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Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C
Cryobiology xxx (2014) xxx–xxx
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Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C q Mehmet Bozkurt Ataman a, Mustafa Numan Bucak a,⇑, Kenan Çoyan b a b
Selcuk University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Konya, Turkey Pamukkale University, Faculty of Medicine, Department of Histology and Embryology, Denizli, Turkey
a r t i c l e
i n f o
Article history: Received 5 January 2014 Accepted 13 March 2014 Available online xxxx This study is dedicated to my beloved mother, Emel Aynur Ataman, who passed away after cancer treatment. Keywords: Ram semen Aflatoxin Glucomannan Liquid storage
a b s t r a c t The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5 °C. Thirty-two Merino rams (12–14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 lg/day of total AF. The EG group received commercial feed plus 2 g/day of EG. The AF + EG group was given commercial feed plus 250 lg/day of total AF and 2 g/ day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electroejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage. During the first two weeks of the trial, the groups did not statistically differ from each other for sperm motility or rates of abnormal and nonviable spermatozoa at 0, 24, 48 and 96 h of storage. As from the third week, the short-term storage of semen produced statistically significant differences between the AF group and the other treatment groups for sperm parameters (p < 0.05). The administration of aflatoxin was observed to have reduced sperm motility and to have increased the rates of abnormal and nonviable spermatozoa in comparison to the control group (p < 0.05), while EG co-administered with AF was determined to have ameliorated the adverse effects of AF on sperm parameters, and this ameliorative effect continued throughout the short-term storage of semen. On the other hand, aflatoxin administration resulted in the deterioration of the sperm parameters in the following weeks, and the combined administration of EG + AF reversed this adverse effect, thus, bringing the sperm parameters closer to the values of the control group. This study demonstrated that, in rams, AF adversely affected sperm, biochemical and testis parameters, and that the combined administration of EG and AF reversibly improved these adverse effects. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Aflatoxins (AF) are a group of metabolites produced by fungi belonging to the genus Aspergillus, and in particular by the species A. flavus and A. parasiticus [23]. The growth of Aspergillus species and the production of aflatoxins require the presence of certain q Statement of funding: This study was financed under a project supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 107 O 866). ⇑ Corresponding author. E-mail address: [email protected] (M.N. Bucak).
favourable environmental conditions. These conditions include among others, the relative humidity of the stored feed material and the storage area, the storage temperature, the rate of oxygen in the environment, the storage time and the quality of the stored feed [11,15,23,28]. The dissemination of semen from a small number of rams with superior genetic merit to remote areas in order to inseminate a large number of ewes requires the preservation of semen in an artificial environment. Two major storage systems (liquid and frozen) are used for sperm preservation. The preservation of sperm generally requires a reduction or arrest of the metabolism of sperm cells,
http://dx.doi.org/10.1016/j.cryobiol.2014.03.007 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
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which prolongs their fertile life [25,39]. Cooled semen suffers from a decrease in motility and membrane integrity, accompanied by a decline of the survival rate in the female reproductive tract, reduction in fertility and increased embryonic loss [4,22,25,30]. The successful short-term liquid storage of ram semen is dependent on the reversible reduction of the survival and metabolic activity of spermatozoa. This is achieved by providing environmental conditions favourable for the survival of cooled ram semen, mainly through the use of extenders that maintain the membrane integrity, motility and fertilizing ability of spermatozoa [16,26,30]. As is the case with all mycotoxins, the general mode of action of aflatoxins is based on the inhibition of DNA, RNA and protein synthesis [19]. The toxicity of the most toxic type of aflatoxins, AFB1, is reported to arise not from the toxin itself but from the metabolites of the toxin generated as a result of the activity of certain enzymes, including cytochrome p-450 and aryl hydrocarbon hydroxylase [18,21]. The maximum residue limit of AFs allowed in food, as adopted in August 1966 by the Codex Alimentarius Commission, jointly formed by the FAO and WHO, is 30 ppb [13]. However, this limit has been reduced to 20 ppb by the Food and Drug Administration (FDA). It has been reported that, when administered to rats at sublethal doses, AFB1 causes the degeneration of the testes and the death of the germinal cells, which in return results in a decrease in sperm production, as well as in the concentration and motility of spermatozoa [9,10]. It has been shown that aflatoxin decreased the motility of sperm obtained by ejaculation or from the epididymis when ram semen was incubated in different concentrations of aflatoxin [35]. In another study, it was also observed that aflatoxin treatment decreased the ejaculate volume, sperm concentration and sperm motility [29]. A different approach to biological detoxification is based on the use of Saccharomyces cerevisiae (SCE) and its cell-wall component (glucomannan) to ameliorate the adverse effects of AF [8,27]. Esterified glucomannan has a capacity of binding AF at very high levels (80–97%) [3,8]. In previous studies, in which EG was administered as an adsorbent at varying doses, this substance eliminated the adverse effects of AF on biochemical [3] and haematological parameters [5], performance [27] and immune response [6] either partly or completely [2,27,31]. To our knowledge, the effects of AF and the protective efficiency of EG incorporated into commercial feed, on the maintenance of sperm parameters during the low temperature liquid storage of sperm has not been investigated before. The aim of this study was to address this need, and thus to investigate the potential effects of esterified glucomannan incorporated into feed, in terms of the amelioration of sperm damage, in rams with high genetic merit, resulting from the consumption of aflatoxin-contaminated feed stored under unhygienic and humid environmental conditions, and in terms of the storage of the semen of these animals.
Materials and methods Animals and diet The present study was approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine of Selçuk University (2008/061). Thirty-two 12–14-month-old Merino rams were used. Animals were examined for their general health status. Ivermectin (Avromec-F, 1 mL/50 kg) and oxfendazole (Okzan-F, 1 tablet/50 kg) were administered for antiparasitic treatment. In addition, enterotoxemia (Pluritoxiven-8, 1 mL) and smallpox vaccinations were performed. For the adaptation of the animals to the environment and the new feeding regimen, a 15-day acclimatization programme was applied prior to the start of the study. Individually weighed
rams were divided into four equal groups. The experimental feeding regimen was continued for ninety-two days. The duration of the treatment (92 days) was based on a possible cumulative toxicity as well as on the duration of spermatogenesis and spermiogenesis in rams. The rams were fed on commercial feed. Water and alfalfa were given ad libitum. Each morning, before given feed; the animals were administered with AF and EG, incorporated into 250 g of commercial feed. Experimental design The experimental design consisted of four dietary treatments. Each treatment group consisted of 8 Merino rams. The control group (C) was fed with commercial feed. The AF group received commercial feed plus 250 lg/day of total AF. The EG group was given commercial feed plus 2 g/day of EG (Mycosorb, Alltech, Australia). The AF + EG group was fed with commercial feed plus 250 lg/day of total AF and 2 g/day of EG. The AF and EG doses given to the animals throughout the study were calculated by pharmacologists. Aflatoxin (AF) The AF used in this study was produced (at the premises of the Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Selçuk University, Konya, Turkey) from the A. parasiticus NRLL 2999 culture (USDA, Agricultural Research Service, Peoria, IL, USA) via the fermentation of rice and using the method of Shotwell et al. [33] with minor modifications by Demet et al. [7]. Fermented rice was sterilized in an autoclave, dried at 70 °C, and ground to a fine powder. In compliance with the method described by Vicam [37], the extraction and purification of AF in fermented rice was performed by applying the rice to an immunoaffinity column (Down Test; Vicam). The amount of AF was measured by high-performance liquid chromatography (HPLC) according to the method described by Stroka et al. [38]. The amount of total AF in the fermented rice was 73.96 ppm. The AF in the rice consisted of 84.15% AFB1, 6.29% AFB2, 9.13% AFG1, and 4.25% AFG2 (rate of return method 97.4%; sensitivity 0.4 ppb). Semen collection and spermatological examination In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. An ejaculate volume of 2–3 mL was obtained with each application. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage. Progressive motility was assessed using a phase-contrast microscope (100 magnification), with a warm stage maintained at 37 °C. A wet mount was made using a 5 lL drop of extended semen (1:10) placed directly onto a microscope slide and covered by a coverslip. Sperm motility estimations were performed in three different microscopic fields for each semen sample. The mean of the three successive estimations was recorded as the final motility score. For the assessment of sperm morphology, at least three drops of semen were added into Eppendorf tubes containing 1 mL of Hancock’s solution (62.5 mL formalin, 150 mL sodium saline solution, 150 mL buffer solution, and 500 mL double-distilled water) [32]. One drop of this mixture was placed onto a microscope slide and covered with a coverslip. The percentage of abnormal sperm (detached heads, acrosomal aberrations, abnormal mid-pieces and tail defects) was recorded by counting a total of 200 spermatozoa under phase-contrast microscopy (1000 magnification; oil immersion).
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
M.B. Ataman et al. / Cryobiology xxx (2014) xxx–xxx
The viability of the sperm cells in the samples was assessed using the nigrosin–eosin stain [12]. The stain was prepared by dissolving 1.67 g of eosin-Y, 10 g of nigrosin, and 2.9 g of sodium citrate in 100 mL of distilled water. The sperm suspension smears were prepared by mixing a drop of the extended semen sample with two drops of the stain on a warm slide and immediately spreading the stain with a second slide. Viability was assessed by counting 200 cells under phase-contrast microscopy (1000 magnification). Sperm cells showing partial or complete purple colorisation were considered non-viable and only sperm cells showing strict exclusion of the stain were considered to be alive. Statistical analysis Sperm parameters obtained from a total of 32 rams for four experimental groups were analyzed by two-way repeated measures ANOVA, followed by Tukey post hoc test to determine significant differences between the groups, storage time and weeks. The arcsine transformation were performed to normalise the data distributions. Differences with values of p < 0.05 were considered to be statistically significant. Statistical analyses were performed by using the SPSS 10.0 package programme. Results Sperm parameters Sperm motility rates at 0, 24, 48, 72 and 96 h of storage for the different weeks are presented in Fig. 1. Statistically significant difference of sperm motility percentage was determined at extending times of storage for same groups and weeks. When weeks were compared at same storage time for each group, changes at motilities were no statistically significant during weeks. But for AF group, it was found that decrease of motility was statistically significant
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as from the fifth (0 h of storage) and ninth (24, 48, 72 and 96 h of storage) weeks (p < 0.05). When difference between groups at same week and storage time was examined, no statistically significant difference was determined between the groups for motility rates at 0, 24, 48 and 96 h of storage during the first two weeks of the trial. As from the third week, significant differences were observed between Group AF and the other groups for motility rates at all time points (24, 48, 72 and 96 h of storage). Aflatoxin administration reduced sperm motility in the subsequent weeks of the study, and the combined administration of glucomannan and aflatoxin reversed this adverse effect and drew the motility rates closer to the values of the control group (p < 0.05). The rates of abnormal spermatozoa at 0, 24, 48, 72 and 96 h of storage for the different weeks of the study are presented in Fig. 2. When weeks were compared at same storage time for each group, increases at abnormal sperm were statistically significant as from the tenth week. Statistically significant increase of abnormal sperm percentage was determined at 96 h of storage for same groups and weeks (p < 0.05). When difference between groups at same week and storage time was examined, the groups were determined not to significantly differ from each other for the abnormal spermatozoa rates at 0, 24, 48 and 96 h of storage during the first two weeks of the study. As from the third week, the rates of abnormal spermatozoa for all time points significantly differed between the AF group and the other treatment groups. While AF administration increased abnormal spermatozoa rates throughout the following weeks of the study, the combined administration of AF + EG reversed this adverse effect and drew the rates closer to those of the control animals throughout the short-term storage of semen (p < 0.05). The rates of nonviable spermatozoa at 0, 24, 48, 72 and 96 h of storage for the different weeks of the study are presented in Fig. 3. Statistically significant difference of nonviable sperm percentage was determined at extending times of storage for same groups and weeks and nonviable sperm increased during the storage
Fig. 1. Rates of sperm motility by weeks at different storage times. ⁄Means are different within week (p < 0.05; n: 8 per group).
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
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Fig. 2. Rates of abnormal spermatozoa by weeks at different storage times. ⁄Means are different within week (p < 0.05; n: 8 per group).
Fig. 3. Rates of dead spermatozoa by weeks at different storage times. ⁄Means are different within week (p < 0.05; n: 8 per group).
period (p < 0.05). When weeks were compared at same storage time for AF group, increases at nonviable sperm were statistically significant as from the fourth week, and the supplementation of
glucomannan could not reversed this adverse effect. When differences between groups at same week and storage time was examined, AF administration increased the rate of nonviable
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
M.B. Ataman et al. / Cryobiology xxx (2014) xxx–xxx
spermatozoa compared to the values of the control group (p < 0.05), and the administration of EG in combination with AF decreased the rate of nonviable spermatozoa that had increased due to AF, and this decrease was observed to have further continued during the storage of semen (p < 0.05).
Discussion Spermatogenesis occurs in the testes, in the wall of the convoluted sac-like seminiferous tubules referred to as the tubuli seminiferi contorti. The Sertoli cells found in these walls support the supply and development of the spermatozoa. Reaching the rete testis by means of the tubuli seminiferi recti, the spermatozoa gain fertilizing ability during epididymal migration and are stored in the cauda epididymis until ejaculated [21]. Certain toxins, including aflatoxin, which are ingested in feed, pass the blood-testis barrier and cause damage to the testicular tissue, negatively affect spermatogenesis, and result in the deterioration of sperm parameters. Multiple environmental, physiological and genetic factors are influential on the impairment of sperm functions and infertility. The determination of how these factors influence sperm functions bears significance for the development of effective treatment methods [34]. EG, which has started to be used in the past few years and has adsorbent activity, has produced promising results in terms of the amelioration of the damaging effects of aflatoxins on cells [2,3,20]. Hafez et al. [14] reported that, in rabbits, aflatoxins adversely affect sperm parameters and inhibit spermatogenesis. Ibeh et al. [17] suggested that aflatoxins cause significant decrease in sperm motility. The decrease observed in sperm motility in the AF group, in comparison to the other trial groups, is in support of aflatoxininduced damage. The acrosome contains the enzymes required for the penetration of the spermatozoon through the ovum layers and zona pellucida during fertilization [36]. Alm et al. [1] determined that the rates of abnormal spermatozoa and acrosomal disorders had increased in bulls suffering from chronic aflatoxicosis. In the present study, it was also ascertained that the rate of abnormal spermatozoa had increased due to aflatoxicosis. Some other researchers have indicated that in broiler flocks, aflatoxin incorporated into feed adversely affected fertility, but did not affect the viability of spermatozoa [24,39]. The differences observed in the results of previously conducted studies are attributed to these studies having been conducted in different animal species and to differences in the metabolism of ingested aflatoxin resulting from differences between animal species, differences in the digestibility of feed and the amount of aflatoxin ingested. In the present study, the deterioration of sperm parameters having been observed as from the third week of the experimental feeding regimen suggested that the adverse effects of aflatoxin develop at this time point. Furthermore, no adverse effect having been observed in the groups administered with EG alone and a combination of AF and EG demonstrated that EG has an ameliorative effect against the adverse effects of AF and that its administration alone does not induce any adverse effect. The progressive decrease of sperm motility and the progressive increase of nonviable and abnormal spermatozoa rates with the advance of the weeks were an indicator of spermatogenesis being affected adversely over time due to chronic aflatoxicosis. In the present study, sperm motility having decreased and nonviable and abnormal spermatozoa rates having increased between 0 and 96 h after the dilution of semen was considered normal. Nonetheless, it was observed that chronic aflatoxicosis accelerated the decrease in sperm motility (Fig. 1), and the increase in the
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abnormal spermatozoa (Fig. 2) and nonviable spermatozoa rates (Fig. 3). As from the third week of the study, differences had started to be observed between the groups for sperm motility, and abnormal and nonviable spermatozoa rates, and these differences increased in the course of time. In the present study, the effects of AF on ram sperm parameters and the potential ameliorative or inhibitory effect of glucomannan co-administered with aflatoxin on aflatoxin-induced adverse effects in the sperm parameters were investigated. The results obtained in the present study demonstrated that, of the sperm parameters investigated, sperm motility had significantly decreased and abnormal and nonviable spermatozoa rates had significantly increased due to chronic aflatoxicosis. In result, given the need for the prevention of peracute, acute and chronic intoxication in both livestock and laboratory animals, this study demonstrated that glucomannan ameliorated spermatozoa damage induced by chronic aflatoxicosis. Therefore, care should be given to the nutrition of breeder rams and the prevention of the contamination of animal feed with aflatoxin, to ensure that sperm quality is maintained and the desired success is achieved with both natural mating and artificial insemination using liquid-stored semen. In the event of aflatoxin production in stored feed, it is suggested that EG can be incorporated into feed as an aflatoxin binder with an aim to prevent aflatoxin-induced sperm damage.
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Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C q Mehmet Bozkurt Ataman a, Mustafa Numan Bucak a,⇑, Kenan Çoyan b a b
Selcuk University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Konya, Turkey Pamukkale University, Faculty of Medicine, Department of Histology and Embryology, Denizli, Turkey
a r t i c l e
i n f o
Article history: Received 5 January 2014 Accepted 13 March 2014 Available online xxxx This study is dedicated to my beloved mother, Emel Aynur Ataman, who passed away after cancer treatment. Keywords: Ram semen Aflatoxin Glucomannan Liquid storage
a b s t r a c t The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5 °C. Thirty-two Merino rams (12–14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 lg/day of total AF. The EG group received commercial feed plus 2 g/day of EG. The AF + EG group was given commercial feed plus 250 lg/day of total AF and 2 g/ day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electroejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage. During the first two weeks of the trial, the groups did not statistically differ from each other for sperm motility or rates of abnormal and nonviable spermatozoa at 0, 24, 48 and 96 h of storage. As from the third week, the short-term storage of semen produced statistically significant differences between the AF group and the other treatment groups for sperm parameters (p < 0.05). The administration of aflatoxin was observed to have reduced sperm motility and to have increased the rates of abnormal and nonviable spermatozoa in comparison to the control group (p < 0.05), while EG co-administered with AF was determined to have ameliorated the adverse effects of AF on sperm parameters, and this ameliorative effect continued throughout the short-term storage of semen. On the other hand, aflatoxin administration resulted in the deterioration of the sperm parameters in the following weeks, and the combined administration of EG + AF reversed this adverse effect, thus, bringing the sperm parameters closer to the values of the control group. This study demonstrated that, in rams, AF adversely affected sperm, biochemical and testis parameters, and that the combined administration of EG and AF reversibly improved these adverse effects. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Aflatoxins (AF) are a group of metabolites produced by fungi belonging to the genus Aspergillus, and in particular by the species A. flavus and A. parasiticus [23]. The growth of Aspergillus species and the production of aflatoxins require the presence of certain q Statement of funding: This study was financed under a project supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 107 O 866). ⇑ Corresponding author. E-mail address: [email protected] (M.N. Bucak).
favourable environmental conditions. These conditions include among others, the relative humidity of the stored feed material and the storage area, the storage temperature, the rate of oxygen in the environment, the storage time and the quality of the stored feed [11,15,23,28]. The dissemination of semen from a small number of rams with superior genetic merit to remote areas in order to inseminate a large number of ewes requires the preservation of semen in an artificial environment. Two major storage systems (liquid and frozen) are used for sperm preservation. The preservation of sperm generally requires a reduction or arrest of the metabolism of sperm cells,
http://dx.doi.org/10.1016/j.cryobiol.2014.03.007 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
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which prolongs their fertile life [25,39]. Cooled semen suffers from a decrease in motility and membrane integrity, accompanied by a decline of the survival rate in the female reproductive tract, reduction in fertility and increased embryonic loss [4,22,25,30]. The successful short-term liquid storage of ram semen is dependent on the reversible reduction of the survival and metabolic activity of spermatozoa. This is achieved by providing environmental conditions favourable for the survival of cooled ram semen, mainly through the use of extenders that maintain the membrane integrity, motility and fertilizing ability of spermatozoa [16,26,30]. As is the case with all mycotoxins, the general mode of action of aflatoxins is based on the inhibition of DNA, RNA and protein synthesis [19]. The toxicity of the most toxic type of aflatoxins, AFB1, is reported to arise not from the toxin itself but from the metabolites of the toxin generated as a result of the activity of certain enzymes, including cytochrome p-450 and aryl hydrocarbon hydroxylase [18,21]. The maximum residue limit of AFs allowed in food, as adopted in August 1966 by the Codex Alimentarius Commission, jointly formed by the FAO and WHO, is 30 ppb [13]. However, this limit has been reduced to 20 ppb by the Food and Drug Administration (FDA). It has been reported that, when administered to rats at sublethal doses, AFB1 causes the degeneration of the testes and the death of the germinal cells, which in return results in a decrease in sperm production, as well as in the concentration and motility of spermatozoa [9,10]. It has been shown that aflatoxin decreased the motility of sperm obtained by ejaculation or from the epididymis when ram semen was incubated in different concentrations of aflatoxin [35]. In another study, it was also observed that aflatoxin treatment decreased the ejaculate volume, sperm concentration and sperm motility [29]. A different approach to biological detoxification is based on the use of Saccharomyces cerevisiae (SCE) and its cell-wall component (glucomannan) to ameliorate the adverse effects of AF [8,27]. Esterified glucomannan has a capacity of binding AF at very high levels (80–97%) [3,8]. In previous studies, in which EG was administered as an adsorbent at varying doses, this substance eliminated the adverse effects of AF on biochemical [3] and haematological parameters [5], performance [27] and immune response [6] either partly or completely [2,27,31]. To our knowledge, the effects of AF and the protective efficiency of EG incorporated into commercial feed, on the maintenance of sperm parameters during the low temperature liquid storage of sperm has not been investigated before. The aim of this study was to address this need, and thus to investigate the potential effects of esterified glucomannan incorporated into feed, in terms of the amelioration of sperm damage, in rams with high genetic merit, resulting from the consumption of aflatoxin-contaminated feed stored under unhygienic and humid environmental conditions, and in terms of the storage of the semen of these animals.
Materials and methods Animals and diet The present study was approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine of Selçuk University (2008/061). Thirty-two 12–14-month-old Merino rams were used. Animals were examined for their general health status. Ivermectin (Avromec-F, 1 mL/50 kg) and oxfendazole (Okzan-F, 1 tablet/50 kg) were administered for antiparasitic treatment. In addition, enterotoxemia (Pluritoxiven-8, 1 mL) and smallpox vaccinations were performed. For the adaptation of the animals to the environment and the new feeding regimen, a 15-day acclimatization programme was applied prior to the start of the study. Individually weighed
rams were divided into four equal groups. The experimental feeding regimen was continued for ninety-two days. The duration of the treatment (92 days) was based on a possible cumulative toxicity as well as on the duration of spermatogenesis and spermiogenesis in rams. The rams were fed on commercial feed. Water and alfalfa were given ad libitum. Each morning, before given feed; the animals were administered with AF and EG, incorporated into 250 g of commercial feed. Experimental design The experimental design consisted of four dietary treatments. Each treatment group consisted of 8 Merino rams. The control group (C) was fed with commercial feed. The AF group received commercial feed plus 250 lg/day of total AF. The EG group was given commercial feed plus 2 g/day of EG (Mycosorb, Alltech, Australia). The AF + EG group was fed with commercial feed plus 250 lg/day of total AF and 2 g/day of EG. The AF and EG doses given to the animals throughout the study were calculated by pharmacologists. Aflatoxin (AF) The AF used in this study was produced (at the premises of the Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Selçuk University, Konya, Turkey) from the A. parasiticus NRLL 2999 culture (USDA, Agricultural Research Service, Peoria, IL, USA) via the fermentation of rice and using the method of Shotwell et al. [33] with minor modifications by Demet et al. [7]. Fermented rice was sterilized in an autoclave, dried at 70 °C, and ground to a fine powder. In compliance with the method described by Vicam [37], the extraction and purification of AF in fermented rice was performed by applying the rice to an immunoaffinity column (Down Test; Vicam). The amount of AF was measured by high-performance liquid chromatography (HPLC) according to the method described by Stroka et al. [38]. The amount of total AF in the fermented rice was 73.96 ppm. The AF in the rice consisted of 84.15% AFB1, 6.29% AFB2, 9.13% AFG1, and 4.25% AFG2 (rate of return method 97.4%; sensitivity 0.4 ppb). Semen collection and spermatological examination In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. An ejaculate volume of 2–3 mL was obtained with each application. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage. Progressive motility was assessed using a phase-contrast microscope (100 magnification), with a warm stage maintained at 37 °C. A wet mount was made using a 5 lL drop of extended semen (1:10) placed directly onto a microscope slide and covered by a coverslip. Sperm motility estimations were performed in three different microscopic fields for each semen sample. The mean of the three successive estimations was recorded as the final motility score. For the assessment of sperm morphology, at least three drops of semen were added into Eppendorf tubes containing 1 mL of Hancock’s solution (62.5 mL formalin, 150 mL sodium saline solution, 150 mL buffer solution, and 500 mL double-distilled water) [32]. One drop of this mixture was placed onto a microscope slide and covered with a coverslip. The percentage of abnormal sperm (detached heads, acrosomal aberrations, abnormal mid-pieces and tail defects) was recorded by counting a total of 200 spermatozoa under phase-contrast microscopy (1000 magnification; oil immersion).
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
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The viability of the sperm cells in the samples was assessed using the nigrosin–eosin stain [12]. The stain was prepared by dissolving 1.67 g of eosin-Y, 10 g of nigrosin, and 2.9 g of sodium citrate in 100 mL of distilled water. The sperm suspension smears were prepared by mixing a drop of the extended semen sample with two drops of the stain on a warm slide and immediately spreading the stain with a second slide. Viability was assessed by counting 200 cells under phase-contrast microscopy (1000 magnification). Sperm cells showing partial or complete purple colorisation were considered non-viable and only sperm cells showing strict exclusion of the stain were considered to be alive. Statistical analysis Sperm parameters obtained from a total of 32 rams for four experimental groups were analyzed by two-way repeated measures ANOVA, followed by Tukey post hoc test to determine significant differences between the groups, storage time and weeks. The arcsine transformation were performed to normalise the data distributions. Differences with values of p < 0.05 were considered to be statistically significant. Statistical analyses were performed by using the SPSS 10.0 package programme. Results Sperm parameters Sperm motility rates at 0, 24, 48, 72 and 96 h of storage for the different weeks are presented in Fig. 1. Statistically significant difference of sperm motility percentage was determined at extending times of storage for same groups and weeks. When weeks were compared at same storage time for each group, changes at motilities were no statistically significant during weeks. But for AF group, it was found that decrease of motility was statistically significant
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as from the fifth (0 h of storage) and ninth (24, 48, 72 and 96 h of storage) weeks (p < 0.05). When difference between groups at same week and storage time was examined, no statistically significant difference was determined between the groups for motility rates at 0, 24, 48 and 96 h of storage during the first two weeks of the trial. As from the third week, significant differences were observed between Group AF and the other groups for motility rates at all time points (24, 48, 72 and 96 h of storage). Aflatoxin administration reduced sperm motility in the subsequent weeks of the study, and the combined administration of glucomannan and aflatoxin reversed this adverse effect and drew the motility rates closer to the values of the control group (p < 0.05). The rates of abnormal spermatozoa at 0, 24, 48, 72 and 96 h of storage for the different weeks of the study are presented in Fig. 2. When weeks were compared at same storage time for each group, increases at abnormal sperm were statistically significant as from the tenth week. Statistically significant increase of abnormal sperm percentage was determined at 96 h of storage for same groups and weeks (p < 0.05). When difference between groups at same week and storage time was examined, the groups were determined not to significantly differ from each other for the abnormal spermatozoa rates at 0, 24, 48 and 96 h of storage during the first two weeks of the study. As from the third week, the rates of abnormal spermatozoa for all time points significantly differed between the AF group and the other treatment groups. While AF administration increased abnormal spermatozoa rates throughout the following weeks of the study, the combined administration of AF + EG reversed this adverse effect and drew the rates closer to those of the control animals throughout the short-term storage of semen (p < 0.05). The rates of nonviable spermatozoa at 0, 24, 48, 72 and 96 h of storage for the different weeks of the study are presented in Fig. 3. Statistically significant difference of nonviable sperm percentage was determined at extending times of storage for same groups and weeks and nonviable sperm increased during the storage
Fig. 1. Rates of sperm motility by weeks at different storage times. ⁄Means are different within week (p < 0.05; n: 8 per group).
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
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Fig. 2. Rates of abnormal spermatozoa by weeks at different storage times. ⁄Means are different within week (p < 0.05; n: 8 per group).
Fig. 3. Rates of dead spermatozoa by weeks at different storage times. ⁄Means are different within week (p < 0.05; n: 8 per group).
period (p < 0.05). When weeks were compared at same storage time for AF group, increases at nonviable sperm were statistically significant as from the fourth week, and the supplementation of
glucomannan could not reversed this adverse effect. When differences between groups at same week and storage time was examined, AF administration increased the rate of nonviable
Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007
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spermatozoa compared to the values of the control group (p < 0.05), and the administration of EG in combination with AF decreased the rate of nonviable spermatozoa that had increased due to AF, and this decrease was observed to have further continued during the storage of semen (p < 0.05).
Discussion Spermatogenesis occurs in the testes, in the wall of the convoluted sac-like seminiferous tubules referred to as the tubuli seminiferi contorti. The Sertoli cells found in these walls support the supply and development of the spermatozoa. Reaching the rete testis by means of the tubuli seminiferi recti, the spermatozoa gain fertilizing ability during epididymal migration and are stored in the cauda epididymis until ejaculated [21]. Certain toxins, including aflatoxin, which are ingested in feed, pass the blood-testis barrier and cause damage to the testicular tissue, negatively affect spermatogenesis, and result in the deterioration of sperm parameters. Multiple environmental, physiological and genetic factors are influential on the impairment of sperm functions and infertility. The determination of how these factors influence sperm functions bears significance for the development of effective treatment methods [34]. EG, which has started to be used in the past few years and has adsorbent activity, has produced promising results in terms of the amelioration of the damaging effects of aflatoxins on cells [2,3,20]. Hafez et al. [14] reported that, in rabbits, aflatoxins adversely affect sperm parameters and inhibit spermatogenesis. Ibeh et al. [17] suggested that aflatoxins cause significant decrease in sperm motility. The decrease observed in sperm motility in the AF group, in comparison to the other trial groups, is in support of aflatoxininduced damage. The acrosome contains the enzymes required for the penetration of the spermatozoon through the ovum layers and zona pellucida during fertilization [36]. Alm et al. [1] determined that the rates of abnormal spermatozoa and acrosomal disorders had increased in bulls suffering from chronic aflatoxicosis. In the present study, it was also ascertained that the rate of abnormal spermatozoa had increased due to aflatoxicosis. Some other researchers have indicated that in broiler flocks, aflatoxin incorporated into feed adversely affected fertility, but did not affect the viability of spermatozoa [24,39]. The differences observed in the results of previously conducted studies are attributed to these studies having been conducted in different animal species and to differences in the metabolism of ingested aflatoxin resulting from differences between animal species, differences in the digestibility of feed and the amount of aflatoxin ingested. In the present study, the deterioration of sperm parameters having been observed as from the third week of the experimental feeding regimen suggested that the adverse effects of aflatoxin develop at this time point. Furthermore, no adverse effect having been observed in the groups administered with EG alone and a combination of AF and EG demonstrated that EG has an ameliorative effect against the adverse effects of AF and that its administration alone does not induce any adverse effect. The progressive decrease of sperm motility and the progressive increase of nonviable and abnormal spermatozoa rates with the advance of the weeks were an indicator of spermatogenesis being affected adversely over time due to chronic aflatoxicosis. In the present study, sperm motility having decreased and nonviable and abnormal spermatozoa rates having increased between 0 and 96 h after the dilution of semen was considered normal. Nonetheless, it was observed that chronic aflatoxicosis accelerated the decrease in sperm motility (Fig. 1), and the increase in the
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abnormal spermatozoa (Fig. 2) and nonviable spermatozoa rates (Fig. 3). As from the third week of the study, differences had started to be observed between the groups for sperm motility, and abnormal and nonviable spermatozoa rates, and these differences increased in the course of time. In the present study, the effects of AF on ram sperm parameters and the potential ameliorative or inhibitory effect of glucomannan co-administered with aflatoxin on aflatoxin-induced adverse effects in the sperm parameters were investigated. The results obtained in the present study demonstrated that, of the sperm parameters investigated, sperm motility had significantly decreased and abnormal and nonviable spermatozoa rates had significantly increased due to chronic aflatoxicosis. In result, given the need for the prevention of peracute, acute and chronic intoxication in both livestock and laboratory animals, this study demonstrated that glucomannan ameliorated spermatozoa damage induced by chronic aflatoxicosis. Therefore, care should be given to the nutrition of breeder rams and the prevention of the contamination of animal feed with aflatoxin, to ensure that sperm quality is maintained and the desired success is achieved with both natural mating and artificial insemination using liquid-stored semen. In the event of aflatoxin production in stored feed, it is suggested that EG can be incorporated into feed as an aflatoxin binder with an aim to prevent aflatoxin-induced sperm damage.
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Please cite this article in press as: M.B. Ataman et al., Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.03.007