British Poultry Science Volume 47, Number 3 ( June 2006), pp. 350—356
PHYSIOLOGY, BIOCHEMISTRY & NEUROBIOLOGY
Vitamin E supplementation reduces dexamethasone-induced oxidative stress in chicken semen Y. EID, T. EBEID
AND
H. YOUNIS
Department of Poultry Production, Kafr El-Sheikh Faculty of Agriculture, Tanta University, Kafr El-Sheikh, Egypt
Abstract 1. We examined the effects of supplemental dietary vitamin E (Vit E) on semen quality and antioxidative status in male domestic fowls exposed to oxidative stress induced by synthetic glucocorticoid, dexamethasone (DEX) injection. 2. Thirty-six Egyptian local cross males, 42 weeks old, were housed individually in cages in an opensided building under 16 h light:8 h dark and were provided with commercial feed and water ad libitum. Birds were divided into 4 groups: DEX (4 mg/bird/d), Vit E (200 mg/kg diet), DEX þ Vit E (4 mg/bird/ d þ 200 mg/kg diet, respectively) and control, n ¼ 9. All treatments lasted for 7 continuous days. 3. Oxidative stress induced by injection of DEX (4 mg/bird/d) resulted in decreased sperm count and motility correlated with an increased percentage of dead sperms. Vit E (200 mg/kg diet) enhanced sperm count and viability when supplemented to stress-induced birds, compared to DEX treatment alone. 4. In seminal plasma, low calcium concentration, high lipid peroxidation and reduced activity of glutathione peroxidase were associated with the oxidative stress. Vit E reduced lipid peroxidation in the seminal plasma. 5. In conclusion, excessive supplemental dietary Vit E improved semen quality when cockerels were subjected to stress conditions. It increased both sperm count and motility, reduced the percentage of dead sperm and enhanced the antioxidative status of seminal plasma.
INTRODUCTION Stress susceptibility of chickens is a major problem in the modern intensive poultry industry. Birds are often subjected to stressors such as fasting, transport and exposure to high or low environmental temperature. During stress the endocrine system is markedly affected. The hypothalamus—pituitary—adrenal axis and thyroid glands are seriously affected by stress (Siegel, 1980). As a result of stress, feed consumption, growth rate, feed efficiency, eggshell, fertility and chick quality decline (Gross and Siegel, 1993; El-Lethey et al., 2000). In chickens, adrenal corticosteroids are secreted shortly after exposure to stress (Siegel, 1980) and elevated plasma corticosterone
concentration has been used as an index of the response to stress in poultry (Freeman, 1971). Heat stress, for example, stimulates the release of corticosterone from the adrenal gland (Edens, 1978) and increases its plasma concentration in chickens (Ben-Nathan et al., 1976), turkeys (El-Halawani and Waibel, 1976) and pigeons (Pilo et al., 1985). Catabolic effects of excessive glucocorticoid hormones from endogenous or exogenous sources are well documented (Akiba et al., 1992; Ohtsuka et al., 1995). Previous studies have shown that glucocorticoid administration induced oxidative stress status in chicken (Eid et al., 2003). Similarly, synthetic glucocorticoid dexamethasone (DEX) administration mimics the adverse effects of increased corticosterone. DEX (doses ranging from 02 to 40 mg/kg) was used
Correspondence to: Dr Yahya Z. Eid, Department of Poultry Production, Kafr El-Sheikh Faculty of Agriculture, Tanta University, 33516 Kafr El-Sheikh, Egypt. Tel.: þ20-127469602, þ20-35372484. Fax: þ20-473232032. E-mail:
[email protected] Accepted for publication 22nd December 2005.
ISSN 0007–1668(print)/ISSN 1466–1799 (online)06/030350—7 ß 2006 British Poultry Science Ltd DOI: 10.1080/00071660600753912
VITAMIN E OXIDATIVE STRESS AND SEMEN
as an immune suppressive agent (Fowles et al., 1993), as a mediator of prenatal stress (Welberg and Seckl, 2001; Maccari et al., 2003) and to induce oxidative stress in laying hens (4 mg/ hen/d, El-Habbak et al., 2005). As a result of accelerated metabolic rates under stress conditions, elevated levels of free radicals (in particular reactive oxygen species, ROS) tend to be formed. ROS can have beneficial roles, as in phagocytes where they protect against bacteria and parasites. However, if natural antioxidant mechanisms are not adequate to quench excess oxygen radicals then they can react with cell structures and attack proteins, lipids, carbohydrates and nucleotides within the cell, a state referred to as oxidative stress (Hidalgo et al., 1988). Biological antioxidants (such as vitamin E—Vit E) are natural molecules which can prevent lipid peroxidation in the biological membranes. The destruction of most free radicals and activated oxygen species relies on the oxidation of endogenous antioxidants, mainly scavenging and reducing molecules. Reduction—oxidation recycling of such antioxidants markedly increases their biological efficiency and this needs greater provision of antioxidant nutrients under oxidative stress conditions. Chicken and vertebrate sperm display high rates of metabolic activity (and consequently ROS production which is believed to be increased under stress conditions) and are rich in polyunsaturated fatty acids (PUFA), which renders them particularly susceptible to oxidation by ROS, especially under stress conditions in humans (Aitken et al., 1989) and also in various domestic birds (Wishart, 1984; Suraı¨ et al., 1998a, b). ROS can modify the spermatozoon cytoskeleton and axoneme which results in reduction of sperm motility (de Lamirande and Gagnon, 1992) and inhibition of sperm—oocyte fusion (Aitken et al., 1989) and consequently leads to reduced fertility (Wishart, 1984). Free radicals can also attack the DNA within the sperm nucleus. Such damage to the genome may be translated into infertility (Roberts, 1998). Hood (1999) reported that heat exposure caused an increase in the percentage of dead sperm. McDaniel et al. (1995) found that when hens were inseminated with semen from stressed males, sperm—egg penetration and fertilised egg production decreased when compared to hens inseminated with semen from control males. As a result, a decrease in reproductive performance followed by significant financial losses occurs. It has been suggested that antioxidants reduce the physiological response to stress in animals (Taniguchi et al., 1999; Eid et al., 2003). Under stressful conditions, the requirement of antioxidants such as Vit E ( -tocopherol) is
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thought to increase to protect tissues from lipid peroxidation (Ohtsuka et al., 1998). Vit E is the most efficient scavenger of peroxyl radicals in phospholipid bilayers. In the membranes of mammalian cells, Vit E scavenges lipid peroxyl radicals LOO. through hydrogen atom transfer (Chaudiea and Ferrari-Iliou, 1999). A high dietary intake of Vit E (200 mg/kg diet) increases its concentration in both blood and seminal plasma and in addition produces beneficial changes in the antioxidant capacity and lipid profile of chicken semen (Suraı¨ et al., 1997a) under normal conditions. The objective of our study is to examine the effects of super-nutritional Vit E supplementation on the quality and antioxidative status of cockerel semen under oxidative stress conditions experimentally induced by DEX injections.
MATERIALS AND METHODS Thirty-six 42-week-old cockerels [Egyptian local cross: Gimmizah (local strain) Lohmann] were obtained from the research farm of the Poultry Production Department, Kafr El-Sheikh Faculty of Agriculture. They were selected from a population of about 108 birds to obtain a uniform body weight (295 011 kg) and sperm count (213 015 109/ml). Birds were housed in individual batteries, and provided with water and a commercial maize—soybean diet ad libitum (160 g/kg crude protein and metabolisable energy 1149 MJ/kg). Diet composition was formulated to meet recommended nutrient requirements (NRC, 1994). Ambient temperature averaged (25 2 C) with relative humidity 60 to 70% and 16 h light:8 h dark. All experiments were performed in accordance with institutional guidelines concerning animal use. Schedule and experimental design Birds were divided into 4 groups (n ¼ 9). The first one was injected each day intramuscularly with 1 ml of the synthetic glucocorticoid, DEX (Amriya Pharmaceutical Industries Co., Alexandria, Egypt) as sodium phosphate 4 mg/ml. The second group received a diet supplemented with Vit E (Cairo Pharmaceutical Industries Co., Egypt) in the form of -tocopherol acetate (200 mg/kg diet) and was similarly injected daily intramuscularly with 1 ml sterile physiological saline solution (09% NaCl). The third group received both DEX injection and Vit E supplementation (4 mg/bird/d and 200 mg/kg diet, respectively). The fourth group served as control and was only injected with the same dose of sterile physiological saline solution. All treatments lasted for 7 successive days.
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Sampling
Statistical analysis
On d 7 of the treatments, and 4 h after the last injection, individual semen samples were taken by the abdominal massage technique, squeezing the copulatory organs (Lake and Stewart, 1978). Semen samples were subjected to fresh analysis and centrifuged at 2500g for 20 min. The seminal plasma (supernatant) was stored at 20 C for subsequent analysis. Blood samples were obtained from the brachial vein using heparinised syringes and subjected to centrifugation at 5900g for 10 min, plasma was collected and stored at 20 C for analysis.
Data were analysed by two-way analysis of variance (ANOVA) using the General Linear Model procedure (SAS Institute, 1988) with multiple-range test. A value of P 005 was considered to be statistically significant.
Sperm count (109 sperm/ml) was estimated by using a haemocytometer (Lake and Stewart, 1978). Sperm motility percentage was measured using a small droplet from each individual placed on a warm slide, covered with a cover slide and examined for sperm motility microscopically at 400 magnification using a stage warmer set at 39 C. Sperm motility was classified as described by Melrose and Laing (1970). Semen was given an arbitrary score from 0 to 5 based on the following assessment: 0 (0%, no motility discernable); 1 (1 to 20% of sperm exhibiting slight undulating movement; mostly weak and oscillatory); 2 (20 to 40% of sperm showing undulating movement; no waves or eddies formed; there may be a number of inactive sperm); 3 (40 to 60% of sperm showing progressive motility; vigorous motion; slowly moving waves and eddies produced); 4 (60 to 80% of sperm showing progressive motility; waves and eddies of great rapidity of formation and movement); and 5 (80 to 100% of sperm in vigorous and progressive movement; extremely rapid formation of eddies and movement). Viability was estimated as the percentage of sperm that were permeable to eosin; these were regarded as dead (Lake and Stewart, 1978). Calcium (Ca2þ) in seminal plasma was assayed by the method of Gindler and King (1972) using the ‘Ca-kit’ produced by bioMe´rieux (Marcy l’Etoile, France). Lipid peroxidation in the blood plasma and seminal plasma was measured in the form of thiobarbituric acid reactive substance (TBARS) as described by Richard et al. (1992). TBARS, in particular malondialdehyde (MDA), is a product of the oxidative degradation of PUFA, and thus used as an index of oxidative stress. The activity of the antioxidative enzyme glutathione peroxidase (GSH-Px) was determined by the method of Levander et al. (1983). One unit of GSH-Px activity oxidises 10 nmole of NADPH/(mg protein/min). Proteins were determined in the seminal plasma by the Biuret method (Armstrong and Carr, 1964).
The influence of DEX and Vit E treatments on blood plasma TBARS is illustrated in Figure 1. DEX injections for 7 successive days elevated (P 005) blood plasma TBARS to more than three times its value in control samples. Vit E supplementation appeared to antagonise the effect of DEX, where blood plasma TBARS in the third group (DEX þ Vit E) was significantly lower than that for the group receiving DEX alone, reaching about 67% of its value. However, it was still significantly higher than the control, being about twice its value. Samples from cockerels given supplemental Vit E alone (group 2) did not significantly differ from the control. Data in the Table show the effect of each of the DEX and supplemental Vit E treatments alone, or together, on semen quality traits. Birds receiving DEX injection had lower sperm counts (P 005), being about 40% of the controls. When DEX injection was accompanied by supplemental Vit E, a significant increase in the sperm count occurred compared to the DEX group. However, it did not reach the values recorded for control birds (only about 72%). DEX significantly impaired sperm motility, and supplemental Vit E antagonised this suppressive effect of DEX on sperm motility (P 005). Semen samples from birds receiving DEX injections contained a significantly higher percentage of dead sperms than any of the other groups. The concentration of Ca2þ in seminal plasma was significantly reduced by DEX treatment, compared to the control or Vit E group; the latter two
(mmole MDA/l)
Measurements and chemical analysis
RESULTS
8 7 6 5 4 3 2 1 0
(6.18) a (4.18) b (1.92) c (1.4) c
DEX
Vit E
DEX x Vit E
Control
Figure 1. Effect of dexamethasone (DEX) administration and excessive dietary vitamin E (Vit E) supplementation on blood plasma TBARS in cockerels. Values are expressed as means standard deviation; means with different letters differ from each other (P 005).
VITAMIN E OXIDATIVE STRESS AND SEMEN
(mmole MDA/l)
(A) 10 9 8 7 6 5 4 3 2 1 0
(8.4) a (5.3) b
(U/g protein)
supplemental Vit E significantly increased seminal plasma GSH-Px compared to the control (Figure 2B).
(3.8) c
DISCUSSION
(22.2) a
(B) 25
(16.4) c
20 15
(4.9) b
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(19.3) b
(11.4) d
10 5 0 DEX
Vit E
DEX x Vit E
Control
Figure 2. Effect of dexamethasone (DEX) administration and dietary vitamin E (Vit E) supplementation on (A) TBARS and (B) glutathione peroxidase activity (GSH-Px) in seminal plasma of cockerels. Values are expressed as means standard deviation; means with different letters differ from each other (P 005).
did not significantly differ from each other. A significant increase in seminal plasma Ca2þ concentration was obtained when DEX-injected cocks were given supplemental dietary Vit E. When calculated as a percentage of control, seminal plasma Ca2þ concentrations in DEX, Vit E and DEX þ Vit E groups were about 72, 100 and 88%, respectively. The influence of DEX and Vit E treatments on seminal plasma TBARS and GSH-Px are graphically presented in Figure 2A and B, respectively. Cocks exposed to oxidative stress via DEX injection had significantly greater lipid peroxidation activity. TBARS values in the DEX group reached about 171% of that in controls. A significant reduction occurred when Vit E was incorporated in the diet of DEX-injected cocks. The TBARS concentration in seminal plasma samples from group 3 (DEX þ Vit E) did not exceed 63% of that of the DEX group and did not differ significantly from the control group. Supplemental Vit E significantly reduced TBARS in seminal plasma samples from males not subjected to DEX-induced oxidative stress (group 2) as compared to the control (Figure 2A). As for GSH-Px activity in seminal plasma, the results were completely opposite to those of TBARS. DEX treatment caused a significant decrease of seminal plasma GSH-Px activity and supplemental Vit E ameliorated this effect. DEX þ Vit E treatment resulted in significantly higher values than DEX alone. When administered to males under control conditions,
Administration of glucocorticoids causes hyperglycemia and hyper-triglyceridemia, resulting in increasing of abdominal fat content, fatty liver and initiating oxidative stress (Eid et al., 2003). A decline in egg production, egg quality and immunity were observed in DEX injected hens (El-Habbak et al., 2005). Using TBARS as an index of oxidative stress, we studied lipid peroxidation in cockerel blood plasma. TBARS in blood plasma was significantly elevated by DEX treatment and this was significantly ameliorated by Vit E (Figure 1). Ohtsuka et al. (1998) and Taniguchi et al. (1999) found that dietary glucocorticoids raised plasma TBARS in rats and broilers, respectively. This could indicate that DEX treatment triggers oxidative stress systemically. Semen characteristics are important in determining the fertility of cockerels. Nongenetic factors such as stress, nutrition, age of the bird and management are believed to influence semen characteristics and subsequently the flock fertility (Kalamah et al., 2000). Climatic stress, especially high ambient temperature, which increases circulating glucocorticoids (Edens and Siegel, 1975), affects semen concentration in chickens. Moreover, sperm motility shows a correlated decline with sperm concentration under heat stress (Kalamah, 2001). The same trend was present in our results. A correlated decline in both sperm concentration and sperm motility was associated with the oxidative stress induced by DEX treatment (Table). Excessive concentrations of glucocorticoids due to stress inhibit reproduction in most species. These anti-reproductive effects can be rationalised as a logical contributor to the stress response through a decline in portal GnRH concentrations and pituitary release of gonadotrophins within minutes, as well as the subsequent rises in sex steroid hormone concentrations (Sapolsky et al., 2000). Therefore, it could be argued that the inverse relationship between plasma concentrations of glucocorticoids and gonadotrophins as well as sex steroid hormones might decrease the maturation of spermatozoa and hence produce a reduction in ejaculated sperms in DEX treatment. At the same time it could be suggested that DEX treatment might enhance lipid peroxidation in the testes and also in mature spermatozoa membranes which reduce the sperm count. Treatment with Vit E significantly enhanced both sperm count
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Table. Effect of dexamethasone (DEX) administration and excessive dietary vitamin E (Vit E) supplementation on semen variables in cockerels. Values are expressed as means standard deviation; means with different superscripts in the same row differ from each other (P 005) Treatment DEX 9
Sperm count (10 /ml) Sperm motility (score) Dead sperm (%) Ca2þ (mg/100 ml)
Vit E c
086 007 411 022b 153 14a 699 04c
and motility compared to the DEX group (Table). Our results show an increased percentage of dead sperms with DEX treatment; however, Vit E treatment reduced this trait significantly (Table). In cockerels Vit E tended to improve semen quality by increasing sperm concentrations and viability (Franchini et al., 2001). One can therefore argue that these positive effects of Vit E on sperm count, sperm motility and reduced percentage of dead sperm under oxidative stress could be linked to the antioxidative properties of this vitamin (Brzezinska-Slebodzinska et al., 1995). It has been suggested that the morphology and the motility of sperm cells would be preserved by binding of this vitamin to endo-peroxides (MarinGuzman et al., 2000). In a recent study in humans, Eskenazi et al. (2005) found that higher antioxidant intake was associated with greater sperm numbers and motility. With regard to electrolyte concentration in seminal plasma, the ions of sodium, potassium, chloride, bicarbonate, calcium, magnesium, phosphate and sulphate are involved in homeostasis of sperm membranes (Darszon et al., 1999). Ca2þ is one of the electrolytes which play an important role in homeostasis. Our data show that Ca2þ in seminal plasma declined with oxidative stress (Table), and Vit E treatment could partly restore this decrease. However, we have no data to explain how dietary supplemental Vit E could ameliorate the negative effect of DEX on seminal plasma Ca2þ concentration. With regard to lipid peroxidation in spermatozoa, lipids are a basic component of semen contributing to the membrane structure of spermatozoa, the metabolism of sperm cells and to their ability to capacitate and fertilise female oocyte (Mann and Lutwak-Mann, 1981). Fatty acids in seminal plasma are also utilised as a source of energy by sperm cells (Nissen and Kreysel, 1983). There is considerable evidence that the lipid composition of the sperm membrane is a major determinant of motility (Roldan and Harrison, 1993). The presence of high concentrations of PUFA (Ravie and Lake, 1985) within the lipid fraction necessitates the presence of an efficient antioxidant system to protect
DEX þ Vit E a
282 012 494 016a 63 08b 965 03a
b
154 024 483 025a 73 01b 853 04b
Control 213 015a 494 016a 80 13b 970 03a
against peroxidative damage and possible associated sperm dysfunction (Aitken, 1994). Because the actual mechanism of lipid peroxide formation by fowl spermatozoa remains unidentified, it is not yet known whether the presence or absence of some factors such as antioxidants could suppress the production of high concentrations of lipid peroxides and subsequently enhance the sperm quality under stress conditions. The lipid content of the seminal plasma and spermatozoa make it a good target for free radical attacks under oxidative stress. This assumption is supported by the elevation of TBARS under DEX treatment and a decline in sperm motility was associated with accumulation of the TBARS. Vit E supplementation decreased TBARS in the seminal plasma because of its antioxidative properties and as a result of this, sperm motility was enhanced by Vit E under DEX treatment (Table and Figure 2A). This is in accordance with the report of Suraı¨ et al. (1997b) who showed that enhancement of the antioxidant capacity of semen could present a major opportunity for improving male fertility. This was clear in our data too: Vit E protects sperms from free radical attacks under oxidative stress, which is translated into enhanced sperm count, motility and reduced dead sperm percentage, in addition to the reduced TBARS value under the stress condition. With regard to the activity of glutathione peroxidase (GSH-Px) in seminal plasma, the selenium-containing enzyme GSH-Px plays an important role in the detoxification of any lipid peroxides which appear in the chicken sperm (Froman and Thurston, 1981). GSH-Px occupies a particularly important role in antioxidant protection of the cell in conversion of hydrogen peroxides to less harmful components (Olafsdottir and Reed, 1988). Suraı¨ et al. (1988) indicated that GSH-Px and Vit E are believed to be the primary components of the antioxidant system of the chicken spermatozoa. Suraı¨ et al. (1997b, 1998a) demonstrated that spermatozoa from 5 avian species, including chickens, are all characterised by high proportions of PUFA. Major isozymes of superoxide dismutase and
VITAMIN E OXIDATIVE STRESS AND SEMEN
GSH-Px, which protect against the peroxidation associated with a high degree of fatty acid unsaturation, were active in spermatozoa from all species. The activity of GSH-Px declined sharply with DEX treatment in the seminal plasma under oxidative stress; this activity was enhanced by Vit E. Moreover, Vit E alone increased the activity of GSH-Px compared to the control group (Figure 2B). These results suggest a synergistic antioxidant effect of Vit E and GSH-Px in protecting chicken sperms under oxidative stress. In conclusion, oxidative stress reduced sperm numbers, reduced motility, increased percentage of dead sperms and raised the level of lipid peroxidation; these changes would lead to lower fertility. Under oxidative stress conditions the efficiency of sperm function can be enhanced by supplemental antioxidants such as Vit E. Because Vit E is naturally present in avian sperm membranes and seminal plasma, it can exert its antioxidative action under oxidative stress in two ways. Firstly, in the testes Vit E can protect the biological membranes from lipid peroxidation during spermatogenesis. Secondly, in the seminal plasma itself, Vit E can reduce the peroxidation of seminal lipids and maintain adequate viability of sperms to complete the fertilisation process. Finally, supplemental supernutritional concentrations of Vit E under stress conditions enhance sperm number and quality.
ACKNOWLEDGEMENTS The authors wish to acknowledge the helpful suggestions of Professor Mohamed El-Habbak, Department of Poultry Production, Kafr El-Sheikh Faculty of Agriculture, Tanta University, Egypt and Dr Refaat El-Naggar, Animal Production Research Institute, Sakha, Kafr El-Sheikh, Egypt for his technical support.
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