Update on Fatty Liver in Dairy Cattle with Major Emphasis on Epidemiological Patterns, Pathophysiology in Relationship to Abdominal Adiposity, and Early Diagnosis

Abstract

Fatty liver is a more common than expected metabolic disease affecting dairy cattle around parturition, which generates high economic losses for the dairy industry. The disease has evolved from a low incidence of moderate cases to a greater increase of severe cases in recent years. This evolution could be explained by the higher rate of genetic selection that has been carried out for milk production, which concomitantly brings pleiotropic genes that determine greater abdominal adiposity, ketosis, and other diseases. Abdominal fat is much more reactive, pro-inflammatory, saturated, and low in adiponectin than subcutaneous fat. In this review, we will mainly address the epidemiological aspects, the pathophysiology concerning the different types of fat depots (subcutaneous and abdominal), and the early diagnosis of the disease to carry out efficient control and preventive strategies.

1. Introduction

The transition period in dairy cows comprises the last 21 days before expected parturition to 21 days postpartum, including the parturition, which is characterized by several physiological (metabolic, endocrine, immune, digestive) adjustments that the cows must experience to achieve adequate lactational and functional performance [1,2].

In this intricate scenario, the liver is considered a pivotal organ in regulating the entire metabolism, particularly in high-producing dairy cows, which may surpass its normal function, leading to a series of local and systemic inflammatory and degenerative processes compromising integral animal health. In fact, fibrosis has been reported as a common pathological finding in cows with fatty liver, associated with oxidative stress, hepatocyte demise, and inflammation [3]. Consequently, the cow becomes more susceptible to developing secondary diseases, including ketosis, displaced abomasum (DA), mastitis, retained fetal membranes (RFM), metritis, decreased fertility, and low milk production [1,4].

The liver, as a set-point metabolic organ, carries out more than 500 vital functions such as bile, plasma proteins, cholesterol, and lipoproteins synthesis; glucose, glycogen, propionate, and lactate metabolism; deamination and synthesis of amino acids; metabolism of hemoglobin; the urea cycle; and clearance of pharmacological products and toxins. The tricarboxylic acid cycle is one of the most important metabolic pathways in the hepatocytes, where acetyl Co-A and related compounds may enter the cycle, regulating its progression for energy production [5].

The transition period is typically characterized by a curvilinear drop and then an increase in dry matter intake (DMI) with the nadir around parturition. Furthermore, energy requirements for lactogenesis increase abruptly right after parturition. As a result, the cow develops a typical postpartum negative energy balance (NEB), characterized by hypoglycemia, because glucose is used to synthesize lactose during the process of milk yield. These metabolic responses are the results of the increased synthesis of IGF-I and growth hormone, reduction of the synthesis and release of insulin, and the surge of glucagon and catecholamines, which in turn will activate the hormone-sensitive lipase and other enzymes in the adipose tissue, triggering the typical breakdown of ester bonds of triglycerides (TG), releasing glycerol and non-esterified fatty acids (NEFA) into the bloodstream [6,7].

This physiological mechanism may become a vicious cycle in which several key compounds are restricted, reducing the available precursors for gluconeogenesis. As a result, major metabolic pathways are shifted, with the typical adaptative nutrient partitioning characterizing transition dairy cows [8]. Glycerol and NEFA from TG breakdown are utilized as energy sources for the liver and other tissues. However, if hepatocytes experience a large uptake of NEFA, the β-oxidation process may be overwhelmed, and excess NEFA may reassemble into TG and generate VLDL for export into the bloodstream. Unfortunately, the export of TG from the liver as VLDL is a slow process in the bovine species and is decreased under severe fatty acid oxidation and ketogenesis, resulting in a net accumulation of TG, with subsequent development of fatty liver. One of the reasons for net TG accumulation in the liver is because of a downregulation of acetyl CoA acetyltransferase 2 in the liver, inhibiting cholesterol synthesis, increasing TG synthesis, and reducing VLDL and low-density lipoprotein-C [9]. If NEFAs are completely oxidized, acetyl-CoA increases and can either continue its oxidation progression in the tricarboxylic cycle or be shifted to the formation of ketone bodies depending upon the amount of oxaloacetate, the NAD+/NADH ratio, and the upregulation of enzymes that allow the Krebs cycle to continue in optimal functioning in the mitochondria. Liver NEFA uptake is boosted when their concentration in the blood is high. When lipolysis is extremely high, particularly in over-conditioned cows, because of hypoglycemia and lower concentrations of gluconeogenic precursors at the Krebs cycle level, ketogenesis and excessive TG accumulation in the liver occur [5,8,9].

This article provides new information about the epidemiology of fatty liver and the evolution of the disease over time, the pathophysiology in relationship to abdominal adiposity, genetic selection, and differences with subcutaneous adipose tissue. Early diagnosis based on fine needle aspiration and molecular techniques (early biomarkers) in dairy cattle is reviewed. For more detailed information about the treatment and control of fatty liver in dairy cows, the reader should study the recent literature review by Gross [10].

2. Basic Concepts

Fatty liver or hepatic lipidosis is a common metabolic syndrome affecting dairy cattle around parturition that leads to decreased milk production, poor fertility, higher risk of culling, and death. Furthermore, fatty liver is related to a higher incidence of infections and inflammation, altering the overall performance of the cow [7,11]. Fatty liver develops when an excessive NEFA uptake by the hepatocytes occurs, surpassing the oxidation capacity and exporting mechanism of newly assembled TG as VLDL by the liver. Elevated concentrations of NEFA in the plasma result from a high rate of lipolysis, particularly occurring in cows with excessive abdominal fat and/or high BCS [7,11]. Moreover, in dairy cows, especially those with high BCS, a progression to a state of insulin resistance around parturition may occur, which is also linked to a higher rate of lipolysis in cattle [6,12]. Insulin resistance may aggravate the presentation of fatty liver. Consequently, excessive lipo-mobilization in early postpartum cows, which leads to a high concentration of NEFA in the blood, must be properly addressed [7].

Categories of Adipose Tissue

Subcutaneous adipose tissue is richer in unsaturated fatty acids than abdominal fat [13]. Abdominal fat is more responsive to lipolysis and dynamic tissue than subcutaneous fat in humans [14] and dairy cattle, through a more upregulated expression of hormone-sensitive lipase in abdominal fat than subcutaneous fat and an excessive lipolysis rate [7,15]. In addition, excess abdominal fat is consistently linked to higher concentrations of NEFA because of low concentrations of insulin [14]. Furthermore, there is a great similarity between the fatty acid profiles of abdominal fat and NEFA in blood compared to subcutaneous fat [16]. This supports the hypothesis of a more marked fat mobilization in the abdominal adipose tissue than in the subcutaneous tissue of dairy cows [16]. Therefore, cows with abundant abdominal adiposity have a greater risk of lipolysis and NEFA release, which is associated with a higher incidence of diseases such as ketosis, fatty liver, and DA. In humans, adipose tissue distribution differs with gender, genetics, diseases, certain pharmacological compounds, development, and the elderly [17]. Nevertheless, a genetic component in the distribution of body fat in cattle is also likely. For instance, in a study in the USA, it was demonstrated for the first time that cows with an extreme accumulation of fat in the omental membrane and the entire abdominal cavity had a genetic component involved. Several SNPs related to four QTLs were reported to be significantly linked to excessive abdominal fat deposition. Most of these genomic areas were detected on BTA 12 and BTA 19 of the bovine chromosomes. It was concluded that a single gene does not define extreme abdominal adiposity in the Holstein breed with mild to normal BCS, and it seems to be moderately heritable to the next generation. Therefore, selection strategies may decrease abdominal adiposity if the parameters of the proper predicted transmitting abilities are well-defined in Holstein cattle [18].

The most important role of adipose tissue is storing and releasing fatty acids to provide energy in response to milk production requirements. However, lipids in the body also have immunological, mechanical, hormonal, regenerative, and thermal functions [17]. Lipids are stored in the subcutaneous tissue and around abdominal organs and annexes such as the omentum, mesenterium, kidney, liver, and retroperitoneal space, playing immunological and mechanical protective functions [19]. Once adipose tissue shifts from lipogenesis to lipolysis, it is more likely to be inflamed, potentially driven by local proinflammatory cytokine release [20]. For example, the inflammatory response of adipose tissue to lipopolysaccharides may boost high concentrations of NEFA through cytokine-induced lipolysis to modulate the infection. Obesity and aging in humans are strongly related to activating an immune response from lipids that play a robust role in proinflammatory cytokine release, further increasing insulin resistance, reducing lipogenesis, and boosting lipolysis [17]. This generates so-called “sterile” systemic inflammation, metabolic dysregulation, and lipotoxicity. In fact, in high BCS nonlactating, nonpregnant cows, interleukin -1β and interleukin-6 expression were upregulated, mostly occurring in abdominal fat tissue [21]. Furthermore, macrophages are a large population of immune cells in bovine adipose tissue [22]. During high rates of TG breakdown, increased trafficking of macrophages within fat depots in dairy cows has been described, resulting in a higher number of subcutaneous and visceral fat macrophages in cows with extreme lipolysis [7,23]. Interestingly, structural proteins such as collagen and tubulins are more expressed in cows with higher lipolysis rates than low lipolysis rates, suggesting that these proteins may impair the trafficking and conformation of GLUT4 receptors, which may suggest a relationship with insulin resistance [24]. Furthermore, the blood adiponectin concentration, an adipokine related to glucose and lipid metabolism, is inversely correlated with plasma NEFA [25]; consequently, adiponectin is high before parturition and decreases after calving. However, adiponectin expression is much lower in abdominal fat than subcutaneous fat in dairy cattle, suggesting large metabolic differences between both adipose tissues [26].

Gene expression for different adipose depots is also variable in dairy cattle [27]. In addition, the replication of preadipocytes shows regional differences, which makes it potentially heritable over several cell generations. This suggests that adipocyte characteristics are consistently linked to several cell molecular mechanisms, reinforcing heritable differences [28]. Other genetic differences include insulin responsiveness, lipase gene expression and activity, cell lipid build-up, and intracellular calcium signaling pathways. In fact, in a study conducted in New Zealand, the results supported the hypothesis that genetic lines from New Zealand Holstein vs. US Holstein cows have dissimilar fatty acid profiles in different fat depots and milk, which may impact the biological functioning of the several adipose depots of the body differently [29]. For instance, the content of adiponectin, which has a significant role in lipid/glucose metabolism, insulin responsiveness, and inflammation, differs conditionally from the nature and distribution of fat tissue (visceral vs subcutaneous). Because adiponectin enhances lipogenesis in adipocytes, decreased concentrations during the transition period are observed, suggesting a potential improvement of glucose partitioning to the mammary gland [26,30]. Furthermore, in a study conducted with German Holstein cattle, a genome-wide association analysis identified a locus related to DA. In the same study, genes involved in calcium homeostasis and insulin responsiveness related to diabetes mellitus in humans were identified, which are important risk factors in the development of DA [31]. In addition, the close desaturase index between abdominal adipose tissue and NEFA in blood implies preferential abdominal lipolysis in dairy cows, particularly with DA presentation [16]. Another study conducted in the US also found a genomic prediction for DA in Holstein cows [32]. These findings imply that some genes related to abdominal adiposity are also linked to the presentation of DA in Holstein dairy cattle. In fact, in a study that analyzed a data set of 28,000 lactations and the frequency of diseases from 14,000 dairy cows, it was found that a region on BTA 20 was significantly related to visceral adiposity and, at the same time, also related to the presentation of DA. Overall, it was concluded that abdominal adiposity in dairy cows is controlled by pleiotropic genes, which affect visceral adiposity, ketosis, and DA development at the same time [33].

With this new evidence, it is noticeable that abdominal fat in dairy cows is much more active and responsive to lipolysis than subcutaneous fat, which implies that abdominal fat is a key regulator of the metabolic status of the modern transition dairy cow [7,21]; however, mesenteric and omental fat accumulation may also occur in animals that are fed concentrate relative to silage-fed animals, providing evidence that fat accumulation distribution it is not only accounted for by genetic variation but also influenced by feed management and diet [34].

3. Epidemiology

Hepatic lipidosis occurs when TG is accumulated beyond its normal threshold, expressed as % of wet liver tissue. Based on the classification of Bobe et al. [11], a normal liver should contain < 1% TG, while a mild fatty liver should contain between 1 and 5%, and a moderate fatty liver between 5 and 10%. A severe fatty liver should contain > 10% [11]. Under this classification, the research bibliography of the last 5 years (

Table 1. Mean triglyceride concentration in the liver of dairy cows from studies from the last 5 years.

Reference	Animals	Mean TG 1 Concentration Wet Basis, (g/100 g)	DIM 2
Mann et al. [36]	Control Holstein cows	10.6	<9
	Treatment Holstein cows	11.9
Shen et al. [40]	Control Holstein Cows	2.6	22
	Fatty Liver Holstein cows	10.5
Fry et al. [37] Holstein cows	26% of cows	<5	<10
	34% of cows	5–10
	8% of cows	10–15
	10% of cows	15–20
	22% of cows	>20
Caixeta et al. [41]	Control Holstein cows	7	23
	Treatment Holstein cows	3.1
Angeli et al. [38] Holstein cows	Prepartum	4.5	−30
	Postpartum	17.5	4
	Postpartum	11.5	14
Caputo Oliveira et al. [42]	Control Holstein cows	12	14
	Treatment Holstein cows	8
Vogel et al. [43] Holstein cows	Prepartum cows	0.5	−21
	Control cows	6.8	+28
	Treatment cows	4.5	+28
Bollatti et al. [44]	Control Holstein cows	3.41	<21
	Treatment Holstein cows	4.05
Leal-Yepes et al. [45] Holstein cows	Control	8.4	5
	Control	9.2	21
	Treatment	5.1	5
	Treatment	5.8	21
Angeli et al. [35] Holstein cows	LBCS 3	5.4	−14
	HBCS 4	6.5	−14
	LBCS	17.3	4
	HBCS	16.5	4
	LBCS	15.5	21
	HBCS	18.0	21
Garcia-Roche et al. [46]	Maximum pasture (M) vs. Fixed pasture (F)	4.3 vs. 4.0 prepartum NZ vs US	−45
		4.5 vs. 4.0 prepartum M vs F
	New Zealand (NZ) vs. US Holstein (US)	8.2 vs. 8.0 postpartum NZ vs US
		11.1 vs. 5.2 postpartum M vs F	21
Zhu et al. [47] Holstein cows	Normal cows BCS:3.6	0.85 ± 0.03	4–12
	Severe fatty liver BCS:3.8	11.9 ± 0.4

¹ Triglycerides wet/wet basis; ² days in milk; ³ low body condition score; ⁴ high body condition score.

) reports that most cows have progressed to a moderate to severe fatty liver during the transition period, regardless of management and nutritional approach. Cows with moderate or mild fatty liver are generally related to a more severe NEB than healthy cows. Consequently, severe NEB and fat infiltration impair dairy cows’ well-being and performance more drastically [11]. According to studies published in the last decade, it is reported that the average liver TG concentration in early postpartum cows is greater than 3%, and more than half of the cows may experience moderate to severe hepatic lipidosis. Even worse, in several studies, it was reported that the average concentration of liver TG was more than 10%, implying that more than 50% of cows, during early lactation, experienced severe fatty liver, based on the classification of Bobe et al. [11]. Indeed, in a recent investigation, cows with high and low BCS at 4 d of lactation showed an average concentration of TG of 17.3% and 16.5%, respectively. The cows at 21 d of lactation showed an average liver TG concentration of 15.5% and 18.0%, respectively [35]. Similar results were found in a group of cows that served as controls in a nutritional pilot study between 3 and 9 days of lactation, with an average liver TG concentration of 10.6%. The treatment group did not differ statistically from the control group, which showed an average hepatic TG concentration of 11.9% [36]. In another study, 8% of cows showed an average liver TG concentration between 10 and 15%, 10% of cows had a TG concentration between 15 and 20%, and 22% of cows had greater than 20% TG, demonstrating that 40% of the cows developed a severe fatty liver [37]. Finally, dairy cows between 14 and 21 d before expected parturition during the prepartum period showed strong evidence of mild fatty liver with an average hepatic TG content > 3% [35,38,39]. Unfortunately, in these studies, the relative influence of abdominal adiposity when compared to subcutaneous fat was not determined, which raises the question of the true impact of subcutaneous fat alone. Considering these investigations, it is suggested that the presentation of hepatic lipidosis has progressed over time, becoming a more common and severe disorder than it was 20 years ago. Furthermore, in most of the studies, even in groups treated with some nutritional additives to modulate the NEB, the carryover effect of fatty liver for these animals has not been elucidated in terms of the presentation of other metabolic and/or infectious disorders and early culling from the herds. Based on Table 1, it is suggested that the normal liver TG content of Holstein cows be reconsidered and that the different degrees of fatty liver be redefined.

A recent study measured liver TG in Holstein cows from New Zealand and the US, starting at 45 d before expected parturition until mid-lactation. In addition, two feeding strategies were assessed. One group received a maximum amount of pasture and supplementation of pelleted concentrate, and the other group received a fixed amount of pasture and supplementation of a total mixed ration. The concentration of liver TG was investigated. It was reported that liver TG increased by 3X from pre-partum to early lactation and decreased by 4X from early to mid-lactation. In addition, liver TG concentrations were 50% higher for the cows fed the maximum amount of pasture than those fed the fixed amount. The authors concluded that intensive grazing is detrimental to liver metabolism in US Holstein cows, highlighting the significance of selecting a proper Holstein line adapted better for pasture-based dairy systems [46].

In light of the evidence, modern dairy cows are genetically more productive with different dynamics of adipose tissue distribution than in the past, with current animals having more abdominal fat content (omental, mesenteric, retroperitoneal). Abdominal fat mobilizes faster and produces a more severe inflammatory state than subcutaneous fat when it is excessive [7]. Based on these assumptions, it is hypothesized that cows with a greater abdominal fat content are more likely to develop fatty liver with higher severity.

4. Diagnosis

Diagnosis of fatty liver is not easy. No analytical test is accurate, fast, and reliable for diagnosing this condition [11,48]. Traditional liver damage diagnostic tests have been used to determine liver enzymes and other blood metabolites; however, the diagnostic utility for these biochemical profiles is often ineffective because of lag time. The increase in liver enzymes and other metabolites occurs when there is already severe liver damage, which does not allow for correcting the problem promptly. Therefore, the diagnosis of fatty liver should be earlier than anticipated in order to take effective therapeutical and preventive actions [11,49]. Fat extraction is the gold standard for hepatic TG content; however, histopathology or flotation of liver biopsies obtained in live animals can be used as an indirect method [11]. Unfortunately, liver biopsies are invasive and associated with risks of hemorrhage, infection, and adhesions [11,50]. Transcutaneous ultrasound is another technique for diagnosing hepatic lipidosis in cattle; nevertheless, this methodology has low sensitivity when TG content is <10% [11,51,52,53,54]. This method is, therefore, more accurate for the diagnosis of severe fatty liver (TG > 10%) [51,52,53,54]. However, a recent study aimed to identify peripartum biomarkers and weight loss fluctuations in cows with changes in hepatic ultrasonographic patterns indicative of fatty liver [55]. This study reported that cows with fatty liver had a thicker backfat layer, higher blood NEFA and glucose concentrations, and altered AST activity compared to normal cows.

Fine needle aspiration cytology (FNAC) is another technique that has been revisited for the last time to diagnose fatty liver in cattle and other species [56,57]. Fine needle aspiration cytology is technically feasible in clinical practice, being minimally invasive and inexpensive and having rapid results compared with biopsy [58]. In fact, two studies have shown promise for FNAC in clinical settings [37,49]. Our study reported a sensitivity, specificity, positive predictive value, and negative predictive value for FNAC of 73%, 85%, 90%, and 63%, respectively [49]. Microscopic cytology scores are shown in Figure 1. Our study concluded that the cytology offers acceptable sensitivity (73%) and specificity (85%) using a cut-off value of 2% for liver TG content [49], by which an early diagnosis of fatty liver (TG infiltration 2–5%) can be achieved. In the study of Fry et al. [37], liver TG content was correlated more strongly with cytology score than with NEFA or BHB concentration in serum. A receiver operating characteristic curve analysis showed that cytology had better diagnostic performance than either NEFA or BHB for correctly categorizing hepatic TG at thresholds of 5, 10, and 15%. Based on these two studies, FNAC is recommended as a rapid and inexpensive fatty liver diagnostic test that can be conducted under field conditions.

Noninvasive techniques that aid in the diagnosis of fatty liver in dairy cattle include the determination of circulating concentrations of ornithine carbamoyl transferase (>25 U/L), glutamate dehydrogenase (>8.9 U/L), NEFA (>1 mmol/L; [48]), decreased liver propionate to glucose conversion [39], and altered transcutaneous ultrasound imaging (TG liver content > 10% [51,52,53,54]. Unfortunately, these methods are only reliable when the hepatic TG content is >5%.

The solute carrier family 27, member 1, also known as SLC27A1, which mediates the translocation of long-chain fatty acids across plasma membranes, was low, with a liver TG content of 2%, and the expression of SLC27A1 was the highest at 1 week postpartum along with the highest NEFA and liver TG concentration. These findings would support the mRNA expression pattern of SLC27A1 during the NEB in both early lactation and in feed-restricted animals as an indicator of enhanced uptake of plasma NEFA and TG accumulation in the liver [59]. In another study [60], proteins from circulating exosomes were compared between cows at 1 week postpartum with a higher risk of metabolic dysfunction (1.8 mEq/L of NEFA; 1.0 mmol/L of BHB and 9.0% hepatic TG) and lower risk of metabolic dysfunction (0.66 mEq/L of NEFA; 0.45 mmol/L of BHB; and 1.8% hepatic TG). Exosomes are small, membrane-bound extracellular vesicles (ECV; 40–100 nm in diameter) exocytosed by cells, containing components from the propagating cell, such as proteins, lipids, and nucleic acids. The aberrant cellular function (particularly pathologic conditions) can alter the rate of release of ECV and their protein composition, such that the contents reflect the physiological state of the originating cell. In addition, as ECVs are transported in blood, they may function as potential early biomarkers of disease state. Because of this relationship, exosomes isolated from cows at risk of metabolic dysfunction during the transition period may exhibit an altered protein composition. They could be used as a potential biomarker of health. Cows in the high-risk category were chosen because their metabolic profile reflected an extreme tendency to mobilize adipose tissue (high NEFA), an inability of the liver to oxidize mobilized NEFA completely (high BHB), and a failure of the liver to export the resultant VLDL to peripheral tissues (high liver TG). A higher concentration of both envoplakin and oncoprotein-induced transcript-3 was elevated in the cows at increased risk of disease compared with cows with a low hepatic TG content (1.8%). These proteins have been identified as important for liver function. Consequently, the authors of this study proposed that these proteins reflect hepatic steatosis and may be useful biomarkers of a more severe pathological condition before clinical signs are evident, starting to increase as early as 2% hepatic TG content [60].

In more recent studies, the determination of miRNA offers a novel alternative for early diagnosis of tissue damage, including fatty liver [61,62]. Among miRNAs determined in bovine plasma, miRNA-122 was the best candidate for liver damage [63]. In addition, miRNA-802 was also enhanced in the liver. Although not previously reported in cattle, data in humans and mice have shown miR-802 to be a key regulator of liver function. Specifically, hepatic concentrations of miRNA-802 were elevated in obese individuals, contributing to insulin resistance and glucose intolerance [64]. Another study reported that miRNA-802, not previously identified in cattle, can regulate insulin sensitivity and lipid metabolism and thus could potentially be used as a specific biomarker of liver function in dairy cows [65]. In this context, further studies should focus on the role of miRNA-802 combined with miRNA-122 as potential biomarkers of NEB and liver damage in dairy cows.

A study investigating the liver PNPLA3 protein showed a negative association between this protein’s abundance and liver TG content in peripartum dairy cows. Consequently, a greater liver PNPLA3 protein abundance is related to lower liver TG accumulation during the peripartum period, potentially preventing the development of fatty liver or accelerating recovery from hepatic lipidosis in dairy cattle [66].

In a recent study, cows around parturition showed extensive changes in acylcarnitine, phosphatidylcholines, sphingomyelins, and bile acids, indicating widespread remodeling of the liver functioning, suggesting altered fatty acid oxidation, likely related to mitochondrial dysfunction or enzymatic imbalance, altered cell membrane configuration, cell signaling, and functionality. This marked change in the liver metabolome may indicate an adaptive change or the onset of a pathological state that can be used as very early biological markers for altering hepatic functionality [67]. Furthermore, a study to determine changes in macro and microelements in cows with fatty liver (mean liver TG content wet tissue basis of 6.0 ± 1.5%) found that serum glucose concentrations were considerably lower in fatty liver cows than in healthy cows. Instead, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transpeptidase, and NEFA concentrations were significantly higher in fatty liver cows than in healthy cows. Interestingly, between 7 and 28 days postpartum, Ca, K, Mg, Sr, Se, Mn, B, and Mo were higher in healthy than fatty liver cows, while Cu was lower in healthy than fatty liver cows. These differences were more pronounced at 7 days postpartum [68].

These latest studies undoubtedly demonstrate that cows with mild to moderate fatty liver, according to the classification of Bobe [11], already present an alteration in the normal functioning of the liver and therefore reinforce the importance of an early diagnosis of the disease and, even better, the establishment of effective preventive interventions to modulate liver functionality during the transition period.

5. Control and Prevention

Despite the advances in the knowledge of the physiology of the prepartum and postpartum dairy cow and the establishment of nutritional strategies that modulate the transition to successful lactation, the incidence of metabolic diseases and related disorders remains high, resulting in an excessive culling rate during early lactation and high economic losses for the dairy industry [7]. Undoubtedly, these responses are associated with genetic selection, mainly for milk production, which brings pleiotropic genes related to greater abdominal adiposity with more lipolytic and pro-inflammatory activity that determines a higher incidence of metabolic diseases such as ketosis, fatty liver, and DA [7,18,33]. Therefore, the control and prevention of diseases related to this elevated metabolic rate will depend on adequate feeding management and cow comfort and the genetic component of metabolic rate through future selection indexes that consider genomic information about abdominal adiposity, metabolism, and diseases [32,45].

Regarding fatty liver and related diseases, the cow requires a moderate level of lipolysis; however, if abdominal adiposity is excessive and environmental management and cow comfort is inadequate, the cow will be more prone to mobilize this fat, generating a substantial pro-inflammatory state. Therefore, the first objective of avoiding excessive fat mobilization is to avoid the over-conditioning of cows during the last third of lactation and the dry period. A single total mixed ration may not be the best approach, and a low-production diet at the end of lactation should be more strategic and lower in energy density [2,4]. In this case, energy intake will be limited more precisely based on the cow’s requirements [1]. Other measures to take are: (i) improving DMI during the prepartum period. This does not necessarily imply increasing energy consumption but rather filling the rumen. A rumen with more content and buoyancy at calving will provide the cow a base for a better postpartum DMI and increase the energetic input [2]; (ii) avoiding stress and maintaining adequate cow comfort. Control of heat stress abatement, stock density, resting time, and feed bunk space is fundamental [7]; (iii) providing well-formulated diets, and adapting the rumen adequately to avoid rumen acidosis and hypocalcemia [2,4]. Considering the use of additives such as protected choline, methionine, niacin, monensin, and other gluconeogenic precursors are appropriate strategies [2,4,45,69]; (iv) maintaining an efficient health monitoring program to diagnose and treat diseases promptly and adequately [1,7,69].

6. Proposed Pathophysiological Hypothesis in Modern Dairy Cows

Because of intense genetic selection for milk production over the past 40 years, modern dairy cows peak in milk yield much earlier, around 25 to 40 days after parturition, compared to early cows, which peaked at 50 to 70 days post-calving [70,71]. This selection has also brought genes that control more than one characteristic, known as pleiotropic genes. In this case, genes for higher milk production also affect characteristics that worsen the severity and duration of the postpartum energy balance, mainly through a less noticeable increase in the postpartum DMI [72]. The same study revealed that the genetic correlation between milk yield and DMI was positive; however, milk yield and energy balance were negatively correlated, particularly during the onset of lactation. Therefore, selecting animals for a higher DMI in early lactation may be a wise strategy to improve the postpartum energy balance of dairy cows [72].

Genetic selection has also led to adipose tissue, particularly abdominal fat, playing a role in providing energy quickly and more efficiently. Many of the genes that control the milk production process have pleiotropic effects on the ketogenesis process in the liver. Additionally, genes associated with abdominal fat also have pleiotropic effects on the presentation of DA. These biochemical mechanisms can be explained more precisely at a molecular level. In cows with higher adiposity, it has been found that the gene encoding adiponectin, which has an insulin-sensitizing effect, is downregulated. This leads to disrupted fat/carbohydrate metabolism, increased insulin resistance, and triggers a strong inflammatory response. These factors contribute to a higher degree of ketogenesis, increased circulation of NEFA in the blood, and higher uptake and accumulation of TG in the liver [32,33,72]. Furthermore, there is a strong genetic correlation between ketosis and DA in Holstein cows. After calving, cows may predominantly begin to mobilize abdominal fat, as their lipases have greater gene expression and are more active than the lipases of subcutaneous tissue. Additionally, NEFAs from abdominal fat, which are more saturated than those from subcutaneous fat, become more concentrated in the portal vein, reaching the liver much faster than systemic blood. This can lead to further oxidation in the Krebs cycle or conversion to ketone bodies. However, if NEFAs are in higher concentration, they will be re-esterified to TG. This can result in a net accumulation of TG in the liver quickly, particularly if the cow is stressed or the overall management and cow comfort is poor [7,9,17,37].

In high-producing dairy cattle, this metabolic burden can be attributed to the uncoupling of the somatotropic axis, which includes the growth hormone (GH), GH receptor, and insulin-like growth factor I (IGF-I). This uncoupling mechanism leads to the liver responding less to GH, synthesizing less IGF-I, and enabling high milk yield as part of the nutrient partitioning process in lactating dairy cows. The somatotropic axis functions differently depending on the genetic level of milk production, as demonstrated by genomic studies comparing different genetic lines of Holstein cattle. These findings may explain why high-producing cows often show high blood BHB concentrations, indicating proper metabolic adaptation to high lactation [73,74].

Moreover, excessive fat accumulation can lead to an intense inflammatory response, causing the infiltration of pro-inflammatory macrophages and the release of cytokines that activate the synthesis and release of acute-phase proteins [7,17]. This vicious cycle can result in a more aggressive accumulation of fat in the liver, inflammation throughout the body, an increased incidence of ketosis and DA, and the disruption of calcium and magnesium signaling effects. It can also lead to the development of other diseases, such as metritis and mastitis. Insulin resistance may worsen, and antioxidant activity can be compromised [6,7,9,33,72,73]. If liver damage is severe and lipotoxicity occurs, the cow may die suddenly or suffer from chronic health conditions, leading to decreased milk production and infertility, which could result in early culling from the herd. [69].

Delving deeper, the NEB results from greater energy output (e.g., high milk yield, increased physical activity, stress) than input (e.g., low feed intake, decreased feed digestibility). It is well-known that a negative genetic correlation between milk yield and DMI exists, and NEB is strongly linked to milk production [75,76,77]. Other investigators have validated these findings, studying the genetic relationship between NEB, DMI, BCS, and solid contents of milk [72,78]. Buttchereit et al. [78] have concluded that a higher milk yield is strongly associated with a more severe NEB. These concepts are not new. In the 1980s, Bauman and Currie [79] laid the foundation for understanding milk production as a postpartum process regulated by homeorhetic mechanisms [79]. They defined this concept as “the orchestrated modifications in the metabolism of body tissues necessary to support a physiological state”. A basic condition for the homeorhesis concept is that “nature has arranged a high priority to the functions of pregnancy and lactogenesis, allowing them to proceed at the expense of other metabolic processes even to the point that a disease state is expected as normal or in some way pathological” [79,80]. Therefore, ketosis may be considered as a “homeorhetic disease” because the cow will attempt to maintain milk production despite nutrient deprivation or imbalances [81,82]. This context is crucial for understanding the current state of knowledge in this field. Given this evidence, the modern dairy cow has evolved to a level of achieving higher milk yields at the cost of metabolic adaptations that in the past may have been considered as an abnormal or pathological state. It seems that the fatty liver classification of Bobe et al. [11] proposed 20 years ago no longer fits with the physiological responses of high-producing modern dairy cows, as, according to that classification, more than 40% of early postpartum dairy cows nowadays should be considered as cows developing moderate to severe fatty liver (>5 to 10% of liver TG wet weight) (Table 1). It appears that modern dairy cows are well adapted to deal with higher amounts of TG accumulated in the liver with greater production of ketone bodies during the first week of lactation without greatly altering the health status of the cow. The liver could be considered a homeorhetic organ for TG reserves, able to slowly release them into the bloodstream in the form of VLDL to provide a continuous supply of TG for the mammary gland to synthesize milk fat. Similarly, BHB could be used as a base molecule for elongating short-chain fatty acids in the mammary gland during the “de novo synthesis” of milk fat.

Considering all the evidence presented in this pathophysiological hypothesis, one of the major arguments is that milk production is consistently related to the uncoupled GH-IGF-I axis in the liver [83]. The question arises whether this uncoupling is “normal” or “abnormal”. Lucy et al. [84] pointed out that “the consequences of an inadequate GH receptor 1A expression are serious”. The liver remains unresponsive to GH, and various GH-dependent processes, including gluconeogenesis, are not activated. This may predispose the cow to fatty liver and ketosis and preclude the normal hepatic mechanism for nutrient partitioning during early lactation. Supporting this hypothesis, studies carried out with knockout mice without GH-receptors in the liver, mimicking an uncoupled GH-IGF-1 axis, caused severe metabolic alterations, resulting in a noticeable increase in liver TG content even when fed a standard diet [85].

7. Integrating the Pathophysiology of Fatty Liver and Management

As we have discussed earlier, fatty liver in dairy cows is caused by an elevated uptake of excessive blood NEFA by the hepatocytes and the resynthesis of TG and export as very low-density lipoproteins, which is a slow process in the bovine species, with a net accumulation of hepatic TG. These mechanisms are modulated by hormones with an increase of growth hormone, the development of insulin resistance in combination with decreased insulin, and IGF-1 concentrations. These are hormonal adaptations that are related to an uncoupling of the growth hormone IGF-1 axis, which enhances fat mobilization. Excessive lipolysis is associated with inflammation and oxidative stress. These metabolic and hormonal alterations are the result of the selection of dairy cows primarily for milk production without considering DMI and energy balance traits with the consequence of fatty liver, ketosis, and production diseases [86]

Dry matter intake is drastically reduced around parturition in dairy cows and many other mammals. This is a crucial point that determines the development of fatty liver in today’s dairy cows. Colostrogenesis occurring 24 to 48 h before calving already triggers an NEB prior to parturition, which begins to become more severe as lactation progresses because DMI slowly increases. Hence, this determines a logical sequence of the beginning of NEB, increases of blood NEFA concentrations and uptake by the liver, development of mild hepatic lipidosis, and finally, ketosis [87].

This concept is very well described and discussed in the review by Martens [87] where he analyzes in great detail how milk production is negatively associated with DMI, partly explained by the genetic selection programs of recent decades that only gave priority to milk production, without considering DMI and NEB. The energy partitioning, physiological adaptations, and priority for milk yield still occur despite the lower DMI, leading to a more profound NEB. If we want to reduce the negative impact of NEB, it is time to seriously consider better genetic selection indices that consider traits such as higher DMI, milk efficiency, digestibility of diets at the ruminal level, and the rest of the digestive tract to name a few. The efforts made with environmental management to improve DMI and milk efficiency, such as increasing feeding frequency during early pp, improving nutritional quality and palatability of diets, and managing adequate cow comfort, evidently seem to be insufficient if this is not accompanied by a genetic improvement of these characteristics. Indeed, Buttchereit et al. [78] stated: “Continued selection for high milk production will lead to a further increase in the postpartum energy deficit unless energy balance is directly or indirectly included in the breeding programs with appropriate economic weights”.

Although liver function has been modified to the higher milk production of the new generations of dairy cows, it seems that this adaptation is partial. In a study conducted by Du et al. [88], it was shown that the protein and mRNA expression of the hepatic sterol regulatory element-binding protein 1c, which facilitates storage of TG in the liver, and the mRNA expression of Acetyl-CoA carboxylase 1, a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA for the biosynthesis of fatty acids [5], and the upregulation of fatty acid synthase, a multi-enzyme protein that catalyzes fatty acid synthesis [5], were higher in dairy cows with mild fatty liver than in control cows. In addition, the protein and mRNA expression of PPARα and the mRNA expression of lipid oxidation genes (ACO and CPT1A) were also significantly increased in dairy cows with mild fatty liver. Furthermore, ultrastructural analysis showed an increased number and volume of hepatic mitochondria in dairy cows with mild fatty liver. The authors concluded that dairy cows with mild fatty liver exhibited increased hepatic lipid synthesis, increased lipid oxidation, and increases in the number of mitochondria and ATP production. These changes are suggested to be an adaptive mechanism of dairy cows with mild fatty liver, which may explain why the incidence of mild to severe fatty liver, without evident health consequences, has increased during the last decade. However, the same study [88] showed that cows with mild fatty liver (3% TG) had a lower DMI, lower blood glucose concentrations, and higher concentrations of BHB, aspartate aminotransferase, and alanine aminotransferase, which may already indicate irreversible damage to the hepatocytes. Are the lower DMI and lower glucose concentrations a response to fatty liver or could they be the cause of fatty liver?

Large amounts of glucose are required for the synthesis of lactose in the mammary gland; consequently, glucose is without doubt a key metabolite during early lactation [1,4,7,8]. In a metabolomic and proteomic study of the liver of transition dairy cows [89], it was found that 44 metabolites and 250 proteins were differentially expressed during the transition period. Furthermore, upregulated gluconeogenesis, tricarboxylic acid cycles, amino acids degradation, fatty acid oxidation, AMP-activated protein kinase signaling pathway, peroxisome proliferator-activated receptor signaling pathway, and ribosome proteins were revealed during the postpartum period. In addition, the upregulated glucagon and insulin signaling pathways demonstrated a significant requirement for glucose in postpartum dairy cows [89]. Great efforts have been made to deal with the low glucose and high BHB concentrations in transition dairy cows. Recommendations for the use of gluconeogenic precursors such as monensin and propylene glycol and the use of membrane stabilizers and methyl group donors to aid liver function such as choline and methionine remain in vogue; however, their positive effects have been very variable [1,2,4,42,44,45]. This can be explained very simply. In dairy cows, glucose is mainly produced by gluconeogenesis, which can scarcely be maintained without an adequate DMI, which leads to a severe NEB and a damaged liver. This sequence can become a vicious circle with a fatal outcome. As a result, a lower incidence of diseases and traits of energy metabolism (NEB, BCS, DMI, and fat-to-protein ratio) should have high priority in the genetic selection indices in dairy cattle today.

8. Conclusions

Liver function has evolved with a greater capacity for TG accumulation. Using the definitions of Bobe et al. [11], the incidence of moderate to severe fatty liver has increased considerably compared to 20 to 30 years ago; therefore, it is proposed to reassess what is the normal content of TG in the liver of contemporary dairy cows and to reconsider the definition, prevalence, and incidence of hepatic lipidosis. Liver TG metabolism is influenced by a complex interplay of factors, including genetic traits. A liver with TG contents > 5% may already show impaired functionality or the cow adapts to a higher homeoretic hepatic content of TG. Excessive hepatic fat accumulation occurs when lipolysis is more severe, especially in cows with high BCS and abdominal adiposity. Abdominal adiposity has a genetic component related to greater milk production and cows that peak early in lactation (<40 days postpartum). This is associated with a greater incidence of ketosis, DA, fatty liver, and carry-over effects. Consequently, management strategies that adequately modulate the NEB of modern dairy cows, especially avoiding excessive BCS and stress, providing adequate cow comfort, and improving DMI, are essential tools for the success of lactational performance. Traits of energy metabolism (NEB, BCS, DMI, and fat-to-protein ratio) should have high priority in the genetic selection indices of modern dairy cattle.