To date, poultry diets have been formulated to meet estimated requirements for total calcium (Ca), while frequently also maintaining a specified ratio of total Ca to available phosphorus (AvP). In vegetable-based diets, limestone can contribute in excess of 50% and 90% of dietary Ca in broiler and layer diets, respectively. In laying hens, it is known that limestone particle size can alter the availability of Ca for shell formation and bone mineralisation, and as a consequence, breed recommendations have included specifications for a minimum percentage of coarse limestone grit (>2 mm) in feed formulation. More recent research in broilers has shown that limestone source and particle size could profoundly alter Ca digestibility, while simultaneously having significant effects on P digestibility and phytase efficacy. In light of recent studies that have documented the effects of limestone quality on the digestibility of Ca and P, it was of interest to quantify differences in the quality of limestone used in commercial feed mills. With this objective, our laboratory collected 255 limestone samples from feed mills in 16 different countries on the European continent. Limestone samples were analysed for mineral content, geometric mean diameter (GMD), as well as dynamic solubility at five, 15, and 30 minutes for samples with GMD<1000 μm and at 30, 90, and 150 minutes for grit limestone with GMD>1000 μm. While analysed Ca in limestone samples was in most cases high, there was large variation in the GMD particle size of either fine or grit limestone used in commercial feed mills. The dynamic solubility results showed that, while there was an inverse correlation with GMD and solubility at all time-points, limestone geology contributed significantly to differences in the solubilisation rates between different sources of limestone. These large differences in the dynamic solubility rate arising from differences in the GMD particle size and geology of limestone samples used in commercial poultry feed mills can be expected to significantly alter the digestibility of Ca and P in practical diets fed to broilers and laying hens. A further consequence of this is that the ratio of digestible Ca:AvP supplied to the bird would vary between diets and/or feed mills, dependent on the quality of limestone used. This highlights that the current practice of formulating to total calcium, or maintaining a fixed ratio of Ca:AvP in feed formulation is inaccurate and that there is a need to transition to a digestible calcium system in poultry feed formulation.
Calcium (Ca) and phosphorus (P) are two minerals of great concern to poultry nutritionists as a result of the relatively large quantities needed in the diet, and the adverse effects on bone formation, shell quality, and overall performance when inadequate amounts of these minerals are supplied. It is further difficult to discuss Ca supply in poultry diets without referring to P, since the dietary requirement of these two minerals has previously been shown to be interdependent.
Should plasma Ca or P concentrations decrease, synthesis of 1,25-dihydroxy cholecalciferol (1,25(OH)2D3) increases and, in turn, promotes increased intestinal Ca absorption while renal excretion is decreased. In broilers, when dietary Ca is increased, typically by increasing the inclusion of
limestone, there is also a progressive decrease in P digestibility. The primary mechanism whereby this occurs has been thought to be directly, or via formation of calcium–phytate complexes that reduce the digestibility of phytate-bound P. Since the utilisation of Ca and P is influenced by the concentration and digestibility of the other nutrients in the diet, common practice in broilers has been to maintain a ratio of Ca to available phosphorus (AvP) when specifying requirements of Ca and P. Conveniently, this ratio has been set at 2:1 Ca:AvP in broilers. The obvious limitation of this approach is that the interdependence of Ca and P homeostasis in the bird is driven by the amount and ratio of these nutrients provided at tissue level and hence by the ratio of digestible Ca to digestible P in the diet, and not by the ratio of total Ca to available P supplied in the diet. In recent years, several research groups have shown that Ca digestibility in broilers can vary dramatically depending on the Ca source provided, the solubility of limestone, as well as by the source of phytate and addition of phytase.
With the knowledge that the digestibility of total dietary Ca can be significantly altered by the aforementioned dietary factors, and that the form of Ca provided and absolute amount can alter P digestibility, the specification of Ca requirements for broilers as total Ca in the diet becomes obsolete, as does the adherence to a fixed ratio of total Ca:AvP. The need to better understand how to predict variation in Ca digestibility, and the influence of Ca on P digestibility becomes even more critical when considering the high incidence of lameness and bone abnormalities observed in the industry, with over 1% of commercial broilers grown to heavy processing weights affected after five weeks of age. A similar case can be made for commercial laying hens for which requirements for Ca are still specified on a total basis. Particle size and solubility of limestone are known to influence the availability of Ca to the hen and can alter shell quality and bone ash. While breed nutrition recommendations for laying hens do specify the supply of a portion of limestone as coarse limestone grit, potential differences in the solubility characteristics of that grit are not considered when formulating to meet the Ca demand of the hen. This observation becomes increasingly important in the context of modern laying hens where, as a result of increasing length of production cycles,
optimising the utilisation of dietary Ca sources as hens age is critical to meet demands for shell formation without compromising skeletal integrity and bird welfare.
Variation in limestone quality
In vegetable-based broiler diets, limestone can contribute over 50% of the total dietary Ca supplied to broilers, and in excess of 90% of the Ca consumed by laying hens. Given the previous observations by multiple research groups that limestone particle size and solubility can affect the utilisation of Ca by broilers and laying hens, as well as P digestibility and phytase efficacy in broilers, it was of interest to characterise the observed variation in particle size and solubility of limestone used in commercial feed production in Europe to quantify differences in limestone quality used in commercial poultry diets.
Methodology and results
A total of 255 limestone samples were collected from feed mills in 16 different countries on the European continent. Of these, 192 samples were classed as fine limestone, with an average geometric mean diameter (GMD) of <1000 μm, and 63 samples as limestone grit, with an average GMD>1000 μm. All limestone samples were analysed for moisture and nine minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc). Limestone particle size was determined using a shaker and a set of 14 sieves plus the base pan using a sample of 100 g of each limestone shaken for 10 minutes. The method for GMD of limestone particles by mass (dgw) was calculated using the equations described by Wilcox et al. (1962). Solubility of limestone was determined in duplicate using the dynamic solubility assay recently published by Kim et al. (2019). For fine limestone, solubility was determined at five, 15, and 30 minutes, and solubility of limestone grit samples was determined at 30, 90, and 150 minutes.
Results in Table 1 reflect the large variation in the quality of fine and grit limestone used in commercial feed mills across Europe. Of the 192 fine limestone samples analysed, Ca levels were generally high, with an average of 37,82%, and only 28 samples having Ca<36%. What is particularly striking is the lack of standardisation of the GMD particle size of so-called fine limestone used in poultry feed. While the average GMD was 248 μm, the standard deviation of 223 μm was almost as high as the average with a CV of almost 90%. Over 48% of the fine limestone samples had a GMD below 150 μm, and 30% below 100 μm, reflecting the very fine nature of limestone frequently used. While there was a significant correlation between the GMD particle size and solubility of the limestone at five minutes (Figure 1), there are many exceptions to this generalisation. For example, two limestone samples originating in the Ukraine and Poland, with similar respective particle sizes of 299 and 285 μm GMD, can have dramatically different initial solubility at five minutes of 87% and 45%, respectively. In a similar manner, two limestone samples from different quarries in Germany had very different average GMD particle sizes of 46 μm and 250 μm, but both reached 94% solubility at five minutes. Similar examples can be drawn from the grit limestone samples analysed, with samples from Germany and Turkey having similar respective GMD particle size of 2 523 μm and 2 587 μm, yet solubilising to 73% and 42% at the first time-point of 30 minutes, respectively. These examples suggest that the geology of the limestone rock, as well as the particle size fractions, can have a profound influence on the rate of solubility of the limestone. Consequently, one cannot define an optimal limestone particle size to achieve a given rate of solubility without understanding the specific solubility characteristics of the limestone sample over time.
Implications of differences in limestone particle size and solubility characteristics on calcium digestibility and phytase efficacy in broilers
Several recent papers have investigated methods to determine ingredient Ca digestibility and described effects of limestone particle size on Ca and P digestibility and phytase efficacy. For example, in the paper by Anwar et al. (2016), fine (<0,5 mm) and coarse (1 mm to 2 mm) limestone had in vitro solubility of 0,60% and 0,33%, and true Ca digestibility coefficients of 0,43 and 0,71, respectively. These findings demonstrated for the first time in broilers that a positive correlation existed between limestone particle size and in vivo Ca digestibility, with Ca digestibility being negatively correlated to limestone solubility. Kim et al. (2018) also showed fine limestone (0,75 μm) to have a higher in vitro solubility that was supported in vivo by a higher gizzard pH in 28-day-old broilers fed diets with 0,8% or 1,0% calcium. However, in that study, the adverse effects of the fine, more rapidly soluble limestone on Ca digestibility were only observed when diets had a lower Ca level (0,6%) and no phytase. Further publications by the same group, Angel (2019) and Kim et al. (2019), have subsequently provided greater insight into the impact that differences in limestone solubility arising from different sources (geology of limestone), or different particle size, can have on Ca digestibility in broilers. When comparing limestone from the same source, a reduction in particle size from 0,8 mm GMD to 0,15 mm GMD reduced standardised ileal digestibility (SID) of Ca from 49,2% to 38,1%. Importantly, in the paper by Kim et al. (2019), differences in GMD particle size alone could only explain <40% of the observed differences in Ca digestibility from limestone; and differences in limestone geology and physical/chemical characteristics were equally important to particle size in their potential effects on Ca digestibility. This observation is supported by our findings in the European limestone survey above, that limestone particle size alone was not able to adequately explain the large variation observed in in vitro solubility between different samples of limestone. Initial models by Kim et al. (2019) further showed that differences in Ca digestibility between limestone sources could be explained by the extent of solubility achieved at 15 and 30 minutes in vitro, highlighting that limestone must be solubilised in the proventriculus/gizzard in order to be digested. In addition to differences in limestone characteristics, the group at the University of Maryland has also shown the phytate source to alter digestibility of Ca from limestone, with phytate from corn tending to be more reactive with Ca from limestone than when diets contained a mixture of corn and soybean meal.
While not the main focus of this paper, one of the outcomes of the recent research focus on Ca digestibility was that the solubility kinetics of limestone could potentially alter P digestibility to a far greater extent than the observed differences in Ca digestibility. Using one example from our laboratory, Taylor (2019) evaluated three different sources of limestone that had been standardised to a GMD of 0,8 mm, or simply included in the same diet at the commercial particle size supplied by each limestone company. For each limestone source, increasing the particle size and thereby slowing down the rate of solubilisation increased P digestibility. Of equal importance was that phytase efficacy and, in particular, matrix values from phytase, were significantly affected by the source of limestone used in the diet and is supported by the previous findings of Kim et al. (2018).
The significance of this and other research on limestone and Ca digestibility in practical broiler diet formulations still needs to be better understood. One of the reasons why we may not have seen large differences in broiler performance when commercial feed mills use different limestone sources is that analysed Ca and P levels in commercial broiler grower and finisher diets are typically far above the birds’ requirements as determined in more recent research. However, given environmental pressures on reducing P excretion in broiler manure in some regions, as well as the recent trend of reducing Ca levels in commercial broiler grower and finisher diets, the frequency whereby broiler performance is compromised as a result of not accurately defining the amount of digestible Ca supplied in diets of broilers will likely increase in the future.
Implications of calcium digestibility and effects of limestone particle size and solubility characteristics in laying hens and broiler breeders
Over short periods, commercial laying hens and broiler breeders are particularly resilient to small variations in Ca supply vs that required for egg formation. This is as a result of homeostatic mechanisms designed to regulate the supply of Ca for shell formation in times of excess or deficiency relative to demand. For example, should the diet contain insufficient digestible Ca to maintain the Ca demand for shell formation, the subsequent reduction in ionised blood calcium (iCa) stimulates parathyroid hormone (PTH) in order to elevate blood iCa to the hen. However, the elevation of blood iCa through elevated PTH is, in part, achieved through the initiation of medullary bone breakdown. If the dietary Ca deficiency persists over extended periods, the hen will start to break down structural cortical bone Therefore, the trade-off that occurs when insufficient digestible Ca is provided is that the hen compromises the structural integrity of her skeletal system, which results in a shorter productive life since the scavenged cortical bone is not replaced. This is a much greater problem in commercial hens for table eggs than it is for broiler breeders, both as a result of the shorter laying cycle and lower egg output of breeders.
A further issue that arises when laying hens utilise Ca from their bone reserves is that eggshell quality decreases as the hen utilises more Ca from her bones. Eggshell breakages form a large proportion of economic losses incurred by egg producers; therefore, the determination of Ca digestibility values for ingredients may pose a solution to mitigate the effects of inaccurate Ca formulation for laying hens.
Since limestone as an ingredient contributes in excess of 90% of the Ca consumed by the hen, any variation in the digestibility thereof can significantly alter the supply of Ca at tissue level. The in vitro solubility of limestone at a single time-point has been reported to be inversely related with its in vivo solubility. Larger limestone particles with a lower solubility would reside in the gizzard longer, thereby increasing the in vivo Ca availability and improving eggshell quality and bone mineralisation in the hen. However, in contrast to these observations in laying hens, Bueno et al. (2016) observed no effects of limestone particle size on egg characteristics and performance of broiler breeders post-moulting. However, this research was done in post-moult broiler breeders from 74–83 weeks, which may not have been a long enough feeding period to show the effects of limestone particle size. Consequently, more research is therefore required to elucidate if limestone particle size is influential on Ca utilisation in broiler breeders that are restrict-fed a single meal a day. In laying hens, there is little doubt that coarser particle limestone with lower solubility will be beneficial the for saleable egg output and the maintenance of bone integrity. Based on this, commercial recommendations for laying hen nutrition have incorporated the provision of a certain percentage of total dietary limestone to be provided as grit, with the balance of limestone being supplied as fine or powdered limestone. While the minimum/maximum particle size of grit was specified between 2 mm and 4 mm, this does not consider potential variation in the solubility of grit as a result of observed differences in the geology or origin of the limestone. As was shown in the survey of limestone grit samples (Table 1), there was a large variation of what particle size is defined as grit in Europe, and equally large variation in the rate of solubility over time. This is illustrated in Figure 2, which depicts the rate of solubility of five samples of limestone within a narrow range in GMD particle size of 2 145 μm–2 522 μm.
The objective of supplying grit to laying hens is to reduce the rate of limestone solubilisation, and therefore slow down the delivery of dietary Ca to coincide with the period of shell formation later in the day/evening. The data in Figure 2 show that limestone with a GMD of 2 522 μm can potentially solubilise in excess of 75% within 30 minutes in vitro at a pH of 3.0. In contrast, a second limestone with almost identical GMD (2587 μm) only reached 42% solubility at 30 min. These two limestones would, therefore, potentially deliver calcium to the hen at very different rates and be less or more beneficial in meeting the objectives of a slow release of calcium for egg shell formation. Further, one of the objectives of still providing a proportion of fine limestone in laying hen diets is to meet the rapid demand for calcium required to replenish the medullary bone supply in the morning. Based on many of the limestone grit samples analysed solubilising over 50% at the first time-point of 30 minutes, one could speculate if there is indeed a need for a separate source of fine limestone, and is the subject of ongoing research in our laboratory.
In terms of determining Ca digestibility in laying hens, a further aspect that should be taken into account is the hens ability to rapidly alter Ca digestibility based on the time of day, or rather the physiological demand for blood iCa for shell formation. This was recently investigated in our laboratory with results showing that ileal calcium digestibility changed from 54,61% at three hours post-oviposition to 64,77% 11 h after oviposition, and during the time of peak shell formation (Sinclair-Black, 2019). This finding has several implications, foremost that when determining Ca digestibility for the purpose of developing matrix values for feed formulation, the timing relative to oviposition time of the hen can dramatically alter results. Further, from a commercial perspective, while Ca requirements of the hen are specified as total Ca to be supplied per day, the timing of when that Ca is supplied will greatly affect its utilisation and, subsequently, the absolute total amount required. This again reiterates the limitations of continuing to specify Ca requirements and feed formulation on the basis of total Ca.
ACKNOWLEDGEMENTS: This work was, in part, supported by Danisco Animal Nutrition, DuPont Industrial Biosciences, and Chemuniqué (Pty) Ltd.
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