MICROBIAL COMPOSITION AND ADAPTATION IN SHORT AND LONG TERM FEEDING OF SAPONINS

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Glucose

Glucose is one of the most widely used blood metabolites to determine the energy status of ruminants. However, some studies have shown a certain difficulty in exploiting the results because the homeostatic mechanism that controls blood glucose levels has become difficult to interpret objectively due to a link between nutritional status and glucose levels. In addition, many tissues use free fatty acids (FFA) and ketones bodies as an energy source, despite the fact that the liver of these animals has a high gluconeogenic function (Payne & Payne, 1987; Peixoto & Osório, 2007).
When it comes to the ruminant species, this metabolite is extremely important due to the central role it has in mammary gland function in supplying components such as carbon, hydrogen and oxygen for the synthesis of lactose. Moreover, the synthesis of lactose is responsible for controlling the volume of milk produced due to the high osmotic pressure it induces. Glucose plasma concentrations are stable during the transition period, with an increase only being seen during labor and immediately after parturition. This increase is linked mainly to rises in glucagon concentrations and glucocorticoids (Kunz et al., 1985; Vazquez-Añon et al., 1994).
In well-fed ruminants, the main precursor for hepatic gluconeogenesis is propionate, one of the major VFA byproducts of pregastric fermentation, which is absorbed via the ruminal epithelium into portal venous blood and almost quantitatively removed by the liver. The rate of ruminal production of propionate and other VFA is directly related to dietary intake of fermentable substrate; propionate synthesis is especially favored by fermentation of starches by amylolytic bacteria. Since the hepatic supply of propionate is a principal determinant of hepatic glucose synthesis; it is not surprising that in all classes of ruminant whole-body glucose production is highly correlated with digestible energy intake. As the supply of propionate dwindles, the importance of other glucogenic substrates, such as lactate, amino acids, and glycerol, increases (Bell & Bauman, 1997; Caldeira, 2005).
Blood glucose levels may be changed as a function of the availability of precursors for the synthesis of glucose. Low metabolizable energy intake can cause a reduction in propionate in the rumen, which is the main factor responsible for a decrease in glucose levels (Reynolds et al., 2003).
Monitoring and/or evaluating the energy status of ruminants via glucose has been identified in previous studies as an imperfect indicator due to its insensitivity to nutritional changes and its sensitivity to stress. Nevertheless glycemia could still be used in situations of severe energy deficit and in animals that are not pregnant and during lactation (Mundim et al., 2007). Previous studies on sheep and goats with pregnancy toxemia confirmed that glucose is not a reliable indicator of metabolic disorders when referring to this disease, since a common finding is that the animals the most affected had hyperglycemia and this was explained by a stress induced increase in cortisol levels (Santos et al., 2011; Souto et al., 2013).
At the end of pregnancy, glucose requirements are absolute and are not controlled by small ruminants when there is transfer of glucose from the dam to the offspring(s), the latter use it and glucose does not return to the maternal bloodstream. Moreover, when feed intake is too low to meet fetal energy demands, especially for females carrying twin fetuses, the dam uses its own body reserves to try to maintain the correct glucose balance (Bruére & West, 1993). In addition, the glycemic profile is the most important indicator during the last days of gestation as a predictor of fetal viability.
This information about glycemia has a great importance in the developement of therapeutic protocols, especially by the intravenously administration of glucose in sick animals (Lima et al., 2012). Glucose in the pregnant goat and ewe is the major source of energy to the fetus(es). Therefore, pregnant goats are at high risk of developing pregnancy toxemia due to the rapid foetal growth (Bergman, 1993). The energy requirement of the pregnant goat increases by a factor of 1.5 when it carries one fetus and by a factor of 2 when it carries two fetuses (Pugh, 2005). Blood glucose levels in pregnant goats are generally low, because of fetal demand. There is little information in the literature addressing the occurrence of hyperglycemia in pregnant does. Studies showed that as the disease progresses in ewes, blood glucose and cortisol levels may increase due to fetal death. Wastney et al. (1983) suggested that hyperglycemia occurs because fetal death removes the inhibitory effect of the fetus on hepatic gluconeogenesis. Smith and Sherman (2009) referred to the existence of a marked hyperglycemia in terminal cases.

Non-Esterified Fatty Acids (NEFA)

In the ruminant, ketones are produced by intermediary metabolism and during the absorption of butyric acid from the rumen. In the peri-partum period the main source of ketones is fatty acids including those with short, medium and long chains. Of course, any compound (glucose, lactate, glycerol, amino acids, etc.) that can be converted to fatty acids and can be considered a source of ketone, but the origin of ketones is considered to be fatty acids, either esterified or nonesterified (Kaneko et al., 2008).
A massive mobilization of NEFA from adipose tissue during and after parturition in high-yielding dairy ruminants is the metabolic hallmark of the transition from pregnancy to lactation (Bell, 1995). There is an abrupt shift in the metabolic demands from crucial nutrients (body reserves and fetal mass) to rapid mobilization of lipid and protein stores in support of the sudden onset of high milk production (Adewuyi et al., 2005).
NEFA are released into blood when adipose tissue is mobilized to supply the metabolic needs of the animal, primarily the need for energy. Although the quantity of NEFA in the blood of ruminants is small, it is an important factor in caloric homeostasis of the body. In other words, high levels of this metabolite reflect the magnitude of fat mobilization of body reserves, usually associated with the period of insufficient energy consumption, and are an effective way to evaluate the energy status of ruminants (Bowden, 1971; LeBlanc, 2010).
Increased lipolysis and decreased lipogenesis are stimulated by negative energy balance (BEN), observed when the animals do not meet their requirements through the diet, in addition to the high demands for fetal growth and milk production. It is necessary to use alternative sources for energy production through increased lipid mobilization, which is used as an energy source by many tissues, including the mammary gland and the liver (Bell, 1995; Grummer, 1995). Furthermore this mobilization results in decreases in body condition score and weight of animals, leading to endocrine and metabolic disturbances (Rodrigues et al., 2006; Artunduaga et al., 2011).
Circulating NEFA comes from the hydrolysis of triglycerides deposited in adipocytes and these metabolites are released and carried into bloodstream by albumin, which provides the necessary solubility for their circulation in the blood (Caldeira, 2005). Their accumulation due to the inability of the liver to metabolise them can trigger metabolic disorders. Accordingly, this will expose the animals to increased risks of diseases such as subclinical and clinical pregnancy toxemia, uterine diseases, decreased milk production and reduced reproductive performance (Pullen et al., 1989; Radostits, 2000). Consequently, the blood concentration of NEFA depends on the degree of fat mobilization in response to NEB (Van Saun, 2000).
An increase in NEFA concentrations is observed as pregnancy progresses and this provides some advantages to the pregnant ruminant, acting as a source of energy for dam metabolism and promoting the development of an insulin resistant state (Regnault, 2004). This mechanism reduces the utilization of glucose by the peripheral tissues, through the inhibition of the action of insulin, thus preserving and providing the glucose for placental and fetal metabolism (Brockman, 1979; Regnault, 2004). An elevation in plasma NEFA occurs commonly during parturition, but it is observed before the animal presents symptoms of metabolic disease (Barakat et al., 2007). Therefore, previous studies reported that the level before parturition is an important tool to predict the mobilization of body reserves due to high energy demands at this stage. This situation allows early detection in ruminants at risk of developing disorders associated with NEB (Grummer, 2002; Souto et al., 2013).
Sheep in the early lactation period have higher energy demands when compared to pregnancy and the dry period due to milk synthesis (Abdelrahman et al., 2002; Karapehlivan et al., 2007). In the post-partum period, the rate of lipolysis overrides lipogenesis, providing greater amounts of NEFA to supply peripheral tissues. Bell (1995) noted that NEFA provide about 40% of the fat in milk during the first days of lactation. In ruminants, mammary uptake of NEFA depends on their circulating concentrations. It is important to realize that fatty acids in milk come from two sources, uptake from the circulation and synthesis within the mammary epithelial cells. The free fatty acids taken up from the circulation by the mammary gland are derived from circulating lipoproteins and NEFA that originate from respectively absorption of lipids from the digestive tract and from mobilization of body fat reserves (Adewuyi et al., 2005).
In the liver, NEFA metabolism depends on the availability of glucose and on the mobilization rate, because they may be completely oxidized for energy production or partially oxidized for the production of ketone or reesterified and stored as triglycerides. The ruminant liver has a limited capacity to export triglycerides as low density lipoproteins, so that high mobilization in relation to low exportation leads to hepatic steatosis and predisposes the body to metabolic disorders (Head & Gulay, 2001).

β-Hydroxybutyrate (βHB)

Ruminants usually have higher ketone body concentrations in blood plasma than monogastric animals due to postprandial production of β-hydroxybutyrate (βHB) in the ruminal epithelium. Even higher concentrations of ketone bodies are present in ruminants suffering from clinical or subclinical ketosis. A NEB during the transition period around parturition is regarded as the primary cause of the development of the disease and the development of hyperketonemia in small ruminants and cattle. Sustained hyperketonemia is probably the most characteristic biochemical sign of spontaneous clinical and subclinical ketosis of sheep, goats and cattle. In ketotic animals, when feed intake has ceased, ketone bodies are almost exclusively produced by the liver from β-oxidation of fatty acids (Schlumbohm & Harmeyer, 2004).
Ketone bodies are energy sources in the absence of carbohydrates and lipids in ruminants and their precursors are lipids and fatty acids from the diet, as well as fat deposits. Butyric acid is produced in the rumen and transformed in the epithelium of the pre-stomachs via acetoacetate to βHB (Wittwer, 2000; Wittwer et al., 2006). βHB assays are important clinical tools for assessing nutritional status and adaptation to the NEB (Chung et al., 2008). Amongst the ketones bodies (βHB, acetoacetate and acetone), βHB is the most widely used indicator of NEB due to its stability in serum, and from the fact that it is not influenced by stress and its blood concentration in NEB situations is not limited by the availability of a carrier (Caldeira, 2005). The severity and duration of NEB is reflected by the increase in circulating NEFA and βHB and the degree of the decrease in glucose concentrations (Drackley, 1999). The decreased DMI prepartum causes NEB and increases NEFA and βHB concentrations (LeBlanc, 2010).

Table of contents :

1. GENERAL INTRODUCTION
2. REVIEW OF THE LITERATURE
2.1 METABOLIC PROFILES
2.1.1 Transition period
2.1.2 Energy profile
2.1.2.1 Glucose
2.1.2.2 Non-Esterified Fatty Acids (NEFA)
2.1.2.3 β-Hydroxybutyrate (βHB)
2.1.2.4 L-lactate
2.1.2.5 Fructosamine
2.1.3 Lipid profile
2.1.3.1 Total Cholesterol (TC)…….
2.1.3.2 Triglycerides
2.1.3.3 High Density Lipoproteins (HDL)
2.1.3.4 Total Bilirubin (TB)
2.1.4 Protein profile
2.1.4.1 Total Protein (TP)
2.1.4.2 Albumin
2.1.4.3 Globulin.
2.1.4.4 Urea
2.1.4.5 Creatinine
2.1.4.6 Haptoglobin (Hp)
2.1.5 Enzyme profile
2.1.5.1 Aspartate Amino Transferase (AST)
2.1.5.2 Gamma Glutamyl Transferase (GGT)
2.1.5.3 Creatine Kinase (CK)
2.1.5.4 Alkaline Phosphatase (ALP)
2.1.6 Mineral profile
2.1.6.1 Total Calcium (Ca)
2.1.6.2 Sodium (Na+)
2.1.6.3 Potassium (K+)
2.1.6.4 Magnesium (Mg3+)
2.1.6.5 Chloride (Cl-)
2.1.6.6 Phosphorus (PO4
2.2 PREGNANCY TOXEMIA
2.2.1 Etiology
2.2.2 Pathogenesis
2.2.3 Diagnostic
2.2.3.1 Clinical signs
2.2.3.2 Laboratory tests
2.2.3.3 Necropsy findings
2.2.4 Treatment
2.2.5 Prevention
2.3 NATURAL PRODUCTS: GLUCAN & SAPONIN
2.3.1 β-glucans: structure, properties and bioactivity
2.3.1.1 Introduction
2.3.1.2 Historical interest in β-glucans
2.3.1.3 Sources and structure
2.3.1.4 β-glucan immunostimulating activity
2.3.1.5 Role of β-glucan on cholesterol and glucose levels
2.3.1.6 Anticarcinogenic activity
2.3.1.7 Future perspectives
2.3.2 Plant secondary metabolites and their interest in ruminants
2.3.2.1 Saponins
2.3.2.1.1 Nature
2.3.2.1.2 Occurrence
2.3.2.1.3 Mechanism of action
2.3.2.1.4 Biological effects
2.3.2.1.4.1 Effects on rumen microorganism
2.3.2.1.4.2 Rumen antiprotozoal activity
2.3.2.1.4.3 Effects on protein digestion
2.3.2.1.4.4 Hypoglycaemic activity
2.3.2.1.4.5 Effects on cholesterol metabolism
2.3.2.1.4.6 Methane
2.3.2.1.4.7 Ammonia concentration
2.3.2.1.4.8 Bacteria and fungi
3. MAIN OBJECTIVES AND APPROACH OF THE STUDY
3.1 GENERAL OBJECTIVE
3.2 SPECIFIC OBJECTIVES
4. PAPERS
4.1 PAPER 1: Do intramuscular injections of β1,3-glucan affect metabolic and enzymatic profiles in Santa Inês ewes during late gestation and early lactation?
4.2 PAPER 2: Effects of a Saponin-based Additive on Two Different Dairy Goat Metabolic Statuses
5. GENERAL DISCUSSION AND PERSPECTIVES
5.1 INTEREST IN THESE NATURAL SUBSTANCES (β-glucan and saponin)
5.2 EXPERIMENTAL DOSE
5.3 JUSTIFYING THE EXPERIMENTAL NUMBERS OF ANIMAL
5.4 DIFFERENT METABOLIC STATUSES
5.5 MICROBIAL COMPOSITION AND ADAPTATION IN SHORT AND LONG TERM FEEDING OF SAPONINS
5.6 EFFECT OF THESE NATURAL SUBSTANCES ON HORMONAL PROFILE
5.7 IMPACT ON THE IMMUNE SYSTEM
5.8 METABOLOMIC BY NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY
5.9 CONCLUSION
6. RÉSUMÉ SUBSTANTIEL
7. GENERAL BIBLIOGRAPHY
8. ANNEXES

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