Vol. 6, Suppl. 1 201 The effect of long term exposure to copper on physiological condition and reproduction of sheep Maciej Murawski 1,2, Gabriela Bydłoń 3, Katarzyna Sawicka-Kapusta 3, Edward Wierzchoś 2, Marta Zakrzewska 3, Sylwia Włodarczyk 3, Edyta Molik 2, Dorota Zięba 2 2 Department of Sheep and Goat Breeding, University of Agriculture, 3 Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland SUMMARY The influence of copper upon some physiological parameters and reproduction in ewes was studied. Four groups of animals were investigated: 1/ control ewes (untreated); 2) ewes receiving copper as a supplement over the recommended amount of copper in food (10, 25 or 50 mg Cu/ ewe/day); 3/ control, superovulated ewes; and 4) ewes treated with 50 mg copper during one month and then superovulated. After 10 months of daily exposure to 10 mg of copper/ewe/day no signs of toxicity on physiological condition and reproduction were found. In ewes exposed to 25 or 50 mg of copper a decrease in blood parameters and increase in concentration of Cu in blood and liver were noticed. The wavy pattern of follicles was disturbed and disorders in fecundity, prolificacy and pregnancy occurred. Significant differences between the number of corpora lutea in superovulated control animals and experimental (Cu 50 mg) ewes were observed. Reproductive Biology 2006 6 Suppl. 1:201 206. Key words: ewes, copper, reproduction, toxicity 1 Corresponding autor: Faculty of Animal Breeding and Biology, University of Agriculture in Cracow, Al. Mickiewicza 24/28, 30-050 Krakow, rzmmuraw@cyf-kr.edu.pl Copyright 2006 by the Society for Biology of Reproduction
202 Exposure to copper and ewe reproduction INTRODUCTION Copper, an essential trace metal for organism plays an important role as a cofactor of crucial enzymes. It is necessary for energy production, connective tissue formation and pigmentation [13]. Sheep belong to domestic animals most susceptible to copper toxicity because of the narrow range between required level of this element and the level at which toxicity can occur [3]. For sheep, 5 to 10 µg of copper/g of diet is recommended together with a normal level of molybdenum (1µg/g dw; [10]). The proper Cu:Mo ratio should be 6:1 [7]. Lower Mo content in the diet can cause copper toxicity even if the amount of Cu is appropriate [3]. Chronic copper poisoning is well recognized as a hazard in sheep production [13]. It is usually associated with decreasing value of production parameters and decreasing adaptation ability to environmental conditions. Dysfunction of sheep reproduction is probably caused by copper which displaces other metal cofactors from their natural ligands. The replacement of Zn(II) by Cu(II) in the zinc-finger DNA binding domain of estrogen receptor renders this protein defective, altering its role in hormonedependent signal transduction in vivo [11]. During chronic ingestion of copper, its passive accumulation may occur over a period from few weeks to more than a year. Copper toxicity is considered to have two distinct phases: first pre-hemolitic phase without clinical symptoms and second phase called hemolytic crisis which is fatal [4]. Copper-induced changes were reported in the blood, liver, kidney and central nervous system of the animals [12]. We investigated the influence of sub-toxic and toxic doses of copper on physiological condition and reproduction of ewes. Effect of copper on blood, liver, kidneys and muscles Three to six year old long-wool ewes were exposed to copper. There were groups: 1/ control-(untreated) ewes; 2/ ewes treated with 10, 25 or 50 mg of Cu for 10 months daily; 3/ control-untreated, superovulated ewes, and 4/ ewes treated with copper (50 mg) for 30 days and than superovulated.
Murawski et al. 203 Copper was applied once a day as a supplement to the natural amount of copper in food (13 µg/g dry weight and molybdenum 2 µg/g; [7]). Routine measurements of blood parameters were performed in control and coppertreated animals every three weeks for 10 months. Copper, zinc, iron, and molybdenum were estimated by Atomic Absorption Spectroscopy (AAS) in the blood, liver, kidneys and muscles. There was neither significant influence of 10 mg copper upon blood parameters nor upon copper, molybdenum, zinc and iron concentrations in blood. A significant decrease in red and white cell number was observed only in the group treated with 50 mg of copper. The blood serum concentration of copper in control animals was between 50 to 84 µg/100 ml. In animals receiving 25 and 50 mg of copper, a significant increase in copper and simultaneous decrease in zinc level was noticed. According to Gough et al. [6] the copper concentrations >500 µg/g of liver indicated poisoning. In the present study, in ewes receiving 50 mg of Cu, its content in the liver was 946 µg/g. In spite of this, such copper accumulation did not modify metabolism of physiological metal elements. The fact that almost all animals survived ten months of chronic copper exposure indicates that copper toxicosis was only in its first phase. Even if copper concentration is almost 1000 µg/g dry weight, the liver maintains its regeneration ability and continues production of new hepatocytes to take up copper released by dying cells [4]. No differences in concentration of zinc and iron between the exposed ewes were found in the liver. The influence of copper upon reproductive function Ovarian growth of follicles and hormonal dynamics during the estrous cycle in ewes were previously reported [5, 14]. It was interesting to study the influence of long term copper supplementation in the diet upon folliculogenesis in ewes. Several authors [1, 2, 3] showed that hyperphysiological doses of copper affected ovarian activity and the estrous cycle in cattle. The ewe ovaries of the control and copper (10 mg)-treated groups were scanned daily by ultrasound equipment for assessment of number and diameter of growing follicles and they were similar to those observed previously by others [3, 7]. Among 24 oestrous cycles studied, nine cycles
204 Exposure to copper and ewe reproduction showed three waves, and 15 cycles had four waves of follicles. There were no differences in growth parameters of follicles between control and copper-treated ewes. We found that continuous exposure to copper (10 mg daily during ten months) had no effect on the wavy pattern of follicular growth [8]. There were no significant differences in duration of pregnancy as well as in prolificacy between control and experimental groups [9]. Ewes treated with 25 or 50 mg of Cu from the onset of breeding season were scanned by USG every day during three consecutive estrous cycles. The folliculogenesis of the group receiving the lower dose of copper did not differ in the pattern from that of controls. However, not all follicles >5 mm ovulated during the last ovulatory wave in the group treated with the highest dose of copper. We were the first to notice that one to three follicles continued to grow and then were seen in the following cycle in the first and the second wave. Some metals are known to cross the placenta barrier and accumulate in fetus [13]. The highest concentration of copper was found in placenta of mothers receiving 50 mg of Cu and the lowest in ewes getting 10 mg of Cu per day. No statistical differences in blood parameters and copper concentrations in blood of offspring were seen. Similarly, no significant changes in concentration of metals in examined lamb tissues were found. Colostrum and milk, the lamb s only food, contained low physiological amounts of copper, so that this metal had no harmful effect on their physiological condition. Fecundity and prolificacy between control and copper (25 or 50 mg)- treated groups differed significantly. In the control group, all mated ewes became pregnant and gave birth to two lambs each, and all their lambs were weaned. However, only 50% of females receiving 50 mg of Cu gave birth to lambs. Interesting results were obtained during monitoring of the pregnancy. No dying embryos or premature termination of pregnancy were noted in the control group, while in copper (25 and 50 mg)- treated groups, a significant decline of pregnancies by day 30 occurred Moreover, in these groups, 14.2% of ewes lost their foetuses before day 90 of pregnancy.
Murawski et al. 205 To test whether copper could affect development of oocytes and their fertilization competence, oocytes were collected from ovaries of slaughtered control and copper-treated ewes. Morphology of cumulusoocyte complexes (COCs) were examined under light microscope. The highest number of oocytes was collected from ovaries of the control group. The lowest number of morphologically healthy COCs was obtained from the animals receiving 50 mg of copper. Ewe cycles were synchronized by insertion of a vaginal sponge impregnated with 40 mg of fluorogestone acetate (Chronogest) for 14 days and then superovulated by 200 IU of FSH. It was applied in eight doses for four days in 12-h intervals. Ewes were mated by rams at 48 and 60 hours after sponge withdrawal. Good-quality embryos in morula and early blastocyst stages were collected six days later. There was highly significant difference between number of corpora lutea in the control and the Cu-treated group. It is still difficult to recognize the mechanism of copper influence upon reproductive disorder in ewe. It is possible that copper causes a dysfunction of endometrium and the hypothalamo-hypophysealovarian axis. According to Al-Gubory et al. [1] copper zinc-superoxide dismutase has an inhibitory action on pulsatile LH secretion, which may suggest that Cu plays an important role, dependently or/and independently on some neurotransmitters, in the neuroendocrine regulation of reproduction in sheep females [1]. The excess of the copper could also cause a dysfunction of estrogen receptors resulting in reproductive disorders and pathology of pregnancy on various levels of the reproductive axis [11]. ACKNOWLEDGMENTS Research was supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN-84/P06/2002.
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