J. COMP. PATH. 1985. VOL. 95.

ULTRASTRUCTURAL CHANGES OF THE LIVER IN SPONTANEOUSLY KETOTIC COWS

BY

Y. GR~FTN* and L.-A. LINDBERGH

* Department of Physiological Scitxccs, School of Vettrinary Medicine, University of California, Davis, U.S.A. f Departments of Anatomy and Embryology, and Biochemishy, and Loboratory of Electron Microscopy, College of

Veterinary Medicine, Helsinki, Finland

INTRODUCTION

Bovine ketosis is a common and economically important disease. The clinical signs (loss of appetite, decrease of milk production, and rapid deterioration of body condition) have been well known for at least 50 years (Shaw, 1956). The more obvious biochemical features, hyperketonaemia and hypoglycaemia, were noted long ago. An inadequate supply of nutrients for high milk production is a widely accepted explanation for the ketosis syndrome (Pehrs- son, 1966; Schultz, 1968; Bergman, 1973; Baird, 1982). This imbalance leads to negative carbohydrate balance, increased fat mobilization, and increased hepatic ketogenesis.

A fatty liver has been a common finding in severe cases of bovine ketosis Saarinen and Shaw, 1950; Ford and Boyd, 1960; Kronfeld, Simesen and Dungworth, 1960; Grohn, Lindberg, Bruss and Farver, 1983), but ultrastruc- tural studies have not been performed on livers of spontaneously ketotic cows. It has been assumed that the changes are the same as of the fatty liver of lactating cows suffering from fasting ketosis (Reid, 1973; Baird, 1982). Although it is possible to induce fatty liver by fasting, typical ketosis does not develop (Hibbit and Baird, 1967; Reid, Harrison and Collins, 1977), and a field study on ketotic fatty liver was needed. Our purpose was to study ultrastructural changes of the liver in spontaneously ketotic cows with samples obtained under field conditions.

MATERIAL AND METHODS

Animals

Thirty lactating dairy cows (21 Finnish Ayrshire, one Finn, and 8 cross-bred cows) were used. Ketotic cows were examined at the request of dairy farmers. During the same farm visit, a healthy cow that was at the same stage of lactation as the ketotic cow was selected as a control. The cows were divided into 3 groups. Groups were designated control, mildly ketotic and severely ketotic. A cow was defined as ketotic if she had the clinical signs (depression, decrease of milk production, and especially, a history of anorexia) and her blood beta-hydroxybutyrate concentration (BHB) was more than l.OmM. Cases of secondary ketosis (i.e., ketosis that appeared to.be secondary to other disease conditions) were excluded. The ketotic group was divided into mild and severe cases by the concentration of blood BHB (cut-off point= 3mOmM).

0021-9975/85/030443+10$03.00/O 0 1985 Academic Press Inc. (London) Limited

444 Y. GRtiHN AND L.-A. LINDBERG

Blood Samples Blood Samples

Heparinized blood samples were taken from the jugular vein just before liver Heparinized blood samples were taken from the jugular vein just before liver biopsies were obtained. Perchloric acid supernatant fluids from whole blood were biopsies were obtained. Perchloric acid supernatant fluids from whole blood were stored at stored at -20°C until analysed. Ketone body and glucose concentrations were - 20°C until analysed. Ketone- body and glucose concentrations were measured according to Tyopponen and Kauppinen (1980). measured according to Tyopponen and Kauppinen (1980).

Liver Samples

Liver samples were obtained from cows by percutaneous needle biopsy. The samples were immediately cut into small (1 mm3) pieces and processed as described previously (Grohn and Lindberg, 1982; Grohn et al., 1983).

Stereological Analyses

Three Epon-embedded blocks, taken at random from liver samples of each cow, were sectioned for light and electron microscopy and used in stereological analysis (Fig. 1, ‘i.

Light microscopy. The volume fraction of trabeculae and parenchyma was estimated by a loo-point eyepiece graticule (mesh size 0*5mm) in a 10 x ocular and 10 x objective. Using the same loo-point eyepiece graticule in a 10 x ocular and a 25 x objective the volume fraction of cell components of hepatic parenchyma was mea- sured. Points overlying sinusoids, nuclei and hepatocytic cytoplasm were counted. In all, for each cow, 10 fields of 100 points each were observed.

Electron microscopy. Volume fractions of cytoplasmic organelles were determined by a loo-point graticule (size 16 cm x 19 cm) at a final magnification of 32 000. The points overlying mitochondria, peroxisomes, lysosomes, lipid, glycogen, rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), Golgi apparatus and cytosol were counted. In addition, the profile density of mitochondria, peroxisomes, and lysosomes per micrograph ( 16 cm x 19 cm + 32000 = 1 mm2), were counted. From these counts, the volume fraction occupied by each of the cytoplasmic components, as well as profile density per field (1 mm2), could be calculated. For each cow a total of 20 micrographs of 100 points each was observed.

Statistics. The counts from the micrographs per block were averaged, and the block means representing each animal were averaged to give values for individual animals. Comparisons of variables were by one-way analysis of variance. When effects of group were statistically significant (P-=0.05), the least significant difference (LSD) method was used for pair-wise comparisons of group means (Kleinbaum and Kupper, 1978).

RESULTS

Comparison of Groups; Ketone Body and Glucose

A comparison of descriptive variables for control, mildly ketotic, and severely ketotic cows is given in Table 1. The 3 groups are comparable because they did not differ for any of the descriptive characteristics. Beta-hydroxybu- tyrate (BHB) was the grouping variable. There were statistically significant differences in blood acetoacetate and glucose concentrations.

Histology

Light microscopy. The volume fraction of trabeculae and parenchyma did not differ significantly between control, mildly ketotic and severely ketotic cows (Table 2). The volume fraction of hepatocytes within the hepatic parenchymal

ULTRASTRUCTURE OF KETOTIC COW LIVER 445

I Trobeculoe

Stage I: LM, x 100

Stage II: LM, x 250

mm 1 Cytoplasmic components

Stage III: EM, x 32 000

Fig. 1. Scheme of sampling procedure of liver structure for stereological analysis in control, mildly ketotic and severely ketotic cows.

volume was significantly increased, and the volume fraction of sinusoids was decreased in the severely ketotic group compared with the other 2 groups (Table 2). A ‘g ‘fi sl m cant increase in the volume fraction of cytoplasm and a decrease in the volume fraction of nuclei within hepatocytes in the severely ketotic group were observed (Table 2).

Electron microscopy. Relative volumes (Vv) of cytoplasmic components within hepatic parenchymal volume are given in Table 3. In the livers of severely ketotic cows there was a significant increase in the volume fraction of lipid and SER as compared to the other 2 groups. A decrease in the profile density of mitochondria per 1 mm2 (Table 4) and an increase of the volume occupied by mitochondria (Table 3) were not significant.

A significant decrease in all groups from control to severely ketotic cows in the volumes occupied by glycogen and Golgi apparatus was found. A corresponding significant increase in the volume of cytosol was also seen. A decrease in the volume of RER in mildly and severely ketotic cows was not significant. Mitochondrial alterations, such as swelling, abnormal cristae, and

TABLE 1

COMPARISONS OF DESCRIPTIVE VARIABLES AND BLOOD CHARACTERISTICS OF CONTROL, MILDLY KETOTIC AND SEVERELY KETOTIC COWS*

Variable

Breed ( Avrshire score = 1, not Ayrshire score = 0) Pa&y GdYs post partum Acetoacetate, mM Beta-hydroxybutyrate, mM Glucose, mM

Control

otlfo~lt

4.2f0.67 30.2f8.37

0.2 f o.ot 0.7f@lt 2.5fO.IT

Mildly ketotic

0.7*0.2t

5.1 l 0.7t 24.4 f 5.87

0.7f0.2: 1.9*0.1: 2.3*0.2t

Severely ketotic

0.6*0.2t

3.4f0.6t 31.6f4.67

2.2f0.25 4.9 f o+ 1~6~0~1~

*The values are means and standard errorx for 30 cows in 3 groups of 10 based on blood beta- hydroxybutyrate concentrations with cut-off points of 1.0 and 3.Omaa.

t :$Means within variable groups sharing common superscripts do not differ (P>O.O5).

446 Y. GRGHN AND L.-A. LINDBERG

TABLE 2

VOLUME FRACTION OF LIVER STRUCTURES 1N CONTROL, MILDLY KETOTIC AND SEVERELY KETOTIC COWS*

component Control Mildly k&tic Severely ketotic

Per cent of liver tissue volume

Trabeculae Parenchyma

2.6f0.2t 2.9f0.37 2.9f0.37 97.4f0.2f 97.1 f 0.3f 97.1 k0.37

Per cent of hepatic parencfzzal volume

Sinusoids Hepatocytes

13.2i0.7t 12.6f0.7t 9.0*0.7: 86.8tO.7t 87.4f0.7t 91*0*0.7:

Per cent of hepatocyte volume

Nuclei 3.3fOcq 3.5*0.3t 2.4*0.3$ Cytoplasm 96.7fO.Zt 965f0.3t 97.6f0.3:

*The values are means and standard errors for 30 cows in 3 groups of IO based on blood beta- hydroxybutyrate concentrations with cut-off points of I.0 and 3.0mM.

+: Means within variable groups sharing common superscripts do not differ jP’O.05).

TABLE 3 RELATIVE VOLUME OF CYTOPLASMIC COMPONENTS WITHIN HEPATIC PARENCHYMAL VOLUME IN CONTROL,

MILDLY KETOTIC AND SEVERELY KETOTIC COWS*

Per cent of hepatic parenchymal volume

Component

Mitochondria P eroxisomes

Control

17.1 l 0.7t 2.4fo~lt

Mildly ketotic

17.5f 1.ot 2.6f0.3t

Severely ketotic

18.7 f l.Ot 2.2*0.2t

Lysosomes Lipid

0.8f02j 0.8kO.tj 4.4* l-4t

0.7fkij 4.6f 1.6) 115*2.9+

19.2 f 2.67 6.6*0.6t

14.1*2.0: 3.6f0.81 6.1 f0.8t 6.1f0.5t

SW 20.2*0.7t Golgi

20.3f 1.4j l~ofoc?t

24.7 f 1.4i

Cytosol 0.7f0.1: 0~5fO.l§

@“2*0.7t 17.6f 1.5: 20.8f 1.2$

Nuclei and sinusoids 16.1 f0.77 15.8f0.87 11.2&0.8$

*The values are derived from volume pervolume = (component per hepatocyte) multiplied by (hepato- cyte per parenchyma).

They are means and standard errors for 30 cows in 3 groups of 10 based on blood beta-hydroxybutyrate concentrations with cut-off points of 1.0 and 3.0rn~.

7 +‘$ Means within variable groups sharing common superscripts do not differ (P> 0.05). WRough endoplasmic reticulum. ((Smooth endoplasmic reticulum.

TABLE 4 PROFILE DENSITY OF MITOCHONDRIA, PEROKISOMES AND LYSOSOMES IN CONTROL, MILDLY KETOTIC AND

SEVERELY KETOTIC COWS

Number per mm’ of cytoplasm

Component Control Mildly ketotic Severely ketotic

Mitochondria g-650-6 9.3f0.7 8.4 f O-6 TyEt;e 2.3f0.1 2.51tO.3 1.7f0.2

0.6fO.l 0.8fO.l 0.7fO.l

The values are means and standard errors for 30 cows ,in 3 groups of 10 based on blood beta- hydroxybutyrate concentrations with cut-off points of 0.1 and,$OmM.

There were no significant differences (P? 0.05) between groups. ‘,’ ’

ULTRASTRUCTURE OF KETOTIC COW LIVER 447

Fig. 2. Cytoplasm of two hepatocytes from a control cow. Flattened cisternae of RER (r), Golgi apparatus (arrows), a bile canaliculus (bc) and glycogen particles (g) among tubules of SER are shown. EM x 20000.

448 Y. GR6HN AND L.-A. LINDBERG

Fig. 3. Cytoplasm of a hepatocyte from a mildly ketotic cow. Distended cisternae of RER (r) and increased amount of SER (arrows) are shown. EM x 20000.

ULTRASTRUCTURE OF KETOTIC COW LIVER 449

Fig. 4. Cytoplasm of a hepatocyte from a severely ketotic cow. Lipid droplets (I), loss of glycogen distended RER (r) and cytosol with polyribosomes (arrows) are shown. EM X 20000.

450 Y. GR6HN AND L.-A. LINDBERG

increased number of matrix granules were seen in the livers of severely ketotic cows. Occasionally, these changes were detected also in mildly ketotic and control cows. The mitochondria were often in close contact with lipid droplets. Compared with control livers, the number of peroxisomes was slightly increased in mildly ketotic cows but slightly decreased in severely ketotic cows. Peroxisomes containing crystalloids were not seen in any of the groups but they often had a single electron-dense plate at the inner surface of the enclosing membrane.

In the livers of severely ketotic cows, the cisternae of RER were often widely distended, compared with the controls; this was also seen in some mild cases. Sometimes it was difficult to distinguish between SER and RER reticuli.

DISCUSSION

Based on previous studies on fatty liver in lactating cows suffering from fasting ketosis, the principal findings are an increase in the size of hepatocytes, a decrease in the volume occupied by giycogen and RER, a decrease in the number of mitochondria per hepatocyte with an enlargement of individual mitochondria and a massive increase in the volume of cell occupied by liposomes and lipid droplets (Reid, 1973; Reid et al., 1977).

In our study, the volume fraction of hepatocytes increased and the volume fraction of sinusoids decreased in severe cases of ketosis. The decrease in the volume occupied by glycogen was significant but the decrease in the volume density of RER was not significant. A decrease in the profile density of mitochondria per field and an increase of volume occupied by mitochondria were not significant either. The volume of individual mitochondria could not be estimated because the number of mitochondria was counted as a profile density (i.e. per field, and not per volume). An increase in the volume fraction of lipid was significant, but the extent of fatty infiltration in these cows was lower than Reid reported in healthy high-yielding dairy cows (Reid, 1980).

An increase in ketone-body concentration and a decrease in blood glucose concentration in early lactation indicate general fat mobilization. Hyperketo- naemia and fatty infiltration of the liver become particularly pronounced during severe ketosis (Grohn et al., 1983). The degree and duration of negative energy balance obviously have an effect on the morphological changes of the fatty liver. The changes in the volume fractions of lipid and glycogen support this view.

Not only the mitochondria but also peroxisomes are involved in the oxidation of long-chain fatty acids (Debeer and Mannaerts, 1983). Increased fatty acid oxidation may cause the liberation of toxic amounts of harmful intracellular oxygen metabolites, causing membrane damage. Mitochondria, peraxisomes and endoplasmic reticulum are important organelles in the defence against the effect of these metabolites. An increase of SER may indicate such an activation, because this organelle is involved both in defence and in the elaboration of new cellular membranes. However, the main reason for an increase of SER may simply be an activation of glycogenolysis and lipid metabolism.

ULTRASTRUCTURE OF KE’tOTIC COW LIVER 451

Endoplasmic reticulum, mainly SER but also RER, is responsible for the transportation of lipids and lipoproteins to other parts of the hepatocytic cytoplasm, where they can be stored as lipid droplets. The Golgi apparatus is probably the principal director of macromolecular traffic in the cell. Many types of molecules pass through some portion of the Golgi at the stage of their maturation, usually shortly after their synthesis in the endoplasmic reticulum. For example, incompletely glycosylated lipoprotein particles are transported by the endoplasmic reticulum- to the Golgi as liposomes. After additional glycosylation in Golgi they form very low density lipoprotein (VLDL) particles (Goldfischer, 1982). In ketosis, there is a deficit of glucose in the hepatocytes. One might speculate whether the lack of glucose disturbs the function of the Golgi; the decreased volume fraction of the Golgi supports this view.

It seems more likely that negative energy balance and ketosis lead to fatty liver than vice versa. It is possible that poor response to treatment in some cases of ketosis may be explained by fatty liver. Like post-parturient fatty liver, ketosis is associated with infertility (Benjaminsen, 1977; Refsdal, 1977). Ultrastructural changes in the liver observed in this study may affect the function of the liver. However, one ought to be cautious in interpreting associations as cause-effect relationships. Additional work is needed to deter- mine the effect of the ultrastructural changes on liver function.

SUMMARY

The ultrastructure of the liver in normal, mildly ketotic and severely ketotic cows was studied using stereological methods. In the liver of severely ketotic cows there is: (1) a significant increase in the volume fraction of hepatocytes and a decrease in the volume fraction of sinusoids, and (2) an increase in the volume fraction of lipid and smooth endoplasmic reticulum and a decrease in the volume fraction of glycogen and Golgi in parenchyma. A decrease in the profile density of mitochondria per 1 mm2 field and an increase of the volume occupied by mitochondria were not significant nor was the decrease in the volume density of rough endoplasmic reticulum.

The degree and duration of negative energy balance obviously affect the morphological changes of the fatty liver. However, additional work is needed to determine the significance of ultrastructural changes in liver function.

The authors wish technical assistance.

ACKNOWLEDGMENTS

to thank Mrs Riikka Santalahti and Mr T. Santamaki for

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452 Y. GRijHN AND L.-A. LINDBERG

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[Receivedfor publication, May 22nd, 19841