Selenium-Enriched Probiotics Improves Murine Male Fertility Compromised by High Fat Diet
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A total of 75 male mice were allotted to five groups of 15 each in a completely randomized experimental design to study the effects of probiotics, inorganic selenium, and selenium-enriched probiotics on male fertility in hyperlipidemic status. The mice in group 1 were fed a normal basal diet and served as negative control. The mice in group 2 were fed a high fat diet and served as positive control. The mice in groups 3, 4, and 5 were fed the high fat diet supplemented with probiotics, inorganic selenium, and selenium-enriched probiotics, respectively. The high fat diet was composed of 15% lard, 1% cholesterol, 0.3% cholic acid, and 83.7% basal diet. Over 90% of the selenium in the selenium-enriched probiotics was present in forms of organic selenium. After the mice were fed these diets for 75 days, serumal total cholesterol, triglycerides, low density lipoprotein, high density lipoprotein, and testosterone levels, plus sperm index (count, motility and abnormalities), penis length, and weight and histopathology of testes were measured. The results showed that in the mice fed the high fat diet were significant (P < 0.01) elevations of serumal total cholesterol, triglycerides and low density lipoprotein, and decreases of high density lipoprotein. The high fat diet caused a decline in serumal testosterone level, reduced semen quality, and atrophy and degeneration of seminiferous tubules. No effects on penis length or relative weight of testis were observed. Supplementation of probiotics, inorganic selenium, or selenium-enriched probiotics to the high fat diet significantly alleviated (P < 0.05) the adverse effects of hyperlipidemia by reducing testicular tissue injury, increasing serumal testosterone level, and improving sperm indexes. It was concluded that hyperlipidemia had significant adverse effects on male fertility, which could be ameliorated at various degrees by feeding the diets supplemented with probiotics, inorganic selenium, or selenium-enriched probiotics. Selenium-enriched probiotics or inorganic selenium supplementation gave better results than probiotics supplementation and may be used to improve animal and human male fertility compromised by hyperlipidemia or obesity.
KeywordsSelenium-enriched probiotics Selenium Probiotics Male fertility Hyperlipidemia Mouse
Colony forming unit
High density lipoprotein
High fat diet
Inorganic form of selenium or sodium selenite
Low density lipoprotein
Organic form of selenium
Selenium-enriched probiotics that contain organic form of selenium
Standard error of the means
It has been reported that high plasma levels of cholesterol and/or triglycerides are associated with testicular dysfunction and poor semen quality in humans, which may lead to male infertility . Hypercholesterolemia may also stimulate oxygen radical production and increase lipid peroxide levels in various tissues as suggested by Ohara et al. in their study with rabbits . Lipid peroxides are cytotoxic to spermatozoa causing sperm morphological change . Diet-induced hyperlipidemia in rabbits has been found to influence sperm lipid composition and adversely affect semen quality including sperm concentration, motility, cap citation, and acrosomal reaction [4-6].
Oxidative damage to cells and cell membranes has been implicated in the pathogenesis of many disorders including male infertility, whereas antioxidants play a major role in preventing or reducing the lipid peroxidation in human and animals. Selenium (Se), an essential trace element, possesses antioxidant activity that is required for maintenance of normal physiological functions [7-9] including the spermatogenesis of mammals [7, 10]. It was found that Se can lower triglyceride (TG), total cholesterol (TC), and free fatty acid concentrations in the serum, and inhibit liver biosynthesis of TG and cholesterols [11, 12]. Since there is a close relationship between Se and lipid metabolism, the lipid peroxidation-related male infertility may be ameliorated by dietary Se intake.
Many studies have also indicated that probiotics (PB) may have health promotion effects on lipid metabolism, including cholesterol level lowering effect [13-15]. However, data on the effects of PB, as well as Se, on male reproductive function are still limited, and more evidence is needed to draw definitive conclusions as to the therapeutic effects of PB and/or Se administration on male fertility .
In light of the aforementioned knowledge, our laboratory has developed Se-enriched probiotics (OSePB) as a new feed additive product for promoting animal industries. To produce this OSePB, two strains of microorganisms, Candida utilis and Streptococcus thermophilus, were cultured under appropriate micro-environmental conditions with sodium selenite (an inorganic form of Se; ISe) being added into the culture media . The conversion of ISe into organic forms of Se (OSe) in the production of OSePB has been proved to be very efficient [17, 18]. Here we considered it worthwhile to investigate whether or not the OSePB administration exerts a synergistic effect of PB and Se on animal health in terms of hypolipidemic action and thus the amelioration of male infertility caused by hyperlipidemia. Thus, this study was designed to compare the effects of oral administration of PB, of ISe, and of OSePB on the male fertility of mice fed a high fat diet (HFD).
Materials and Methods
Cholesterol, bile salt, and an ISe product, sodium selenite (Na2SeO3), were all purchased from Beijing Chemical Reagent Co. (Beijing, China). Pork lard was purchased from a local farmers’ market, Tong Wei Road Market, in Nanjing city (Jiangsu, China).
Two probiotic strains, C. utilis (Cu.M02) and S. thermophilus (St.S07), were provided by the Institute of Nutritional and Metabolic Disorders of Domestic Animals and Fowls, Nanjing Agricultural University (Jiangsu, China). Both the PB and the OSePB products used in this study were produced in this laboratory with the two aforementioned probiotic strains. The colony forming units (CFU) of St.S07 and Cu.M02 in both products were approximately 1010/mL and 109/mL, respectively. The total Se content in the OSePB was 5.0 mg/L, with over 90% of Se being present in forms of OSe and over 75% as l-selenomethionine.
Animals and Feeding
Seventy-five male ICR mice at age of 4 weeks (body weights around 20 g) were purchased from the Center of Laboratory Animals, Nanjing Medical University (Nanjing, Jiangsu, China). As a general purpose stock, the strain of outbred ICR (abbreviation for Imprinting Control Region) or Swiss mice is widely used in pharmaceutical and oncological research . After purchase, the mice were housed in this laboratory animal room with standard management conditions. The room temperature was maintained at 20–25°C, and light exposure time was 12 h/day. The normal basal diet for the mice was also purchased from the Center of Laboratory Animals, Nanjing Medical University. The basal diets in a pellet form were formulated to meet or exceed the nutrient requirements as recommended by NRC  for normal laboratory mice.
An HFD was prepared by grinding the pellets of the basal diet into powder. Then this powder was fully mixed with lard, cholesterol, and cholic acid at a weight ratio of 83.7:15.0:1.0:0.3. The doses of PB, ISe, and OSePB were calculated, measured out, and fully mixed with the HFD to make three types of new pellets , which served as treatment diets and were respectively provided to the corresponding groups of mice as fresh.
Experimental Design and Sample Collection
According to a completely randomized experimental design, 75 mice were divided into five groups of 15 mice in each. The five groups of mice were then randomly assigned to five dietary treatments as follows:
To group 1, a negative control (NC) group, a normal basal diet containing 0.05 μg/g of Se was fed. To group 2, an HFD group (as a positive control), the HFD containing 0.04 μg/g of Se was fed. To group 3, a PB treatment group, the HFD supplemented with PB was fed. To group 4, an ISe treatment group, the HFD supplemented with ISe was fed. To group 5, an OSePB treatment group, the HFD supplemented with OSePB was fed. The number of viable microbial cells in the PB-supplemented and the OSePB-supplemented diets was approximately 109 CFU/g, and the final total Se concentration in the ISe-supplemented and the OSePB-supplemented diets was 0.3 μg/g.
The five treatment diets prepared for the five experimental groups of mice were replenished daily, and fresh water was accessible all the time throughout the trial which lasted for 75 days. At the end of the experiment, mice were fasted for 12 h before anaesthetized with ether inhalation. Blood was then collected from the orbital plexus using capillary tubes for serum separation. After that, the mice were euthanized by cervical dislocation, abdominal cavity opened, and male reproductive organs removed.
Total serumal testosterone concentration was determined by a radioimmunoassay using a commercial assay kit (Beijing Atom Hightech Co, Ltd., Beijing, China) on an automated analyzer (UniCel™ DxI 800, Beckman, USA). The commercial kit used for determination of serumal T3 and T4 concentration was the Coat-A Count RIA total T3 & T4 kit purchased from Beijing North Institute of Biological Technology (Beijing, China) and used according to the manufacturer’s instructions.
Sex Organ Pathology and Semen Evaluation
Both the left and right testes were taken from the removed reproductive organs and weighed. A relative testis weight (percent) was calculated by dividing the absolute weight of a testis by its corresponding absolute total body weight. Penis lengths were measured in centimeter. The epididymis was dissected in 20 ml saline (0.9% NaCl) and incubated at 37°C, and semen was then collected from it. Semen smears were made from the suspension and stained with 1% eosin to measure sperm index parameters, which include the number of sperms, sperm motility, and sperm abnormalities . Sperm numbers were counted using a haemocytometer, and the sperm motility was evaluated under a light microscope. For each animal, 300 sperms were examined to count and record different types of abnormality. The types of abnormal sperm heads include small, pig, amorphous, banana, hammer, pin, folded, hookless, and double heads. The types of abnormal sperm tails include short, folded, and double tails.
For testis histopathology examination, small pieces of tissue samples were taken from the removed testes and fixed in 10% neutral buffered formalin (3.7% formaldehyde in H2O). After samples were trimmed and embedded in paraffin, sections of 4–6 μm thick were prepared and stained with haematoxylin and eosin (H & E stain) . Under a light microscope, the shape, the size, and the cell normality of the seminiferous tubules of the testes were examined, and so were the size and the cell density of the interstitial tissue. These histopathological features in the mice of groups 2 through 5 were all compared and scored against those in group 1 NC mice.
Data were analyzed with the statistical model of one-way analysis of variance (ANOVA) using the SPSS computer program (version 19.0) for Windows (IBM SPSS Statistics). The differences among the five dietary treatment groups were compared using a Least Significant Difference (LSD) post hoc test. Final data for each treatment group were expressed as mean±standard error of the means (SEM). A level of P value less than 0.05 was considered statistically significant.
Serumal Lipid Profile and Testosterone Concentration
Effect of probiotics (PB), inorganic selenium (ISe), and Se-enriched probiotics (OSePB) on the serumal lipid profile and the testosterone level of the mice
Serumal testosterone levels (Table 1) exhibited a significant decrease (P < 0.01) in the HFD group when compared to the NC group. When compared to the HFD group, although the increase in the testosterone level of the PB group was not significant (P > 0.05), the increases in the levels of both the ISe and OSePB groups were highly significant (P < 0.05). Furthermore, it can be seen that the serumal testosterone levels in either the ISe or the OSePB group were increased up to the level that was close to the level in the NC group.
Sex Organ Pathology
Effect of probiotics (PB), inorganic selenium (ISe), and Se-enriched probiotics (OSePB) on relative weights (percent) of testes and penis length of mice
Penis length (cm)
Left testes (%)
Right testes (%)
Effect of probiotics (PB), inorganic selenium (ISe), and Se-enriched probiotics (OSePB) on the testis histopathology of mice
Effect of probiotics (PB), inorganic selenium (ISe), and Se-enriched probiotics (OSePB) on murine sperm count and motility
Sperm count (×106/epididymis)
Sperm motility (%)
Effect of probiotics (PB), inorganic selenium (ISe), and Se-enriched probiotics (OSePB) on murine sperm abnormality (percent)
For sperm tail abnormality, it can be seen also from Table 5 that feeding HFD to the mice increased (P < 0.01) the percentages of all three types of tail defect, which include folded (2.3%), short (0.88%), and double (0.35%) tails. Dietary supplementation of PB, ISe, and OSePB almost all significantly reduced (P < 0.05) the percentages of folded, short, and double tails when compared to the HFD group, except that dietary PB supplementation increased (P < 0.05) the percentage of folded tail. In addition, the percentages of short and double tails in the PB, ISe, and OSePB groups were close (P > 0.05) to that in the NC group.
The overall percentages of sperm head and tail abnormalities in the HFD group were higher (P < 0.01) than the NC group (Table 5). The overall percentages of sperm head abnormality in the PB group were lower (P < 0.05) than those in the HFD group. The overall percentage of sperm tail abnormality in the ISe group was further lower (P < 0.05) than the PB group, and the overall percentages of both sperm head and tail abnormalities in the OSePB group were further lower (P < 0.05) than those in the PB group.
Hyperlipidemic Effects on Murine Male Fertility
The increased levels of serumal TC, TG, and LDL and decreased level of HDL following the feeding of HFD to group 2 mice in this study are in agreement with the previous findings by other investigators [24, 25], and this agreement validated that the HFD feeding resulted in a hyperlipidemic murine model to produce adverse effects of hyperlipidemia on male fertility for this study. In these hyperlipidemic mice, both the degenerative changes in testicular tissues and the decrease in serumal testosterone level were observed, and these changes might result from the degeneration of Leydig cells since it has been shown that cholesterol-rich diets can produce detrimental effects on the secretary capacity of Leydig and Sertoli cells .
Histopathological examination of the testicular tissues of the hyperlipidemic mice in this study revealed marked misshapen and atrophied seminiferous tubules, and degeneration of spermatocytes (evidence of decreased spermatogenesis), as were also observed in a previous study . In hyperlipidemic rat it was found that the population of testicular cells including spermatocytes and spermatids was significantly reduced . The reduction in sperm count in the hyperlipidemic mice of this study may be attributed to the direct effect of hyperlipidemia on the hypothalamo-pituitary-gonadal axis thus affecting spermatogenesis , or the increased oxidative stress. Both hypertriglyceridemia and hypercholesterolemia can increase the production of oxygen radicals and thus increase the levels of lipid peroxides in tissues and simultaneously decrease the antioxidant effect of glutathione [2, 29]. The oxidative stress-induced DNA damage may accelerate the process of germ cell apoptosis  leading to a decline in sperm counts.
The significant decrease of sperm motility observed in the group of HFD-fed mice of this study agrees with the result of Mohammad et al. , and this decrease could be attributed to the increased lipid peroxidation that causes morphological changes in sperms . The increase in reactive oxygen species may explain the increased number of abnormal sperm cells in our hyperlipidemic mice. The epididymal dysfunction in hypercholesterolemic animals may also result in decreased sperm motility and increased morphometric abnormality . In short, the HFD-induced hyperlipidemia or hypercholesterolemia adversely affected the indexes of murine male fertility, which is supported by previous research on this matter [4, 6, 24, 25].
Effect of Probiotics on Male Fertility
In the PB group of this study, the serumal TC, TG, and LDL levels were decreased when compared to the HFD group. A similar result was reported earlier by Fukushima and Nakao  that S. thermophilus, Saccharomyces cerevisiae, and C. utilis mixed with rice bran at rates of 107–108 CFU/g lowered the serumal levels of TC and LDL, as well as the liver level of cholesterol in hypercholesterolemic rats. Our results plus some other previous studies [34-37] suggest that the incorporation of a mixture of Bacillus, Lactobacillus, Streptococcus, Clostridium, Saccharomyces, and Candida in animal diets can improve the balance of intestinal flora and metabolites and lower the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, a rate-controlling enzyme for cholesterol biosynthesis, and thus suppress the levels of liver and serumal cholesterol. However, the two probiotic strains used in the PB product of this study did not significantly increase the serumal HDL level, a result that does not agree with that of Fabian and Elmadfa .
Probiotics may decrease the systemic levels of blood lipids by inhibiting hepatic cholesterol biosynthesis as the bacteria in the lower intestines ferment and produce short chain fatty acids . One short chain fatty acid, propionic acid in particular, has been shown to decrease cholesterol synthesis in the liver and/or to redistribute cholesterol from plasma to the liver . Some bacteria may interfere with cholesterol absorption from the gut either directly through assimilation of cholesterol  or indirectly by deconjugating bile salts and thus affecting the metabolism of cholesterol .
Further health benefits of probiotics include the stimulation of animal and human immune function [41, 42] and the improvement of animal and human antioxidant defense system [43, 44]. Although there is a scarcity of reports in the literature regarding the direct link between probiotics and animal male fertility [45, 46], the significant improvement in sex organ histology and semen index parameters was found in this study with those mice fed the HFD diet supplemented with PB. And this beneficial effect may have resulted from an indirect consequence of the hypolipidemic and antioxidant bioactivities of PB. It was reported that oral administration of probiotics can improve animal antioxidative status , which is important in that it can protect sperm cells from the damage by harmful free radicals induced by lipid peroxidation.
Effect of Selenium on Male Fertility
Se has several essential biological functions including hypolipidemic action [11, 12, 47-49]. Dietary Se supplementation can protect LDL from oxidative modification and atherogenic change [50, 51] and can also increase the fraction of HDL-C . In this study, an ISe compound was supplemented to the HFD diet at 0.3 μg/g, and the serumal lipid profile in the mice was shifted. The hypolipidemic effect of Se may be attributed to the decreased synthesis as well as the increased catabolism of lipids . Dhingra and Bansal  concluded that dietary Se supplementation up to 1.0 μg/g led to decreased apolipoprotein B expression via modulation of type-I 5′-iodothyronine deiodinase (a selenoenzyme) expression. Such a mechanism may explain the protective role of Se against hypercholesterolemia.
The male fertility index improvement by ISe may be attributed to the hypolipidemic effect and/or the antioxidant action of Se through various selenoproteins. It has been reported that Se is essential in the development and production of sperm cells. Absence of Se in the testicular tissues induced degeneration that resulted in an active impairment of sperm motility . Se may be incorporated into the sperm mitochondria capsule and thus affect the development, behavior, and function of spermatozoa . The critical role of Se in spermatogenesis is mainly mediated by two selenoproteins, namely phospholipid hydroperoxide glutathione peroxidase and selenoprotein P, with the former being the major selenoprotein expressed by germ cells and representing the pivotal link between Se, sperm quality, and male fertility . These roles of Se could explain our finding that the histopathological changes in the testicular tissue of the ISe treated mice were less severe than the changes in the pure HFD-fed mice. Increased Se intake might have activated Leydig cells and influenced the secretion of testosterone.
Although Hawkes et al.  reported that Se supplementation had no effect on human serumal androgen concentration, sperm count, sperm morphology, and sperm motility or progressive velocity, Kaushal and Bansal  found that the fertility status of male mice was significantly reduced after the mice were fed either the Se-deficient or the Se-excess diets. In this study, the supplemental dose of Se was 0.3 μg/g, which was in a safe and adequate range for normal mice, and our results indicated that dietary supplementation of Se at this dose can improve murine male fertility compromised by feeding an HFD to the mice.
Effect of Selenium-Enriched Probiotics on Male Fertility
This research was the first to study the effect of OSePB supplementation to an HFD diet fed to male ICR mice on their hyperlipidemic and fertile parameters. The OSePB product used in this study was made from the same microbial strains as those used for making PB product, but enriched with Se, and over 90% of the Se was in the form of OSe. The results showed that the serumal levels of TC, TG, and LDL in the OSePB-fed mice were all lower than the levels in the PB-fed mice and that the TG level in the OSePB-fed mice was lower than in the ISe-fed mice. For serumal HDL level, it was only increased in the OSePB-fed mice when compared to the pure HFD-fed mice. Therefore, it can be concluded that OSePB supplementation is more effective in improving serumal lipid profile than the supplementation of ISe or PB alone.
Histopathological examination of the testes of the mice showed that the testicular tissue structure and the cell population in the OSePB-fed mice were better than the PB-fed mice and were almost normal when compared to the NC mice. Both sperm count and sperm motility values in the OSePB group were higher than in the PB group. The overall percentages of sperm head and tail abnormalities in the OSePB group were lower than those in the PB group. The serumal testosterone level in the OSePB group was higher than in the PB group. In short, it can be concluded that for improving murine male fertility indexes, the OSePB is definitely more effective than the PB alone although it is not dramatically more effective than the ISe alone.
The form of OSe is being increasingly used in diets for animals and humans as the supplemental Se in OSe form can be retained more efficiently in tissues [57-59]. On the other hand, the use of ISe is limited in many regions or countries because it may pollute the environment and be toxic to animals and humans when the intakes are high.
In summary, this study showed that feeding an OSePB-supplemented diet, which combines the hypolipidemic effects of PB and Se, was more effective in improving murine blood lipid profile than feeding the diet supplemented with ISe or PB alone. For improving male fertility indexes that could be compromised by hyperlipidemia and/or hypercholesterolemia, both OSePB-supplementations and ISe-supplementations were more effective than the supplementation of PB alone. With a consideration of the potential environmental pollution, it is herein suggested that the emerging product of OSePB should be a dietary supplementation of choice to improve animal and human fertility compromised by hyperlipidemia or obesity.
This research was supported by the National Natural Science Foundation of China (grant numbers: 30871892, 31011130155) and by the Priority Academic Program Development of Jiangsu Higher Education Institutions. We thank Professor A. A. Jameel, Department of Pathology, Faculty of Veterinary Medicine, University of Khartoum, Sudan, for reviewing the manuscript. Appreciation also goes to Ms. F. Tang for her help in diet preparation and Mr. M. O. A. Abdelrahim for his available support throughout the study.
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