At birth, the rumen is non-functional, making calves pre-ruminants. By weaning, the rumen is the principal site of volatile fatty acid (VFA) absorption, responsible for over 50% of all VFA being absorbed (Allen, 1997). Consequently, the calf rumen must undergo a ruminant transition, where the rumen goes from non-functional to functional in 6-10 weeks, coinciding with the period when most dairy calves are weaned (USDA, 2012). The transition of the rumen from non-functional to principal site of energy absorption is the biggest physiological change the rumen will undergo, therefore requiring considerable attention and management.
A functional rumen requires an intact and functional epithelium. To be fully functional, an epithelium must carry out two principal functions: 1) the exchange of nutrients across the epithelial barrier; and 2) preventing the translocation of bacteria into the bloodstream. During the ruminant transition, changes in nutrient absorption capacity and epithelial barrier integrity occur simultaneously, so nutrient absorption and health are closely linked in early life during the ruminant transition of rumen development.
In a neonatal calf, the rumen wall is smooth, with little vasculature and musculature (Figure 1). The development of papillae and vasculature stems predominantly from the consumption and fermentation of rapidly fermentable calf starter, which calves will begin to eat in the second or third week of life. Through fermentation of calf starter, microbes in the rumen will produce butyrate, the VFA most effective at stimulating rumen development (Warner et al., 1956). As the link between calf starter intake and rumen development was discovered, maximizing calf starter intake became the primary goal of calf raising(USDA, 2012). In the NRC (2001) for dairy cattle, weaning of calves is recommended once daily calf starter intake is 680 g/day (1.5 lb/day), though this threshold was recently adjusted to 1000 g/day (Stamey et al., 2012). Crucially, calf starter intake is the only weaning indicator currently recommended.
Figure 1. Rumen development at 28 days of age of calves fed milk replacer only (LEFT) and milk replacer with calf starter (RIGHT). Calves fed calf starter show more rumen development, evidenced by dark, prominent papillae, which increase absorptive surface area of the rumen. Modified from https://extension.psu.edu/photos-of-rumendevelopment.
To maximize calf starter intake, feeding a low plane of nutrition (low amounts of milk or milk replacer) was popular. When calves were fed a low plane of nutrition of 750 g/d, their calf starter intake reached 680 g/d by 7 weeks of age (Laarman and Oba, 2011). In another study, calf starter intake increased once the weaning transition began, regardless of whether calves were fed a high or low plane of nutrition, or whether they were weaned at five or eight weeks of age (de Passillé et al., 2011). What these studies show is that starter intake and milk intake are inversely linked. Furthermore, milk and starter play two distinct roles and are digested and absorbed at different sites.
When suckled from a bottle, milk bypasses the rumen via the esophageal groove and is shunted directly to the omasum and abomasum. In the abomasum, milk is coagulated via rennin, and then further digested and absorbed in the small intestine. The absorption of nutrients from milk is key to meeting the calf’s energy needs early in life. Feeding colostrum, for instance, increases lactase activity needed to digest and absorb lactose in milk (Hammon et al., 2012). Additionally, fat oxidation increases dramatically in the first day of life, especially medium chain fatty acids most common in milk fats (Girard et al., 1992). At birth, the calf is therefore geared (Girard et al., 1992). Consequently, closure of the esophageal groove, abomasal rennin, and lactase activity in the small intestine are crucial for meeting the calf’s energy needs until the shift to solid feed is complete.
Unlike milk, calf starter does not use the small intestine as the primary site for digestion and absorption. Instead, calf starter is primarily digested, fermented, and absorbed in the rumen, although rumen-undegradable-protein, -fat, and -fibre bypass the rumen and are still digested and absorbed in the lower gut. The difference in primary site of digestion and absorption, however, does have big implications for the importance of optimal rumen function in a weaned calf. For the rumen to become the principal site of nutrient absorption, it must develop the rumen epithelium from birth to weaning.
If the rumen is insufficiently developed prior to weaning, nutrient absorption is compromised and growth suffers. In one study, scientists weaned calves completely at 42 days of age, with the weaning transition lasting anywhere from 0-22 days (Sweeney et al., 2010). In calves where weaning transition began at day 20 of life, the decrease in milk intake led to an increase in calf starter intake, as expected (Figure 2). Even though calf starter intake in the 22-day weaning group ate as much starter as the 10-day weaning group, the total digestible energy intake and body weight never kept up.
Figure 2. Impact of premature weaning on growth. Calves whose weaning transition began on day 21 (A; black squares) increased their starter intake (B) but were unable to make up for the loss of milk, resulting in lower energy intake (C) and lower growth (D). Modified from Sweeney et al. (2010).
Over time, the rumen’s ability to ingest, digest, and absorb nutrients improves, resulting in considerably improved performance. Waiting to wean calves until 8 weeks of age reduces the weaning performance gap often noticed in early-weaned calves (Eckert et al., 2015). When calves were weaned at 6 weeks, their metabolizable energy (ME) intake did not recover to preweaning intakes until 14 days after weaning (Figure 3). Calves weaned at 8 weeks, however, saw a recovery of ME intake to pre-weaning levels within 4 days. What these differing responses to the same dietary change demonstrate is the readiness of a calf’s gut to adapt to a sudden change in the primary site of digestion and absorption.
Beyond intake, these data also show the immaturity of the nutrient absorption mechanisms. During the weaning transition, the ME:gain ratio, a measure of energy intake to body weight gain, was twice as high for calves weaned at 6 weeks (18.60 Mcal/kg) compared to calves weaned at 8 weeks (9.17 Mcal/kg; Eckert et al., 2015). These changes indicate that the 6-week old calves were not only struggling with ME intake, but were also unable to effectively turn their feed intake into growth. Therefore, calves weaned too early are less effective at converting metabolizable energy intake into growth.
Figure 3. Change in metabolizable energy (ME) intake in calves weaned at 6 weeks (A) or 8 weeks (B). Calves weaned at 6 weeks showed a greater and longer drop in ME intake than calves weaned at 8 weeks. From Eckert et al. (2015).
Boosting Rumen Development Using Nutritional Stimuli
If calves are unable to turn their ME intake into growth, there is likely a compromised absorption ability. Little is known about how nutrient absorption mechanisms develop early in life and which factors promote them. Currently, the primary metric used to stimulate rumen development is the feeding of rapidly fermentable carbohydrates in calf starter. The impact of calf starter fermentation on rumen papillae development is clear (Figure 1) and well-documented in past research.
Fermentation of calf starter leads to production of butyrate, one of the VFA long linked to the development of rumen papillae (Warner et al., 1956; Figure 1). Butyrate is very bioactive, acting to alter gene expression of multiple genes involved in tissue adaptation (Baldwin et al., 2012). In adult cows, adding extra butyrate to the rumen improves VFA absorption capacity (Laarman et al., 2013a) and improves barrier function (Baldwin et al., 2012; Laarman et al., 2013b). Because of butyrate’s diverse impact on rumen development and function, increasing butyrate production in young calves is a sought-after nutritional goal.
Several avenues exist for improving butyrate supply in the gut. Direct feeding of butyrate is the most familiar, as its inclusion in milk replacer and calf starter increases growth and rumen papillae development in week-old calves and prior to weaning (Gorka et al., 2018). When fed during the weaning transition to eight-week old calves, calf starter intake increases, and remains 800 g/d higher after butyrate supplementation is withdrawn (McCurdy et al., 2019). Interestingly, the impact of butyrate on rumen development is noticeable even when the butyrate is rumen-protected (Gorka et al., 2018; McCurdy et al., 2019). Through the stimulatory effects of butyrate, the rumen’s nutrient transport mechanisms develop and ready the calf for weaning. Despite the clear impact of butyrate on morphological development and growth, its impact on nutrient absorption is far more nuanced, likely because of the complexity and plasticity of nutrient absorption dynamics.
Nutrient Transport Mechanisms
Nutrient absorption is a multi-factorial, multi-pathway system that takes place across every gastrointestinal epithelium. In young calves, the rumen epithelium is of principal interest, as it is non-functional. Currently, VFA absorption mechanisms are categorized primarily through transport pathways and transport proteins embedded in the epithelium that physically transport the VFA across the membrane. Transport pathways use kinetics measurements to determine the rate of VFA transport. Transport proteins, meanwhile, use abundance measurements to determine the capacity of VFA transport and potential targets for nutritional intervention to improve VFA transport.
Nutrient transport is accomplished through one of two mechanisms: passive diffusion or protein-mediated flux. Passive diffusion is the unregulated flow of VFA across the rumen epithelial membrane. For passive diffusion to occur, there needs to be a high VFA concentration in the rumen and a lower concentration in the epithelium, and the VFA need to be associated with a proton (H-VFA), as opposed to an anionic VFA (VFA-). As rumen pH drops, H-VFA become more prevalent (Table 1), and there is more opportunity for H-VFA to freely diffuse across the lipid membrane of the epithelial cells.
Table 1. Prevalence of unregulated (H-VFA) and regulated (VFA-) available for transport across the rumen wall at physiological pH (6.8), subacute ruminal acidotic pH (5.8), acute ruminal acidotic pH (5.2), and the pKa (4.8).
Several constraints limit the amount of passive diffusion. Firstly, the pKa, the pH at which 50% of VFA are dissociated, is 4.8. Therefore, at physiological pH of 5.8 – 6.8, the abundance of H-VFA is below 10% of all VFA present in the rumen and does not provide an adequate avenue to meet the cow’s energy needs. Even at acidotic pH (4.8 – 5.8), the abundance of H-VFA never exceeds 50%, so physiological rumen pH poses serious constraints on the ability of cows to effectively absorb ruminal VFA through passive diffusion alone. This is supported by research showing that the rate of VFA diffusion at decreasing pH does not match what would be predicted from biochemistry and lipophilicity of the VFA (Sehested et al., 1999). In a more recent study, a five-fold increase in VFA concentration resulted in only a 2.4-fold increase in VFA transport rates (Schurmann et al., 2014).
Part of the reason passive diffusion is more limited is because of the impact passive diffusion has on the epithelial cell. While rumen pH can vary from 6.8 to 5.2 without clinical incidence of ruminal acidosis (Aschenbach et al., 2011, Laarman et al., 2012b), the intracellular pH (pHi) of epithelial cells is tightly regulated at 7.4, with programmed cell death occurring when pHi drops below 7.0 (Lagadic-Gossmann et al., 2004), leading to compromised barrier integrity.
When a H-VFA enters the cell via passive diffusion, its pKa of 4.8 means it will immediately dissociate, adding a proton to the cell and thus lowering intracellular pH. The cell must then return pHi to 7.4 by modifying H+ and HCO3 - in the cytosol. Although H-VFA may diffuse freely across the lipid membrane of the epithelium, H-VFA can ultimately only be transported completely across the epithelium via protein-mediated transport mechanisms.
Consequently, passive diffusion of VFA across the rumen epithelium is overshadowed by regulated, protein-mediated flux of VFA- (Figure 4). Protein-mediated VFA transport is mediated predominantly through bicarbonate-mediated transport, which comprises up to 50% of all VFA transport (Aschenbach et al., 2011). This involves transporters such as anion exchanger (AE), which export one HCO3 - from the epithelial cell while importing one VFAinto the cell. There is also a bicarbonate-independent pathway, which is predominantly found in ruminants more efficient in VFA absorption, and consists of a nitrate-sensitive and nitrateinsensitive pathway (Penner et al., 2009). The bicarbonate-dependent and bicarbonateindependent comprise the two principal pathways of protein-mediated transport in the rumen.
Figure 4. Principal VFA absorption mechanisms across the rumen epithelium; arrows indicate direction. Passive diffusion can occur at the luminal (rumen-facing) side, but HVFA will dissociate upon entry into the epithelial cell (top right). Common transportbased mechanisms are shown, including bicarbonate-dependent mechanism (AE2), and a bicarbonate-independent mechanism (MCT1). Transport of VFA is intertwined with H+ and HCO3 - transport, therefore is intertwined with regulation of intracellular pH (NHE1, NHE3, NBC1). Not shown is a nitrate-sensitive transport pathway, the nature of which is not well-understood (Aschenbach et al., 2011). Schematic is simplified, merging strata of the rumen epithelium into one layer. From Laarman et al. (2015).
Development of Nutrient Transport Capacity
Currently, little is known about how the facilitated diffusion mechanisms develop in the calf. In the past, most research focused on the morphological and histological development of rumen papillae. Developed rumens, with greater papillae size and number, have a greater absorptive surface area, presumed to be indicative of VFA absorption capacity. Recently, however, the function of the epithelial cells was discovered to follow a trajectory quite different from previously held assumptions. Furthermore, the nutrient transport pathways are regulated differently at different stages of early life.
Prior to weaning, calves fed milk and starter, as opposed to milk only, have greater expression of MCT1 mRNA (Laarman et al., 2012a), but not MCT1 proteins (Yohe et al., 2019; Hiltz et al., unpublished), or VFA clearance rates from the rumen (Yohe et al., 2019). Between abundance of mRNA, which code the instructions to build more proteins, and abundance of protein, which do the actual transport, there is clearly considerable regulation that is currently not well understood. Much potential lies in targeting these cellular processes to improve VFA transporter abundance in young calves.
During weaning, nutrient absorption capacity at the genomic level continues to change. At weaning, for instance, there is differential expression of over 20 genes involved in ion transport (e.g. VFA, H+, HCO3 -; Connor et al., 2013). Despite changes in mRNA of VFA transporters, there is no difference in protein abundance of VFA transporters during and after weaning (Hiltz et al., unpublished). Reasons for the disconnect between mRNA and protein likely relate to post-transcriptional regulation, an area that is poorly understood in food production animals. Nevertheless, the gastrointestinal tract can adapt considerably in early life.
Post-weaning, VFA absorption capacity remains adjustable, especially in response to dietary changes. In one study using seven-month-old crossbred weanling steers, dietary supplementation of a Lactobacillus fermentation product increased butyrate production in vitro and increased rates of gain in vivo (Hall et al., 2018). In another study, 7-month-old Holstein steers, feed restriction to 25% of their ad libitum intake resulted in increased passive diffusion of acetate and butyrate (Laarman et al., 2016). Throughout early life, plasticity in VFA absorption remains, suggesting much research remains to be done in developing optimal strategies for changing diets.
Integration of VFA Absorption and Intracellular pH
One constant theme in rumen epithelial adaptation is that adaptations in VFA transport are intertwined with adaptations in pHi regulation. In a study on 7-month-old steers, VFA transport was positively correlated to pHi regulation capacity (Laarman et al., 2016). In a different study, low pH without VFA did not damage rumen epithelia, while adding VFA to a low pH environment caused breakdown of the epithelium (Meissner et al., 2017). At low pH, the increase in passive diffusion of VFA put a downward pressure in pHi, leading to cellular death and damage to the epithelium.
The link between VFA and pHi can also be found in the adaptation of the rumen epithelium to dietary changes. For instance, prior to weaning, the introduction of calf starter changes abundance of both NHE3 (Laarman et al., 2012a) and NBC1 (Hiltz et al., unpublished), both of which regulate pHi. Similarly, during the weaning process, when diet fermentability increases dramatically, NHE3 expression changes and decreases pHi regulation (Laarman and Oba, 2012a; Hiltz et al., unpublished). In adult cows, the lactation transition also exhibits decreased pHi regulation (Laarman et al., 2015), suggesting the metabolic adaptation of the rumen epithelium is more centered around changes in pHi regulation than direct VFA transport.
Despite nutrient absorption capacity historically being thought of as a function of absorptive surface area, research in the past decade showed definitively that nutrient absorption is closely linked to cellular physiology. The ability of epithelial cells to transport VFA and regulate their pHi are intertwined, and are regulated at the mRNA, protein abundance, and protein activity level. Throughout the first months of a calf’s life, major changes in development are brought on by the consumption of rapidly fermentable solid feed and major dietary changes such as weaning. These major changes include not only changes in absorptive surface area, but considerable and consistent changes in pHi regulation more so than changes in VFA absorption. Fostering improved nutrient absorption in young calves may therefore need to focus more on cellular physiology.
Nutrient Absorption & Health
Another reason why nutrient absorption is key to health is that the preferred energy substrate for the cells lining the gastrointestinal tract come from the lumen, not the blood. In the small intestine, the preferred energy substrates are the amino acids glutamine (Blachier et al., 2009), and, in the neonate, arginine (Wu et al., 2009). Unlike the small intestine, the large intestine and rumen use butyrate as a preferred energy substrate (Bergman, 1990). As a result, different preferred energy substrates are used for dietary supplementation in cows and calves. Another reason why nutrient absorption is key to health is the preferred energy substrate for the cells lining the gastrointestinal tract are sourced from the lumen, not the blood. In the small intestine, the preferred energy substrates are the amino acids glutamine (Blachier et al., 2009), and, in the neonate, arginine (Wu et al., 2009). Unlike the small intestine, the large intestine and rumen use butyrate as a preferred energy substrate (Bergman, 1990). As a result, preferred energy substrates are used for dietary supplementation in cows and calves.
Of the preferred energy substrates, the most abundant work involves butyrate supplementation. Used as rumen-protected supplement for calf starter, butyrate can have positive impacts on rumen development (Gorka et al., 2018) as well as starter intake (McCurdy et al., 2019), especially when fed around the weaning transition. Furthermore, butyrate is linked to decreased localized immune response (Dionissopoulos et al., 2013) and improved tight junction formation (Baldwin et al., 2012, Laarman et al., 2013b), indicating its ability to improve gastrointestinal health.
Likewise, glutamine is used to improve intestinal health. During the first months of life, glutamine and arginine are essential amino acids, playing major roles in intestinal energy supply and health (Wu et al., 2009, Wu et al., 2016). In calves, providing supplemental arginine or glutamine at 1% w/w in the milk replacer improved villus height, width, and surface area in the duodenum and jejunum when calves were fed a high plane of nutrition (van Keulen et al., 2020). Interestingly, the positive effects of glutamine or arginine supplementation are absent when calves are fed low amounts of milk replacer or a soy-protein concentrate (Drackley, 2008, Ahangarani et al., 2020, van Keulen et al., 2020). Although more research is needed on optimal supplementation strategies, conditionally essential amino acid supplementation can improve gut health in young calves.
Ultimately, supplementation strategies will only be successful if the preferred energy substrates can be absorbed by the intestine. Intestinal absorption of glutamate and glutamine requires facilitated transport (Howell et al., 2001), while absorption of butyrate is predominantly through facilitated transport (Sehested et al., 1999). Therefore, the health of gastrointestinal epithelial cells is dependent on functional absorption capacity, leaving nutrient absorption and gut health very much intertwined.
Optimizing Nutrient Absorption and Health
Optimizing nutrient absorption and health in young calves are key for preparing calves for the health challenges common in early life, principally weaning and disease pressure. During weaning of 7-month-old beef calves, neutrophil counts increase and phagocytic function of the immune system decreases, suggesting impaired immune function (Lynch et al., 2010). Likewise, dairy calves often exhibit reduced circulating neutrophils and monocyte function during the weaning transition (Hulbert and Moisá, 2016). In both dairy and beef calves, weaning is a stressful event.
One of the principal ways of optimizing health during weaning is to ensure weaning occurs independent from other stressors. Oftentimes, weaning is concurrent with changes in housing (e.g., feedlot in beef, comingling in dairy), which can leave calves vulnerable to secondary disease pressures. In one study, beef calves that were weaned two weeks before transport to a new facility had a lower mortality rate from a secondary bacterial infection than calves that were weaned and transported on the same day (Hodgson et al., 2012). In another study, gradual weaning using a 2-week stepdown increased solid feed intake and growth post-weaning (Khan et al., 2007), highlighting the importance of stress management in increasing nutrient intake.
Through effective management strategies in early life, calves improve performance later in life. For instance, calves exposed to a Salmonella enterica Typhimurium challenge five weeks after weaning had improved immune response and 10% higher solid feed intake if they had been fed a high plane of nutrition prior to weaning (Ballou et al., 2015). In a different study(Ballou et al., 2015). Alternatively, dairy calves with greater liquid and solid feed intake prior to weaning produce more milk in their first lactation (Soberon et al., 2012, Rauba et al., 2019). In all, emphasizing nutrient intake and absorption as a principal goal early in a calf’s life confers considerable health and productivity benefits in subsequent life stages.
Calves are born as pre-ruminants, with an underdeveloped and non-functioning rumen. In a mature rumen, nutrient digestion and absorption play critical roles in meeting the energy needs of the cow. Consequently, bolstering nutrient absorption capacity in young calves is an import goal of calf management strategies. Key to development of nutrient absorption capacity is ensuring a higher plane of nutrition to ensure growth requirements are met pre-weaning. Supplying a higher plane of nutrition will also directly benefit the cells lining the gastrointestinal tract. The gastrointestinal tract is fueled by energy substrates from the lumen, so having an adequate supply of butyrate and glutamine, arginine, and glutamate is essential for meeting the energy supply of the gastrointestinal epithelium.
In the rumen, feeding rapidly fermentable feeds such as calf starters will play an important role in developing the rumen epithelium to prepare it for the weaning transition. The development of the rumen epithelium involves changes in pHi more so than changes in VFA transporters directly. Furthermore, these transitions continue beyond weaning, so the freshly weaned calf is not yet a mature ruminant. In all, weaning imposes a major stress on the calf. Managed successfully, weaning should occur in isolation from other stressors like housing changes.
Together, both beef and dairy systems have similar goals for calf-raising programs. That is, the development of nutrient absorption capacity while maintaining a healthy gastrointestinal tract is one of the key concerns in preparing calves for weaning, regardless of age at weaning. Physiologically, weaning is a shift in the primary site of nutrient absorption from the small intestine to the rumen, which is inherently stressful. While cow/calf systems benefit from allowing calves more time on liquid feed before initiating weaning, dairy heifer systems benefit from aggressively pushing ruminal development prior to weaning through feeding rapidly fermentable calf starters. Through continued research into optimal feeding and absorption strategies, more effective calf raising strategies can be developed that further improve nutrient absorption capacity and health in young calves.
Published in the proceedings of the Animal Nutrition Conference of Canada 2020. For information on the event, past and future editions, check out https://animalnutritionconference.ca/.