LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY
Faculty of Veterinary Medicine
Omri Azarzar
Blood mineral status of Suffolk sheep influenced by season,
sheep lines and geographical location
Sufolkų veislės avių kraujo mineralinių medžiagų kiekio
svyravimai priklausomai nuo sezono, veislės linijos ir
geografinės padėties
MASTER THESIS
of Integrated Studies of Veterinary Medicine
Supervisor: Veterinary doctor Jurgita Autukaitė
2 THE WORK WAS DONE IN THE DEPARTMENT OF LARGE ANIMAL CLINIC,
CONFIRMATION OF THE INDEPENDENCE OF DONE WORK
I confirm that the presented Master Thesis “Blood mineral status of Suffolk sheep influenced
by season, sheep lines and geographical location”
1. has been done by me;
2. has not been used in any other Lithuanian or foreign university;
3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.
12/13/2019 Omri Azarzar
(date) (author’s name, surname) (signature)
CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE ENGLISH LANGUAGE IN THE DONE WORK
I confirm the correctness of the English language in the done work.
12/13/2019 Omri Azarzar, Colman Kelliher
(date) (author’s name, surname) (signature)
CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESIS
12/13/2019 Jurgita Autukaitė
(date) (supervisor’s name, surname) (signature)
THE MASTER THESIS HAVE BEEN APPROVED IN THE DEPARTMENT/CLINIC/INSTITUTE
Arūnas Rutkauskas
(date of approval) (name, surname of the head of
department/clinic/institute)
(signature)
Reviewer of the Master Thesis
(name, surname) (signatures)
Evaluation of defence commission of the Master Thesis:
(date) (name, surname of the secretary of the defence
commission)
3
TABLE OF CONTENT
SUMMARY ... 5 SANTRAUKA ... 6 ABBREVIATIONS ... 7 INTRODUCTION ... 8 1. LITERATURE REVIEW ... 10 1.1 Nutrient requirements ... 10 1.1.1 Water ... 10 1.1.2 Energy ... 10 1.1.3 Proteins ... 10 1.1.4 Vitamins ... 11 1.2 Minerals ... 11 1.2.1 Phosphorus ... 12 1.2.2 Calcium ... 14 1.2.3 Magnesium ... 16 1.2.4 Copper... 17 1.2.5 Iron ... 19 1.2.6 Zinc ... 201.2.7 Copper differences in sheep breeds ... 22
2. MATERIAL AND METHODS ... 23
2.1 Experimental animals, housing and feeding ... 23
2.2 Study design ... 24
2.3 Collection and Processing of Blood ... 24
2.4 Statistical Analysis ... 24
3. RESULTS ... 25
4. DISCUSSION OF RESULTS ... 30
4 RECOMMENDATIONS ... 34 REFERENCES ... 35
5
SUMMARY
Blood mineral status of Suffolk sheep influenced by season, sheep lines and geographical location
Omri Azarzar
This study was carried out with the objective to determine the variation in blood macro (Ca, P, Mg) and micro (Cu, Fe, Zn) minerals depending on the different seasons, geographical location and diet differences within two lines of Suffolk breed and finally mineral correlation.
The study was conducted in Lithuania, using sheep which comprise of two Suffolk breed lines: Germany and United Kingdom origin. Blood samples were taken from the animals in three periods: spring of 2018 (May), autumn 2018 (November) and winter of 2019 (January). These three periods represent different diets and geographical location of the animals. In the spring (placed in Vievis region) the sheep were grazing and, in the autumn (also placed in Vievis region) and winter (placed in Valentai region) the diet consisted of hay and oats fed in the barn. In spring and autumn, blood serum samples were taken from 20 randomly selected female sheep. In the winter, the blood samples were taken from 17 sheep.
The findings of mineral concentrations over the seasons revealed that calcium (Ca) and phosphorus (P) was relatively low in the spring (p<0,001) and winter (p<0,001) in comparison to autumn. Magnesium (Mg) was significantly lower in the spring (p<0,01) and in autumn (p<0,05) relative to winter. Iron (Fe) was significantly lower in the spring relative to autumn (p<0,05) and winter (p<0,05). Zinc (Zn) concentration in winter was significantly higher in comparison to spring (p<0,01) and autumn (p<0,01). The German Suffolk line had higher concentration of zinc than the UK (United Kingdom) line by 10.4% (p<0,05). There was a significant increase in Ca (7.83%), Mg (5.7%), Fe (15.3%) and P (22.04%) in the transition from grazing to being fed hay and oats (in the barn). Calcium and phosphorus were significantly higher in Vilnius than in Druskininkai (p<0,001) and zinc was lower by 10.1% during the time in Vilnius relative to Druskininkai (p<0,05). Magnesium was lower by 6.1% in Vilnius relative to the time in Druskininkai (p<0,05). A positive correlation between calcium and phosphorus was found (p<0,01). A positive correlation of magnesium with iron and zinc (p<0,01), and iron had a positive correlation to magnesium (p<0,01) and phosphorus (p<0,05).
6
SANTRAUKA
Sufolkų veislės avių kraujo mineralinių medžiagų kiekio svyravimai priklausomai nuo sezono, veislės linijos ir geografinės padėties
Omri Azarzar
Tiriamojo darbo tikslas nustatyti Sufolkų veislės avių makro- (Ca, P, Mg) ir mikro- (Cu, Fe, Zn) elementų kaitą kraujo serume priklausomai nuo sezono, geografinės padėties šėrimo tipo, veislės linijos. Nustatyti mineralinių medžiagų tarpusavio koreliacijas.
Tyrimas buvo atliktas Lietuvoje, tiriant skirtingas Sufokų veislės avių linijas, kilusias iš Vokietijos ir Didžiosios Britanijos. Tyrimui atsitiktine tvarka buvo atrinkta 20 ėriavedžių. Atrinktos avys buvo panašaus amžiaus ir kūno svorio. Kraujo mėginiai iš avių imti trys kartus : 2018 m. pavasarį (gegužės mėn.), 2018 m. rudenį (lapkričio mėn.) ir 2019 m. žiemą (sausio mėn.). Šiuo periodu avys buvo šeriamos skirtingu pašaru bei laikomos skirtingose geografinėse vietovėse. Pavasarį avys buvo ganomos ganyklose (Vievis); rudenį avys buvo laikomos tvarte ir šeriamos šienu ir avižomis (Vievis); žiemą avys buvo laikomos tvarte ir šeriamos šienu ir traiškytomis avižomis (Valentai).
Nustatyta, kad mineralinių medžiagų koncentracija avių kraujyje buvo didesnė tvartiniu laikotarpiu, perėjus iš ganyklinio laikotarpiu (pavasarį) prie šėrimo šienu ir avižomis (rudenį bei žiemą). Kalcio ir fosforo buvo santykinai mažiau pavasarį (p <0,001) ir žiemą (p <0,001), lyginant su rudeniu. Magnio buvo reikšmingai mažiau pavasarį (p <0,01) ir rudenį (p <0,05), lyginant su žiema. Geležies buvo reikšmingai mažiau pavasarį, lyginant su rudeniu (p <0,05) ir žiema (p <0,05). Cinko koncentracija žiemą buvo reikšmingai didesnė, lyginant su pavasariu (p <0,01) ir rudeniu (p <0,01). Nustatyta, kad Vokietijos veislės linijos avių kraujyje cinko koncentracija 10.4% didesnė nei Didžiosios Britanijos veislės linijos (p <0,05).
Avis pradėjus šerti šienu ir avižomis (rudenį ir žiemą), reikšmingai padidėjo Ca (7,83%), Mg (5,7%), Fe (15,3%) ir P (22,04%) koncentracija kraujo serume, bei santykinai padidėjo visų mineralinių medžiagų kiekis kraujyje, lyginant su mineralinių medžiagų kiekiu nustatytu ganyklinio sezono metu (pavasarį). Nustatytas reikšmingai didesnis kalcio ir fosforo santykis Vilniaus rajone laikomų avių kraujo serume (p <0,001), tuo tarpu cinko kiekis buvo 10.1% (p <0,05) mažesnis. Nustatytas 6.1% mažesnis magnio kiekis Vilniaus rajone laikomų avių kraujo serume (p<0,05). Nustatyta teigiama koreliacija tarp kalcio ir fosforo (p<0,01). Nustatyta teigiama koreliacija tarp magnio - geležies ir cinko (p <0,01) bei tarp geležies - magnio (p <0,01) ir fosforo (p <0,05).
7
ABBREVIATIONS
Ca - Calcium P - Phosphorus Mg - Magnesium Cu - Copper Fe - Iron Zn - Zinc Mo - molybdenumBCS - Body condition score EBW - Empty body weight ME - Metabolizable energy NPN - Non-protein nitrogen PTH - Parathyroid hormone DM - Dry matter
AST - Aspartate aminotransferase GDH - Glutamate dehydrogenase LDH - Lactate dehydrogenase Hb - Hemoglobin
O2 - Oxygen
ppm - parts per million UK - United Kingdom
8
INTRODUCTION
Sheep throughout the world mostly obtain their nutrient requirements from sown pasture, natural pasture and browse. Therefore, the nutritional management of sheep largely involves the management of the quality and amount of the forage resource, as it is influenced by seasonal weather conditions, regional climate and the current plant species. In winter, feed can be scarce, but in spring (the main period of vegetative growth in most sheep-grazing systems), rich-quality forage usually accumulates in excess compared to animal requirements and in summer drought, there may be a large quantity of forage reserve, but this is likely to be of poor quality relative to sheep requirements. In comparison to housed animals, the nutritional resource for the grazing sheep is always changing (1). On top of energy importance in grazing sheep diets, a sufficient quantity of essential minerals are crucial to maximizing the animals health (2).
All members of the animal and plant kingdom in nature require minerals. There are twenty two minerals which are considered to be “essential” for all animals (3). These essential minerals can be divided into two groups based on the concentration required in the diet. Minerals that are required in relatively large concentrations include magnesium, calcium, phosphorous, sulfur, potassium, sodium, and chlorine and these are referred to as macro minerals. Minerals that are required in small concentrations are referred to as trace elements or micro minerals. These micro minerals include iron, copper, zinc, cobalt, fluorine, iodine, manganese, molybdenum, chromium, selenium, and vanadium (4,5).
These minerals exist in specific concentrations in the cells and tissues of the animal, which is require for the normal development and health of the animal. The 3 most important functions of the minerals are: the physical composition of tissues, organs and the body; as constituents of fluid and tissues - electrolytes in the body are concerned with maintenance of acid-base balance, osmotic pressure, tissue irritability and membrane permeability; finally, as catalysts in the hormonal system and enzyme as an integral and special component of structure in metallic-enzyme or as activators inside those systems (3, 4, 5) .
In a similar way to carbohydrates, proteins, lipids, and vitamins; minerals must to be provided in optimal amounts and based on the requirements that change during the development and rapid growth of the animal. Ideal nutrition should contain adequate mineral concentration, which guarantees normal function of the animal (6).
9
An insufficient supply of these elements in the diet may alter physiological functions, cause biochemical defects, and structural disorders. This occurs when tissue mineral concentrations become excessive or deficient (5, 4).
Ensuring adequate mineral supply depends on many factors including age, lactation stage, breed, stage of growth and the balance with other nutrients (2). Feed sources of minerals are usually divided into feedstuffs (e.g. harvested forages, concentrates, pasture plants) and mineral supplements (7). The amount of individual minerals in pasture herbage, which constitute the majority of the diet of grazing sheep can greatly depend on management factors, soil, plant type, the stage of the plant maturity and climate. In turn, mineral availability in forages can affect their concentration found in the sheep, which can lead to mineral disorders associated with elements such as phosphorus, potassium, sulfur, sodium, molybdenum, iron, copper, zinc, and selenium varying widely between season and locations (2).
Studies on the variations of mineral levels in the blood due to the different seasons, feeds and geographical locations in the Suffolk breed are few, furthermore the Suffolk breed has several blood lines which can also play a role in mineral concentrations.
Thus, this study was carried out with the objective to determine the variation in blood serum concentrations of macro (Ca, P, Mg) and micro (Cu, Fe, Zn) minerals depending on the different seasons, geographical location and diets within two lines of Suffolk breed and finally mineral correlation.
Tasks of the work:
1. Evaluate blood macro (Ca, P, Mg) and micro-minerals (Cu, Fe, Zn) changes over the different seasons in Suffolk sheep.
2. Discover peculiarities in blood serum minerals between two lines of Suffolk sheep breed. 3. Analyze how different diets can impact blood mineral levels.
4. Find how different geographical location impacts on blood mineral status in the same animals. 5. Detect correlation between the evaluated macro (Ca, P, Mg) and micro-minerals (Cu, Fe, Zn) in Suffolk sheep.
10
1. LITERATURE REVIEW
1.1 Nutrient requirements
1.1.1 Water
The importance of water as an element of the diet, is equal to foodstuffs in the health and life of the animals (8). For best health and production results an adequate supply of fresh and clean water should be available at all times. The water consumption for sheep is 74-200 ml / kg body weight / day (9). Long periods of water shortages may have an impact on lamb birth weight and even survival, in addition it can lead to reduction in milk production. Lack of drinking water, often combined with heat stress, is a common problem that grazing sheep face, causing physiological disturbances that affect the sheep’s health and productivity. For this reason it is important to remember that ewes in heat stress, late gestation and during lactation may consume double the amount of water then ewes at maintenance (10).
1.1.2 Energy
The energy requirements of sheep are highly dependent on quantity and quality of the grassland (11). Furthermore it can vary with age, genotype, sex, physical activity, physiological condition and environmental temperature (12). Metabolizable energy (ME) status of grass varies with the grazing season and is affected by rainfall and temperature. In the warm season, the ME concentration in grass is greater than the sheep’s requirement for maintenance, while ME is in shortage in the cold season. Therefore, grazing sheep must deposit reserves in the warm season and then during the cold season mobilize the reserves (through body weight) to maintain health. For this reason, the best option to evaluate adequacy of energy in the feed is by the body condition score evaluation system (11).
1.1.3 Proteins
A minimum of 7% dietary crude protein is required for proper rumen bacterial growth and function for sheep (13).Under conditions of grazing system, the protein content of green pasture usually has a sufficient amount of protein to meet the requirement of sheep (1).However, protein content varies widely over the various types of feedstuff. Perennial grass hay content can range from less than six percent to more than twelve percent crude protein, while legumes in the vegetative stage may occasionally have more than 28 percent crude protein. With maturity, the protein content of plants declines. As for energy requirements, crude protein needs vary with the sheep’s stage of production. For maintenance, ewes of most weight classes need a diet containing
11
7% to 8% protein. During lactation, ewes require 13% to 15% crude protein from the diet, depending on how many offspring are suckling. Supplementation of protein can be necessary for fattening lambs. Growing or lactating sheep should be especially monitored for protein deficiency whenever the diet contains grass hay.Often protein supplements include oilseed meals (soybean meal, cottonseed meal), commercially blended supplements have both natural protein together with non-protein nitrogen (NPN). Finally, protein should be fed to meet, but not exceed the requirements (13).
1.1.4 Vitamins
Classification of vitamins is according to their solubility as water-soluble and fat-soluble. In the group of fat-soluble are the vitamins A, D, E, and K, and in the group of water-soluble vitamin C and those of the complex B (8). Because the rumen usually synthesizes B vitamins in healthy sheep and vitamin C is produced in the same cells of the organism, the only vitamins which require in the diets of non-stressed sheep are the fat-soluble vitamins (8, 13). Vitamins may play a key role in the maintenance of sheep health. In this respect, the action of the antioxidant vitamins (especially beta-carotene, vitamin E and vitamin C) in improving the immune system efficiency is of special interest. These vitamins are the ones which contribute to the maintenance of the functional and structural integrity of the cells, opposing the mechanism of free radicals. Green forage has plenty of carotenes (retinol precursors) which means that deficiency syndromes of vitamin A rarely manifests in grazing sheep. The most important forms of vitamin D are cholecalciferol (D3) and ergocalciferol (D2), whose precursors (7-dehydrosterol and ergosterol) are converted into vitamins at the subcutaneous level by the sun’s ultraviolet rays. Same as carotenes, vitamin E is widely found in green forages. Its levels in hays can actually be reduced by as much as 90 percent, depending on the season of cutting of the hay, the period of storage, along with the time between mowing and the process of drying. The leaves of either hay or green forage are a good source of vitamin K. Vitamin K2 is also found to be synthesized in the rumen, which means that under proper conditions its addition to the diet isnot needed (8).
1.2 Minerals
Veterinarians generally consider 7 macro-minerals and 8 micro-minerals (or trace elements) when assessing mineral nutrition for sheep (13). This classification is based on the levels needed in the diet and in the tissues of sheep, and does not reflect the mineral importance (5). Macro-mineral needs are usually expressed as percentage in the diet, whereas micro-Macro-mineral
12
requirements are generally expressed as mg/kg or ppm. The 7 commonly assessed macro-minerals are phosphorus, sodium, calcium, magnesium, potassium, chlorine and sulfur. The 8 micro-minerals are molybdenum, cobalt, copper, iron, iodine, manganese, zinc and selenium (13). For humans, plants and animals, these minerals are important for all biological processes. They function as structural components in tissues, act in fluids as electrolytes, and function as catalysts in the endocrine system and/or in enzymes. Furthermore, they support the immune system, optimal growth, health condition, reproduction and productivity (4, 14, 15). Mineral excesses and deficiencies may cause some nutritional changes which will impair animal performance. Thus, ensuring proper mineral nutrition is fundamental to optimize animal health and performance (16).
1.2.1 Phosphorus
Function of phosphorus
Phosphorus is one of the main structural components of the animals body, due to its role in the formation of bones and tissues, as a component of cell membranes and its role in most metabolic pathways (17). Most of the body’s phosphorus is found in teeth and bones (17, 18); the rest is found in soft tissues and body fluids where it is involved in plenty of essential functions with regard to the utilization of carbohydrate, fat, protein and other nutrients in the body. Furthermore, P is a part of nucleic acids, which are carriers of the genetic information. Inorganic phosphates play a role in buffering the body’s fluids, including those of the rumen. For normal functioning of rumen micro-organisms, phosphorus is needed especially for those that consume plant cellulose. Finally, P seems to be part of the appetite control. In conclusion, it can be stated that P plays an important role in the body’s metabolic processes and especially in the rumen where phosphorus is needed for proper microbial activity (17). A key component in the function of P is Calcium. P and Ca are interrelated in body functions, and therefore keeping their homeostasis will verify their proper function in the body (19). A Ca:P ratio of 1:1 to 2:1 is usually recommended for proper utilization of the minerals by sheep. Dietary Ca:P ratios ,<1:1 or >7:1 are expected to adversely affect growth and the feed efficiency of sheep (20). P and Ca homeostasis regulation in ruminants and other species is still not clearly understood. Some endocrine substances, such as parathyroid hormone (PTH), insulin, vitamin D3, calcitonin, and cathecholamines, are known to play a role in phosphorus homeostasis in mammals (21).
13
Phosphorus in feed
Sheep fed with a diet that contains high levels of grain or concentrate typically do not require additional supplements of phosphorus. Grains contain moderate to high concentrations of phosphorus. Furthermore, browse and plants tend to accumulate phosphorus, but range sheep are less attracted to them, hence they may be deficient in P. Deficiency of P is mostly encountered in range- or winter-pastured sheep.For the lactating dairy ovine, supplementation of phosphorus is recommended to meet with the high demands for milk production (13). Soil P concentration is also an important factor to consider, due to its ability to accumulate in plants and cause toxicity in sheep (20).
Phosphorus deficiency
Phosphorus deficiency is a major problem in many places around the world. P is the most common deficiency for grazing ruminants after sodium (22). Hypophosphatemia is defined as the decline of the serum P below the established reference value and can be the result of inadequate dietary supply of P, increased losses of P (through feces, milk, or urine), a compartmental movement of phosphorus from the extracellular into the intracellular space, or a combination of 2 or even more of these mechanisms. Although the first 2 mechanisms are consistent with P decline in the animal, a compartmental movement of P to the intracellular space results in hypophosphatemia without a concomitant decline of the total P concentration (21). On the other hand, P is mobilized from the bones to keep normal concentrations in the blood when sheep suffer severe P deficiency (23). It is, therefore, important to consider different etiological reasons when investigating subnormal serum P (21, 24). Plenty of clinical symptoms and conditions, such as anorexia, pica, disrupted growth and fertility, unthriftiness, muscle weakness, recumbency, osteomalacia, intravascular hemolysis and many others have been related with P deficiency in ruminants. Because signs attributed to chronic P decline differ from acute P deficiency, it seems reasonable to differentiate these two forms from each other. While bone demineralization, fertility and decreased growth, are primarily related with chronic P deficiency, muscle weakness, recumbency or intravascular hemolysis are commonly seen in acutely hypophosphatemic individuals (21).
14
Phosphorus Toxicity
With ruminant diets limited in P content, and the high cost of supplements containing P, toxicity of phosphorus is rare in ruminants. The defensive mechanism of ruminants fed with large amounts of P orally is decreased intestinal absorption of the mineral and as a result, an increase of the fecal excretion of P (21). Furthermore, the feeding of a high concentrate diet to growing sheep results in an increase of renal and salivary excretion of P (21, 23). Depending on the urine pH, which determines the solubility of P in the urine, hyperphosphaturia can result in urinary calculi formation. Acute hyperphosphatemia in sheep is seen mostly because of dehydration and ensuing hemoconcentration. Hyperphosphatemia can occur as a consequence of massive releases of intracellular phosphorus after rhabdomyolisis or hemolysis (21). Moreover, plasma P concentrations may be falsely increased as a result of hemolysis during storage of blood samples tubes (25). In most cases, clinical and subclinical symptoms observed in hyperphosphatemia are because of the interference of hyperphosphatemia with the normal regulation of other electrolytes, namely calcium and possibly magnesium. Chronic phosphorus over feeding has physiologically relevant effects on calcium metabolism through inhibition of renal vitamin D3
hydroxylation that reduces intestinal calcium absorption and deposition primarily as crystals of calcium phosphate in soft tissue (21).
1.2.2 Calcium
Function in the body
Calcium is an essential component of the bones and, therefore, of great importance for proper bone growth. Most (98-99%) of Ca in the body is found in bones (18, 26). Within the bone tissue, calcium is present in hydroxyapatite crystals ((Ca10(PO4)6(OH)2). Extraskeletal
calcium (1-2% of body Ca) is found as either free ions or bound with serum proteins and complexed to inorganic and organic acids. The free calcium ions (50-60% of plasma Ca) function in nerve conduction, blood clotting, cell signaling, and muscle contraction mechanisms (26).
Calcium in feed
Most of the forage tends to have high calcium concentration. Beet pulp and legumes (such as alfalfa and clover) are considered a sufficient to excellent sources of calcium. Grains on the other hand are relatively low in calcium. Sheep fed high concertations of grain or concentrate
15
diets typically need supplementation of calcium. As for phosphorus, supplemental calcium is necessary to meet high demands for milk production in lactating dairy sheep (13).
Calcium deficiency
Calcium deficiency in sheep can appear either as an acute or chronic form (26). Acute Ca deficiency can occur from 6 weeks prior to 10 weeks post lambing (26, 27). Highly conditioned ewes have been found to be more prone than lean ones. The condition onset is usually sudden, and up to 30 percent of a flock can be involved in an outbreak. Although the etiology is not completely understood, sharp changes in feed composition, weather or other circumstances (stress) are usually involved. Affected sheep display slight hyper-excitability, experience muscle tremors and a stilted gait. Later on, dullness, recumbency with the hind legs stretched backward, regurgitation and tympani, staring eyes and tachypnea is dominant. Finally, coma and death may occur. In sheep in advanced pregnancy, a condition called transport tetany may occur and some form of calcium deficiency is suspected to be involved. Symptoms during or after transportation (stress factor) can be hyperactivity, restlessness, paralysis and a staggering gait. Later on, it appears to be more or less like milk fever in dairy cows. The pulse rate may increase to 100-120 and labored breathing with tachypnea. Pale mucous membranes and extreme thirst may develop. Unless treated, the sheep may die within several days (26).
Currently, chronic Ca deficiency in sheep is rare. At the event, poor appetite, growth and later on, bone mineralization disorders can be observed. In young sheep, enlarged and painful joints, malformations of teeth, jaws and legs and a stiff gait can be observed. This condition is named rickets and can develop within one month from birth. This condition can occur when the calcium to phosphorus ratio is < 1:1. In mature animals, a stiff gait, painful joints and fractures of the bones may be the symptoms of the condition named osteomalacia (26).
Calcium toxicity
Due to the extensive capacity of sheep to decrease calcium absorption from the gastrointestinal tract, Ca itself can be considered as barely toxic. Only when the Ca uptake regulation is dominated by massive doses of vitamin D3, a life-threatening tissue calcification due to calcium toxicity may occur. Hypercalcaemia can occur secondary to a phosphorus deficiency. Finally, long-term excessive Ca concentrations in the diet, can disrupt the absorption
16
of other elements, such as P, Zn and Cu and clinical symptoms can occur from deficiencies of these minerals (26).
1.2.3 Magnesium
Function of magnesium
Magnesium is a co-factor of many enzymes and function in the activity of more than 300 cellular enzymes that are responsible for cell growth and reproduction, energy metabolism, protein synthesis, stabilization of mitochondrial membranes and synthesis of RNA and DNA. Furthermore, magnesium acts together with calcium ions, for proper cardiac- and skeletal- muscle function. Furthermore, these minerals also function in the transmission of signals in the nervous system (28, 29). Apart from its function in signal transmission, magnesium also plays a role in the secretion of parathyroid hormone and the sensitivity of PTH receptors. Magnesium may also be of great importance in vitamin D metabolism (29).
Magnesium in feed and availability
Pastures containing legumes and legume-grass are good sources of magnesium (13). On the other hand, many heavily fertilized and/or fast growing cereal grains or grass pastures are deficient in magnesium (6, 13). Magnesium absorption is disrupted by rumen ammonia or high concentrations of potassium and nitrogen in plants (13, 29, 30). In contrast, increasing starch content (like rolled barley) in sheep diet has been shown to increase magnesium absorption (29).
Magnesium deficiency
Clinical hypomagnesaemia is known as lactation tetany, grass tetany, grass staggers, wheat pasture poisoning or winter tetany. Before tetany occurs, sheep appear to be nervous or excited, with trembling, especially in the facial muscles; onset is rapid and if untreated, the disease may result in death (29, 31). Secondary disorder manifests in poor glycemic control and insulin
resistance by impairing both insulin secretion and its effect on peripheral tissue cells (29, 31, 32). Clinical hypomagnesaemia is less common in sheep than cows, this may be due to the high
magnesium absorption and retention in sheep. While the condition is generally rare in sheep, hypomagnesaemia can be a problem in individual grazing herds at the peak of lactation, in the first 4 to 8 weeks post lambing. It is more prevalent in older ewes with two lambs, particularly if insufficiently fed. This increased susceptibility may relate to decreased bone turnover rates in older sheep and higher loss of magnesium associated with increased milk production in ewes,
17
having multiple lambs. Grass tetany may increase in periods of a high growth rate of grass in the pasture. This is, apart from climatological factors, related to the degree of fertilization of the pasture. High potassium content in the diet is an additional risk factor for hypomagnesaemia as it reduces magnesium absorption from the rumen and lowers calcium in serum (29, 32). Subclinical hypomagnesaemia is associated with an increase in incidences of clinical hypocalcemia. The mechanism underlying this relationship is not completely clear, but it is considered to be related to an inadequate production of parathyroid hormone, and increased bone resistance to PTH or an impaired vitamin D metabolism (29, 33).
Magnesium toxicity
In practice, magnesium toxicity is not a major problem as the Mg balance is well regulated by the kidneys, which can excrete large amounts of the absorbed mineral. However, accidental cases of overconsumption of magnesium by sheep has been reported in the past. The consumption of concentrates with high magnesium content is associated with severe diarrhea in sheep (29, 6). It seems that most of the occurrence of diarrhea is limited when the dietary magnesium content does not exceed a level of about 10 g/kg dry matter (DM). However, in order to avoid the risk of a disruption to the digestibility of the diet, it may be recommended to set the maximum tolerable level of at 6 g/kg DM (29).
1.2.4 Copper
Function of copper
Copper is an essential component of many enzymes. As such, Cu plays a role in the synthesis of connective tissues (lysyl oxidase, formation of cross links in collagen and elastin), hemoglobin formation and iron transport. Moreover, it is involved in the electron transport during aerobic respiration and the protection of the body against oxygen radicals. Finally, copper is needed for the production of melatonin (tyrosinase). The liver is the main site of copper storage in the body. In the newborn lamb, about 50 % of the total body Cu is located in this organ. Outside the liver, the highest concentrations are found in the kidney and heart (34).
Copper in feed and availability
High quality grass forages have less available copper than that typical for most hays, and legumes have more available copper than most grasses. High concentrations of dietary cadmium, molybdenum, sulfur, zinc, iron, selenium, and vitamin C as well as alkaline soils will reduce
18
copper absorption in the gastrointestinal tract (GI). Roughage grown on “improved” (fertilized, limed) pastures is expected to be deficient in the mineral (13). The concentration of copper in pasture is lowest in the winter, and the addition of lime, with the purpose of increasing soil pH from acidic to more neutral, increases molybdenum (Mo) in plants, which in turn can reduce copper availability to 30 to 60 %. The Cu/Mo ratio should be greater than 4.5:1 (35).
Copper deficiency
Due to the relatively low copper concentrations in forages and the vulnerability of copper absorption to impairment by molybdenum and sulfur , copper deficiency can occur in sheep (34). Furthermore, parasites in the gastrointestinal tract have been observed to decrease Cu status and may lead to Cu deficiency (35). An early sign of copper deficiency is a loss of the wool, resulting in straight, “hair-like” wool. Neonatal ataxia (“swayback”) in newborn lambs is additional clinical sign of Cu deficiency, which cannot be cured but only prevented by copper supplementation (34). This condition occur in lambs until the age of 6 months and is characterized by demyelination of the central nervous system and with symptoms such as lack of coordination in the hind legs and, possibly the forelegs, flaccid or spastic paralysis, balance problems while walking, total inability to walk and possibly even death (36). Moreover, small and dead fetuses are often associated with anemia, osteoporosis, osteochondrosis, fragile bones, widening of epiphyses, poor growth, cardiac failure, reduced fertility or infertility. Finally, disturbed immune function can be observed (34, 37).
Copper toxicity
The toxicity due to copper depends on the source, route, and quantity of the intoxicant (37). In addition, some sheep breeds seem to be more susceptible to copper toxicity then others. Excess Cu by ingestion can originate from pollution of the pasture, consumption or application of any Cu-containing feeds or supplements, such as salt licks or concentrates which are formulated for cows (34). The expected pathologies in an acute poisoning due to ingestion are necrosis, intravascular hemolysis, liver failure, gastrointestinal irritation and shock while if injection occurs it results in renal and liver failure (37). First signs of copper toxicity are rather unspecific (reduced weight gains, dullness, decreased feed intake, dark urine, anemia, jaundice and diarrhea). Sheep suffering from chronic copper intoxication can show biochemical and histological evidence of liver tissue damage (34). This occurs when a prolonged ingestion of copper exceeding the nutritional requirements of 5-7 ppm DM (38). Following chronic
19
accumulation of copper in the liver, an acute hemolytic crisis can occur. This condition is characterized by methaemoglobinemia, hemolysis, hemoglobinuria and jaundice. An early sign is an increase in the activities of serum lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and glutamate dehydrogenase (GDH) 5-8 weeks before the crisis occurs (34). As a result, in most cases sheep die within a short time (34, 37). Pathological signs mainly include yellow to orange discoloration of liver and carcass and necrosis of the liver tissue. The ingestion of pyrrolizidine containing plants such as heliotrope (Heliotropium europaeum) can result in liver damage and excessive Cu accumulation in the liver of sheep and as a result, Cu toxicity can occur. However, heliotrope is not present in Lithuania. In sheep, considerable breed differences exist in the sensitivity for copper toxicity. Therefore, giving one maximum tolerable level for all breeds is difficult, even though 15 ppm (DM) is suggested (34).
1.2.5 Iron
Function of iron
Iron is one of the most abundant metals of the body. The majority (60–70%) of the mineral is present in the hemoglobin of red blood cells. About 10% of the body’s iron is a constituent of cytochromes, myoglobin and enzymes, while the remaining 20–30% of this mineral is stored as ferritins and hemosiderins in hepatocytes (liver parenchyma) and reticuloendothelial macrophages. Iron is an essential mineral for many physiological functions. It takes part in DNA synthesis, metabolic processes, oxygen transport and electron transport (6). The main importance of iron is related to its role as a component of heme, which is present in hemoglobin (Hb) and myoglobin. Both Hb and myoglobin are essential for oxygen (O2) transport from the lungs to the
tissues in the body. Hemoglobin is bound to erythrocytes, whereas myoglobin is found in muscle tissue. Because myoglobin has a higher affinity for O2 than Hb, the result is an efficient transport
of O2 from the blood into the cells. Moreover, iron is necessary for proper function of enzymes of
the cytochrome oxidase, electron transport chain, succinate dehydrogenase ferredoxin, myeloperoxidase, catalase, and finally, cytochrome P-450 enzymes. Thus, Fe is involved in all stages of energy metabolism in body tissues (39).
Iron in feed and availability
Cereal grains are the poorer in iron, while common roughages contain a high to variable, iron concentration since this depends mainly on the plant species and the Fe content in the soil.
20
(6). New born lambs are born with minimal iron stores, hence milk replacers need to contain sufficient amount of this mineral (13).
Iron deficiency
Iron deficiency is quite rare under grazing conditions (13, 38). Lambs raised in total confinement and deprived of access to feed from the pasture and earth-floored stalls or paddocks can become deficient (6, 13). Iron deficiency can occur when young animals are fed a milk replacer deficient in iron. Iron is an important component of hemoglobin, and a deficiency can result in microcytic-hypochromic anemia. In addition, adult sheep with excessive parasitism may have iron deficiency (13).
Iron toxicity
Free iron is cytotoxic because of its ability to create free oxygen radicals, which can cause cell membrane damage. Therefore, the degree to which tissues are affected depends on the animal’s antioxidant status (Se, Cu, Zn, vitamins E and A and carotene). Moreover, a high intake of polyunsaturated fatty acids (found in spring grass) from iron-rich soils may present a hazard. Finally, plants such as lupines and brassicas may increase the liver Fe content. For sheep maximum tolerable levels of Fe 500-1200 ppm is recommended. (39). Death may occur in sheep that are fed with excessive amounts of ferric ammonium citrate and other Fe preparations. Apparently, two syndromes are involved in the mechanism of toxicity. Firstly, a per-acute syndrome characterized by sudden death within several minutes to hours after iron injection. Secondly, a sub-acute syndrome which is characterized by severe depression and coma followed by death. This syndrome is related directly to the toxic effects of the iron resulting from excessive consumption (40).
1.2.6 Zinc
Zinc function
There are plenty of physiological functions to zinc. It play a role in fertility, normal appetite and proper gene expression (41, 42). Zinc function in gene expression influences many processes of the body, including cell replication. Disorders in cell replication may affect growth of hair and hooves, bones, skin as well as immune function. Furthermore, Znplays a role in the formation of insulin and is a component of metalloenzymes such as alkaline phosphatase, carboxypeptidase, alcohol dehydrogenase, carbonic anhydrase, RNA polymerase and Cu-Zn superoxide dismutase
21
which has antioxidant effects – it catalyses the conversion of superoxide anions to O2 and
hydrogen peroxide (42, 43). Besides this, zinc is a component of thymuline and takes part in the regulation of protein kinase C, calmodulin, thyroid hormone binding and the synthesis of inositol phosphate and prostaglandin (42).
Zinc in feed and availability
Zinc is usually found in higher concentrations in legumes than in grasses, but legumes may contain large concentrations of calcium, which can depress zinc availability. Zinc is less available from cereal grain, and is greater with the presence of lactose, vitamin C, and citrate in the diet. Oxalates, phytates, and large dietary concentrations of iron, calcium, cadmium, molybdenum, and orthophosphate all decrease zinc availability (13).
Zinc deficiency
Low zinc content in earth and feeds, deficiency of carbohydrates, high intake of phosphates, vitamin A and amino acids in feed, can lead to the occurrence of zinc deficiency. Absorption of zinc is also reduced by a deficiency or excess of proteins in the meal (43). One of the earliest and most striking signs of zinc deficiency is anorexia, which can stimulate several other symptoms. Growth arrest occurred within two weeks and plasma Zn concentrations fell within one week of feeding a diet containing 1-2 ppm Zn to lambs. Parakeratosis lesions in the eyes area, above the hooves and on the scrotum can be observed, this comes as late sign of the condition (41, 42). In lambs, wool fibers lose their crimp, and thin staples and shedding of the whole fleece, wound healing is retarded, reversible cessation of spermatogenesis may occur(42).
Zinc toxicity
Zinc toxicity may principally be caused by the use of large amounts to combat facial eczema or the wrong use or preparation of mineral supplements (42). Zinc poisoning due to mineral imbalance is conditioned primarily by the antagonistic relationship of zinc to iron and copper (43). Although zinc is not very toxic, reduced feed intake and weight gain have been reported as symptoms of Zn toxicity. Zn levels of 750 ppm can cause severe copper deficiency and increase the incidence of abortions and stillbirths in sheep, whereas levels as high as 1500 and 1700 ppm (DM) can induce pica and anorexia. The anorexia is apparently due to decreased numbers of ruminal microorganisms, which cause reductions of ruminal digestion of dietary amino acids and cellulose. Zinc levels up to 150 ppm (DM) can be considered safe (42).
22 1.2.7 Copper differences in sheep breeds
Several breeds have been studied with regards to differences in copper metabolism. some sheep breeds have a tendency for copper deficiency while others have a tendency for copper accumulation and hence, toxicity. It has been demonstrated that some Finnish- Landrace sheep may have lower serum copper concentrations than in Merinos, which in turn have lower serum copper levels than in British breeds at similar levels of intake (13). Another comparison, found extreme tendency of the Texel breed to accumulate copper. On the other hand, the Scottish Blackface breed in this study has the lowest concentrations (34). A third comparison according to hepatic copper concentration, found that lambs sired by Finn, Romanov, and Montadale rams were lowest, Dorset sired were intermediate, and Texel sired had highest values of the minerals (37). According to a study made on subclinical copper toxicity, comparing Mutton Merino and Blackhead Suffolk cross breed found that the Suffolk breed had significantly higher copper liver concentration then the Merino breed (44). In conclusion, it is clear that there is a large variation in the sensitivity for Cu. The Texel breed was found to be the most sensitive to Cu exposure, while Finn, Romanov, Montadale and Scottish Blackface are the least sensitive. Suffolk is found to be of an intermediate sensitivity in comparison to other breeds.
23
2. MATERIAL AND METHODS
2.1 Experimental animals, housing and feeding
The study was conducted from 2018 until 2019 in two different regions of Lithuania, using sheep which comprised of two Suffolk breed lines: line of Suffolk imported from Germany and a line which was imported from United Kingdom. At the time of measurement, the herd comprised of 37 adult sheep and 12 lambs. The animals were kept for meat production. All sheep received the same feeding ration. In the winter months the sheep were housed in a barn and were fed hay and 300 grams of oats and during the spring and summer months were at pasture feeding off the grass, which was in a mature stage of growth at the time of the experiment. At all times the sheep had free access to clean drinking water and mineral salt. During the spring and autumn, the herd was located in theVievis region, near the city of Vilnius and during the winter the same herd was moved to the Valentai region, near the city of Druskininkai. Sheep included in this study were dewormed regularly, were vigilant, and appeared in good health with body condition score (BCS) roughly equal of 4 and without any clinical signs of illness. The research was conducted following the provisions of the Law of the Republic of Lithuania No. 11-2271 on Protection, Keeping and Use of Animals, dated 03/10/2012 (Valstybės žinios No. 122- 6126 dated 20/10/2012) and of the by-laws, and Education and training purposes of animals used in storage, maintenance and conditions of use No. B1-866, dated 31/10/2012 (Valstybės žinios No. 130-6595 dated 10/11/2012).
24
2.2 Study design
Blood samples were taken from the animals in three periods: spring of 2018 (May), autumn 2018 (November) and winter of 2019 (January). These three periods represent different diets and geographical location of the animals which were used in this study. In spring and autumn, blood serum samples were taken from 20 randomly selected female sheep. In the winter, the blood samples were taken from 17 sheep. At the time of the experiment, some of the tested animals were in varying stages of gestation.
2.3 Collection and Processing of Blood
Blood samples were collected from the jugular vein of each ewe in the morning before access to feed and water and placed into tubes without additive. For the biochemical analyses, blood samples were centrifuged for 10 min and blood serum was collected. Blood biochemical parameters calcium (Ca), phosphorus (P), magnesium (Mg), copper (Cu), zinc (Zn), iron (Fe) were measured using commercial kits on the Selectra Junior analyser (Netherlands, 2006).
2.4 Statistical Analysis
Statistical characteristics in the sample (n) - arithmetic mean (M), standard error (SE), P - value (P) - were calculated using IBM SPSS Statistics, version 20. Data analysis was performed by using Student-t and Chi-Squared statistical significance tests. The results were considered statistically significant when P≤0.05.
25
3. RESULTS
Sheep’s blood serum (n = 20) were evaluated for their concentrations of macro-minerals and trace elements during three seasons. In total, 3 different macro-minerals (Ca, P, Mg) and trace elements (Cu, Fe, Zn) were analyzed.
Evaluating phosphorus distribution (Fig. 2.) in spring (1.5 mmol/L) relative to autumn (2.25 mmol/L), P was significantly lower in the spring 0,75±0,162 (p<0,001). There were no significant differences of P between spring and winter (1.55 mmol/L) (p>0,05). Phosphorus in the autumn was significantly higher 0,707±0,169 (p<0,001) in comparison to winter.
Fig. 2. Phosphorus distribution in each season.
Evaluating calcium distribution (Fig. 3.) in the spring season (2.37 mmol/L) relative to autumn (2.75 mmol/L) showed that it was significantly lower 0,374±0,047 (p<0,001). There were no significant mean differences of calcium between spring and winter seasons (p>0,05). Calcium in the autumn was significantly higher 0,376±0,049 (p<0,001) in comparison to winter (2.37 mmol/L). In the spring the calcium to phosphorus ratio (Ca:P) was 1.6:5, in autumn 1.2:1 and in the winter 1.7:1.
Fig. 3. Calcium distribution in each season.
0 0.5 1 1.5 2 2.5
Spring Autumn Winter
P (m m o l/ L) Season Phosphorus distribution 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8
Spring Autumn Winter
C a (m m o l/ L) Season Calcium distribution
26
There were no significant mean differences (p>0,05) when evaluating magnesium distribution (Fig. 4.) in spring (0.97 mmol/L) relative to autumn (1 mmol/L). However, magnesium in spring comparing to winter (1.06 mmol/L) was significantly lower 0,093±0,029 (p<0,01). Magnesium was significantly lower in autumn 0,065±0,029 (p<0,05), relative to winter.
Fig. 4. Magnesium distribution in each season.
Evaluating iron distribution (Fig. 5.) in spring (24.47 umol/L) with autumn (28.76 umol/L), spring concentration was significantly lower 4,29±1,931 (p<0,05). Iron in spring relative to winter (29.07 umol/L), was significantly lower 4,6±2.014 (p<0,05). There were no significant mean differences of iron status between autumn and winter seasons (p>0,05).
Fig. 5. Iron distribution in each season.
0.92 0.94 0.96 0.98 1 1.02 1.04 1.06 1.08
Spring Autumn Winter
M g (m m o l/ L) Season Magnesium distribution 22 23 24 25 26 27 28 29 30
Spring Autumn Winter
F e (u m o l/ L) Season Iron distribution
27
Evaluating copper distribution (Fig. 6.) in spring (142.04 ug/dl) relative to autumn (145.58 ug/dl), spring relative to winter (147.35ug/dl) and autumn in comparison to winter, there were no significant mean differences between the seasons (p>0,05).
Fig. 6. Copper distribution in each season.
There were no significant mean differences (p>0,05) in the zinc status (Fig. 7) of spring (185.7 ug/dl) in comparison to autumn (183.53 ug/dl). Zinc status in spring was significantly lower 18,428±6,977 (p<0,01) relative to winter (204.12ug/dl). Zinc in autumn was also significantly lower 20,593±6,977 (p<0,01) in comparison to winter.
Fig. 7. Zinc distribution in each season.
139 140 141 142 143 144 145 146 147 148
Spring Autumn Winter
C u (u g/ d l) Season Copper distribution 170 175 180 185 190 195 200 205 210
Spring Autumn Winter
Zn (u g/ d l) Season Zinc distribution
28
Comparing mineral variation between different lines of breeds (Table 1), there was no statistically significant difference in calcium, magnesium, iron, phosphorus and copper (p>0,05). However, zinc was significantly higher by 10.4% in the German line than UK line (p<0,05).
Table 1. Mineral variations in different line of breeds.
Mineral name Breed Mean ± Std. Error Range Calcium mmol/l German Suffolk 2,51±0,03 2,09-2,93 UK Suffolk 2,48±0,1 2,12-2,85 Magnesium mmol/l German Suffolk 1,01±0,01 0,77-1,23 UK Suffolk 0,96±0,03 0,87-1,08 Iron umol/l German Suffolk 27,48±0,85 10,70-42,30 UK Suffolk 26,4±3.38 12,70-42,50 Phosphorus mmol/l German Suffolk 1,81±0,08 0,90-3,38 UK Suffolk 1.58±0,34 0,61-2,88 Copper µg/dl German Suffolk 146,18±4,34 103,10-248,90 UK Suffolk 135,49±10,3 109,60-180,20 Zinc* µg/dl German Suffolk 192,67±3,15 138,00-236,80 UK Suffolk 174.44±7.61 138,00-196,60
*. Statistically significant at the 0.05 level.
Comparing minerals status in different diets (Table 2), calcium when housed in the barn was significantly higher by 7,83%, relative to the time at pasture; (p<0,001). Magnesium was 5.7% higher during the housed period (p<0,05). Iron was higher by 15.3% during the housed period (p<0,01). Phosphorus was higher by 22.04% during the housed period than the time at pasture (p<0,01). There were no statistically significant differences between copper and zinc (p>0,05).
Table 2. Mineral variations in different diets (Pasture– spring, Barn– autumn and winter).
Mineral name Feed Mean ± Std. Error Range Calcium mmol/l Pasture 2,37±0,04 2,09-2,93 Barn 2,57±0,04 2,23-2,92 Magnesium mmol/l Pasture 0,97±0,02 0,77-1,20 Barn 1,03±,014 0,87-1,23 Iron umol/l Pasture 24,47±1,78 10,70-42,30 Barn 28,9±0,78 19,30-42,50 Phosphorus mmol/l Pasture 1,5±0,14 0,61-2,82 Barn 1,93±0,094 1,08-3,38 Copper µg/dl Pasture 142,04±8,74 103,10-248,90 Barn 146,39±4,06 119,40-218,80 Zinc µg/dl Pasture 185,7±2,53 158,70-201,10 Barn 192,99±4,39 138,0-236,8
29
Evaluating the minerals according to geographical location (Table 3), calcium was significantly higher during the period in Vilnius by 15.8% compared to the feeding in Druskininkai (p<0,001). Magnesium was lower by 6.1% in Vilnius relative to the time in Druskininkai (p<0,05). Phosphorus was higher by 45.8% in Vilnius then Druskininkai (p<0,001). Zinc was lower by 10.1% in Vilnius relative to Druskininkai (p<0,05). In iron and copper there was no significant differences (p>0,05).
Table 3. Mineral variation in different places, during autumn (Vilnius) and winter (Druskininkai).
Mineral name Place Mean ± Std. Error Range Calcium mmol/l Vilnius 2,75±0,028 2,43-2,92 Druskininkai 2.37±0,02 2,23-2,61 Magnesium mmol/l Vilnius 1,0±0,02 0,87-1,16 Druskininkai 1,06±0,02 0,92-1,23 Iron umol/l Vilnius 28,76±1,09 22,50-42,50 Druskininkai 29.07±1.14 19,30-38,00 Phosphorus mmol/l Vilnius 2.25±0,13 1,48-3,38 Druskininkai 1,55±0,07 1,08-1,95 Copper µg/dl Vilnius 145,58±5,2 119,40-187,50 Druskininkai 147,35±6,56 119,90-218,80 Zinc µg/dl Vilnius 183,53±6,84 138,00-236,80 Druskininkai 204,12±3,82 165,40-225,0
Analyzing mineral correlation over the whole experiment (Table 4), calcium and phosphorus had a statistically significant positive correlation (p<0,01). Magnesium had a significant positive correlation with iron and zinc (p<0,01). Iron had a significant positive correlation with phosphorus (p<0,05) and with magnesium (p<0,01).
Table 4. Mineral correlation in sheep blood.
Calcium Magnesium Iron Phosphate Copper Zinc Calcium Correlation Coefficient 1,000 -0,009 0,052 0,418** 0,006 -0,083 Magnesium Correlation Coefficient -0,009 1,000 0,470** 0,109 0,066 0,344** Iron Correlation Coefficient 0,052 0,470** 1,000 0,264* 0,077 0,249 Phosphate Correlation Coefficient 0,418** 0,109 0,264* 1,000 0,127 0,031 Copper Correlation Coefficient 0,006 0,066 0,077 0,127 1,000 0,045 Zinc Correlation Coefficient -0,083 0,344** 0,249 0,031 0,045 1,000
30
4. DISCUSSION OF RESULTS
When phosphorus and calcium levels over the different seasons were evaluated, concentration of both minerals were relatively low compared to the reference values defined by D. G. Pugh et. al (13). Approximately 90% of body calcium and 80% of the body phosphorus are found in the bones and teeth. Low levels of these minerals can be found in fat tissues (24). As the body weight increases, there is a decrease in the proportion of bones and an increase in the proportion of fat in the empty body weight (EBW). Therefore, the decrease in the concentration of these minerals as the body weight increases are expected (18). The animals were of high body condition score (BCS) as they were bred for meat production and this may explain the reason for the low measurements of calcium and phosphorus. Considering the fact that the animals were dewormed regularly, chronic parasitism infestation can be excluded as a reason for the general low Ca and P measurements as suggested by D. G. Pugh et. Al (13).
Incorporating grains (oats) and hay into the diet of the animals during autumn and winter helped to increase P and Ca blood concentration in the animal’s body since grains are a good source of P and most hay has sufficient Ca (12).
The absorption of Ca from the rumen can be hindered by a Vitamin D3 deficiency. Vitamin D3
comes from sunlight and is essential for the efficient utilization of Ca and P. This may explain why the sheep had a decline in Ca and P measurements in the winter (30, 31).
Magnesium levels over the different seasons rise in the serum when moving from the pasture (spring) into the barn (autumn and winter) and a further rise from autumn to winter. This is probably due to the fact that grass pasture has less content of Mg (6, 13). Furthermore, according to Schonewille JT. et. al (29), increase in starch content in the diet of sheep (found in hay and oats in this study) increase Mg absorption.
When iron levels over the different seasons were evaluated, a rise in Fe concentration in autumn and winter compared with spring season was observed. This rise could be due to the fact that the sheep moved to the barn and their diet changed from pasture grass to hay and oats. This implies that the grass in the pasture had less Fe when compared with the hay and oats. Furthermore, it is possible that the animals had better access to the mineral bucket in the barn compared to the pasture.
When zinc levels over the different seasons were evaluated, in both spring and autumn Zn levels were significantly lower when compared with the winter. The transition of the sheep to the barn led to
31
an improvement in the mineral values. It can be postulated that the high serum levels of P and Ca during autumn was causing the delayed rise of Zn during this season as high P and Ca were interfering with Zn absorption (13, 43). Furthermore, when P and Ca are low in sheep serum during winter, in contrast, a rise in Zn concentration can be observed.
According to the findings when comparing mineral variation between different lines of the breed, one might assume that the German Suffolk has a greater ability for absorption of Zn or a greater tendency to accumulate the mineral than the UK line, based on the higher concentration found in that breed line. In the literature, several studies investigated breed mineral variation, but none of them mention minerals other than copper (34, 37, 44).
When the influence of different diets over the year on blood mineral values was evaluated, it was apparent that a significant increase in Ca, Mg, Fe and P and a relative increase in all minerals had occurred after the transition of the sheep from pasture (spring) and the changes in diet from feeding of grass to feeding of hay and 300 grams of oats in the barn (autumn and winter). In spring 2018 (time of measurement), there was little to no rain at the grazing place and as a result the grass was of a low quality. However, the diet of the sheep was sufficient to keep normal health. In comparison to the pasture, in the barn the sheep had better access to the mineral buckets. Thus, according to these results, the barn diet was superior because all minerals showed increase in serum concentration.
When the same group of animals ate the same diet in different places, Ca and P was significantly higher in Vilnius then in Druskininkai and Zn was higher in Druskininkai. As noted earlier, the decline in these minerals over the winter can be attributed to the shortage in sunlight, which causes low levels of vitamin D3 and consequently affects the absorption of Ca and P. It is important to note that this is an
estimation only because measurements of vitamin D3 levels were not taken in this study. Furthermore,
it can be suggested that the high serum levels of P and Ca during the autumn time (Vilnius farm) contributed to the delayed rise of Zn during this season as high P and Ca can interfere with Zn absorption. Accordingly, when P and Ca were low during the winter in Druskininkai, a rise in Zn levels was observed. The changes in C, P and Zn are related to the absorption of these minerals and not to the geographical place nor to the management system. When Mg levels was evaluated, it was higher during the housing of the animals in Druskininkai (winter). It is possible that due to farm management in Druskininkai, the sheep had better access to feed which allowed them to eat more starch which is known to enhance Mg absorption (29). Furthermore, J.Th. Schonewille et. al (29) stated that high dietary P can disrupt Mg absorption and even can cause a deficiency of this mineral in ruminants.
32
When P and Mg serum levels corresponding to the different places were analyzed, it was apparent that during the decline of P in the winter period (Druskininkai) there was a rise of Mg in serum levels. In contrast, the levels of P during spring and winter were identical while Mg in the spring was at its lowest levels. Whether P affects Mg absorption in this study or not, is again related to the mineral absorption and not to the geographical location or farm management.
When evaluating the correlation of calcium with other minerals in the experiment, a rise in Ca concentration was followed by a rise in P. This can be contributed to the fact that phosphorus and calcium absorption from the gastro-intestinal tract is increased by PTH secretion which is responsible for stimulating the kidney to produce the hormone 1,25 dihydroxycholecalciferol (Vitamin D3
metabolite) (21, 45). It must be remembered that PTH is secreted in response to hypocalcemia, not hypophosphatemia (46). Furthermore, PTH increases renal excretion of Ca and resorption of P and Ca (45). Hence, Ca homeostasis is tightly related to P, and a rise in calcium concentration is to be expected with a rise in P.
33
CONCLUSIONS
1. The mineral concentration improved after the transition from pasture (spring) to feeding with hay and oats (autumn and winter). Calcium and phosphorus were relatively low in the spring (P: 33.4%, Ca: 13.8%) and winter (P: 31.1%, Ca: 13.8%) in comparison to autumn (p<0,001). Magnesium was significantly lower in the spring by 8.5% (p<0,01) and in autumn by 5.7% (p<0,05) relative to winter. Iron was significantly lower in the spring relative to autumn by 15% (p<0,05) and winter by 15.8% (p<0,05). Zinc concentration in winter was significantly higher in comparison to spring by 9% (p<0,01) and autumn by 10.1% (p<0,01).
2. The German Suffolk line had a higher 10.4% (p<0,05) concentration of zinc than the UK line. 3. There was a significant increase in Ca (7.83%), Mg (5.7%), Fe (15.3%) and P (22.04%) and a
relative increase in all minerals when the sheep transitioned from feeding off the grass in the pasture (spring) to feeding from hay and oats in the barn (autumn and winter).
4. Calcium and phosphorus levels were significantly higher (Ca: 15.8%, P: 45.8%) in Vilnius than in Druskininkai (p<0,001) and zinc was lower by 10.1% during the time in Vilnius relative to Druskininkai (p<0,05). Magnesium was lower by 6.1% in Vilnius relative to the time in Druskininkai (p<0,05).
5. A positive correlation was found (p<0,01) between calcium and phosphorus. A positive correlation of magnesium with iron and zinc was found (p<0,01) and finally, iron had a positive correlation to magnesium (p<0,01) and phosphorus (p<0,05).
34
RECOMMENDATIONS
By analyzing the mineral distribution over the seasons, it is apparent that there was an improvement in all minerals status when the animals transitioned from grazing to feeding inside the barn with hay and oats. This reflects the situation in Lithuania and perhaps the whole region, that the soil does not get sufficient amount of rainfall in springtime as expected, and therefore the pasture quality as a feed source declines. Furthermore, calcium and phosphorus concentrations decline during winter, possibly due to lack of sunlight.
According to this, it is recommended:
1. To supply the animals with mineral buckets which are available at all time for the grazing animals and during autumn and winter confinement.
2. An evaluation of the pasture grass, either by laboratory equipment or even by visual inspection when the animals are grazing, will reflect if the land is suitable for grazing. 3. Exposing the animals to sunlight as much as possible during the winter will increase
utilization of calcium and phosphorus.
35
REFERENCES
1. Dove H. Balancing nutrient supply and nutrient requirements in grazing sheep ଝ. Small Rumin Res [Internet]. 2010;92(1–3):36–40. Available from:
http://dx.doi.org/10.1016/j.smallrumres.2010.04.004
2. Xin GS, Long RJ, Guo XS, Irvine J, Ding LM, Ding LL, et al. Blood mineral status of grazing Tibetan sheep in the Northeast of the Qinghai – Tibetan Plateau. Livest Sci [Internet].
2011;136(2–3):102–7. Available from: http://dx.doi.org/10.1016/j.livsci.2010.08.007 3. Ranjith D, Pandey JK. Mineral Profiles in Blood and Milk of Sheep. Int J Sci Res.
2015;4(10):821–6.
4. Poppenga RH, Ramsey J, Gonzales BJ, Johnson CK. Reference intervals for mineral
concentrations in whole blood and serum of bighorn sheep ( Ovis canadensis ) in California. J Vet Diagnostic Investig. 2012;24(3):531– 538.
5. Schweinzer V, Iwersen M, Drillich M, Wittek T, Tichy A. Macromineral and trace element supply in sheep and goats in Austria. Vet Med (Praha). 2017;2017(02):62–73.
6. T. Studzin´skia*, J. Matrasb ERG, J.L. Valverde Piedraa JT, Tataraa* and MR. 16 Minerals : functions , requirements , excessive intake and toxicity. Biol Nutr Grow Anim. 2006;4:467–509. 7. Khan ZI, Ahmad K, Danish M, Mirzaei F. Minerals Profile Of Forages For Grazing Ruminants
In Pakistan. Int J Livest Res. 2012;2(2).
8. Infascelli F, Moniello G, Cutrignelli MI, Infascelli F, Moniello G, Cutrignelli MI, et al. Vitamin and water requirements of dairy sheep Vitamin and water requirements of dairy sheep. Ital J Anim Sci. 2016;4(sup 1):75–83.
9. Spengler D, Strobel H, Axt H, Voigt K. Wasserbedarf , Wasserversorgung und Thermo - regulation kleiner Wiederkäuer bei Weidehaltung *. Tierärztl Prax. 2015;43(G):49–59. 10. Behrendt A, Fischer A, Kaiser T. Studies on drinking water intake of fallow deer , sheep and
mouflon under semi-natural pasture conditions. Grassl Sci. 2017;63(1):46–53.
11. Song S, Wu J, Zhao S, Casper DP, Zhang L, He B, et al. The effect of periodic energy restriction on growth performance , serum biochemical indices , and meat quality in sheep. J Anim Sci. 2018;29;96(10):1–13.
