Abstract: The aim of the present study was to compare the hepatic Triacyiglycerols (TAG) and serum Non-esterified Fatty Acids (NEFA) and serum vit. E and selenium in cows in different stages of productivity slaughtered at Tabriz abattoir. So, a total of 204 blood and liver samples were collected from the indigenous hybrid cows immediately after being slaughtered at the abattoir. The cows were divided into 4 groups according to their pregnancy statuses: 1-8 months pregnant, 8-9 months pregnant, less and more than 1 month of partunition. Percentage of TAG were measured in the liver and NEFA, vit. E and selenium were measured in the blood circulation. All the liver samples, in this study, show some degrees of fatty infiltration. The accumulation of that in the liver rose significantly (p<0.001) during the 1st month after parturition and returned to the same level of pregnancy after 1 month. We also noticed that serum NEFA concentration was significantly higher (p<0.001) during the 1st month of parturition and serum vit. E and selenium level was significantly lower (p<0.001) in newly calved group (<1 month after parturition). These results reveal that fatty liver is quite common in all the animals in the region especially in their early lactation. These findings indicate the necessity of reconsideration in the animal’s food policies in the area and some especial approaches during the early phase of lactation.

INTRODUCTION

Dairy cows undergo tremendous changes during the transition period, i.e., from 3 weeks before to 3 weeks after parturition (Drackley, 1999). This critical important period is characterized by dramatic changes in nutrient demand that necessitate coordinated changes in body tissue metabolism to meet requirements of energy (Bell, 1995; Overton et al., 2001). During times of energy deficiency, animals break down triglycerides stored in their adipose tissue and produce Non-esterified Fatty Acids (NEFA) which enter the blood circulation and are transported to organs and tissues throughout the body (Emery et al., 1992).

Extensive and prolonged mobilization of body fat leads to fat accumulation in the liver which is called hepatic lipidosis or fatly liver (Gruffat et al., 1996; Goff and Horst, 1997). Fatty liver occurs when the hepatic uptake of lipid exceeds the rate of disappearance of triglyceride from the liver through either oxidation or secretion via VLDL (Bremmer et al., 2000).

Overfeeding during the non-lactating stage reduces feed intake and stress near patunition accelerates the release of NEFA from adipose tissues, resulting in an excess uptake of NEFA by the liver (Rukkwamsuk et al., 1998). Circulating NEFA are absorbed by the liver and re-estrified to Triacylglycerols (TAG) which are then secreted into the blood in the form of VLDL (Herdt et al.,1988). Excess hepatic lipids are stored as TAG and are associated with decreased metabolic functions of the liver (Drackley, 1999). In dairy cows, fatty liver occurs primarily in the 1st month of lactation (Grummer, 1993) when up to 50% of all cows have some accumulation of TAG in liver (Jorritsma et al., 2001). Approximately, severe and moderated fatty liver develop in 15 and 35% of the dairy cows, respectively (Rehage et al., 2006).

Severity of fatly liver can be defined on the basis of amount of TAG accumulation. Bobe et al. (2004) categorized fatty liver into normal (<1% liver TAG on wet weight, bases), mild (1-5% liver TAG), moderate (5-10% liver TAG) and severe (>10% liver TAG). Although, fatty liver is associated with increased incidence of health disorders (Jorritsma et al., 2000). Recent studies have showed no relationship between liver function and the liver’s fat content (Rehage et al., 2006). It is believed that the majority of cows even those with severe fatty liver. May not reveal impaired liver function in the blood biochemistry tests (Rehage et al., 2006) therefore, liver biopsy is the only reliable method to determine severity of fatty liver in the dairy cows. The objectives of this study were to assess the percentage and severity of fatty liver among cross breed cows in Tabriz in the east of Iran. An attempt was made to confirm the previous mentioned problem by measuring TAG in the liver and NEFA, vit. E and selenium concentration in the blood circulation.

MATERIALS AND METHODS

The research was carried out in Tabriz, the capital of Azarbaiejan province which is placed in the 600 km to Teheran, the country’s capital. During a period of 3 months from the beginning of July to the end of September 2010, blood and liver samples were collected from 204 cross breed cows (Holsteinxindigenous) at Tabriz’s abattoir and were classified into 4 groups regarding the cow’s pregnancy status: 1-8 months pregnant, 8-9 months pregnant, less and more than 1 month of parturition.

The cows were sent to the pastures with inferior quality grasses during the day and in their return to their barns they were nursed with 2-3 kg of a locally made concentrate containing barley, wheat and maize. The cows had access to ad lib straw. The daily milk production of these cows was 16-20 kg day^-1. Blood was taken for the determination of NEFA, vit. E and selenium while the calves were being slaughtered. After collection, the blood samples were allowed to stand for 20-30 min and then they were transferred to the laboratory where they were centrifuged at 2000-3000 rmp.

The serum was separated and stored at -20°C till analysis. Liver samples of at least 10 g weight were taken from a certain area of the right lobe after the animal’s abdomen was opened. For TAG determination, the liver samples were placed in physiological saline and carried to the laboratory in a thermostatic (0-4°C) container. Lipid extraction from the liver carried out by the method of Folch et al. (1957). TAG concentrations were measured as described by Neri and Firngs (1973) and Frings et al. (1972), respectively.

Serum vit. E and selenium concentrations were measured as explained by HPLC and atomic absorbtion spectrophotometery, respectively. Serum NEFA level was determined using also spectrophotometer with a commercially available Kit (Randox). Analysis of variances and post-hoc Tukey’s honestly significant difference tests were used to analyze the data. All statistical analyses were performed using the SPSS software, version 13.0. Significance was declared at p<0.001.

RESULTS AND DISCUSSION

As it is shown in Table 1 in newly calved group (<1 month after parturition), the percentage of TAG accumulation in the liver was significantly (p<0.001) higher than other 3 groups. Mean serum NEFA concentrations were also significantly (p<0.001) higher for cows with <1 month of parturition. Mean concentration of vit. E was significantly lower (p<0.001) for cows with <1 month of parturition. Also mean concentration of selenium was significantly lower (p<0.001) for cows with <1 month of parturition. The prevalence of moderate as well as severe fatty liver in this cohort of 204 cows was 16.2% (Table 2). The total percentage of cows with mild fatty liver was 65.5%, the moderate group 15.5% and the cows with severe fatty liver composed 0.7% of the animals.

Based on the Bobe et al. (2004), classification about 44.3, 51.3 and 3.4% of the cows in the present study had mild, moderate and severe fatty liver, respectively. Reid (1980) reported that the incidence of moderate and severe fatty liver in Holstein cows in England was 48 and 15%, respectively and in Guernsey cows, he showed a 33% moderate and 5% severe fatty liver. In the USA, incidences of 20% for moderate and 15% (Gerloff et al., 1986) to 24% (Herdt, 1991) for severe fatty liver were reported. In a review of the literature, Bobe et al. (2004) indicated that in the 1st month after calving, 5-10% of dairy cows show severe and 30-40% show moderate fatty liver which means that up to 50% of dairy cows are at a higher risk of the disease. In present study, we noticed that about 90.9 and 4.6% of the indigenous dairy cows had moderate and severe fatty liver, respectively in the 1st month after parturition. This can be due to the nutritional mismanagement during dry period and stressful condition after parturition. In contrast to the Reazai Saber et al. (2007) and Gerloff et al. (1986) reports who showed that severe fatty liver occurred prior to parturition, we in line with the finding of Van den Top et al. (1996) observed that severe fatty liver mainly developed in the 1st month after calving.

It has been shown that postpartum accumulation of TAG in the liver is a possible consequence of a postpartum negative energy balance which originates from a reduction or diminished pre-parturient dry matter intake and higher demands of energy due to the start of milk production (Bertics and Grummer, 1999). In these conditions, NEFA are mobilized from the adipose tissues and transported to the liver where some are re-estrified to TAG which are then secreted into the blood circulation in the form of VLDL (Mazur et al., 1989). However when the intra-hepatic NEFA concentrations increase, the production of VLDL is impaired and the liver accumulates a great amount of TAG.

Table 1:	Percentage of TAG in the liver and the levels of serum NEFA, vit. E and selenium
*p<0.001 within the column

Table 2:	Prevalence of different grades of fatty liver changes among the studied groups

It has been shown that (Grummer, 1993) the concentrations of NEFA increase gradually during the final weeks prior to parturition and sharply elevates at parturition however, the greatest increase was observed between 1-3 weeks after parturition (Rukkwamsuk et al., 1998). It is believed that the rapid rise in NEFA at parturition could be due to the stress of calving. serum NEFA decrease after calving but remain higher than what they were before parturition (Grummer, 1995).

In present study, a significant increase (p<0.001) in serum NEFA was observed in the newly calved cows which was in accordance with the finding of Reid et al. (1983) who showed that plasma NEFA concentrations 1 week after parturition were approximately twice as high in cows with moderate fatty liver and they concluded that the increased serum NEFA could be responsible for fatty liver. In present study, we noticed 4.6% of the recently calved cows had severe fatty liver with concomitant high serum NEFA concentrations.

These results are in accordance with the finding of Gerloff et al. (1986) who showed that cattle with severe hepatic lipidosis had the greatest serum NEFA. A high serum NEFA is commonly seen during the negative energy balance of post partum dairy cows due to the shortage of energy and subsequent body fat mobilization (Jorritsma et al., 2001).

In the current study, the cows during their dry period were fed mostly straw ad lib and small amount of wilted grass and after calving, a mixture of 2-4 kg of barley, bran and dried bread were added to the straw feeding. It has been reported that both over-conditioning and underfeeding of cows during late pregnancy could severely affect the animal productivity and may cause perparturient metabolic disorders (Forbes, 1986; Bell, 1995; Tesfa et al., 1999). Gerloff and Herdt (1984) indicated that feeding only straw for 5 days at late pregnancy induced prepartal fatty liver. In present study, we noticed that about 100% cows in their late pregnancy had a mild fatty liver (>1% liver TAG). We believe that the reason for high percentage of fatty liver among the studied cows was a long period of feed restriction before their parturition and feeding them with poor quality foods after their calving. One of the numerous changes may occur in fatty liver is a drop in serum vit. E and selenium (Bobe et al., 2004) therefore, serum vit. E and selenium could be used as a marker of the liver’s function. Vitamin E blood levels which reflect the dietary vit. E intake for cows (Maplesden et al., 1960; Tikriti, 1969) where significantly lower in 1st month after calving as compared to another groups (p<0.001).

In fatty liver syndrome cows, until the 2nd week after calving, blood vit. E levels were lower than those levels reported as normal (Bayfield and Mylrea, 1969; Mukhtar, 1996; Hidroglou and Hartin, 1982; LeBlanc et al., 2004; Tappel, 1962). Blood vit. E functions as a source for tissue vit. E and the liver is the major storage or metabolic site of this vitamin. In the liver, many oxidation-reduction reactions require vit. E as a biological antioxidant (Tappel, 1962; Mudron et al., 1999). Excess accumulation of fat in parenchymal cells could be related to some interference with the process of fatty acid oxidation or with the synthesis or the intracellular transport of lipoproteins. Vitamin E has a function in promoting the stability and integrity of the lipoprotein membrance cells (Diplock, 1981; Mudron et al., 1997). The criteria of successful treatment of fatty liver syndrome cases in which liver dysfunction is thought to be present because of excessive hepatic lipogenesis are rapid reduction of fatty infiltration of liver tissues and restoration of normal metabolism. It was observed (Harrill et al., 1965; Prodouz and Navari, 1975; Spratt and Kratzing, 1971) that in laboratory animals fatty liver of diverse origin are alleviated by administration of vit. E. In the present study, the observation of lower blood vit. E in the 1st month after calving (Table 1) suggests a possible cause-effect relationship between fatty liver syndrome and vit. E status of cows and encourages further evaluations of the possible preventive administration of massive doses of vit. E for alleviation of the fatty liver in the periparturient cows. In present study, a significant decrease (p<0.001) in serum selenium concentration was observed in the newly calved cows which was in accordance with the findings of Bobe et al. (2004), Hidroglou and Hartin (1982) and West (1990).

It was reported (Backall and Scholz, 1979) that difficulties arise when attempting to predict the magnitude of Se inadequacy in dairy cattle which could result in clinical manifestations of Se deficiency. It was found (Perry et al., 1976) that plasma Se levels <20 ng mL^-1 which is equivalent to at least 56 ng mL^-1 of whole blood are indicative of Se deficiency in dairy cattle. However, a problem of retained placentas is reported in dairy herds with Se levels of 61-73 ng mL^-1 of whole blood (Trinder et al., 1973). Serum selenium concentration, shown in Table 2 was suggestive of relation with Se responsive disease in the dairy cow (Conrad et al., 1976; Julien et al., 1976)

CONCLUSION

Results from the present study showed that fatty liver existed either in pregnant or non-pregnant cows and in the 1st 4 weeks after calving was severe than any other times. The researchers believe poor quality of food can be a major factor in this high percentage of fat infiltration in the liver. The high frequency of hepatic lipidosis that was found in the present study may have a great impact on health and fertility of the cows, since it has been shown that even mild fatty liver can be associated with decreased health status and fertility of dairy cows. To reduce fatty liver incidence in cows in Tabriz, it is suggested to supply sufficient nutrients before and after calving.

ACKNOWLEDGEMENT

The researchers wish to thank F. Farhangpajouh of Uromia University (Iran) for his assistance in preparing this manuscript.