4.1. Energy Status: Fasting Contrasted with the Postprandial Period
The metabolic profile of the animals during the postprandial period was generally consistent with reports from Kaneko et al. [11
]. As a typical example, the glycemic curve was characterized by a mean glucose concentration that was lower at the first postprandial hour than at the subsequent hours. This was to be expected because the absorption of propionate in the rumen causes a sudden release of insulin and a consequent moderate reduction in blood glucose [21
Glycemia is considered a basic but important variable for the evaluation of energy status [10
]. The blood glucose concentrations during the postprandial period of this study, considering two standard deviations around the mean (3.4 to 4.8 mmol/L; mean of 4.1 mmol/L), were well above the reported by Kaneko et al. [11
] (2.5 to 4.2 mmol/L) and Payne and Payne [10
] (2.0 to 3.0 mmol/L). These data were obtained in dairy, beef, lactating, or pregnant cows, which are known to have lower blood glucose concentrations than young cattle [12
]. There should be two reference values for blood glucose—one each for adult and young cattle. This was also supported by Otto et al. [13
] in Paraguay; they reported reference values of glucose (4.32 ± 0.72 mmol/L) in young beef cattle that were similar to those obtained in this experiment.
Blood glucose concentrations decreased significantly during the fasting period, which were more intense with a longer duration of food abstention. This was also observed by other researchers [25
]. Despite the sharp decrease in blood glucose, the fasting animals maintained energy through indirect homeostasis; energy was generated through lipolysis and proteolysis. These were directly or indirectly proven in this study.
Fat is stored in the form of triacylglycerol. The lipase enzyme breaks this compound into glycerol and three molecules of free fatty acids. Under experimental conditions, it has been shown that fasting induces a decrease in insulin secretion and an increase in glucagon secretion in bovines. This hormonal condition is highly stimulating for the higher activity of lipase, as insulin is an inhibitor of this enzyme, while glucagon is a stimulator [28
]. In this experiment, lipolysis was manifested by the substantial increase in serum levels of free fatty acids, which were higher with longer durations of fasting (r
= 0.92; p
Blood circulating glycerol and volatile fatty acids can generate glucose through gluconeogenesis in the liver and kidney. The first substrate enters the glycolytic pathway and converts into triose-phosphate, which in turn converts into glucose; this is a route widely used during fasting [29
]. The second substrate enters the liver and oxidizes the mitochondria to generate ATP and acetyl-CoA, which combines with oxaloacetate and enters the Krebs cycle. Finally, glucose is formed through a pathway called gluconeogenesis [31
When large amounts of free fatty acids are generated, oxaloacetate levels decrease and ketone bodies form; approximately 65% comprise βHB [31
]. This compound can also be generated by butyrate produced in ruminal fermentation, which is absorbed into the rumen epithelium and transformed into βHB to be used as energy [21
]. Normally, the βHB detected in plasma is generated from rumen fermentation. In the present study, the plasma concentrations of βHB during the postprandial period ranged from 0.35 mMol/L (minimum value before diet offer) to 0.67 mmol/L, which are similar to those obtained by Herdt et al. [34
]. The concentration of butyrate in the portal vein of cattle that fasted for two days was 0, demonstrating that during fasting, ruminal butyrate did not contribute to the formation of blood βHB [30
]. In this study, there was an increase in plasma βHB concentrations between 24 and 48 h of fasting. Although the final increase (0.57 mmol/L) of the βHB concentration was still within normal limits, βHB was predominantly influenced by the generation of ketone bodies, as a high positive correlation was observed (r
= 0.68; p
< 0.0001) between the plasma levels of FFA and βHB. This indicates that ketogenesis occurred, but it was not enough to cause ketosis, which can be caused in cattle when the concentration of βHB exceeds the plasma concentration of 1 mmol/L [31
The ketone bodies produced during fasting were not sufficient to be detected in urine, which is usually characteristic of cases of ketosis. Rule et al. [28
] subjected steers to fasting for eight days and demonstrated that plasma βHB increased during the second fasting day, reaching 0.6 mmol/L. This is similar to the findings of our study; the level was maintained until the 8th day of fasting. These results bring the classic but generic statements that ketonuria is common in ruminants submitted to fasting into question [4
]. In particular, ketonuria can occur more frequently in fasting parturient cows; they have a characteristic hormonal profile and a high requirement for milk synthesis, which causes a notorious negative energy balance that favors lipolysis and generates much higher amounts of ketone bodies that exceed the renal threshold and allow its detection during the Rothera test [31
]. Ketonuria was not observed in steers submitted to prolonged dietary energy deficiency [15
The overall medians for cholesterol and AST during fasting showed a slight significant increase when compared to the postprandial period. Despite this significant increase, both variables were within the reference values [10
]. In other words, the mobilization of free fatty acids for the liver did not cause lesions in hepatocytes and did not alter the esterification mechanisms of triglycerides; hepatic lipidosis was not induced.
The fasting cattle maintained energy through the mobilization and use of lipids and amino acids from their body reserves. In normal cattle, the serum urea concentrations are maximal between 3 and 9 h of the postprandial period, mainly due to ammonia absorption induced by the digestion of dietary nitrogen in the rumen, which is duly converted in the liver to urea in the cycle of the same name [35
]. In this experiment, longer fasting duration was associated with higher serum urea concentrations (r
= 0.52; p
< 0.0001).This increase in urea may be attributed to a decreased peripheral uptake of amino acids and increased catabolism of labile protein reserves, resulting in an increased burn of amino acids to produce energy and urea synthesis, which was observed by Rule et al. [28
] until the second day of fasting.
In this experiment, it was demonstrated that food fasting caused a moderate increase in plasma volume deficit rate (i.e., mild hypovolemia), evidenced by increases in albumin, total protein, and serum globulins of 6.1%, 9.3%, and 11.8%, respectively, after 48 h. Unlike in previous studies that detected high plasma volume deficit rates (around 35%), dehydration, and oliguria in cattle with ruminal lactic acidosis [6
], we did not observe dehydration at the end of the fasting period. We observed a moderate hypovolemia, which was probably a consequence of the lower water intake caused by fasting, since a significant portion of the water consumption came from the water in the food composition. In a controlled experiment, Bond et al. [17
] submitted 300 kg steers to food fasting and evaluated daily water intake; while during the control period the average water intake was 41 L, at the end of 48 h of fasting the consumption had reduced to only 8.5 L.
In summary, the 48 h food fasting caused moderate hypoglycemia in the animals, accompanied by increased lipolysis, which triggered a slight increase in the concentration of serum βHB of ketogenic origin, without the occurrence of ketonuria or hepatic lipidosis.
4.2. Energy Status: Re-Feeding Contrasted with the Fasting Period
On the one hand, if the fasting caused a significant change in energy metabolism in the short term, the re-feeding generated immediate recovery of the energy state. The results indicated that there was marked gluconeogenesis on the first day of re-feeding, accompanied by a drastic reduction in lipolysis and the generation of βHB by the ketogenic pathway. There was a transitional period characterized by a higher serum urea concentration during the first 6 h. Lomax and Baird [30
], based on the amount of propionate, lactate, βHB, glycerol, and some amino acids in the hepatic artery, theorized that the main precursor substrate of glucose during re-feeding was lactate (54.4%), followed by propionate (16.9%) and some amino acids (4.4%). The high levels of serum urea obtained during the sixth hour of re-feeding may indicate that amino acids have a more important role in the formation of glucose.
The concentrations of FFAs decreased dramatically during re-feeding, and it would be logical to state that insulin secretion increased while glucagon secretion decreased, as this hormonal condition would lead to a reduction in lipolysis and also a decrease in amino acid burn [31
]. Therefore, this situation may not have occurred, because at the beginning of re-feeding the plasma glucose was at its minimal concentration, which would stimulate the secretion of glucagon and growth hormone.
In summary, during re-feeding there was a rapid recovery of the mild hypoglycemia detected during fasting, with a marked drop in lipolysis and ketogenesis that was temporarily accompanied by increased amino acid burn for energy. The mild intravascular dehydration observed during fasting was resolved during re-feeding.