Hepatic Ultrasonography and Biochemical Alterations in Barki sheep under negative energy balance

Abstract

As little is known about the ultrasonographic features of pregnancy toxemia in sheep. The present study was designed to evaluate the significance of ultrasound as well as markers of negative energy balance for the diagnosis of pregnancy toxemia in Barki sheep. A total of seven apparently healthy Barki sheep, in late stage of pregnancy, were subjected to daily clinical examination. Of all ewes, six animals completed the study. The selected ewes were exposed to feed-restriction for five consecutive days to induce a state of negative energy balance. Serum and urine samples were collected on the day before feed-restriction (day 0) and then daily for the next five days. The collected serum samples were analyzed for glucose, cholesterol, triglyceride, beta-hydroxyl butyrate (BHBA), Non-esterified fatty acids (NEFA), insulin and leptin. Urine samples were also tested for the presence of ketone bodies using reagent strips. In parallel, 5-MHz linear and convex transducer was used to examine the liver and associated structures at different time points of the study. All ewes showed no detectable clinical findings throughout the study period. Biochemical analyses showed significant decrease in serum values of glucose and cholesterol with a significant increase (p˂0.05) in values of triglyceride, BHBA, NEFA and insulin, while values of leptin showed no significant difference. Ketone bodies were also negative in the urine of all examined animals throughout study period. Ultrasonographic findings showed a significant increase (P˂ 0.05) in the mean liver size at the 3 rd day of feed-restriction and onward as compared with day 0, while the highest value was at the 5 th day of feed-restriction. However, the other ultrasonographic liver measurements did not show any significant variations. The diameter of portal vein and caudal vena cava were significantly decreased (P˂ 0.05) at the 3 rd day of feed-restriction and ongoing as compared with day 0. There were also a focal hyper echogenic areas in the liver parenchyma observed at the 5 th day of feed-restriction .It can be concluded that ultrasonography as well as metabolic profile could be used as an adjunctive tool for the diagnosis of negative energy balance in Barki sheep. Keyword: Ultrasonography, Metabolic profile, Barki Sheep, negative energy balance Egypt. J. Chem. Environ. Health, 2 (2):265-281 (2016) On line ISSN: 2536-9164. 566 Introduction Barki sheep, which dominate the north western desert of Egypt with population of 470,000 heads (11% of the total Egyptian sheep population) are known to be well –adapted to the desert harsh conditions and scarce vegetation (El-Wakil et al., 2008) including poor feeding, heat stress and disease. Barki ewes produced about 40.6 kg of milk; in a lactation period of 18 weeks (El-shahat, 1970).The basic information on their body conformation is available (Ragab and Ghoneim, 1961).However, the major information regarding the genetics of their body weight growth is still lacking. Pregnancy toxemia (gestational ketosis), caused by a negative energy balance, is commonly observed in ewes and goats in the late gestation period (Henz et al., 1998; Rock, 2000; Van Suan, 2000andKulcsar et al., 2006).During pregnancy, fetuses often have a large glucose demand that is satisfied by the mother. If the fetal demand and the mother supply become imbalanced due to fasting of the mother or increased nutritional demands of the rapidly developing fetal placental unit, the females could suffer from a negative energy balance thereby causing a severe hypoglycemia (Batchelder et al., 1999 and Dalrymple, 2004). Ovine pregnancy toxemia develops frequently during the last 4 to 6 weeks of gestation, primarily in pregnancies with more than one fetus, about 60% of fetal growth takes place in this last gestation period. Besides, approximately 33 to 36% of the circulating glucose is directed to the fetoplacental unit in order to satisfy its energetic demands (Hay et al., 1983). Hyperketonemia usually develops when, for yet, the capacity of maternal endogenous glucose production cannot cope with the increased demand of glucose, which is present in the pregnant ewe. Goats suffering from pregnancy toxemia become anorexic, depressed and recumbent and some affected animals become constipated, grind their teeth, and have acetone smell to their breath and suffering from dystocia. Neurologic signs include blindness, circling, in-coordination, stargazing, tremors and convulsions. Death can occurred if the case is left untreated (Pough, 2002). Hyperketonemia and hypoglycemia are more common obvious biochemical features. The main ketone bodies in the blood are acetoacetate and β-hydroxybutyrate. Most of the acetoacetate produced by the liver is reduced to β-hydroxybutyrate by hydroxybutyrate dehydrogenase enzyme accounting for the higher blood concentration of βhydroxybutyrate (Grohn et al., 1983). Pregnancy toxemia in sheep is a major metabolic disorder of energy metabolism. It occurs because of a decline in the plane of nutrition, especially in ewes carrying twins or triplets in the last month of pregnancy (Rock, 2000 and Radostits et al., 2007). Failure to identify and separate ewes bearing twins or triplets, over fat condition, prolonged starvation, intercurrent diseases, and stress are Egypt. J. Chem. Environ. Health, 2 (2):265-281 (2016) On line ISSN: 2536-9164. 562 known to predispose the pregnant ewes to this disorder (Rock, 2000; Radostits et al., 2007 and Sargison, 2007). Ultrasonographic diagnosis of pregnancy has gained great attention due to its ability to diagnose the early pregnancy which has been known as important reproductive management practice in sheep flocks (Bazer et al ., 2007 and Ganaie et al., 2009). It has been shown that the energy deficits often results in pregnancy toxemia in ewes and does particularly during the late stage of pregnancy (Pugh and Baird, 2012).The diseases often follow a state of negative energy balance with subsequent impaired gluconeogenesis, hypoglycemia, fat mobilization, ketonemia, and finally ketonuria (Rook, 2000).When hepatic uptake of lipids exceeds its oxidation and secretion, the excess lipids are stored in the liver as tricycle glycerol resulting in fatty liver syndrome with subsequent reduction of hepatic functions. The severe cases of fatty liver can be subdivided into non-encephalopathic severe liver and hepatic encephalopathy (Bobe et al., 2004).The tentative diagnosis of pregnancy toxemia is usually based on a competent history, clinical findings and the characteristic alterations of serum biochemical analyses. In general, ultrasonographic examination has been considered as one of the most important principle imaging techniques used in veterinary practice. It can permit the clinician to gain instant information about a wide variety of body systems and in some cases; the dynamic function of organs can be evaluated. It has been used previously for the examination of the liver in healthy conditions (Braun and Steininger, 2011) and in several pathological conditions encompassing fatty liver in goats (Gonenci et al., 2003), hepatic abscesses in cattle (Abdelaal et al., 2014) and hydatid cyst in sheep (Guarnera et al., 2001; Hussein and Elrashidy, 2014).The purpose of the present study was to evaluate the significance of ultrasound as well as markers of metabolic profile for the diagnosis of experimentally induced negative energy balance in Egyptian Barki sheep. Materials and Methods Animals and feeding and management: A total of seven apparently healthy Barki sheep, of similar ages (mean: 4.2 years, range: 3.5-5 years) and body weight in a range of 23 and 36 kg (mean: 29.5 kg), were randomly selected and were subjected to daily clinical examination (Pugh and Baird, 2012). All investigated ewes were naturally mated and the pregnancy was detected 45 days later by using ultrasonography (Samsung Medison SONOACE R3 ultrasound system, Korea). This study was conducted at Mariut Research Station, Desert Research Center, Elamria, Alexandria, Egypt. All the procedures were done with the approval of the Ethical Committee of the Desert Research Center (Egypt).The animals received aprophylactic treatment against internal and external parasite regularly. Animals were housed in semi open shaded pens. The feed Egypt. J. Chem. Environ. Health, 2 (2):265-281 (2016) On line ISSN: 2536-9164. 562 consisted of concentrate ration plus berseem hay (Trifoliumalexantrinum) and rice or wheat straw all the round of the year. The concentrate feed mixture consisted of cottonseed cake, maize, wheat or rice bran, calcium carbonate, and sodium chloride. All ewes fed on 500 gm of concentrate /day at 2 month gestation, increasing to 750 gm/day for the last 4 weeks of pregnancy. The average crude protein was 14 % this mixture was fed once a day and water was available twice daily around noon after feeding and in late afternoon. Rational, natural pasture (green herbage, grass and remnant of plant, barseem and darawa) was fed when available. The induction of a negative energy status: Four weeks prior to parturition, the selected ewes were housed in separated pens containing straw bedding. After a period of two weeks adaptation time, the animals were subjected to feed restriction for five consecutive days to induce a state of negative energy balance according to the method described by Gonzalez et al. (2011 & 2012), while water was offered ad libitum throughout the study period. During the fasting procedure, one of the ewes lambed and hence eliminated from the study so only six ewes completed the study. Blood Sample: Blood samples were collected daily every morning at 10:00 AM. The first sampling was taken before feed restriction (day 0) and continued for 5 consecutive days. Blood samples were collected by the jugular venipuncture without anticoagulant. After centrifugation of the blood, serum was collected and then frozen at -20 0 Cfor 1week and subsequently anal