Studying weaning at different ages is challenging for research approaches. The regimen of weaning at younger age has different requirements compared to weaning at older age. In this study, for instance, calves at 15 wk of age voluntarily ingest more concentrate than calves at 5 wk of age (8). To provoke an increase in concentrate intake at this early age, MR consumption has to be reduced. As a result, concentrate intake increased to 1 kg/d. In calves at 15 wk of age, the concentrate intake was twice as high compared to the early-weaned calves. Thus, at the beginning of weaning, the concentrate intake was limited to 1 kg/d in the late-weaned group. After weaning, all calves received hay and a TMR. The effects of two different rearing regimens and mother’s parity are discussed in the following.
Rumen maturation at early age was assessed by behavioral observations, rumen sounds and scoring of rumen fill. Closely related to rumen maturation, acid base homeostasis and N metabolism have to adapt to ruminant status. Thus, the pH in blood, urine, feces and saliva was measured to assess changes in systemic acid–base-metabolism going along with rumen maturation. Furthermore, the concentration of several N-containing compounds (creatinine, allantoin, uric acid, hippuric acid and urea) in urine was determined to estimate the onset of microbial activity in the gastrointestinal tract, especially in the rumen, and to assess the development of early N metabolism in growing calves.
Mother’s parity had no effect on any parameter except rumen fill score (p = 0.018). This indicated that rearing conditions have a greater impact on rumen development, maturation of acid–base homeostasis and N metabolism than mother’s parity. Rumen fill score was greater in PCs, which might be caused by their smaller body seize paired with the same feed intake as MCs.
Rumen maturation at early age
Late-weaned calves increased their solid feed intake before weaning . As a consequence, rumen fill score and rumen sound increased before weaning as well. Rumen sounds per 2 min were assessed to get insights into progress of rumen motility, as it is possible to notice the sound of ingesta movements in the rumen with a stethoscope. Biphasic contractions of the reticulum were observed in milk-fed calves at the age of 16 d through ultrasonographic examinations . In the current study, some calves had detectable rumen sounds as early as during the first wk of the trial, that corresponds to an age of ~ 14 days. This indicated that rumen sound and therefore rumen contraction were not only influenced by solid feed intake, but might be determined, at least partly, by an evolutionary blueprint. This might indicate that the nuclear genome was partly determining the age and velocity of maturation of organs and tissues of newborn and young calves with regard to their structure and function. Adverse nutritional interventions, especially during this fragile time window, could deteriorate this process leading to metabolic stress due to immature organs and tissues.
Confirming this assumption, early-weaned calves belatedly increased their time spent chewing from experimental d 70 on, which was 4 wk after weaning. This happened despite the ad libitum supply of hay and TMR, which is as solid feed known to increase rumination. However, late-weaned calves increased chewing during weaning at experimental d 105 already. The immature rumen might have contributed to lower solid feed intake in early-weaned calves. Thus, these calves were undernourished over several wk which was reflected in a lower live weight gain . Concomitantly, early-weaned calves expressed a more active behavior after weaning up to experimental d 70, most likely due to seeking for liquid feed. In late-weaned calves, weaning was not associated with higher activity levels afterwards.
Calves that could freely access different solid feed and MR spent 20% of time with chewing both at the age of 3 and 6 mo . As these calves displayed a low level of non-nutritive oral behavior, such as tongue playing and oral manipulation of the pen structure or other calves, a chewing level of 20% appeared to be enough to satisfy needs for chewing and rumination. This level was reached by our feeding groups at d 21 (earlyMC and earlyPC), 28 (lateMC) and 49 of the trial (latePC) and remained stable or increased most of the time (Fig. 1). Therefore, weaning at 17 wk of age may not trigger abnormal behaviour. The results of the aforementioned and the current study were similar, although we observed behavior only in a small time frame during the day because chewing was found to have no circadian rhythm in calves .
Maturation of acid–base homeostasis in growing calves
Maturation of the gastrointestinal tract might be associated with maturation of systemic acid–base homeostasis. To assess its development, the pH values in saliva, blood, urine, and feces were measured during early life in all calves. As pH in saliva increased over time, being unaffected by weaning age or mothers’ parity, it seemed to be determined solely by the evolutionary blueprint. The first significant increase in pH compared to experimental d 1 was observed at d 42 (p < 0.001) and then again from d 42 to d 140 (p = 0.01) in all groups. Therefore, there might be an ontogenetic window for development of ruminant salivary buffer composition during these time points. The salivary gland responsible for secreting large amounts of buffer in ruminants is the Glandula parotis. The main chemical components of Parotis saliva are Ca2+, Na+, K+, Urea, HCO3−, HPO42−, Cl− and water. In general, the concentration of these components did not vary much between healthy adult cows and were unaffected by diet. Only urea concentration was affected by diet in adult cows . The saliva buffering capacity and alkaline pH is mainly achieved by HCO3− and HPO42− . Therefore, these components are vital for an adequate rumen function. The change in salivary pH indicated that the adequate composition needed time to develop. However, the capacity of salivary glands, especially of the Parotis, to produce adequate volumes of saliva to buffer VFA production in the rumen was not assessed in this study.
As a consequence of the associated slow rise of salivary buffer capacity, early weaning, particularly before the age of 7 wk, could lead to acidification of rumen fluid. Ingestion of starch-rich concentrate might have increased rumen VFA concentration and thereby, decreased rumen pH value. This possible acidotic condition was most likely not balanced by a sufficient availability of saliva, due to low buffer concentrations and saliva volume, respectively. Additionally, absorption of VFA by the rumen epithelium might not be expressed with a sufficient capacity leading to an accumulation of acids in the rumen at this age. Furthermore, chewing activity, which is strongly influencing saliva secretion rate, is low after weaning in early-weaned calves. Others noticed different rumination patterns attributable to weaning age as well. Calves weaned at the age of 8 wk displayed more rumination before weaning than calves weaned at the age of 6 wk . Therefore, it was hypothesized that early-weaned calves were at risk for ruminal acidosis, and for higher acid load in plasma as well. Several authors observed an acidic ruminal pH in early-weaned calves [14, 15]. Calves that were weaned at 4 wk of age had a ruminal pH under 5.5 for at least 4 wk after weaning (15). Weaning at 6 wk of age reduced ruminal pH below 5.5 at least for 3 wk as well . As this might be a sign for chronic rumen acidosis at least for adult cows , it is possible that these calves suffered from this disease as well.
To maintain plasma pH is vital, thus, the lung and the kidneys are important regulators of acid–base homeostasis. While in the lung protons were eliminated as CO2 and H2O, kidneys excrete protons or reabsorb bases to maintain acid–base balance. Physiologically, urinary pH of healthy fully ruminating cattle is neutral to slightly alkaline [17, 18] due to the high amounts of excreted bicarbonate in herbivores. In the condition of metabolic acidosis, the kidney conserved bicarbonate by reabsorption . As a consequence, the urinary pH value declined with decreasing bicarbonate concentration . Consequentially, urinary pH value correlated positively with ruminal pH value . Therefore, urinary pH might reflect the ruminal pH value and the decrease of urinary pH during early-weaning might have indicated rumen acidosis during this time. In early-weaned calves a potentially higher acid load needed more bicarbonate, thus this buffer was reabsorbed in the kidney. Consequently, pH of urine decreased (experimental d 42, 6.6 ± 0.1) directly after early-weaning (Fig. 4D). Furthermore, a higher NH4+ and phosphate concentration may also be responsible for the lower urinary pH value after early weaning, which might result from still low utilization of N-containing compounds in rumen microbial metabolism.
Furthermore, low urinary pH value was associated with greater calcium excretion, probably bound to acid phosphate . As calcium is important for growth and bone development, this could have contributed to the impaired growth of early-weaned calves .
In adult cows, values of NABE and BAR in urine were used to diagnose a mild metabolic acidotic burden . In calves this might not be advisable, as both variables are low in milk-fed calves (Fig. 6). The values of NABE and BAR increased over time, which reflected the transient change in dietary composition. In early-weaned calves, the increase was steep after weaning from experimental d 42 until d 70, but not during their weaning. The metabolic adaptation to an adult status seemed to occur after weaning was already done. On the other hand, the increase of NABE values in late-weaned calves was not as steep but occurred before weaning. They might have experienced a smoother transition to adult status.
The pH value in feces decreased during weaning (Fig. 4 B). Lohakare et al.  measured a pH of 7 in feces of calves at 40—42 d of age. Confirming, in the current study calves were 37 ± 2 d of age at experimental d 28 and had a faecal pH around 7 as well. Lohakare et al.  observed a decrease of faecal pH through weaning as well, but this increased again to a pH of 6.8 – 7.0 at 42 d after weaning. This increase was not detected in the current study, in which faecal pH continued to decline. Adult cattle had a faecal pH from 7 – 8 [24, 25], which, in this study, was not reached by any group until the end of the trial. This may be a result of high intake of starch through concentrate and TMR which could not be digested in rumen and therefore was transported into the large intestine. In the hindgut, it could be fermented, but emerging VFA could not be fully absorbed, subsequently leading to a decrease in pH value. The decrease in faecal pH through increased energy intake and carbohydrate infusion has been tested in steers , as well as a strong correlation between faecal starch content and pH . Calves had a higher percentage of faecal starch after weaning, especially when weaned early at the age of 6 wk . Hence, the low fecal pH could be a sign for inadequate digestion in the rumen, especially of starch. As the concentration of starch was common for calve and heifer feed (concentrate 371 g/kg dry matter (DM); TMR 182 g/kg DM) the rumen might not have been developed sufficiently to digest a typical adult cow diet even at the age of 5 months.
Maturation of N metabolism in growing calves
Nitrogen-containing metabolites in blood and urine indicate changes in the diet, endogenous protein metabolism, and in developing microbial metabolism in the rumen. Dietary effects on N-containing metabolites were most likely associated with changes in microbial metabolism. Urea-N was measured in plasma  and urine, while non-urea-N was measured only in urine. Creatinine is the only N-containing metabolite in urine which is derived solely from muscle protein break-down, thus of endogenous origin . Early weaning resulted in a sudden increase in urinary creatinine and urea, which indicated a catabolic status. Due to the change of highly digestible milk protein and energy to less digestible solid feed protein and energy, calves failed to adapt to weaning at early age. A fasting status was most likely established at the period of early weaning. Concomitantly, this was confirmed by a strong increase in plasma urea and beta-hydroxybutyrate and a low insulin concentration at early weaning  Furthermore, the quick rise in excretion of allantoin and uric acid, end products of metabolism of endogenous nucleic acids, might also be attributable to a catabolic state and/or a more stimulated microbial activity compared to late-weaned calves at this time. While the latter group showed a continuous increase of these compounds the calves weaned at experimental day 42 and changed to a TMR onwards, failed to further increase allantoin and uric acid concentration in urine. This might indicate a relative deficiency of energy available for adequate ruminal microbial protein synthesis due to a lower supply of easily fermentable substrates. Later, urinary urea concentrations decreased steadily in early-weaned calves, most likely due to recycling into the developing rumen to serve as N source for the microbial growth. Due to the prolonged high availability of milk protein and energy in late-weaned calves, urea concentrations in blood  and in urine were stable on a high level until weaning. At weaning, urea decreased by recycling into the rumen to compensate a lower dietary crude protein (CP) supply.
The purines from rumen microbes were metabolized and the end products hypoxanthine, xanthine, uric acid and allantoin were excreted in the urine . Xanthine and hypoxanthine were only found in small amounts in cows’ urine, whereas uric acid and allantoin were the abundant purine derivatives . This is due to the fact that, xanthine and hypoxanthine are converted to uric acid, which is further converted to allantoin . Therefore, urinary concentrations of allantoin and its precursor, uric acid, were used as a predictor for microbial CP production in the rumen . Both increased in early-weaned calves during weaning, but as they decreased again until the end of trial, their importance as a marker for rumen microbial activity can be contested. This has already been refuted for cows in different stages of lactation . As stated above, the steep initial increase in allantoin and uric acid might be provoked by endogenous catabolic processes and only to a small extent by the increase in microbial activity at early weaning. Late-weaned calves expressed steady increases in urinary allantoin and uric acid concentrations, which slightly peaked around weaning. Therefore, it can be assessed that provision of liquid feed together with voluntary solid feed intake promoted a slow but effective rumen development and microbial activity. However, for the full functioning of rumen, the capacity for fibre digestion must be developed. For that, hippuric acid is a well-known marker . It is formed in the liver from benzoic acid and glycine. Benzoic acid is synthesised by microbial metabolism of plant-derived phenolic cinnamic acids in the rumen . Therefore, urinary hippuric acid concentration was strongly linked to diet composition in adult cows [32, 33], which seemed to be the same in calves as urinary hippuric acid concentration increased after weaning in both groups. Early-weaned calves, however, did not reach mature hippuric acid concentrations before experimental d 70, which could indicate an insufficient fibre digestion in the rumen, although they were on solid feed from d 42 on. Concomitantly, time spent chewing was increased only after experimental d 70, supporting the assumption that fibre digestion was not developed after early weaning and fibre intake was low, respectively. The potential restriction was reflected in a lower live weight gain and growth rates after early-weaning .
In addition, urinary N is a source of N2O emission , which affects the environment and can influence surface water, biodiversity and climate change in a negative way . Therefore, attempts should be pursued in optimal rearing strategies to lower the excretion of urinary N-containing compounds.
Rumen maturity at the age of 5 months
All variables linked to rumination, such as chewing behavior, rumen fill and sound, increased over time in both groups, despite the high MR allowance. These variables as well as VFA concentration and pH of ruminal fluid were not significantly different between the weaning groups at the end of the trial (d 140), which indicated that both groups reached a concordant status of rumen development. Therefore, rumen and its functions appeared to be equally developed at the age of 5 months, which corresponds to experimental d 140. Late-weaned calves showed no sign of impaired rumen development at that age despite of the higher weaning age. Ad libitum intake of MR in the first 5 wk of life did not retard rumen development . High MR allowance and higher weaning age did not avert the transition to a functional ruminant status. Additionally, this transition was smoother in late-weaned calves . On the contrary, in early-weaned calves these metabolic changes occurred relatively abrupt, which might have put a lot more strain on organs and tissues. In mammals, development of organs and tissues and their physiological functions in adulthood can be affected by early nutrition [36, 37]. As mentioned before, metabolic imprinting might occur through nutritional experiences in early life and can have a great impact on later health and performance . Therefore, these abrupt changes in nutrition and consequent metabolic adaptations might lead to future impairment of health and performance. Increased MR intake by calves resulted in greater relative kidney weights [38, 39]. Hence, kidney development seemed to be affected by early-life nutrition. The secretion of protons and the rapid change of urinary composition during early-weaning might strain the calves’ kidney massively. This might also result in coping mechanisms that imprint these vital organs and alter their functions later in life. Although there was no difference in the measured variables at the age of 5 months, early-weaning might have caused a metabolic imprinting which might result in negative outcomes later in life. This might be true for calves born to primiparous mothers as well , even though we did not observe a considerable effect of parity on measured parameters. To assess the metabolic imprinting, these animals were further monitored in an ongoing observational study.