Relationship of blood and seminal plasma ceruloplasmin, copper, iron ...

Small Ruminant Research 133 (2015) 135–139

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Relationship of blood and seminal plasma ceruloplasmin, copper, iron and cadmium concentrations with sperm quality in Merino rams Pınar Peker Akalın a,∗ , Bülent Bülbül b , Kenan Çoyan c , Nuri Bas¸pınar d , Mesut Kırbas¸ b , Mustafa Numan Bucak e , S¸ükrü Güngör e , Caner Öztürk e a

Mustafa Kemal University, Veterinary Faculty, Department of Biochemistry, Hatay, Turkey Bahri Da˘gdas¸ International Agricultural Research Institute, Konya, Turkey c Pamukkale University, Faculty of Medicine, Department of Histology and Embriology, Denizli, Turkey d Selcuk University, Veterinary Faculty, Department of Biochemistry, Konya, Turkey e Selcuk University Veterinary Faculty, Department of Reproduction and Artificial Insemination, Konya, Turkey b

a r t i c l e

i n f o

Article history: Received 6 January 2015 Received in revised form 28 August 2015 Accepted 28 August 2015 Available online 29 August 2015 Keywords: Sperm parameters Seminal plasma Ceruloplasmin Copper Iron Cadmium Merino rams

a b s t r a c t The aim of the current study was to investigate the concentrations of ceruloplasmin, copper, iron, zinc and cadmium concentrations in blood serum and seminal plasma obtained from Merino rams. In addition, their relationship with sperm parameters, fertility rate and litter size were also studied. Blood and ejaculate samples (6 replicates) were taken in October from 19 Merino rams, aged between 18 and 24 months. Ceruloplasmin, copper, iron, zinc and cadmium in blood serum and seminal plasma were determined. Sperm parameters including volume, mass motility, motility, concentration, Hos-test, viability, abnormal sperm and acrosome abnormality in semen, fertility rate and litter size were also evaluated. Highly positive correlation was found between blood ceruloplasmin and blood copper concentrations (r = 0.812, p < 0.001), whereas negative correlation were determined between these parameters in seminal plasma (r = −0.195, p < 0.05). Seminal plasma copper concentration was positively correlated with seminal plasma cadmium (r = 0.206, p < 0.05) and seminal plasma iron (r = 0,305, p < 0.01) concentrations. Negative correlation was determined between blood ceruloplasmin level and acrosomal defect (r = −0.443, p < 0.05). Seminal plasma ceruloplasmin level was positively correlated with volume (r = 0.255, p < 0.01) and negatively correlated with abnormal sperm (r = −0.186, p = 0.058) and acrosome abnormality (r = −0.213, p < 0.05). Seminal plasma iron concentration was positively correlated with other abnormality (r = 0.257, p < 0.01). Seminal plasma cadmium concentration was positively correlated with sperm abnormality (r = 0.207, p = 0.052) and other abnormality (r = 0.262, p < 0.05) and negatively correlated with fertility rate (r = −0.449, p = 0.054). Blood cadmium concentration was negatively correlated with litter size (r = −0.579, p < 0.01). In conclusion, blood and seminal plasma ceruloplasmin may be suggested to have positive influence regardless of copper with its antioxidant property whereas iron and cadmium have negative influence on sperm parameters and fertility in Merino rams. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Seminal plasma is an important indicator with its content and quality on monitoring the male infertility. In this context, the important role of reactive oxygen species (ROS) is of interest because excess semen ROS have negative effects on sperm quality including sperm movement and its ability for fertilization (Aitken

∗ Corresponding author at: Department of Biochemistry, Faculty of Veterinary Medicine, Mustafa Kemal University, Tayfur Sökmen Kampüsü, 31040 Antakya, Hatay, Turkey. Fax: +90 326 2455704. E-mail address: [email protected] (P.P. Akalın). http://dx.doi.org/10.1016/j.smallrumres.2015.08.019 0921-4488/© 2015 Elsevier B.V. All rights reserved.

and Curry, 2011). As sperm membrane of rams has a higher polyunsaturated/saturated fatty acids ratio than other species such as rabbit, bull and human, it is more vulnerable to peroxidative damage (Evans and Maxwell, 1987; Saleh and Agarwal, 2002). Ceruloplasmin, mainly synthesized in hepatocytes, has multilateral function in the organism—is important in transporting copper (Cu), involved in iron (Fe) metabolism, antioxidation and acute phase response during inflammation (Halliwell, 1991). About 70 to 90% of the Cu is associated with Cp in blood plasma (Blakley and Hamilton, 1985). Copper is involved in biochemical reactions especially oxidation–reduction processes. The Cu2+ in diet is absorbed from duodenum with aminoacids or small proteins, competitively with Fe2+ , zinc (Zn) and cadmium (Cd) (Craig et al., 2009) and Cu

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antagonists include sulphide, molybdenum (Mo), Zn and Fe (Suttle, 2012). Copper homeostasis is maintained carefully by regulation of Cu+ specific membrane transporter and several metallochaperones (Prohaska, 2011). Among the farm animals, sheep are the most susceptible species to Cu toxicity because they do not have ability to increase biliary Cu excretion (Saylor and Leach, 1980). Ceruloplasmin can be inactivated during oxidative stress and Cu can be released leading to excess hydroxyl radical generation. Therefore ionic Cu is extremely low in the organism (Choi et al., 2000; Prohaska and Gybina, 2004). Many of the toxic effects of Cu, including increased LPO levels in cell membranes and DNA damage, are related to its role in generation of ROS (Bremner, 1998). Negative influence of high semen Cu concentrations were reported on spermatozoa motility and viability in water buffaloes (Tabassomi and Alavi-Shoushtari, 2013). Zinc deficiency results in disorders of testes development and course of spermatogenesis (Cigankova et al., 1998). Excess of cadmium was reported to have degenerative alterations in testes (Toman and Massanyi, 1996). To our knowledge, the relationship of blood serum Cp and Cu with seminal plasma Cp and Cu in ram and their relationship with sperm parameters and fertility rate was not reported, so far. Additionally, the relationship of Cp and Cu with Fe, Zn and Cd remain absent. The aim of the study was to determine the concentrations of Cp, Cu, Fe, Zn and Cd in blood serum and seminal plasma and their correlation with sperm parameters, fertility rate and litter size in Merino ram. 2. Materials and methods Semen and blood samples from 19 Merino rams (18–24 months of age) were used in the current study. The rams, belonging to the Bahri Da˘gdas¸ International Agricultural Research Institute, Konya–Turkey (located at 37.857063 north latitude and 32.567036 east longitude) were maintained under uniform feeding, housing and lighting conditions. Rams were fed with a same ration composed of alfalfa hay, concentrated feed and dried grape, had ad libitum access to fresh water. Group feeding was accessed. Starting 15 days prior to blood and sperm sampling (n = 4 for each type of feed; alfalfa hay, concentrated feed, dry grape), feed samples were taken with an interval of 1 week. By determining the total amount of consumed feed by the group, the average amount of feed consumed per animal was determined. Refusal of the feed was not collected. The study was approved by Bahri Da˘gdas¸ International Agricultural Research Institute Local Animal Research Ethics Committee (No: 22.07.2013/2). 2.1. Evaluation of sperm parameters Ejaculates were collected from the rams using an artificial vagina, in October as 6 replicates (with an interval of 1 day) according to AI standard procedures (Paulenz et al., 2002). The volume of ejaculates was evaluated in a conical tube graduated at 0.1 ml intervals. Immediately after collection, semen was assessed for semen wave motion (mass motility) at 40× magnification using a phasecontrast microscope, graded on a subjective scale ranging from 1 to 5, where 1 was scored when there was no mass movement and 5 represented vigorous waves of sperm motion (Evans and Maxwell, 1987). Spermatozoa motility was estimated subjectively using a phasecontrast microscope (100×), with a warm stage maintained at 37 ◦ C. Semen was diluted with PBS (1/10 w/w) and, a wet mount was made using a 5 ␮l drop of this dilution, placed directly on a microscope slide and covered by a cover slip. Sperm motility

estimations were performed in three different microscopic fields for each semen sample by the same researcher. The mean of the three successive estimations was recorded as the final motility score. Sperm concentration was determined via Hemositometric method, briefly sperm was diluted at ratio of 1:200 with Hayem solution (5 g Na2 SO4 , 1 g NaCl, 0.5 g HgCl2 , 200 ml bicine) and density was determined using a 100 ␮m deep Thoma haemocytometer (TH-100, Hecht-Assistent, Sondheim, Germany) at 400× magnification with using a phase-contrast microscope and expressed as spermatozoa ×109 ml−1 (Bearden et al., 2004). The hypo-osmotic swelling test (Hos-Test) was used to evaluate the functional integrity of the sperm membrane. This was performed by incubating 30 ␮L of semen with 300 ␮l of a 100 mOsm hypoosmotic solution (9 g fructose + 4.9 g sodium citrate per liter of distilled water) at 37 ◦ C for 60 min. After incubation, 0.2 ml of the mixture was spread with a coverslip on a warm slide. Four hundred sperms were evaluated for each sample and the percentage of spermatozoa with swollen and twisted tails were recorded underphase-contrast microscope (400×) (Revel and Mrode, 1994). For the assessment of sperm abnormalities, a minimum of three drops of each sample were added to Eppendorf tubes containing 1 ml Hancock solution (62.5 ml formalin (37%), 150 ml saline solution, 150 ml buffer solution and 500 ml double-distilled water) (Schafer and Holzmann, 2000). One drop of this mixture was put on a slide and covered with a cover slip. The percentage of total sperm abnormalities (acrosomal and other abnormalities) was recorded by counting a total of 400 sperm under phase-contrast microscopy (1000× magnification, oil immersion). Sperm viability rate was determined using eosin–nigrosin staining method (Evans and Maxwell, 1987). The sperm suspension smears were prepared by mixing a drop of the semen sample with 2 drops of the stain on a warm slide and spreading the stain with a second slide immediately. The viability was assessed by counting 200 cells under the phase-contrast microscope. Sperm showing partial or complete purple colourisation were considered nonviable and only sperm showing strict exclusion of the stain were considered to be alive. Then, the ejaculates were centrifuged at 800 × g 20 min at 4 ◦ C and seminal plasma was separated from spermatozoa for the analysis of trace element concentrations within 2 h. Blood samples were collected from the jugular vein prior to sperm collection.

2.2. Evaluation of trace elements Serum and seminal plasma samples were stored at −86 ◦ C until the analysis of Cp, Cu, Fe and Cd. Serum, seminal plasma and feed Cu, Fe, Zn and Cd concentrations were determined with ICP-AES (Inductively Coupled Plasma Atomic Emission Spectrometer—Varian-Vista AX, Australia) using reference material European Reference Materials-LGC (ERM DA120a, Teddington, UK). The samples of serum and seminal plasma were diluted with deionized water (total volume 1 ml) and 5 ml 65% HNO3 + 2 ml 30% H2 O2 (Merck) was added before digestion in the microwave oven (CEM MarsXpress, Matthews, NC, USA) at 210 ◦ C at 200 PSI. Food samples were digested with 7 ml 65% HNO3 + 3 ml 30% H2 O2 . The flame conditions were those recommended by the instrument manufacturer for Cu, Fe, Zn and Cd (wavelength 327.396 and 238.204, 213.856 and 228.802 nm and detection limit 0.6, 0.35, 0.3 and 0.3 ppb, respectively). All data were obtained using 10 s integration time based on 3 standard deviations and in general compromise conditions were used. Analyzing reference material ERM-DA120a tested the reproducibility of the method. Reference values for Cu and Zn are presented to be 1130 and 658 ppb,


respectively in the reference material. In the current study Cu and Zn levels were 1138 and 665 ppb, respectively. 2.3. Evaluation of ceruloplasmin Serum and seminal plasma were incubated with pphenylenediamine dichloride in optimum acetate buffer. The enzymatic oxidation of p-phenylenediamine dichloride eresults in the formation of a pink product with the maximum absorption at 546 nm in spectrophotometer (UV 2100 UV–vis Recording Spectrophotometer Shimadzu, Japan). Cp concentration was calculated using the formula reported by Colombo and Richterich (1964). 2.4. Evaluation of fertility rate and litter size of each Ram Fertility rates of rams were calculated using data for rams mating around time of study and were evaluated 3 weeks before and 3 weeks after the ejaculate collection for the current study (6 weeks in total). Healthy ewes, 2–5 years old, were checked for estrus three times a day (30 min each) and ewes in estrus were naturally mated with rams. Pregnancies were checked on d 30 of pregnancy by Bmode real time ultrasound using transrectal linear probe (Scanner 480 Vet, Esaote Pie Medical, Maastrich, The Netherlands). Calculation of fertility rate (individual): Pregnant ewes mated with individual ram/Total mated ewes by individual ram. Calculation of litter size: Lamb number of the ewe mated with individual ram/Total mating number of the ewe with individual ram. 2.5. Statistics Statistical analyses were performed with the SPSS (version 12.00, Chicago IL) program. Results were expressed as the mean ± S.E.M. Correlation between blood and seminal plasma parameters and sperm parameters were performed with Pearson correlation and considered significant at p < 0.05. 3. Results Total daily intake of Cu, Fe, Zn and Cd levels of rams were 17.24, 720.7, 123.34, 0.074 ppm/ram/day, respectively. As regards sperm quality parameters (n = 112; 1 replication of 2 animals were failed to collect), semen volume was 1.99 ± 0.05 ml; sperm concentration was 2104 ± 53 × 106 ; Hos-T was 78.41 ± 1.04%; viability was 86.53 ± 0.41% and abnormal sperm rate was 9.27 ± 0.30%. Motility parameters were 3.01 ± 0.03 (1–5) mass motility and 81.54 ± 0.41% motility. Blood serum Cp, Cu, Fe, Zn and Cd concentrations were 8.66 ± 0.60 mg/dl, 0.66 ± 0.05, 1.65 ± 0.154, 0.64 ± 0.279 and 0.0018 ± 0.0003 ppm, respectively and seminal plasma Cp, Cu, Fe, Zn and Cd concentrations were 1.86 ± 0.18 mg/dl, 0.21 ± 0.02, 1.63 ± 0.147, 3.81 ± 0.363 and 0.0051 ± 0.0005 ppm, respectively. Only the blood plasma Cd concentrations in 15 rams and seminal plasma Cd concentrations in 92 replications were above detection limit. As regards correlation results, highly positive correlation was determined between blood Cp and blood Cu concentration (r = 0.812, p < 0.001) whereas negative correlation was determined between seminal plasma Cp and seminal plasma Cu concentration (r = −0.195, p < 0.05). There was a positive correlation between seminal Cp level and seminal plasma Zn (r = 0.325, p < 0.001) concentration. There was a positive correlation between blood Cu and blood Zn (r = 0.684, p < 0.01) concentrations. Blood Fe was positively correlated with seminal plasma Fe (r = 0.663, p < 0.01), blood Zn (r = 0.822, p < 0.001) and blood Cd

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Table 1 Individual fertility rate of rams and litter size. Ram no:

% Fertility rate (individual)

Litter size

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Mean ± SE

80.00 75.00 84.21 80.65 69.23 73.33 70.00 85.71 66.67 95.00 77.78 72.22 69.23 61.54 72.73 55.56 45.45 73.68 75.00 72.79 ± 2.53

1.00 1.06 1.58 1.03 0.92 0.87 1.00 1.29 0.67 1.14 0.78 0.94 0.92 0.92 0.73 0.81 0.73 0.79 0.88 0.95 ± 0.05

(r = 0.710, p < 0.01) whereas seminal plasma Fe was positively correlated with blood Zn (r = 0.575, p < 0.01), blood Cd (r = 0.524, p < 0.05), seminal plasma Cu (r = 0.305, p < 0.001) and seminal plasma Cd (r = 0.286, p < 0.01) concentrations. Seminal plasma Cu concentration was positively correlated with seminal plasma Cd (r = 0.206, p < 0.05) concentrations. Individual fertility rate (%) of rams and litter size were given in Table 1. Correlations between blood serum and seminal plasma Cp, Cu, Fe, Zn, Cd concentrations, sperm parameters and fertility rate and litter size were given in Table 2. Negative correlation was determined between blood Cp concentration and acrosomal defect (r = −0.443, p < 0.05). Seminal plasma Cp was positively correlated with semen volume (r = 0.255, p < 0.01) and negatively correlated with acrosome abnormality (r = −0.213, p < 0.05) and abnormal sperm (r = −0.186, p = 0.058). Seminal plasma Fe concentration was positively correlated with other abnormality (r = 0.257, p < 0.01). Seminal plasma Cd concentration was positively correlated with sperm abnormality (r = 0.207, p = 0.052) and other abnormality (r = 0.262, p < 0.05) and negatively correlated with fertility rate (r = −0.449, p = 0.054). There was also a strong negative correlation (r = −0.579, p < 0.01) between blood Cd concentrations and litter size. 4. Discussion Ceruloplasmin is the major Cu-containing protein in the plasma and in addition it has antioxidant properties with its ferroxidase activity. Serum Cp levels in the current study are similar to those obtained (3–40 mg/dl) by Blakley and Hamilton (1985) in rams. Erdo˘gan et al. (2003) reported serum Cp levels between 13.79 and 21.12 mg/dl in Da˘glıç and Ivesi breed between March and June and suggested that serum Cp levels are influenced by breed, mineral composition of the soil, feeding conditions and climate. To the best of our knowledge, this is the first study to represent seminal plasma Cp levels in rams. It was reported to be 12.92 ± 9.1 mg/dl in cocks (Ulutas¸ et al., 2005). High positive correlation between Cp and Cu in blood serum in the current study is in accordance with those given in cattle and sheep (Blakley and Hamilton, 1985). However, we determined a significant negative correlation between these parameters in seminal plasma. The relation of seminal plasma Cu and Cp in rams was not reported, so far. It may be suggested that seminal plasma Cu is related with other Cu carrier proteins in seminal plasma rather than Cp. Namely, Cu is carried mainly with Cp in blood but it is also transported


−0.365 −0.362 0.022 −0.073 0.221 −0.186*** 0.064 0.183 0.065 0.207‡ 0.140 0.162

−0.443* −0.345 −0.353 −0.322 −0.336 −0.213* −0.052 -0.126 0.013 −0.094 −0.006 0.092

−0.192 −0.245 0.226 0.093 0.457 −0.107 0.092 0.257** 0.065 0.262* −0.129 0.144

−0.218 0.030 0.150 0.054 −0.191 0.074 -0.151 0.000 −0.079 −0.131 0.125 −0.117

0.250 0.130 −0.128 −0.133 −0.383 −0.268 0.034 −0.098 −0.091 −0.449† 1 0.619**

−0.072 −0.131 −0.299 −0.380 -0.579* −0.229 −0.055 0.028 −0.261 −0.217 0.619** 1

with albumin and transcuprein (approximately 15% and 10% of the total Cu, respectively) (Gray et al., 2009). Seminal plasma Cu concentrations were reported to be 0.5 ppm in ram (Cole and Cupps, 1969) which were higher from the results in the current study. Data obtained in the current study suggest that seminal plasma Cp may be independent from that of blood Cp from liver origin, thus sertoli cells secrete Cp-like protein (testicular cp) that is immunologically similar to serum Cp (Skinner and Griswold, 1983). Iron concentrations in blood serum were reported to be 1.56 ± 0.23 ␮g/ml by Burriel and Heys (1997) in sheep which were similar to our results. Increased blood Fe concentrations negatively influence sperm viability and morphology (Massanyi et al., 2003, 2004). Huang et al. (2001) reported that, incubation of spermatozoa with Fe2+ ions (at 5 ppm) significantly inhibited sperm motility by increasing MDA levels in vitro. Positive correlation between seminal plasma Fe with other abnormality in the current study may be attributed to its oxidative properties, because also it was correlated with blood Cd (p < 0.05) and seminal plasma Cd (p < 0.01) concentrations, positively. Seminal plasma and blood serum Zn concentrations were reported to be 488.6 ± 76.4 ␮g/dl and 75.6 ± 8.3 ␮g/dl, respectively in Merino ram by Bas¸pınar et al. (1998) which were in accordance with our results. Zinc is an essential element for production of sex hormones, attachment of head to tail in spermatozoa and its deficiency results in disorders of testes development and spermatogenesis (Hambidge et al., 1986). In this study, blood and seminal plasma Zn concentrations were not correlated with sperm parameters. Abou-Shakra et al., (1989) had also reported that seminal plasma Zn concentration was not correlated with sperm density and motility. However, Chia et al. (2000) reported lower (p < 0.05) seminal plasma Zn concentrations in infertile men. It is suggested that high Zn concentration in boar semen protect spermatozoa from the impairing effects of toxic elements (Massanyi et al., 2003). To our knowledge, seminal plasma Cd concentrations were not reported in rams but semen Cd concentrations were reported to be 0.12 ± 0.12 ppm (Massanyi et al., 2003) which were higher from our results in seminal plasma, suggesting most of the Cd in semen to be in spermatozoa. Previous studies reported that Cd decreased spermatozoa motility, progressive spermatozoa motility and the percentage of spermatozoa with the highest motility. Progressive motility, path velocity and straightness are negatively affected with the Cd concentration at 2.0 mg CdCl2 /ml in cows (Massanyi et al., 1996). Furthermore, semen Cd was positively (p < 0.05) correlated with separated flagellum, and retention of cytoplasmic drop in foxes (Massanyi et al., 2005). The correlation of Cd with other abnormality, fertility rate and litter size in the current study supports the negative effects of Cd in fertility of rams.

−0.093 −0.140 0.187 0.126 0.427 0.008 −0.165 0.181 −0.067 0.097 −0.114 −0.124 0.036 0.024 0.375 0.379 0.442 0.086 −0.059 0.219 0.021 0.02 0.130 −0.085 −0.064 0.044 0.049 0.053 0.315 0.255** −0.076 0.155 −0.109 0.060 0.002 −0.143

−0.250 0.050 0.126 0.007 −0.096 0.071 −0.163 0.054 −0.099 −0.145 0.084 −0.170

−0.113 −0.225 0.054 −0.040 −0.146 −0.010 −0.099 0.119 0.065 0.033 0.079 0.476*

Blood and seminal plasma Cp may be suggested to have positive influence regardless of Cu in seminal plasma with its antioxidant effects, whereas Fe and Cd have negative influence on sperm parameters in Merino rams. The relationship of Cp and Cu needs further studies in order to investigate their roles in fertility of rams. Conflict of interest Authors declared that they have no conflict of interest.

‡

†

***

p < 0.05. p < 0.01. p = 0.058. p = 0.054. p = 0.052.

Acknowledgements

*

Blood Cp Blood Cu Blood Fe Blood Zn Blood Cd Seminal Cp Seminal Cu Seminal Fe Seminal Zn Seminal Cd Fertility rate, individual (%) Litter size

5. Conclusion

**

Acrosome abnormality Abnormal sperm Hos-T (%) Concentration (x106 ) Motility (%) Mass activity (1–5) Volume (ml)

Table 2 Correlations (r-values) of blood serum (n = 19) and seminal plasma (n = 112) Cp, Cu, Fe, Zn and Cd with sperm parameters and fertility rate in Merino rams.

Other abnormality

Viability (%)

Fertilty rate individual (%)

Litter size

138

This study was supported by Ministry of Food Agriculture and Livestock, General Directorate of Agricultural Research and Politics (GDAR) (Project No: TAGEM/HAYSUD/11/08/01/04).


References Abou-Shakra, F.R., Ward, N.I., Everard, D.M., 1989. The role of trace elements in male infertility. Fertil. Steril. 52, 307–310. Aitken, R.J., Curry, B.J., 2011. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid. Redox. Signal. 14, 367–381. Bas¸pınar, N., Kaya, A., Altunok, V., Güven, B., Kurto˘glu, F., Ataman, M.B., 1998. Relationship between some biochemical parameters and sperm quality in rams. Vet. Bil. Derg. 14, 91–100. Bearden, H.J., Fuquay, J.W., Willard, S.T., 2004. Applied Animal Reproduction. Prentice Hall, US. Blakley, B.R., Hamilton, D.L., 1985. Ceruloplasmin as an indicator of copper status in cattle and sheep. Can. J. Comp. Med. 49, 405–408. Bremner, I., 1998. Manifestations of copper excess. Am. J. Clin. Nutr. 67, 1069–1073. Burriel, A.R., Heys, V., 1997. Serum and milk iron levels during sheep intramammary infection caused by coagulase-negative staphylococci. Biol. Trace Elem. Res. 59, 153–158. Chia, S.E., Ong, C.N., Chua, L.H., Ho, L.M., Tay, S.K., 2000. Comparison of zinc concentrations in blood and seminal plasma and the various sperm parameters between fertile and infertile men. J. Androl. 21, 53–57. Choi, S.Y., Kwon, H.Y., Kwon, B., Eum, S., Kang, H., 2000. Fragmentation of human ceruloplasmin induced by hydrogen peroxide. Biochimie 82, 175–180. Cigankova, V., Mesaros, P., Bires, J., Ledecky, V., Ciganek, J., Tomajkova, E., 1998. The morphological structure of the testis in stallions with zinc deficiency. Slovak. Vet. J. 23, 97–100. Cole, H.H., Cupps, P.T., 1969. Reproduction in domestic animals. In: Mann, T. (Ed.), Physiology of Semen and of the Male Reproductive Tract. Academic Press, Inc., Fifth Avenue, New York, U.S.A, p. 300. Colombo, J.P., Richterich, R., 1964. Ceruloplasmin analysis in plasma. Schweiz. Med. Wschr. 94, 715–720. Craig, P.M., Galus, M., Wood, C.H.M., Mcclelland, G.B., 2009. Dietary iron alters waterborne copper-induced gene expression in soft water acclimated zebrafish (Daniorerio). Regul. Physiol 296, 362–373. Erdo˘gan, S., Erdo˘gan, Z., S¸ahin, N., 2003. Investigation of serum ceruloplasmin, copper, zinc and wool copper and zinc levels in sheep grown in pasture. AÜ. Vet. Fak. Derg 50, 7–11. Evans, G., Maxwell, W.M.C., 1987. Handling and examination semen. In: Salamon’s Artificial Insemination of Sheep and Goat. Butterworths, Sydney, pp. 93–106. Gray, L.W., Kidane, T.Z., Nguyen, A., Akagi, S., Petrasek, K., Chu, Y.L., Cabrera, A., Kantardjieff, K., Mason, A.Z., Linder, M.C., 2009. Copper proteins and ferroxidases in human plasma and that of wild-type and ceruloplasmin knock-out mice. Biochem. J. 419, 237–245. Halliwell, B., 1991. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am. J. Med. 91, 14–22.

139

Hambidge, K.M., Casey, C.E., Krebs, N.F., 1986. Zinc. In: Mertz, W.D. (Ed.), Trace Elements in Human and Animal Nutrition 2. Academic Press, Orlando, FL, pp. 1–138. Huang, Y.L., Tseng, W.C., Lin, T.H., 2001. In vitro effects of metal ions (Fe2+ , Mn2+ , Pb2+ ) on sperm motility and lipid peroxidation in human semen. J. Toxicol. Environ. Health A. 62, 259–267. Massanyi, P., Lukac, N., Trandzik, J., Toman, R., 1996. Invitro inhibition of the motility of bovine spermatozoa by cadmium chloride. J. Environ. Sci. Health 31, 1865–1879. Massanyi, P., Trandzik, J., Nad, P., Toman, R., Skalick, M., Kornekov, B., 2003. Seminal concentrations of trace elements in various animals and their correlations. Asian J. Androl. 5, 101–104. Massanyi, P., Trandzik, J., Nad, P., Korenekova, B., Skalicka, M., Toman, R., Lukac, N., Halo, M., Strapak, P., 2004. Concentration of copper, iron, zinc, cadmium, lead, and nickel in bull and ram semen and relation to the occurrence of pathological spermatozoa. J. Environ. Sci. Health 39, 3005–3014. Paulenz, H., Söderquist, L., Perez, R., Berg, K.A., 2002. Effect of different extenders and storage temperatures on sperm viability of liquid ram semen. Theriogenology, 823–836. Prohaska, J.R., Gybina, A.A., 2004. Intracellular copper transport in mammals. J. Nutr. 164, 1003–1006. Prohaska, J., 2011. Impact of copper limitation on expression and function of multicopper oxidases (ferroxidases). Am. Soc. Nutr. Adv. Nutr. 2, 89–95. Revel, S.G., Mrode, R.A., 1994. An osmotic resistance test for bovine semen. Anim. Reprod. Sci. 36, 77–86. Saleh, R.A., Agarwal, A., 2002. Oxidative stress and male infertility: from research bench to clinical practice. J. Androl. 23, 737–752. Saylor, W.W., Leach, R.M., 1980. Intracellular distribution of copper and zinc in sheep: effect of age and dietary levels of the metals. J. Nutr. 110, 448–459. Schafer, S., Holzmann, A., 2000. The use of transmigration and spermac stain to evaluate epididymal cat spermatozoa. Anim. Reprod. Sci. 59, 201–211. Skinner, M., Griswold, M.D., 1983. Sertoli cells synthesize and secrete a ceruloplasmin-like protein. Biol. Reprod. 28, 1225–1229. Suttle, N.F., 2012. Copper imbalances in ruminants and humans: unexpected common ground. Adv. Nutr. 3, 366–374. Tabassomi, M., Alavi-Shoushtari, S.M., 2013. Effects of in vitro copper sulphate supplementation on the ejaculated sperm characteristics in water buffaloes (Bubalus bubalis). Vet. Res. Forum 4, 31–36. Toman, R., Massanyi, P., 1996. Cadmium in selected organs of fallow-deer (Dama dama), sheep (Ovis aries), brown hare (Lepus europaeus) and rabbit (Oryctolagus cuniculus). J. Environ. Health A Tox. Hazard. Subst. Environ. Eng. 31, 1043–1051. Ulutas¸ P.A., Serin I˙ ., Ceylan A., 2005. Relations between seminal plasma lipid peroxidation, extracellular antioxidants and some spermatological features in cocks. I˙ stanbul Univ. Vet. Fak. Derg. 31 (2005) 67–74.