Ultrasonographic evaluation of liver, back-fat, and muscle in dairy cows affected by suclinical ketosis

The transition period in dairy cattle is commonly defined as the period of time from three weeks before calving to three weeks postpartum [1]. After calving, the cow enters in a condition of negative energy balance (NEB), which results in the mobilization of body reserves, particularly adipose tissue [2]. A mild state of NEB is considered para-physiological during the postpartum period [3]. If excessive, it is considered a negative factor for the animal's health and productivity due to metabolic diseases and their link with immunosuppression [4]. Ketosis is one of the most common postpartum-metabolic disorders characterized by elevated levels ketone bodies, especially β-hydroxybutyrate (BHB). A BHB concentration of 1.0 mmol/L is the common cut-off for subclinical ketosis. The incidence of subclinical ketosis within the first month of lactation is estimated at 26–56% [5]. One of the key predisposing factors for excessive lipid mobilization and subsequent metabolic dysfunction is the cow’s body condition at calving [6]. In this context, the evaluation of body reserve dynamics is crucial for understanding how cows cope with the metabolic changes. Particularly, liver size can be indicative of hepatic lipid accumulation, while back fat and muscle thickness reflect the extent and type of tissue mobilization occurring during the early postpartum period [7,8]. By integrating metabolic and ultrasonographic data, it is possible to identify potential phenotypic markers useful for early detection and prevention of metabolic disorders such as ketosis. These non-invasive measurements provide valuable insights into both fat mobilization and muscle catabolism, two key processes involved in the adaptation to NEB. The aim of the study was to determine the changes in ultrasound imaging of liver, back fat and muscle in both healthy and ketotic animals. Study procedures were approved by Ethical Committee for Animal Welfare of University of Padua (protocol n. 103549/2024). A total of 60 Holstein-Friesian multiparous dairy cows were enrolled from a single farm (CERZOO ltd, Piacenza, Italy). To be included in the study, cows must have had calved between 5 and 9 days prior (5–9 days in milk, DIM), be free from ruminal acidosis, abomasal displacement, metritis, or mastitis, and must not be undergoing antimicrobial treatment. All animals were evaluated with blood BHB measurement on field, ultrasound examinations of the liver, back-fat and longissimus dorsi at 7, 14, 21 and 28±2 DIM. The BHB field measurement were conducted using a portable digital meter (Abbott Precision Xtra™ meter, Oxon, UK) and blood ketone test strips (Abbott Precision Xtra™ Blood Ketone test strips, Oxon, UK). Animals were divided into two groups according to BHB: CTR or control (BHB<1.0 mmol/L; n=43); KET or subclinical ketosis (BHB≥1.0 mmol/L; n= 17). The ultrasound examination of the liver was performed on the right side of the animal at the level of the 10th intercostal space using the MylabTM OneVET portable ultra-sound scanner (ESAOTE S.p.A., Genoa, Italy) equipped with a multifrequency convex probe (Animal Science Probe, SC3421 2.5-6.6 MHz; ESAOTE S.p.A., Genoa, Italy). Next, measurements of portal vein diameter (PVD, mm), depth of portal vein (DPV, mm), portal vein area (PVA, mm2) and liver depth (LD, mm) [20] were performed for each animal and evaluation time using MyLab Desk software (ESAOTE S.p.A., Genoa, Italy). In the same way, ultrasonography of back-fat and longissimus dorsi muscle thickness were performed and the respective thicknesses were obtained. The back fat represents the thickness (mm) of subcutaneous fat between the skin and the fascia trunci profunda located above the gluteus medius muscle. Statistical analysis of the data was performed using a mixed model to assess statistical differences among the groups. A Spearman correlation matrix was calculated to assess the relationships among parameters. Statistical significance was defined as a p-value ≤ 0.05. P-values greater than 0.05 but less than or equal to 0.10 were considered indicative of a trend towards significance. The BHB concentrations were higher in KET for all time points. Regarding liver measurements, the PVD showed a significant group effect. KET had lower PVD and PVA at 7, 14, and 21 days (KET=35.40, 35.50, 35,50 mm; CTR=39, 39, 39 mm). In all the groups of this study, the PVD value did not deviate significantly from the lower limit of the physiological range of 29-53 mm, as indicated by Braun (2009) [7]. These findings indicate that healthy cows had a larger PVD, which can be attributed to a more efficient hepatic circulation and metabolic function during early lactation. This rising might be related to increased blood flow required to support liver function. In contrast, the ketotic cows showed a reduced portal vein diameter and area, potentially due to impaired liver function or a lower need for hepatic circulation as a result of the metabolic disruptions associated with ketosis. This difference could also be attributed to different milk production. Since, it has been reported that animals in ketosis have lower milk productions [9]. Similarly, the portal vein area was significantly larger in the CTR group throughout the study period (KET=906, 895, 950 mm2; CTR=1072, 1059, 1072 mm2). The DPV showed a time effect only in the CTR group with the highest depth value at 7 days post-partum (DPV = 122 mm). This value decreased in subsequent weeks. The KET showed a greater LD at 14, 21, and 28 days (KET=156, 160, 161 mm; CTR=149, 150, 148 mm) with a significant time-effect. Furthermore, the KET group showed average values over the cut-off of 152.6 mm reported by Fiore et al. (2018) [10] for the ultrasound identification of hepatic lipidosis in all the time point (KET=153, 156, 160, 161 mm). Positive moderate correlation was observed between DPV and milk production (r=0.52; p-value= 0,004). This confirm the fact that liver function and circulatory efficiency play an important role in dairy productivity Regarding back-fat thickness, KET group was greatest at 7 days (KET=8.09 mm; CTR=6.59) followed by a marked reduction over time. The CTR group had stable back-fat thickness over the trial, with values ranging from 6.59 mm at day 7 to 6.60 mm at day 28. The thickness of longissimus dorsi showed no significant differences between, with no marked increase or decrease. The reduced back-fat thickness in the ketotic group reflects the increased lipolysis and fatty acid mobilization that occurs during the disease. This suggests that KET mobilize fat reserves more rapidly than healthy cows. Notably, the cows that developed ketosis had greater back-fat thickness at calving, indicating that they were in a higher nutritional state. These cows likely entered the post-partum period with more fat reserves, which were subsequently mobilized to support lactation and energy needs, but this also put them at higher risk for metabolic disorders such as ketosis. In fact, the risk of developing hyperketonaemia increases by +7% for each additional mm of BFT [8]. At the contrary, muscle mobilization was not as pronounced as fat mobilization during the early post-partum period, suggesting that muscle reserves were preserved even in cows experiencing ketosis. The findings of this study highlight the valuable role of ultrasonography in monitoring the metabolic health of dairy cows, particularly in detecting early signs of ketosis. Cows that developed ketosis had greater back-fat thickness at calving, indicating that they entered lactation with higher fat reserves, which were later mobilized to meet the energy demands of lactation. This fat mobilization was accompanied by an increase in LD, suggesting fatty liver development. Additionally, ketotic cows exhibited significant alterations in hepatic blood flow, as shown by changes in PVD and PVA. In conclusion, these results emphasize the importance of using ultrasonography to detect early metabolic imbalances, allowing for timely intervention and management of ketosis, which is crucial for improving both animal health and productivity in dairy farming.