For most species, the absorption site of trace minerals (TMs) is located in the small intestine ^[1][2][1,2]. However, in ruminants and especially in the rumen, some interactions between microorganisms, minerals and other substances within the diet can occur resulting in reduced final mineral intestinal absorption [1]. The nutritional feeding systems for ruminants focus mainly on the global animal requirements ^[3][4][3,4], indicating dietary optimum levels of 10, 50 and 50 mg/kg DM and regulatory maximum limits of 35, 150 and 120 mg/kg DM for Cu, Mn and Zn, respectively ^[5][6][5,6]. These TMs are essential in animal feed, given their important physiological functions, such as participation in keratin, collagen and elastin synthesis; components or activators of enzymes; in addition to playing an important role for reproductive and immune systems ^[7][8][9][7,8,9]. In addition to the contribution to these metabolic functions, TMs can also affect the ruminal environment and rumen function, given that rumen microorganisms require minerals for their growth (microbial protein synthesis) and fermentative activity ^[10][11][12][10,11,12]. Intestinal bioavailability of the dietary TMs for ruminants is relatively low, as reported levels are at 4–5%, 1–4% and 15–30% for Cu, Mn and Zn, respectively ^[3][13][14][3,13,14], while selecting the most optimal mineral source for supplementation is quite difficult. Furthermore, ruminal microbial uptake levels are not known. Insights on the ruminal solubility, microbial uptake, and effects on rumen environment of the various existing TM sources could support specialists from the animal feed industry when choosing TM products for dietary inclusion.

2. Available Trace Mineral Forms for Dietary Inclusion

Trace mineral sources for supplementing ruminants are numerous and include inorganic (ITM), organic (OTM) or hydroxy (HTM) mineral forms ^{[2][6][15][16]}[2,6,16,17]. The ITM salts such as carbonates, chlorides, oxides and sulfates are characterized as a specific metal (Cu, Mn or Zn) bound to a non-carbon-containing ligand ^{[17][18][19][20][21]}[18,19,20,21,22], and they are widely available at a low cost. These sources are traditionally used for livestock supplementation ^[22][23]. The OTMs such as glycinates, amino acid complexes, amino acid chelates or different proteinates are formed through specific processes, binding the metal component (Cu, Mn or Zn) to a carbon-containing ligand ^[23][24]. The HTMs are defined as a specific metal (Cu, Mn or Zn) bound via a coordinated covalent bond with a hydroxyl ligand and are considered inorganic [14]. However, the covalent bond to an OH group instead of carbon-containing ligands makes HTMs seem like OTMs ^[24][25]. In ruminant feed, HTMs (Cu, Mn and Zn) are provided in the form of copper hydroxy chloride (Cu-Hyd) ^[25][26], manganese hydroxy chloride (Mn-Hyd) ^[26][27] and zinc hydroxy chloride (Zn-Hyd) ^[27][28]. Other TM forms and sources are studied for ruminant supplementation, including nano-minerals ^[28][29] or even different seaweeds ^[29][30][30,31]; however, little research is available on these last forms considering rumen solubility.

3. Trace Mineral Rumen Solubility and Effects on the Ruminal Environment

There are numerous studies addressing the overall effects of TMs in ruminants; however, still little is known about their ruminal solubility and effects on the rumen microbial populations. Ruminal solubility is to be considered when choosing a TM source for dietary inclusion, given that it was identified as one of the factors closely related to TM relative bioavailability in ruminants ^[2][31][2,32]. The solubility of ITMs in rumen fluid was found to be in close relation to their mineral chemical form: sulfates are considered highly soluble compared to oxides ^[32][33]. OTMs like glycinates, amino acid complexes or different proteinates have a high stability in the 6.0–7.0 pH range (similar to the one found in the rumen environment ^[33][34]), showing a minimum effect of enzymatic hydrolysis ^[23][24]. However, their solubility in rumen fluid, exposed to bacterial fermentative activity, could be significantly affected ^[32][33]. When considering the ruminal solubility of HTMs, they appear to be relatively insoluble; however, results are not equivocal for the hydroxy forms of Cu, Mn and Zn ^[34][35]. In the following, after an overview of different solubility evaluation methods, the ruminal solubility of various sources (inorganic, organic and hydroxy) of Cu, Mn and Zn are presented while also considering effects on rumen fermentation parameters and microbial population changes.

3.1. Ruminal Solubility Evaluation Methods

For an overview of TM solubility, the assessment can be performed through in vitro models with deionized water or a 0.1 N HCl solution as a solvent ^[35][36][36,37]. However, even though TM solubilization in deionized water or a HCl solution may be a good indicator of the overall solubility of a specific TM, the ruminal solubility of TMs may be affected by the rumen environment, and it was shown to be significantly lower when compared to solubility in deionized water ^[32][33]. Considering this, when assessing ruminal solubility of TMs via in vitro studies, for a more factual representation of the various interactions in the ruminal environment, rumen fluid-based models are to be privileged. Key aspects, like donor animals, rumen fluid processing as inoculum, incubation substrate and buffer choice for in vitro fermentation techniques, are well established for the assessment of rumen function (fermentation activity, gas production and nutrient degradation) ^[37][38]; however, for TM ruminal solubility evaluation, the techniques are not yet harmonized. One of the applied methods for the assessment of ruminal solubility of TMs following in vitro fermentations (of 24, 48 h or continuous fermentations) is the analysis of TM concentration in a centrifugation supernatant ^[38][39][39,40]. Based on this method, the final fermentation medium (mix of rumen fluid, buffer, substrate and various TMs) is centrifuged (12,000–18,000× g at 23 °C for 15 min) to separate the particulate matter (feed particles, protozoa, bacteria and insolubilized TM), obtaining a supernatant (containing the solubilized minerals), which is analyzed for TM concentration. Next, the ruminal solubility of a specific TM may be expressed as an absolute value (based on the whole TM quantity recovered in the supernatant) or a relative value (related to a sulfate TM, considered as 100% rumen-soluble). In recent studies ^[40][41][41,42], the ruminal solubility of various minerals was assessed based on a separation of the final fermentation medium (after 70 h of fermentation) by multiple centrifugations: at 100× g (5 min at 4 °C), to separate an insoluble fraction (containing feed particles, protozoa and insolubilized minerals); the obtained supernatant is then further centrifuged at 18,500× g (20 min at 4 °C) to separate a bacteria-enriched fraction and a final supernatant, containing only solubilized minerals; and the mineral concentration of each centrifugation fraction is then analyzed. Next, the ruminal solubility of TMs can be expressed as a percentage of the solubilized mineral in the final supernatant (based on the total mineral analyzed in the different centrifugation fractions). The ruminal solubility of TMs can also be determined using in vivo models, supplementing rumen-cannulated animals (direct supplementation in the diet or a pulse dose via the cannula) ^[42][43]. In a study by Arelovich et al. ^[43][44], the ruminal fluid (100 mL) sampled from rumen-cannulated heifers (supplemented with different levels of TMs), was first filtered with a cheesecloth, acidified (addition of 2 mL of 20% sulfuric acid solution) and centrifuged (16,000× g) to obtain a supernatant (containing the solubilized minerals). The ruminal solubility of TMs was expressed in absolute values based on the TM concentration of the supernatant. In other in vivo studies ^[34][44][35,45], the ruminal solubility of different TM sources was assessed following a supplementation with different levels of TMs and the analysis of the mineral concentration of the rumen content. Samples of rumen fluid were separated by ultracentrifugation (28,000× g for 30 min at 4 °C) in solid pellets (containing the insolubilized minerals) and a supernatant, considered to contain the rumen-soluble minerals. The ruminal solubility of TMs was expressed as an absolute value (based on the mineral concentration of the supernatant) or a relative value (as a percentage of whole ruminal mineral, calculated based on the total amount of rumen content samples and mineral concentration of the centrifugation fractions). Other in vivo TM solubility evaluation methods include the in sacco method ^[45][46], evaluating the rumen disappearance rate of a mineral substrate from rumen-incubated nylon bags (50 µm pore size). However, the method does not allow for the assessment of truly solubilized minerals, only the disappearance from the nylon bags, hence it is not best suited for the evaluation of TM rumen solubility, especially for fine TMs.

3.2. Copper Ruminal Solubility

One of the most commonly used inorganic Cu sources for ruminant supplementation is CuSO₄ [14], often used as a comparison basis for rumen solubility with other Cu sources. Table 1 summarizes the available literature data on the ruminal solubility of various Cu sources. In a study by Deters et al. ^[35][36], the solubility of CuSO₄ and glycinate bound Cu (Cu-Gly) in deionized water (at 5.2 pH) was 100% and 68.9%, respectively. ^34][35], a lower ruminal solubility was reported for ZnSO₄ when compared to Zn-Hyd (7 and 11%, respectively) when animals were supplemented with 120 mg/kg DM of Zn. Another study on the ruminal solubility of TMs reported a high ruminal solubility when supplementing steers with 30, 250 and 470 mg/kg DM of Zn as ZnCl₂ ^[43][44]. The solubility of Zn might also be affected by clay minerals ingested with different feeds, as shown in an in vitro study by Schlattl et al. ^[52][53]: the addition of a clay mixture (90% bentonite and 10% kaolinite) reduced Zn (as nitrous Zn) solubility by 42, 49 and 52% under ruminal (pH 7.02), abomasal (pH 2.00) and duodenal (pH 3.58) conditions.

3.5. Trace Mineral Effects on Rumen Fermentation Parameters

When considering rumen fermentation, Cu was identified as a TM with complex responses. Table 4 presents a compilation of various Cu sources’ effects on rumen fermentation parameters.