This study comprehensively analyzed the metabolic status of Japanese Black steers according to fattening phase, including various physiological parameters such as the liver transcriptome, blood metabolites, hormones, and rumen fermentation characteristics. The major physiological changes associated with fattening period and productivity are discussed below.
Changes in physiological parameters in Japanese Black steer according to fattening phase
Here, we reported the changes in metabolite profiles, hormones, and rumen fermentation characteristics during the fattening period in Japanese Black steer bred and raised in Japan. The triglyceride and NEFA concentrations (associated with lipid metabolism) increased with fattening phase due to the changes in the feed, including higher amounts of concentrated diet and increased dry matter intake. Increasing amounts of the concentrated diet from T1 to T2 elevated the total cholesterol, phospholipids, glucose, and γ-GTP levels in T2; however, the levels of these metabolites in T3 were similar to those in T1. Japanese Black cattle are fed large amounts of high-energy concentrate diets in the middle and late fattening phases to improve their growth performance and productivity. A study on Yellow breed × Simmental cattle showed that lipid metabolism-related blood metabolites (triglycerides, cholesterol, and high-density and low-density lipoprotein) were significantly higher in the high-energy group (6.64 MJ/kg) than in the low-energy group (5.98 MJ/kg), and that blood insulin levels increased with increasing energy levels8. Blood glucose and insulin levels also increase significantly with high-starch diets in Angus and Angus × Simmental cattle9. In ruminants, concentrate diets are a major source of glucose, either through increased production of rumen propionate (a gluconeogenic precursor), or—to a smaller extent—through glucose absorption from rumen-bypassed diets10. Glucose synthesized via gluconeogenesis is the primary energy source in ruminants11, and excess glucose is converted into fatty acids that circulate to other parts of the body and are stored as fat in adipose tissue12. Accordingly, the high-energy feeding boosts rumen propionate production and liver gluconeogenesis, and may explain the increased concentrations of blood lipid metabolites during the middle and late fattening phases. In cattle, NEFAs—indicators of fat mobilization—increase due to energy deficiency13 or pathological problems such as liver dysfunction, ketosis, and fatty liver14. In a state of negative energy balance, NEFAs are produced via lipolysis from triglycerides stored in adipose tissue and transported to organs and tissues15. In the present study, high blood levels of NEFAs in the middle and late fattening phases were likely due to relative increases in body fat reserves as fattening progressed, rather than energy deficiency. Samra et al.16 reported that cortisol—an indicator of stress response—promotes lipolysis, thus increasing blood NEFA concentrations. The increased cortisol levels during the fattening period in this study may have promoted lipolysis, resulting in increased concentrations of NEFA in the blood. The blood insulin level depends on age, weight, diets, and feeding management17, and is generally known to increase as fattening progresses. Ruminants are less sensitive to insulin than monogastric animals since insulin secretion in ruminants is mainly stimulated by VFAs18,19. Propionate is a strong stimulator of insulin secretion in sheep20. The increase in insulin secretion from T1 to T3 may be related to the increase in rumen propionate during the fattening period, and may have facilitated the intracellular influx of glucose, thus reducing blood glucose levels. Generally, BHB and other ketone bodies increase during energy deficit and are produced as metabolites in the lipid oxidation process of NEFAs in the liver. Here, the ketone concentrations decreased significantly in the middle and late fattening phases. Particularly, the decrease in BHB levels was likely due to the decreased levels of its precursor, rumen butyrate, during the middle and late fattening phases. To evaluate the liver function in Japanese Black steers, we analyzed the blood levels of ALP, AST, ALT, and γ-GTP. These metabolites are present in hepatocytes and released into the blood stream when hepatocytes are damaged by stimulation such as mold toxins and long-term high-energy feeding. Blood ALP—mainly released from the liver and bones—is related to liver dysfunction and bone development in beef cattle21,22. In growing animals, blood ALP is mainly released from bone tissue; the main tissue that releases the ALP into the blood stream transforms into liver tissue and the overall release decreases with growth23,24. In Japanese Black calves, blood ALP concentrations are significantly lower in the late growing stage than in the early growing stage, likely due to the slower rate of bone growth with increasing age23. In accordance with the findings of the previous study, our results showed that the blood ALP concentration decreased with fattening phases, suggesting that blood ALP concentrations decrease with age unless there is a severe abnormality in liver function. The blood γ-GTP is mainly released from the liver and bile duct and reflects chronic liver function because changes in its blood concentration occur at a slower rate than those of AST and ALT25. Blood γ-GTP concentration is positively correlated with BUN levels in Japanese Black cattle during the fattening period. Similar to the results of the previous study, our results showed that the blood γ-GTP levels were comparatively higher in T2 and T3 than in T1, and the reduction was likely caused by increased feed intake in T2 and T3. The above 4 enzymes increased or decreased slightly with the fattening period, but all were within the normal range.
Amino acids are not only the organic components of proteins, but are also involved in various physiological mechanisms, such as glucose synthesis via gluconeogenesis, fatty acid synthesis, and hormone regulation26. Amino acid metabolism is closely related to liver function because the absorption and transformation of amino acids occur in the liver. Thus, the blood concentrations of amino acids can be used to determine the nutritional and metabolic conditions of animals. Here, 22 of the 29 blood amino acids showed differences in concentration between fattening phases, and 13 decreased during the late fattening phase. Samantha and Connolly2 also reported that the progression of the feeding period in the feedlot was associated with decreasing blood concentrations of amino acids such as proline, leucine, isoleucine, histidine, whereas aspartate, and glutamic acid (a typical gluconeogenic acid) increased in wagyu crossbred steers.
Liver transcriptome changes in Japanese Black cattle according to fattening period
We conducted the GO pathway analysis for enriched DEGs to define the functional roles of DEGs according to fattening period. Here, we focused on comparisons between early and late fattening phases, which showed the greatest differences in gene expression in the DEG analysis. The DEGs were significantly enriched in metabolic processes such as glucose and lipid metabolic process.
The expression of genes related to glucose metabolism—such as SESN3, insulin receptor (INSR), leptin receptor (LEPR), forkhead box O1 (FOXO3)—was decreased in late fattening phases. LEPR is a liver-specific leptin receptor and regulates energy balance. Hepatic LEPR expression is positively correlated with plasma leptin concentration27; serum leptin concentration gradually increases from 9–23 months of age (with slight changes until 30 months) in Japanese Black steer28,29. Additionally, leptin level changes with the body fat mass and stimulation of insulin30. Thus, LEPR regulation in the late fattening phases may be attributed to fat accumulation and increased insulin secretion, and the increased leptin levels may lead to a decrease in leptin receptor levels. The insulin receptor plays a key role in glucose homeostasis and is closely regulated by hormones such as insulin, IGF-I, and IGF-II. Fisher et al.31,32 reported that plasma insulin levels were elevated in liver insulin receptor knockout mice, and the insulin regulation of receptors was related to the inhibition of glycogenolysis and gluconeogenesis. Previous studies in cattle have reported that the mRNA expression of insulin receptor in hepatocytes decreased with increasing insulin concentration in a dose-dependent manner32, and that dairy cattle with ketosis and fatty liver showed low mRNA expression of hepatic insulin receptor33. FOXO3 is also inactivated by the binding of insulin and its receptor, regulates the expression of gluconeogenesis-related genes, and promotes gluconeogenesis. The hepatic suppression of FOXO1 and FOXO3 causes hypoglycemia and hyperlipidemia in FOXO1 and FOXO3 knockout mice; moreover, the expression of hepatic FOXOs affects the reduction of gluconeogenic gene expression and insulin resistance34. Additionally, FOXO3 also activates the SESN3, which seems to be related the reactive oxygen species rescue pathway35.
We also found that the expression of genes related to lipid metabolism such as fatty acid-binding protein 4 (FABP4) was increased in late fattening phases, whereas fatty acid desaturase 1 (FADS1) and FADS2 expressions were decreased. FABP4 encodes the fatty acid binding protein, which is considered to be related to the uptake and metabolism of fatty acids. The FABP4 genotype significantly affects the marbling score in Wagyu and Limousin crossbred cattle36; FABP4 may be associated with the fatty acid composition of intramuscular fat tissue37. Here, FABP4 expression was higher in the late fattening phases than in the early phases. This increase in gene expression related to fat absorption and metabolism is thought to reflect the active accumulation of fat or other energy source in the liver during the late fattening phases. FADS1 and FADS2 are associated with the composition of fatty acids such as arachidonic (C20:4n-6), linoleic (C18:2n-6), alpha-linolenic (C18:3n-3) and eicosadienoic acids38. Supplementation of dietary fat in lambs results in increased expression levels of hepatic FADS1, along with increased intramuscular docosahexaenoic acid (DHA) levels in the DHA supplemented group39. Therefore, here, the changes in FADS1 and FADS2 expression may be due to changes in fat composition with changes in feed.
To identify the significant pathways involving the DEGs between the early and late fattening phases, we performed the KEGG pathway analysis. In total, 33 pathways were significantly enriched for the identified DEGs. The pathway analysis suggested that the main physiological changes associated with fattening period were related to energy and lipid metabolism in Japanese black steers.
Blood metabolic profiles and liver transcriptomes related with carcass traits in Japanese Black cattle
Here, blood triglyceride contents were significantly positively correlation with rib eye area and BMS in T1. Blood triglyceride levels are negatively correlated with the level of intramuscular fat accumulation (BMS)40 which may explain the inconsistent results in prior studies. In our study, the blood ALT level was positively correlated with carcass traits in T1, and the ALT concentration was higher in steers with high carcass weight than in steers with relatively low carcass weights in all fattening periods (Supplementary Table 12 online). This indicates that lipid accumulation with changing fattening diets may contribute to carcass traits such as BMS and carcass weight.
The blood aspartic acid had a positive correlation with rib thickness and BMS, and α-amino adipic acid, 1-methylhistidine, and hydroxyproline showed a strong positive correlation with rib thickness in T1. A study on the wagyu crossbred reported that aspartic acid showed positive correlation with growth rate, no significant correlation with other properties, and that methyl histidine showed a negative correlation with the rump fat41. The α-amino adipic acid is potentially correlated with insulin resistance. Lee et al.42 reported that α-amino adipic acid is correlated with adipogenic differentiation, and that the α-amino adipic acid levels were increased in cell and mouse models of obesity-related insulin resistance. Here, the blood α-amino adipic acid sharply increased between T1 and T2, corresponding to the changes in insulin levels. Thus, the correlation with carcass traits appears to be similar to that with insulin, likely because the blood α-amino adipic acid level reflects changes in insulin levels.
Interestingly, our results showed that ketogenic amino acids such as isoleucine, leucine, tyrosine, and phenylalanine were negatively correlated with most carcass traits in early fattening phases. The ketones were used as an energy source in early fattening phases and more amino acids were consumed, which may have led to a negative relationship between the blood amino acids and the carcass traits of Japanese Black steers. This result suggested that Japanese Black steers in the early fattening phases consumed a lot of amino acids to replenish the considerable amount of energy needed through ketones, resulting in a negative correlation between amino acids and productivity.
In the late fattening phases, glucose had a strong positive correlation with subcutaneous fat. Similar to the results of the present study, some previous studies have reported that glucose level is positively correlated to rump fat and glucose in wagyu crossbred steers41. Glucose is known as the precursor to fatty acid biosynthesis in ruminants, and blood glucose level is deeply related to the accumulation of intramuscular and subcutaneous fat10. Here, the decreased glucose level in late fattening phases may have been to be due to the increase in intracellular influx of glucose by insulin. The positive correlation between blood glucose and subcutaneous fat suggested that more fat is accumulated in Japanese Black cattle with high blood glucose levels. Blood insulin showed a strong positive relationship with carcass weight, growth rates, rib thickness, and BMS. The steers with high carcass weight and BMS had higher concentrations of insulin compared to those with low carcass weight and BMS (Supplementary Tables 12 and 15 online). As noted in an earlier study, insulin promotes the intracellular influx of glucose for fat accumulation, and blood insulin concentrations are associated with carcass traits such as carcass weight, growth rate, rib thickness, and BMS10,40. This suggests that insulin is a more important regulator of lipid metabolism in the T3 phase than in the T1 phase in Japanese Black steers. We also investigate hepatic DEGs according to carcass weight (High vs Low) and BMS (High vs Low) to investigate the physiological changes with productivity in Japanese Black cattle. In total, 14 genes were differentially expressed when compared between carcass weights (High vs Low) and were mainly involved in organ development, cell growth, and muscle contraction. In comparison between BMS (High vs Low), 5 genes related to calcium channel and cytoskeleton components were found. This study revealed slight changes in the liver transcriptome according to carcass characteristics. No differences in energy- and fat metabolism-related gene were found in each fattening period, likely due to long-term adaptation and small metabolic changes into 2 different carcass weight and BMS categories. However, we found differences in the candidate genes and pathways responsible with large metabolic changes between the T1 and T3 phases.
The results suggest that the main physiological difference in Japanese Black cattle is that the high-production steers show high blood insulin concentration. As with high insulin levels in the late fattening phases, the steers that consumed a lot of high-energy diets had high energy intake (Supplementary Tables 12 and 15 online) compared to other steers with relatively low intake, resulting in the secretion of insulin to store excess energy.