Phosphorus (P) is the chemical element whose biological functions are well established. Among the numerous functions attributed to phosphorus, the main ones are the formation of bone structure, participation in the formation of cell membranes, and the use and transfer of energy in the form of ATP (Lehninger, 1994) making it a critical component in almost all metabolic pathways (Suttle, 2010; McDowell, 2003). Reduced food intake is the earliest effect of phosphorus deficiency. Deficiencies of this element have also been associated with reduced growth, anorexia, decreased milk production, and reproductive disorders (Preston et al., 1977; Butterworth, 1985; Underwood, 1996). According to Durand and Kawashima (1980), low dietary P levels can reduce rumen microbial growth, resulting in lower microbial protein production, lower diet digestibility and lower energy supply. The reduction in dry matter intake is also a consequence of the decreased degradability of fiber by rumen microorganisms and the lower metabolic activity of cells, which negatively influence the satiety center (Minson, 1990).

The efficiency of the diet provided to ruminant animals influences both economic viability and environmental sustainability. This is due to its presence in excessive and eutrophic quantities in water bodies, due to the disposal of untreated or unsatisfactorily treated sanitary effluents, causing environmental damage (Sasabuchi et al., 2023). In addition to balanced formulation and animal-diet interactions, diet efficiency also depends on the quality of the ingredients used. In other words, the choice of a particular ingredient or nutrient source depends, among other factors, on the availability of the nutrient for animal metabolism. This is a principle that applies to all sources of nutrients in the diet. However, decision-making is not a simple process. Therefore, in addition to financial cost, the availability of the nutrient in the source options must be considered.

P is essential to metabolism, with mandatory inclusion in diets. This mineral can be obtained from different sources (Satter et al., 2005). Therefore, the choice of source must be based on efficiency criteria, which in this context, may be the one that has the phosphorus best used by the microorganisms in the rumen. Assessing the comparative efficiency of phosphorus sources is not a simple task, as it depends on the characteristics of the source itself (such as origin and processing), its interaction with other nutrients in the diet, the animal category, and productivity. Furthermore, phosphorus can be stored in the body’s reserves and mobilized when necessary. The storage and mobilization process is a complicating factor in comparison tests between sources. However, there are some ways to carry out scientific experiments with the necessary accuracy to compare different phosphorus sources; for example, assays that evaluate ruminal events highly influenced by phosphorus availability.

It is extremely important that the phosphorus meets the requirements of the animal and rumen microorganisms, as its deficiency in the diet can impair digestibility and transit kinetics, which reduces dry matter consumption and, consequently, the overall supply of nutrients (Van Soest, 1994). Thus, an understanding of the availability of phosphorus directly (radioactive tracers) or indirectly (physical, chemical, or biological digestion parameters) is essential for the development of strategies that can increase its efficiency of use. Since the low phosphorus content is one of the reasons for the low degradation of fiber in roughage such as pasture and by-products, fiber transit kinetics can be an important tool to evaluate differences in digestion depending on phosphorus supplementation with different sources.

Therefore, our objective was to compare the potential of different phosphate sources to promote improvements in fiber digestibility. For this we used the technique of evaluation of fiber transit kinetic parameters.

Use of experimental animal statement: i) All animal experimental procedures were approved by the Committee on Animal Use and Care at the Minas Gerais Agricultural Research Company (CEUA EPAMIG01/2019). ii) All experiment procedures were also performed according to relevant guidelines and regulations (ARRIVE guidelines).

Five ruminally fistulated crossbred heifers (Holstein x Gyr) at six months old were used in a 5 × 5 Latin square and randomly distributed into 5 treatments and periods (each 21-d period, being 7-d for adaptation to treatment and 14 for sampling). The animals had a body weight (BW) of 150 ± 8.2 kg (mean ± SD) at the beginning of the experiment and 214 ± 31.9 kg at the end. The heifers were in individual pens, consuming a balanced standard diet to meet the requirements to gain 0.5 kg/day (ingredients: mature grass hay, potato starch, soybean oil, urea, and mineral supplement – Table 1 ).

These diet ingredients were chosen because they are low in phosphorus, this allowed for a greater challenge to the phosphorus sources under study. The mature grass hay offered to heifers was ground and pressed to form briquettes.

The phosphorus sources under study are characterized by differences in the raw rocks, in the acidification processes of these rocks, in the phosphoric acid neutralization bases and in the form of presentation of the product (Powder or Micro-granulated), in addition to the negative control.

Thus, the treatments under study were: 1. negative control (NC) without supplementary phosphorus; 2. dicalcium phosphate from the igneous rock with sulfuric acidulation, neutralization with limestone, and the physical form of microgranules (ISLG); 3. Dicalcium phosphate from the igneous rock with sulfuric acidulation, neutralization with hydrated lime, and powder physical form (ISLhP); 4. Dicalcium phosphate from the sedimentary rock with hydrochloric acidulation, neutralization with limestone, and the micro-granulated physical form (SeCLG); 5. Dicalcium phosphate from the monocalcium phosphate and hydrated lime reaction adjusted with phosphoric acid, and powder physical form (MLhPaP). The phosphorus sources were provided to heifers in premixes (in the same amount: 100 g of P/kg) and included in the total mixed ration (TMR), which had a final concentration of 2 g of P/kg of DM. There was one premix without phosphorus (negative control).

The voluntary dry matter intake was obtained by the difference between the weight of offered feed and the weight of leftovers.

Total phosphorus was determined by Method 965.17 of AOAC (2012), total DM (method 976.03; AOAC, 1990), ash (method 942.05; AOAC, 1990), total nitrogen (method 981.10; AOAC, 2012), fat (method 996.06; AOAC, 2010) and lignin (method 973.18; AOAC, 1997). The CP content was calculated by multiplying the percentage of N by 6.25. Neutral detergent fiber (aNDF) was determined according to Mertens (2002), without the addition of sodium sulfide and with the addition of thermostable alpha-amylase. The aNDF content was corrected for protein and ash for all samples (Licitra et al., 1996) ( Table 2 ).

The highest (p<0.05) transfer rate of particles from the mat or raft (digesta that floats in the dorsal part of the rumen) to the pool of escaping particles dispersed in the liquid phase of the rumen (λ) was detected in animals that received the diet containing dicalcium phosphate from igneous rock with sulfuric acidulation, neutralization with limestone, and presented in microgranule form (ISLG) ( Table 3 ). Fiber from the ISLG diet also showed greater (p<0.05) escape of eligible particles from the rumen-reticulum to the rest of the gastrointestinal tract (k) than the NC diet (without supplemental phosphorus); however, it did not differ (p>0.05) of the other diets, but the k of NC did not differ (p<0.05) from those of SeCLG, ISLhP, and MLhPaP ( Table 3 ).

The transit time of indigestible particles leaving the rumen-reticulum until their appearance in the feces (τ) did not differ (p>0.05) according to the phosphorus sources ( Table 3 ). This parameter has little effect on dry matter intake since the events take place in the digestive compartments after the rumen-reticulum. The longest retention time (p<0.05) of solid particles in the rumen reticulum was detected in animals that received the diet without supplemental phosphorus ( Table 3 ).

The heifers that received the supplemental phosphorus sources ISLG and SeCLG exhibited the highest (p<0.05) dry matter intakes (kg/heifer/day, and kg/100 kg of body weight). There was no difference (p>0.05) for these variables between heifers that did not receive supplemental phosphorus and those that received ISLhP and MLhPaP ( Table 4 ).

It is important to provide a general summary of the phosphates manufacturing process: 1. An acid attack is carried out on the phosphate rock (apatite), with the product being the phosphoric acid and calcium sulfate; 2. Neutralize this phosphoric acid with a basic source; 3. The final solid product is processed in the following ways: a) grinding into powder form or b) going through a granulation process so that the phosphate is presented in micro-granulated form. Therefore, some phosphates differ in terms of the origin of the rock (igneous or sedimentary), the form of acidification (sulfuric or hydrochloric), and the neutralizing base.

The higher transfer rate of particles from the mat to the pool of escaping particles dispersed in the liquid phase of the rumen (λ), in diet with ISLG, allows us to infer that there was better processing of the fibrous mass by the animal (rumination) or by ruminal microflora or both effects. This indicates that the indigestible fraction has the possibility of leaving the rumen more quickly, which is desirable when the objective is to increase the animal’s dry matter intake and consequently, increase the overall supply of nutrients (Vieira et al., 2020). Greater escape of particles from the rumen compartment may be necessary when animals are fed a low-digestibility diet (Dufreneix et al., 2019).

The greater availability of phosphorus from ISLG for the ruminal microbiota, in relation to diets containing other phosphates sources, may have been the factor that provided the improvement in λ values. Phosphorus is an essential mineral for microbial growth, required by ruminal microorganisms to digest cellulose (Yoshihara et al., 2013; Suttle, 2010; Wu et al., 2000). Therefore, more available phosphorus in the diet improves microbial growth, enhancing cellulose degradation and, consequently, increasing the particle transfer rate (Suttle, 2010; Van Soest, 1994). In this study, forage was the basal diet of the heifers; thus, the predominant ruminal microbial population was cellulolytic bacteria (Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens), which have a high phosphorus requirement. Preston and Pfander (1964) reported that these microorganisms have higher phosphorus requirements than the host animal itself. Durand and Komisarczuk (1988) state that ruminal available phosphorus should be at least 5 g/kg OMD (organic matter digestible) to optimize fiber degradation.

It is noteworthy that regardless of the treatment, the fiber of mature grass hay showed high transfer rates of particles from the solid to the liquid phase, in the rumen-reticulum (λ), when comparing this parameter with that of other roughage feeds. The high transfer rate (λ) observed in mature grass hay is due to its smaller particle size in relation to silages. This smaller particle size is consequence of the grinding to enable the briquetting process that was done to prepare the grass hay into pressed briquettes. Roughages with smaller particles tend to be transferred from the solid to the liquid phase in the rumen-reticulum (λ) more quickly. This occurs because the particles have their density increased through the comminution process, resulting from rumination and the action of ruminal microorganisms (Vieira et al., 2020; Rohem Júnior et al., 2020). Smaller particles require less rumination and are more quickly acted upon by rumen microorganisms, due to the greater surface exposure for microbial attack (Regadas Filho et al., 2014; Vieira et al., 2008b). This microbial action increases the digestion rate of potentially digestible cell walls (Processi et al., 2016; Allen, 2014; Durand and Kawashima, 1980). Rumination time is considerably influenced by the physical characteristics of the diet, such as particle size (Van Soest, 1994).

In low-digestibility roughages, the transit time that is computed from the compartment containing the liquid phase until escape through the reticle-omasal orifice is important, as the faster the indigestible particles leave the rumen-reticulum (RR) the lower the negative effect of rumen repletion on the dry matter intake (Allen, 2014; Vieira et al., 2008a). Thus, higher particle transfer rates from the liquid phase to the RR exhaust (k) can result in greater dry matter consumption, consequently greater nutrient input, and greater animal performance (Dixon et al., 2020a, 2018; Dixon and Coates, 2011; Vieira et al., 2008a, b).

The lack of significant difference between the diets for ( τ ) passage rate is possibly due to the fact that phosphorus has effects on the microorganisms that degrade fiber, that is, it has an effect on events that happen within the rumen compartments. In the rest of the gastrointestinal tract, there is no effective action of microorganisms that have effect on fiber kinetic parameters.

The longer mean particle retention time in the rumen-reticulum (MRT) of animals that did not receive supplemental phosphorus in the diet was probably due to the low amount of phosphorus in the rumen environment. This impaired microbial growth and consequently cellulose degradation (Yoshihara et al., 2013; Suttle, 2010; Wu et al., 2000; Hungate, 1966). Possible reductions in the fibrolytic bacterial population due to the drop in ruminal pH caused by the treatments are unlikely since several studies did not find changes in the pH of the medium as a function of phosphorus source (Coneglian et al., 2010; Barreto et al., 2009; Komisarczuk et al., 1987; Witt and Owens, 1983). There are reports that in conditions of low phosphorus concentration (< 4 mg/L), there is a drop in VFA production, with a consequent increase in pH. However, this is not an effect of the phosphorus source but rather a consequence of the low fermentation of fiber resulting from the poor growth of cellulolytic bacteria (Komisarczuk et al., 1987). In other words, the best parameters of fiber transit kinetic in the diets with ISLG and SeCLG can be attributed to the greater phosphorus availability in these sources.

When there is low phosphorus availability in the diet, ruminants normally mobilize the phosphorus stored in the skeleton and send it to the rumen via saliva or the ruminal wall (Schatz et al., 2023; Dixon et al., 2020a; Underwood and Suttle, 2001). This recycling of phosphorus appears not to have been sufficient in this study, probably because animals in the growth phase were used and this meant that phosphorus was prioritized for bone growth and not for recycling.

The MRT is also related to dry matter intake capacity (Processi et al., 2016). The lower the MRT, the greater the possibility of the animal increasing its dry matter intake per unit of time without the rumen-fill regulation system acting, enabling a greater supply of nutrients to the animal (Araújo et al., 2014).

The higher dry matter intake by heifers receiving supplementation with ISLG and SeCLG supports the results of the ruminal kinetic parameters of fiber (λ and k) in the diets that have included these phosphorus sources. Also, as with the transit kinetics, the high dry matter intake, regardless of the treatment, can be justified by the small particle size of the hay. In order to make the briquetting process viable, the hay was finely ground.

Phosphorus supplementation improved fiber transit kinetics, regardless of the phosphate source. ISLG and SeCLG demonstrated the most significant improvements in fiber transit kinetic parameters and dry matter intake among the sources tested. Phosphorus sources that promote better parameters of ruminal fiber transit kinetics could improve animal growth, milk production, and reproductive performance.