmann Tierzucht GmbH. 2018. Lohmann LSL-Lite Management Guide. Vol. 2019. Germany: Lohmann Tierzucht GmbH. https://www.ltz.de.
- Masse, P. G., A. L. Boskey, I. Ziv, P. Hauschka, S. M. Donovan, D. S. Howell, and D. E. C. Cole. 2003. “Chemical and Biomechanical Characterization of Hyperhomocysteinemic Bone Disease in an Animal Model.” BMC Musculoskeletal Disorders 4. doi:10.1186/1471-2474-4-2.
- Masse, P. G., C. M. Rimnac, M. Yamauchi, S. P. Coburn, R. B. Rucker, D. S. Howell, and A. L. Boskey. 1996. “Pyridoxine Deficiency Affects Biomechanical Properties of Chick Tibial Bone.” Bone 18: 567–574. doi:10.1016/8756-3282(96)00072-5.
- Nicol, C. J., S. N. Brown, E. Glen, S. J. Pope, F. J. Short, P. D. Warriss, P. H. Zimmerman, and L. J. Wilkins. 2006. “Effects of Stocking Density, Flock Size and Management on the Welfare of Laying Hens in Single-tier Aviaries.” British Poultry Science 47: 135–146. doi:10.1080/00071660600610609.
- Petrik, M. T., M. T. Guerin, and T. M. Widowski. 2015. “On-farm Comparison of Keel Fracture Prevalence and Other Welfare Indicators in Conventional Cage and Floor-housed Laying Hens in Ontario, Canada.” Poultry Science 94: 579–585. doi:10.3382/ps/pev039.
- Petronini, P. G., E. M. De Angelis, P. Borghetti, A. F. Borgetti, and K. P. Wheeler. 1992. “Modulation by Betaine of Cellular Responses to Osmotic Stress.” Journal of Biological Chemistry 282: 5.
- Raymond, B., A. M. Johansson, H. A. Mccormack, R. H. Fleming, M. Schmutz, I. C. Dunn, and D. J. De Koning. 2018. “Genome-wide Association Study for Bone Strength in Laying Hens.” Journal of Animal Science 96: 2525–2535. doi:10.1093/jas/sky157.
- Rojas, I. Y. M., C. L. Coello, E. A. Gonzalez, J. A. Menocal, and G. A. Gomes. 2018. “Effect of Phytase Dose on Productive Performance and Bone Status of Layers Fed with Graded Levels of Digestible Lysine.” Veterinaria Mexico 5. doi:10.22201/fmvz.24486760e.2018.3.564.
- Selle, P. H., and V. Ravindran. 2007. “Microbial Phytase in Poultry Nutrition.” Animal Feed Science and Technology 135: 1–41. doi:10.1016/j.anifeedsci.2006.06.010.
- Simon, J. 1999. “Choline, Betaine and Methionine Interactions in Chickens, Pigs and Fish (Including Crustaceans).” Worlds Poultry Science Journal 55: 353–374. doi:10.1079/Wps19990025.
- Taylor, A. E., M. R. Bedford, S. C. Pace, and H. M. Miller. 2018. “The Effects of Phytase and Xylanase Supplementation on Performance and Egg Quality in Laying Hens.” British Poultry Science 59: 554–561. doi:10.1080/00071668.2018.1483575.
- Thaler, R., M. Agsten, S. Spitzer, E. P. Paschalis, H. Karlic, K. Klaushofer, and F. Varga. 2011. “Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation.” Journal of Biological Chemistry 286: 5578–5588. doi:10.1074/jbc.M110.166181.
- Toscano, M. J., I. C. Dunn, J. P. Christensen, S. Petow, K. Kittelsen, and R. Ulrich. 2020. “Explanations for Keel Bone Fractures in Laying Hens: Are There Explanations in Addition to Elevated Egg Production?” Poultry Science 99: 4183–4194. doi:10.1016/j.psj.2020.05.035.
- Van Meurs, J. B. J., R. A. M. Dhonukshe-Rutten, S. M. F. Pluijm, M. Van Der Klift, R. De Jonge, J. Lindemans, L. De Groot, et al. 2004. “Homocysteine Levels and the Risk of Osteoporotic Fracture.” New England Journal of Medicine 350: 2033–2041. doi:10.1056/NEJMoa032546.
- Whitehead, C. C. 2004. “Overview of Bone Biology in the Egg-laying Hen.” Poultry Science 83: 193–199. doi:10.1093/ps/83.2.193.
- Whitehead, C. C., and R. H. Fleming. 2000. “Osteoporosis in Cage Layers.” Poultry Science 79: 1033–1041. doi:10.1093/ps/79.7.1033.
- Wilkins, L. J., S. N. Brown, P. H. Zimmerman, C. Leeb, and C. J. Nicol. 2004. “Investigation of Palpation as a Method for Determining the Prevalence of Keel and Furculum Damage in Laying Hens.” Veterinary Record 155: 547–549. doi:10.1136/vr.155.18.547.
- Wilkins, L. J., J. L. Mckinstry, N. C. Avery, T. G. Knowles, S. N. Brown, J. Tarlton, and C. J. Nicol. 2011. “Influence of Housing System and Design on Bone Strength and Keel Bone Fractures in Laying Hens.” Veterinary Record 169: 414. doi:10.1136/vr.d4831.
- Zhou, J., J. Moller, C. C. Danielsen, J. Bentzon, H. B. Ravn, R. C. Austin, and E. Falk. 2001. “Dietary Supplementation with Methionine and Homocysteine Promotes Early Atherosclerosis but Not Plaque Rupture in apoE-deficient Mice.” Arteriosclerosis, Thrombosis, and Vascular Biology 21: 1470–1476. doi:10.1161/hq0901.096582.