Biochemical functions and application of thiamine in ruminants

Thiamine is an essential nutrient for dairy cows and other mammals. By serving as a cofactor of enzymes, including transketolase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and branched chain α-keto acid dehydrogenase, thiamine plays a critical role in carbohydrate metabolism⁽ Reference Subramanya, Subramanian and Said ^{12 )}. Independent of its role as a co-enzyme, thiamine also has specific roles in neuronal communication, immune system activation, signalling and maintenance processes in cells and tissues⁽ Reference Subramanya, Subramanian and Said ^{12 )}. Severe thiamine deficiency in cattle leads to various clinical effects, from anorexia to polioencephalomalacia⁽ Reference McDowell ^{13 )}, while the clinical polioencephalomalacia is not the main point of this review and will not be discussed in detail. For rumen bacteria, thiamine is indispensable for the growth of the rumen bacteria Ruminococcus albus ⁽ Reference Bryant and Robinson ^{14 )} and Ruminococcus flavefasciens ⁽ Reference Ungerfeld, Rust and Burnett ^{15 )}, serves as a cofactor of phosphoenolpyruvate decarboxylase in Bacteroides fragilis ⁽ Reference Zhang, Dai and Lu ^{16 )} and participates in the production of acetyl-CoA via pyruvate-ferredoxin oxidoreductase in Megasphaera, Selenomonas ruminantium, Butyrivibrio fibrisolvens and Ruminococcus ⁽ Reference Russell ^{17 )}.

Due to the key functions of thiamine in dairy cows and rumen bacteria, a thorough understanding of thiamine status in dairy cows is crucial, and there has been more focus on supplying thiamine in ruminant diets. For example, Shaver & Bal⁽ Reference Shaver and Bal ^{9 )} found that the milk and component production tended to be increased by thiamine supplementation (300 mg/d) when dairy cows fed diets high in non-fibre carbohydrate. Similarly, Kholif et al. ⁽ Reference Kholif, Hanafy and El-Shewy ^{10 )} also found that feeding lactating cows a daily ration supplemented with 340 mg of thiamine increased milk yield, milk fat and protein yields. The improvement in milk performance may be related to an increase in precursors of milk components due to thiamine supplementation, since Solouma et al. ⁽ Reference Solouma, Kholif and Hamdon ^{18 )} demonstrated that adding 40 mg/d thiamine to the diet of sheep significantly increased the blood concentrations of albumin, globulin and glucose. However, there are discrepancies surrounding the effects of thiamine on ruminant’s metabolism. Rowghani et al. ⁽ Reference Rowghani, Zamiri and Ebrahimi ^{19 )} found that thiamine supplementation (0, 4 and 6 mg/kg DM) in lambs fed a high concentrate diet had no effect on blood glucose level. Silzell et al. ⁽ Reference Silzell, Hellwig and Kegley ^{20 )} reported that supplemental thiamine did not improve zantibody response and cell-mediated immune response. The inconsistent results concerning the effect of thiamine on ruminant metabolism may be related to differences in thiamine supplementation levels, dietary nutritional composition and physiological stage.

Microbial synthesis, degradation and absorption of thiamine in the gastrointestinal tract of ruminants

The requirement for thiamine in ruminants is mainly met by microbial synthesis, with a small portion coming from feedstuffs degradation. The thiamine content is higher in cereal feeds than in forage feedstuffs (Table 1). Similarly, Tafaj et al. ⁽ Reference Tafaj, Schollenberger and Feofilowa ^{25 )} reported a higher thiamine content in concentrate than in hay (3·78 v. 0·4 mg/kg DM), and different concentrate:hay ratios influenced dietary thiamine intake. However, ruminal thiamine concentration was negatively correlated with dietary thiamine intake and was assumed to be more closely related to the concentrate level than to dietary thiamine content because of the effect of concentrate on rumen conditions⁽ Reference Tafaj, Schollenberger and Feofilowa ^{25 )}. The actual ruminal thiamine synthesis by microbes is extremely difficult to measure as the rumen is a dynamic system in which thiamine synthesis, degradation and absorption occur simultaneously during passage along the digestive tract⁽ Reference Miller, Meiske and Goodrich ^{5 ,} Reference Schwab, Schwab and Shaver ^{7 ,} Reference Beaudet, Gervais and Graulet ^{23 ,} Reference Kon and Porter ^{26 ,} Reference Castagnino, Kammes and Allen ^{27 )}. Therefore, ruminal thiamine AS is calculated by subtracting daily orts-corrected thiamine intake from the amount reaching the duodenum. This calculation does not reflect actual thiamine synthesis, as it ignores ruminal degradation, microbial use, or potential absorption across the rumen wall. The thiamine AS has been measured in some studies and data are provided in Table 2. Schwab et al.⁽ Reference Schwab, Schwab and Shaver ^{7 )} reported that the average ruminal thiamine AS in lactating dairy cows is 50·6 mg/d, which is similar to the value of 51·7 mg/d reported by Breves et al. ⁽ Reference Breves, Brandt and Hoeller ^{2 )} and >26·0 mg/d reported by Santschi et al. ⁽ Reference Santschi, Berthiaume and Matte ^{29 )}. In the recent studies by Beaudet et al. ⁽ Reference Beaudet, Gervais and Graulet ^{23 )}, Castagnino et al. ⁽ Reference Castagnino, Kammes and Allen ^{27 )} and Seck et al. ⁽ Reference Seck, Linton and Allen ^{28 )}, the ruminal thiamine AS was negative (from −39·8 to 0·8 mg/d), which they hypothesised was due to thiamine destruction by thiaminase enzymes or degradation by the ruminal microflora. Based on the above studies, we can conclude that dietary factors such as feed type and nutrient composition influences thiamine synthesis. Castagnino et al. ⁽ Reference Castagnino, Kammes and Allen ^{27 )} reported that long cut alfafa silage reduced thiamine AS which proved that feed type affects thiamine synthesis in the rumen. In addition, Buziassy & Tribe⁽ Reference Buziassy and Tribe ^{30 )} found that the thiamine concentration in rumen fluid was significantly reduced when dietary protein levels decreased. Breves et al. ⁽ Reference Breves, Brandt and Hoeller ^{2 )} showed that ruminal actual thiamine synthesis and the amount of thiamine flowing to the duodenum decreased when 26 % of dietary N was deducted from the diet, and the amount of ruminal thiamine synthesis positively correlated with duodenal microbial N flow (R 0·76), indicating that dietary N levels and microbial metabolism in the rumen affect thiamine synthesis.

Table 1 Summary of thiamine concentrations of several feeds

Table 2 Duodenal flow and ruminal apparent synthesis of thiamine in dairy cows

ND, data are not detected.

* Composition of concentrate: 32·8 % barley, 24 % wheat, 14 % tapioca meal, 2·6 % maize, 23·6 % soyabean meal, 1·0 % soyabean oil, 2·0 % minerals.

† Composition of concentrate: 32·5 % barley, 6·1 % wheat, 36·8 % tapioca meal, 15·7 % maize, 5·9 % soyabean meal, 1·0 % soyabean oil, 2·0 % minerals.

‡ Composition of concentrate: 31·0 % barley, 5·8 % wheat, 35·0 % tapioca meal, 14·9 % maize, 5·5 % soyabean meal, 1·0 % soyabean oil, 2·0 % minerals, 4·8 % urea.

§ Composition of concentrate: the same as concentrateFootnote ‡ but an increased frequency of feeding.

In addition to dietary N, dietary carbohydrate sources and levels also affect ruminal thiamine synthesis. Schwab et al. ⁽ Reference Schwab, Schwab and Shaver ^{7 )} discovered that increasing non-fibre carbohydrates from 30 to 40 % tended to decrease daily ruminal thiamine AS in cows fed a 35 % forage diet, and the opposite effect occurred for cows fed a 60 % forage diet. Castagnino et al. ⁽ Reference Castagnino, Kammes and Allen ^{27 )} reported that thiamine AS was negatively correlated with the amounts of organic matter and ruminally digested starch. Tafaj et al. ⁽ Reference Tafaj, Schollenberger and Feofilowa ^{25 )} also found that ruminal thiamine content was higher under conditions of 40 and 25 % concentrate in the diet than under conditions of 50 and 60 % concentrate, despite higher thiamine content in concentrate than in forage feedstuff. Thus, Tafaj et al. ⁽ Reference Tafaj, Schollenberger and Feofilowa ^{25 )} assumed that the ruminal thiamine concentration was more closely related to the concentrate level, that is the intake of energy and digestible organic matter, than to the dietary thiamine content. The effect of dietary carbohydrates on thiamine concentration may be due to its influence on rumen fermentation and the microbial community. Castagnino et al.⁽ Reference Castagnino, Kammes and Allen ^{27 )} and Seck et al. ⁽ Reference Seck, Linton and Allen ^{28 )} reported a positive correlation between ruminal pH and thiamine AS. The impact of pH on thiamine status may be related to the increasing thiaminase production at low ruminal pH, since pH values below 5·8⁽ Reference Boyd and Walton ^{31 )} are optimal for thiaminase-producing bacteria (C. sporogenes and a few species of Bacillus; Brent & Bartley⁽ Reference Brent and Bartley ^{32 )}), as a result, thiamine degradation by microbial thiaminase increased. Besides, thiamine synthesis by Bacteroidetes, Fibrobacter and Pyramidobacter decreased under low ruminal pH condition⁽ Reference Pan, Xue and Nan ^{33 )}, which also contribute to their positive correlation between ruminal pH and thiamine AS. However, Schwab et al. ⁽ Reference Schwab, Schwab and Shaver ^{7 )} reported a negative correlation between thiamine AS and ruminal pH values, whereas Beaudet et al. ⁽ Reference Beaudet, Gervais and Graulet ^{23 )} didn’t find any relationship between these two parameters. This discrepancy may be related to the different extent of pH decrease under different dietary conditions: in the study of Castagnino et al.⁽ Reference Castagnino, Kammes and Allen ^{27 )}, the average ruminal pH values for dairy cows fed alfalfa silage and grass silage are 6·26 and 5·84, respectively; Seck et al. ⁽ Reference Seck, Linton and Allen ^{28 )} reported that pH values of dairy cows fed high forage and low forage are 6·0 and 5·86, respectively; whereas the ruminal pH values in the study by Schwab et al. ⁽ Reference Schwab, Schwab and Shaver ^{7 )} were approximately above 6·0, thus there was less thiaminase available to degrade thiamine.

Ruminal thiamine exists in a free or bound form or contained in rumen microorganisms. Currently, the proportion of thiamine absorbed in the rumen and intestine is difficult to determine and is unclear. The disappearance rate was used to indicate thiamine fate in the gastrointestinal tract in the study by Santschi et al. ⁽ Reference Santschi, Berthiaume and Matte ^{29 )}, who found that 67·8 % of thiamine disappears before the duodenum and that there is almost no disappearance of postruminally infused thiamine before the duodenum, suggesting extensive ruminal destruction or utilisation of thiamine. Zinn et al. ⁽ Reference Zinn, Owens and Stuart ^{6 )} reported the ruminal disappearance rate of thiamine is 47·7 %. The fate of thiamine that disappears from the rumen is unclear, and it is unknown whether thiamine absorption can occur in the rumen. Through permeability measurements on sheep rumen mucosa at several thiamine concentrations (0·1–12·8 μg/ml), Hoeller et al. ⁽ Reference Hoeller, Fecke and Schaller ^{34 )} found that the rumen wall mucosa has a low permeability to thiamine. Similarly, Smith & Marston⁽ Reference Smith and Marston ^{35 )} demonstrated that thiamine is not absorbed in appreciable amounts through the rumen wall. However, Rérat et al. ⁽ Reference Rérat, Champigny and Jacquot ^{36 )} and McDowell⁽ Reference McDowell ^{37 )} reported that the rumen wall is just not permeable to bound thiamine or thiamine contained in rumen microorganisms, but ruminants can absorb free thiamine from the rumen wall by an active transport mechanism. Regarding intestinal thiamine absorption, the intestinal disappearance of thiamine is 75 %⁽ Reference Santschi, Berthiaume and Matte ^{29 )}, and 90–96 % of thiamine that reaches the duodenum in sheep is in a microbial fraction⁽ Reference Breves, Hoeller and Harmeyer ^{38 )}, indicating the high absorption of microbially produced thiamine in the intestine. Free thiamine can be degraded by ruminal microbes or absorbed through the rumen wall, while the absorption of bound thiamine or thiamine in ruminal microorganisms mainly occurs in the intestine and plays an important role in supplying this nutrient to host ruminants.

Subacute ruminal acidosis induction altered thiamine status in ruminants

As mentioned above, increasing dietary non-fibre carbohydrate levels can decrease the daily AS of thiamine in the rumen⁽ Reference Schwab, Schwab and Shaver ^{7 )} and may cause thiamine deficiency. SARA caused by overfeeding a high-grain diet is known to reduce ruminal pH and microbial activity⁽ Reference Mao, Huo and Liu ^{39 )} and may, therefore, affect thiamine production. The relationship between thiamine status and SARA has attracted increasing attention. Recently, researchers⁽ Reference Pan, Yang and Xue ^{11 ,} Reference Dabak and Gul ^{40 ,} Reference Karapinar, Dabak and Kizil ^{41 )} have reported that thiamine deficiency occurs when sheep or cattle have subacute or acute ruminal acidosis. To better understand how SARA induction interacts with thiamine status, the methods for diagnosing thiamine deficiency and the impact and possible mechanisms of SARA on ruminal and blood thiamine concentrations are reviewed in the following section and summarised in Fig. 1.

Fig. 1 The potential mechanisms by which high-grain-induced subacute ruminal acidosis alters thiamine status in the rumen and blood of dairy cows. When dairy cows are fed a high-grain diet, more starch and less neutral-detergent fibre (NDF) reach the rumen. Then, carbohydrates are decomposed to volatile fatty acids (VFA) through pyruvate by bacterial degradation. During this process, pyruvate accumulates, and more thiamine (in the form of the cofactor thiamine pyrophosphate (TPP)) is needed for the conversion of pyruvate to acetyl-CoA. As a result, thiamine concentrations decrease, and more pyruvate flows to lactate and propionate, resulting in decreased pH. In addition, the abundance of thiamine-synthesising bacteria, including Bacteroidetes, Fibrobacter and Pyramidobacter, decrease, whereas those of thiamine-degrading bacteria, such as Bacillus and C. sporogenes, increase under high-grain feeding; these changes also contribute to decreased thiamine concentrations in the rumen. Decreased ruminal thiamine content and hampered thiamine transport in response to low ruminal pH and lipopolysaccharide (LPS) accumulation act together to alter thiamine status in the blood. Red text, increase; blue text, decrease.

Thiamine supplementation helps to attenuate subacute ruminal acidosis

It is well known that SARA induced by overfeeding a high-grain diet decreases the ruminal pH, alters the rumen microbial population, and increases the concentration of LPS in rumen fluid⁽ Reference Khafipour, Krause and Plaizier ^{54 ,} Reference Plaizier, Li and Le Sciellour ^{65 )}. Specifically, SARA challenge leads to a reduction in the abundance of cellulolytic bacteria⁽ Reference Fernando, Purvis and Najar ^{55 )}, an increase in the proportion of starch-fermenting and lactic acid-producing bacteria⁽ Reference Khafipour, Li and Plaizier ^{66 )}, and enhanced lysis of gram-negative bacteria associated with increased ruminal LPS⁽ Reference Khafipour, Krause and Plaizier ^{54 )}. The alterations in ruminal LPS and pH could act synergistically to disrupt barrier function⁽ Reference Emmanuel, Madsen and Churchill ^{67 )}, once the epithelium has been breached, mucosa-associated lymphoid tissue cells respond by triggering local inflammation via the LPS/toll-like receptor 4 (TLR4) signalling pathway, leading to the excessive production of pro-inflammatory cytokines⁽ Reference Kurashima, Goto and Kiyono ^{68 ,} Reference Zhang, Zhu and Mao ^{69 )}. Moreover, an impaired gastrointestinal epithelium facilitates the translocation of LPS from the digestive tract into circulation, causing metabolic alterations and systemic inflammation in host cattle⁽ Reference Gressley ^{70 )}, which greatly impacts the production and health of dairy cows.

Currently, there is an increasing focus on SARA prevention. Interestingly, our recent studies have shown that dietary thiamine supplementation (180 mg/kg DM intake) may be a new strategy for SARA prevention. As illustrated in Fig. 2, thiamine supplementation attenuates SARA mainly by improving rumen fermentation⁽ Reference Pan, Yang and Xue ^{11 )}, balancing the ruminal bacterial community⁽ Reference Pan, Xue and Nan ^{33 ,} Reference Wang, Pan and Wang ^{71 )} and exerting anti-inflammatory effects⁽ Reference Pan, Yang and Beckers ^{62 )} in dairy cows. Specifically, Pan et al. ⁽ Reference Pan, Xue and Nan ^{33 )} showed that thiamine administration promotes the growth of the bacterial community associated with the degradation of fibre (Ruminococcus 1, Pyramidobacter and Succinivibrio)⁽ Reference Bainbridge, Cersosimo and Wright ^{72 )} and polysaccharides (Bacteroides)⁽ Reference Flint, Bayer and Rincon ^{73 )}, and decreases the abundance of bacteria positively related to ruminal lactate content (Succiniclasticum and Ruminococcaceae NK4A214). As a result, ruminal fermentation and lactate degradation improved, and ruminal pH increased⁽ Reference Pan, Yang and Xue ^{11 )}. Meanwhile, ruminal acetate content in SARA cows supplemented with thiamine increased with a higher abundance of fibre-digesting bacteria, and acetate was then transported to the mammary gland to produce more milk fat⁽ Reference Pan, Yang and Beckers ^{62 )}. On the other hand, during a SARA challenge, the death and lysis of gram-negative rumen bacteria, especially Bacteroidetes spp., are the main sources of free LPS in the rumen⁽ Reference Mao, Zhang and Wang ^{53 )}. Then, free LPS triggers the release of pro-inflammatory cytokines by activating the TLR4/NFκB signalling pathway⁽ Reference Lin, Wang and Xue ^{74 )}. In the study by Pan et al. ⁽ Reference Pan, Yang and Beckers ^{62 )}, the proportion of Bacteroides increased, with a subsequent decrease in free LPS, upon infusion of thiamine in SARA cows. The decreased LPS content and inhibited NFκB activation by exogenous thiamine act together to reduce the production of pro-inflammatory cytokines, and thereby attenuate local inflammation in the rumen epithelium. The anti-inflammatory effects of thiamine have also been demonstrated in rats⁽ Reference Shoeb and Ramana ^{75 )} and humans⁽ Reference Gonzalez-Ortiz, Martinez-Abundis and Robles-Cervantes ^{76 )}.

Fig. 2 The potential mechanisms by which thiamine supplementation attenuates high-grain-induced subacute ruminal acidosis in dairy cows. Thiamine supplementation can balance the bacterial community by increasing the abundance of cellulolytic bacteria, including Bacteroides, Ruminococcus 1, Pyramidobacter, Succinivibrio, and Ruminobacter. Such increases enhance fibre degradation and ruminal acetate production; then, increased concentrations of acetate are transported to the mammary gland to increase milk fat synthesis. On the other hand, thiamine supplementation suppresses the ruminal epithelium inflammatory response by decreasing ruminal lipopolysaccharide (LPS) production and repressing NFκB protein activation. TPP, thiamine pyrophosphate; LBP, LPS-binding protein.

Conclusions and future research

Notwithstanding the knowledge gaps described above, the vital function of thiamine in dairy cows is obvious, including its participation in carbohydrate metabolism in the rumen and host ruminants, regulation of rumen fermentation conditions, and stimulation of milk performance. However, several issues must be addressed to thoroughly understand the thiamine nutritional status in dairy cows. The following are some of the limitations of the current knowledge that need to be resolved:

1. (1) There is no conclusive dietary recommendation of thiamine in dairy cows. To establish a recommendation, the minimum requirement of thiamine must be estimated. During this process, the amount of thiamine from dietary sources that escapes degradation in the rumen and the amount of thiamine synthesised in the rumen must be ascertained. Unfortunately, knowledge of the factors controlling the amount of thiamine escaping the rumen and thus available for absorption by dairy cows is limited. Therefore, the major challenge in determining the thiamine requirement in dairy cows is predicting thiamine supply dynamically according to different dietary chemical compositions. Hence, more studies are needed to reveal how diet composition (e.g. type of forage, starch concentration and protein concentration) affects the fate of thiamine in the rumen and the amount available for cows at different physiological stages. In addition, the simultaneous determination of ruminal, duodenal, blood and milk biomarkers of thiamine would be useful to understand the fate of thiamine in dairy cows.

2. (2) Although Pan et al. ⁽ Reference Pan, Xue and Nan ^{33 )} deduced the possible genera associated with thiamine synthesis based on the positive correlation between thiamine content and the abundance of a particular genera, whether these genera actually participate in thiamine synthesis is still inconclusive. Hence, more experiments, such as the isolation and culture of related bacteria in vitro, are necessary to verify their thiamine synthesis ability. In addition, the rumen is inhabited by a multitude of microorganisms, including bacteria, protozoa, and yeast. Ruminal bacteria on the rumen epithelium, in the liquid or solid fraction, are different because of their distinct ecological niche and metabolic function. Thiamine supplementation has a positive impact on the liquid-associated bacterial community, but whether solid-attached bacteria are affected by thiamine supplementation and whether the anti-inflammatory effect of thiamine is related to its regulation of rumen wall-adherent bacteria are unknown. Moreover, protozoa represent 50 % of the total microbial mass in the rumen and may play crucial roles in thiamine metabolism; therefore, more research on the relationship between ruminal protozoa and thiamine is necessary to understand thiamine metabolism in the rumen.

3. (3) Most studies on the response of dairy cows to thiamine supplementation have been confined to rumen fermentation and milk performance. In non-ruminant research, the activity of non-thiamine-dependent enzymes, such as succinate dehydrogenase, succinate thiokinase and malate dehydrogenase in the TCA cycle, has been shown to be altered by thiamine deficiency. Therefore, it is necessary to explore the systematic changes in carbohydrate metabolites under conditions of different thiamine status in dairy cows using metabolomics, which will improve the understanding of thiamine function in dairy cows and identify a more sensitive biomarker for thiamine status. In addition to its role in carbohydrate and energy metabolism, the effects of thiamine on cell regulation, immune function, and oxidative damage should be evaluated. The discovery of unknown functions and cattle responses to thiamine will help to improve cow health and productivity and enhance the nutritional value of milk and other dairy products.