Impact of in vitro inoculation and dietary supplementation with Bacillus subtilis on sperm quality of aged White Leghorn roosters

Impact of in vitro inoculation and dietary supplementation with Bacillus subtilis on sperm... SUMMARY The probiotic Bacillus subtilis improves broiler performance; however, its effects on rooster reproduction are unknown. Therefore, 2 experiments (EXP) were conducted to evaluate impacts of B. subtilis on poultry semen quality. In EXP 1, B. subtilis was cultured for 48 h to a concentration of 108 CFU/mL. Semen from 72-week-old White Leghorn roosters was pooled and diluted 10-fold with the following treatments: 1) saline control, 2) sterile broth, 3) culture of B. subtilis, 4) supernatant from the culture, and 5) bacterial pellet from the culture. Semen pH and the sperm quality index (SQI) were obtained at 0 and 10 min post dilution to analyze effects of exposure length. The entire experiment was replicated 3 times. Semen pH and SQI were not affected by the B. subtilis pellet as compared to saline control. However, pH and SQI for every treatment containing broth were lower than the saline or pellet treatments. For EXP 2, 42 individually caged White Leghorn roosters, 74 wk old, were fed either 0 or 4.5 × 104 CFU of B. subtilis/g of feed. For each of 4 wk, individual semen samples were analyzed for pH, semen volume, sperm concentration, sperm viability, and the SQI. Additionally, semen concentrations of Na+, Ca2+, K+, Cl−, CO2, and O2 were measured. In the last week, ejaculates were serially diluted and plated to determine Bacillus spp. counts. The dietary addition of B. subtilis did not alter sperm quality characteristics, seminal ion concentrations, or Bacillus spp. counts. In conclusion, neither direct exposure of sperm nor dietary exposure of roosters to B. subtilis alters sperm quality, possibly because this bacterium is indigenous to the rooster's reproductive tract and semen. Therefore, feeding B. subtilis to roosters may not negatively affect fertility and may be an acceptable method to decrease pathogens, because B. subtilis alters intestinal microbiota. Future studies should investigate the effect of this probiotic on semen microbiota, especially regarding the presence of pathogenic bacteria that threaten public health. DESCRIPTION OF PROBLEM The use of antibiotics in broiler production has been widely practiced for decades to prevent diseases, improve broiler performance, and reduce mortality. In fact, apart from their therapeutic and prophylactic use, antibiotics have been supplemented into animal diets as antimicrobial growth promoters (AGP) for years [1]. The addition of antibiotics as AGP to livestock and poultry feed has been reported to improve animal performance by interacting with intestinal microbiota and by decreasing the population of pathogenic bacteria [1]. However, the risk associated with antimicrobial resistance has led to the use of alternatives to AGP, such as probiotics [2]. Probiotics are live microorganisms, including bacteria and fungi, that when adequately supplemented in the diet benefit host health [3, 4]. Supplementation with these feed additives helps to meet the consumer demand for antibiotic-free livestock and poultry products, decreases the risk to human health, and potentially alleviates the reduction in animal performance caused by the removal of antibiotics from animal feed [5]. Bacillus spp. are examples of microorganisms commonly exploited as probiotics for livestock and poultry [2, 6]. Bacillus spp. are Gram positive, aerobic or facultative anaerobic, and endospore-forming bacteria [7]. The genus encompasses a few pathogenic species and especially non-pathogenic bacteria, such as B. subtilis. This bacterium is commonly used as a dietary supplement to prevent gastrointestinal disorders and enhance growth performance [6, 8]. Because the population of B. subtilis gradually decreases after supplementation, its constant addition in the diet is required [9]. Unlike other non-pathogenic and Gram-positive bacteria, such as Lactobacillus and Bifidobacterium, B. subtilis can form spores (dormant life forms). In fact, these spores are predominantly provided in feed (rather than vegetative cells) due to their ability to resist heat, dehydration, and storage prior to consumption, as well as the low pH and bile salts found in the gastrointestinal tract [10]. Despite its complex and diverse effects, the modulation of intestinal microbiota by B. subtilis is an important mechanism of action to improve animal performance. For example, a previous study conducted on broiler chickens showed that the supplementation of cultured B. subtilis improved broiler intestinal microbiota by increasing the population of Lactobacilli and decreasing the population of E. coli as compared to the control group [11]. Furthermore, improvements in average daily gain and feed conversion rate were reported. Similar results were reported by Knap et al. [12] in broilers fed cultured B. subtilis, which showed a reduction of 58% and 3 log units in Salmonella-positive drag swabs and ceca counts, respectively, as opposed to the untreated group. Furthermore, a numerical improvement was reported in feed conversion rate and body weight gain at 42 days. In layer hens, the supplementation of a commercial probiotic containing B. subtilis was associated with improvements in egg quality by enhancing yolk color, albumen quality, shell strength, and shell thickness [13]. Although research concerning the effects of B. subtilis on poultry growth performance has been well documented, scarce information is available concerning the effect of this probiotic on rooster reproductive performance. However, B. subtilis is partially excreted through the cloaca, where the semen is also released during ejaculation. Therefore, it is possible that the microorganisms present in the cloaca can contaminate semen [14]. In fact, Bacillus spp. have been found in contaminated turkey semen, along with other bacteria species, such as Staphylococcus spp., coliforms, and Streptococcus spp. [15]. In addition, Wilcox and Shorb [16] also described the presence of different bacteria in rooster semen at a concentration of 2.2 × 106 CFU/mL. These findings suggest that semen contains several species of bacteria; however, their impacts on semen quality and fertility were not elucidated. Alternatively, research has demonstrated the direct effect of some species of bacteria on semen quality. For example, a previous in vitro study revealed that Salmonella and Campylobacter were apparently attached to different parts of spermatozoa when rooster semen was exposed to these bacteria [17]. In addition, the in vitro exposure of rooster semen to pathogenic bacteria (Salmonella, E. coli, Campylobacter, and Clostridium) decreased sperm motility, but the detrimental effect of bacteria on sperm motility was even more evident in the presence of Lactobacillus and Bifidobacterium, classified as non-pathogenic bacteria that are commonly used as probiotics [18]. However, research analyzing the effects of B. subtilis on semen quality is scarce. As a result, 2 experiments were conducted. The first objective was to evaluate if sperm motility was altered when rooster semen was directly exposed to B. subtilis or its metabolites, in vitro. The second objective was to determine the impact of dietary supplementation of B. subtilis on sperm quality as well as on semen pH, ionic composition, and Bacillus concentration. MATERIALS AND METHODS Experiment 1 Housing and care. In this experiment, semen from 30 White Leghorn roosters, 72 wk old, was obtained. Feed and water were provided ad libitum, and the birds received 16 h of light per day. The birds were fed a common basal diet (Table 1) for 4 wk before and also during the experimental period. Each rooster was caged in raised-wire cages and treated in accordance with the Guide for Care and Use of Laboratory Animals in Agricultural Research and Teaching [19]. Table 1. Experimental diet composition provided to 74- to 78-week-old White Leghorn roosters in Exp. 1 and 2. Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 1Sand was included to replace B. subtilis and maintain the inclusion level for remaining ingredients provided in the basal diet. 2The vitamin and mineral premix provided the following per kg diet: vitamin A, 7717 IU; vitamin D3, 2756 UI; vitamin E, 17 UI; vitamin B12, 0.01 mg; vitamin B6, 1.38 mg; niacin 28 mg; d- pantothenic acid, 6.6 mg; menadione, 0.83 mg; folic acid,0.69 mg; thiamine,1.1 mg; biotin 0.007 mg; choline, 386 mg; riboflavin, 6.61 mg; zinc;100 mg; iron, 50 mg; manganese, 100 mg; copper, 11.25 mg; iodine, 1.25 mg; selenium, 0.15 mg. View Large Table 1. Experimental diet composition provided to 74- to 78-week-old White Leghorn roosters in Exp. 1 and 2. Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 1Sand was included to replace B. subtilis and maintain the inclusion level for remaining ingredients provided in the basal diet. 2The vitamin and mineral premix provided the following per kg diet: vitamin A, 7717 IU; vitamin D3, 2756 UI; vitamin E, 17 UI; vitamin B12, 0.01 mg; vitamin B6, 1.38 mg; niacin 28 mg; d- pantothenic acid, 6.6 mg; menadione, 0.83 mg; folic acid,0.69 mg; thiamine,1.1 mg; biotin 0.007 mg; choline, 386 mg; riboflavin, 6.61 mg; zinc;100 mg; iron, 50 mg; manganese, 100 mg; copper, 11.25 mg; iodine, 1.25 mg; selenium, 0.15 mg. View Large Semen collection and analysis prior to treatment. On each of 3 alternating d, ejaculates from 10 White Leghorn roosters (30 roosters total), 72 wk old, were collected by the abdominal massage method of Burrows and Quinn [20] and pooled into a sterile scintillation vile. Before the addition of treatment solutions, semen was examined to determine if sperm concentration and viability were within the normal range, using a photometer [21] and fluorometer [22], respectively. B. subtilis culture One wk prior to the experiment, 1 g of B. subtilis probiotic product [23] was cultured in 9 mL of sterile fresh nutrient broth [24]. To provide appropriate growth conditions, 1 mL of the culture was aseptically transferred to 9 mL of sterile fresh nutrient broth every 48 h. The culture was incubated under aerobic conditions at 37°C [25] and simultaneously kept in constant motion on an Orbit Junior Shaker [26]. Immediately before inoculation of semen samples, B. subtilis counts for the product were found to be 108 CFU/mL after 24 h of incubation on mannitol egg yolk polymyxin agar (MYP) [27]. Treatments. The pooled semen samples were exposed to the following 5 treatments: phosphate buffered saline (PBS) control, sterile nutrient broth, B. subtilis culture of 108 CFU/mL, supernatant from the B. subtilis culture, and pellet from the B. subtilis culture. To create the supernatant and bacterial pellet treatments, 1 mL of the B. subtilis culture was placed in a microcentrifuge tube and centrifuged for 5 min in a microcentrifuge [28] at 8400 rpm (4700 x g). After centrifugation, the supernatant was aspirated and used for the supernatant treatment. The pellet in the bottom of the microtube after centrifugation was reconstituted with PBS to the original volume and then added to the neat semen. For all treatments, semen was diluted 10-fold (50 μl of semen and 450 μl of treatment solution) and thoroughly mixed in a microcentrifuge tube before the tests were performed. Semen analysis after treatment. After the addition of treatments, diluted semen was analyzed for the sperm quality index (SQI), using a sperm quality analyzer (SQA) [29], and pH [18] was determined by pH indicator strips [30]. Two readings for SQI and pH were obtained for each treatment at both 0 and 10 min after exposure of semen to each treatment under aerobic conditions. The experiment was replicated 3 times, on alternate days. Experiment 2 Housing and care. A total of 42 White Leghorn roosters was used in this experiment. Feed and water were provided ad libitum, and the birds received 16 h of light per day. All the roosters were fed a basal diet (Table 1) for an adaptation period of 5 wk. Roosters were individually caged in raised-wire cages and treated in accordance with the Guide for Care and Use of Laboratory Animals in Agricultural Research and Teaching [19]. Experimental diets and procedures. The concentration of B. subtilis (QST 713) in the commercially available product used in this current study was previously evaluated in the first experiment and determined to be 108 CFU/g. One wk before the beginning of the study, 42 White Leghorn roosters were divided into 2 equal groups, with 21 males per group. For 4 wk, males were fed, ad libitum, the following experimental diets: a control conventional rooster basal diet with no inclusion of B. subtilis or a Bacillus diet with inclusion of 4.5 × 104 CFU of B. subtilis/g of feed (0.045% of Opti bac S- manufacturer recommendation). Both diets (Table 1) were formulated to meet or exceed the NRC recommendations [31]. Semen collection and analysis. Individual semen samples from 42 White Leghorn roosters, 74 wk old, were collected by abdominal massage [20] weekly, for 4 wk. Immediately after semen collection, semen analysis was performed. Semen volume was obtained with a graduated microcentrifuge tube [32]. The SQI, sperm concentration, and sperm viability also were obtained by using the same methods described in Experiment 1. Two readings were obtained for each parameter. Additionally, pH and semen concentrations of Na+, Ca2+, K+, Cl−, CO2, and O2 were measured using an ABL77 gas and electrolyte analyzer [33]. Live performance. Every week, unconsumed feed was weighed for each rooster to determine feed intake. Because all the roosters were over 70 wk old and no longer in the growth stage, body weight and body weight gain were individually obtained only every 2 wk, at 74, 76, and 78 wk of age. Seminal microbial analysis. During the last wk (wk 4) of semen collection and immediately after the semen parameters were estimated, semen samples were kept on ice for a maximum of 2 h and analyzed to determine Bacillus concentrations. The variables measured to determine the concentration of Bacillus in semen samples included log CFU of Bacillus per mL of semen and per billion sperm in the ejaculate [34]. Statistical analysis Data from Experiment 1 were analyzed using a randomized complete block design with a split plot in time [35]. In Experiment 2, data were analyzed using a split plot design, with individually caged roosters serving as the experimental units and dietary treatments split over wk of the study [36]. RESULTS AND DISCUSSION Experiment 1 Semen analysis is a useful tool to predict rooster reproductive performance, by determining the number of viable and motile sperm in the ejaculate that is capable of fertilizing the egg and ultimately producing offspring [37]. In this current study, neat semen analysis performed before addition of any treatments revealed that the semen samples contained 3.3 billion sperm/mL and 7.4% dead sperm, which were similar to values reported in previous studies [22, 38]. Due to semen being collected from old roosters, it was expected that these parameters could be slightly worse as compared to younger roosters [39]. When the different treatments were added to semen, the overall main effect revealed that all treatments containing broth (sterile broth, Bacillus culture, and supernatant from the culture) had similar SQI values that were all drastically lower than those of the saline control or bacterial pellet treatments (P = 0.0001; Figure 1A). However, a time by treatment interaction was found for the SQI (P = 0.0007; Figure 1B). The interaction was due to an increase over incubation in the SQI of the saline control and pellet of B. subtilis treatments. However, a reduction in the SQI was observed in all remaining treatments between 0 and 10 min of exposure of semen to the treatments. During both 0 and 10 min of incubation, no difference was detected between the saline control and pellet of B. subtilis. However, at each of these time periods, the SQI was reduced in all the remaining treatments (P < 0.0001). Figure 1. View largeDownload slide Sperm quality index (SQI) for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on SQI. Means with no common superscript are significantly different at P < 0.0001; SEM = 14.22; n = 6 per treatment (3 blocks * 2 incubation times). (B) SQI interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0001; SEM = 10.552; n = 3 blocks. Figure 1. View largeDownload slide Sperm quality index (SQI) for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on SQI. Means with no common superscript are significantly different at P < 0.0001; SEM = 14.22; n = 6 per treatment (3 blocks * 2 incubation times). (B) SQI interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0001; SEM = 10.552; n = 3 blocks. The SQI is a measure of general sperm movement that is influenced by how often and how many sperm move across a light path [29]. Because the same original pool of semen, with a constant sperm concentration, was utilized to create all in vitro treatments in the present study, the SQI could have been affected only by sperm motility changes among treatments. The lack of a detrimental effect on sperm motility when semen was exposed in vitro to the reconstituted bacterial pellet suggests that B. subtilis does not directly have a negative effect on sperm movement. Additionally, because the SQI of the supernatant was actually greater than that of the broth, it is unlikely that B. subtilis metabolites negatively affect sperm motility. The detrimental effect on sperm motility of the treatments containing broth was possibly due to components of nutrient broth that could inhibit sperm motility. Similarly, Haines and cohorts [18] described a decline in SQI when rooster semen was exposed in vitro to tryptic soy broth. Additionally, the authors also revealed a decline in pH as compared to the saline control when rooster semen was incubated with tryptic soy broth at both 0 and 10 minutes. This decline in pH possibly contributed to the reduction observed in the SQI. In fact, similar to the SQI, the main effect for in vitro treatments, in the current study, revealed that all treatments containing broth yielded lower pH (P = 0.0013; Figure 2A) values as compared to the saline control and pellet treatments. However, a time by treatment interaction also occurred due to a decrease in pH over incubation when semen was exposed to the saline control, but an increase in pH over incubation when semen was diluted in bacterial culture (P = 0.016; Figure 2B). At 0 min, semen exposed to the saline control exhibited the highest pH compared to the other treatments, whereas the bacterial pellet exhibited a higher pH than sterile broth, bacterial culture, or the supernatant. By 10 min of incubation, no significant difference in pH was found between the saline control and the bacterial pellet, whereas semen pH was lower in all the remaining treatments, with the broth diluent exhibiting the lowest pH. These data suggest that the nutrient broth used to culture B. subtilis is mostly responsible for not only the reduction in sperm motility, but also a reduction in pH, whereas the direct exposure of semen to B. subtilis cells only does not alter the SQI or semen pH. Figure 2. View largeDownload slide pH for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on pH. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.062; n = 6 per treatment (3 blocks * 2 incubation times). (B) pH interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.063; n = 3 blocks. Figure 2. View largeDownload slide pH for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on pH. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.062; n = 6 per treatment (3 blocks * 2 incubation times). (B) pH interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.063; n = 3 blocks. However, the presence of other species of bacteria has been described to have a negative effect on sperm motility and semen pH. For example, a previous study revealed that sperm motility is reduced when rooster semen is directly exposed in vitro to Salmonella, E. coli, Campylobacter or Clostridium. However, in the same study, sperm motility was eliminated with exposure of rooster semen to Lactobacillus and Bifidobacterium, which, similar to B. subtilis, are Gram-positive bacteria commonly supplemented as probiotics in animal feed [18]. Furthermore, the direct exposure of rooster semen to all bacteria, except Salmonella, significantly lowered pH as compared to the saline control, and the greatest reduction in pH was again observed in semen exposed to Lactobacillus and Bifidobacterium as compared to the pathogenic bacteria. The reduction in pH upon exposure to Bifidobacterium and Lactobacillus was probably due to the production of lactic acid by these bacteria [40]. Because semen pH plays an important role in sperm function and movement, it is possible that this reduction in sperm motility was partially attributed to the reduction in pH [41]. In fact, in our study, the sterile broth treatment showed the lowest SQI and pH after 10 min of incubation, suggesting that the decrease in pH negatively affected sperm motility. Alternatively, the direct exposure to the pellet from the culture of B. subtilis did not alter sperm movement or pH after 10 min of incubation, as compared to the saline control. Because in the present study, the direct in vitro exposure of rooster semen to B. subtilis did not alter pH or motility, it is possible that B. subtilis do not use the damaging mechanisms described in other species of bacteria to reduce sperm function and semen quality. Similar to this current study, the presence of other Gram-positive bacteria, such as Micrococci and alpha-haemolytic Streptococci, in the ejaculate also did not alter human sperm movement or semen quality [42]. Perhaps B. subtilis does not have any detrimental effect on sperm quality, because Bacillus naturally occurs in the rooster reproductive tract and semen [15, 43]. Experiment 2 Throughout the study, no significant interactions were observed between dietary treatments and time (wk) for any parameter evaluated; therefore, only results for the main effect of diet will be discussed. Dietary supplementation of B. subtilis did not significantly alter SQI (P = 0.320), percentage dead sperm (P = 0.609), total sperm concentration (P = 0.929), live sperm concentration (P = 0.918), semen volume (P = 0.657), total sperm concentration per ejaculate (P = 0.727), or live sperm per ejaculate (P = 0.740; Table 2). These data suggest that the manufacturer recommended inclusion of B. subtilis (0.045% of Opti Bac S) does not alter rooster semen quality. Although the manufacturer claims that this probiotic contains 109 CFU of B. subtilis/g of product, in the current study the concentration of this bacterium was determined to be 108 CFU/g of product. Therefore, the concentration of B. subtilis added in the feed was about 4.5 × 104 CFU/g of feed. Because B. subtilis is provided in the feed as spores, which have been reported to survive harsh gastrointestinal tract conditions [9, 10], possibly the majority of B. subtilis included in the diet was still viable in the intestine. However, because in this current study the intestinal and fecal concentration of B. subtilis was not determined from control or roosters supplemented with B. subtilis, it is not possible to estimate the exact percentage of viable B. subtilis in the gut and feces that could have potentially contaminated rooster semen through the cloaca. Table 2. Semen quality parameters from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 1n = 42 (21 roosters per treatment). 2Sperm quality index. View Large Table 2. Semen quality parameters from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 1n = 42 (21 roosters per treatment). 2Sperm quality index. View Large In contrast to this study, previous research suggests that the addition of B. subtilis and B. licheniformis in the rooster's diet improves semen volume, sperm concentration, and sperm motility, and decreases the percentage of abnormal and dead spermatozoa in comparison to a control group [44]. However, in that work, both B. subtilis and B. licheniformis were supplemented together in the rooster's diet. Hence, it is unknown if an individual bacteria species or the interaction between both bacteria species improved semen quality. Additionally, semen samples were collected only once from 43-week-old Al–Salam roosters (a local Egyptian strain), whereas in this present research, ejaculates were obtained weekly from 74- to 78-week-old White Leghorn roosters. In fact, in poultry species, intestinal microbiota concentration and composition have been found to be affected by age. For example, Awad et al. (2016) [45] found a more diverse microbiota in 28-day-old chickens as compared to younger birds, whereas in laying hens, a higher concentration of Firmicutes and Bacteroidetes as well as a more diverse bacterial community were found in 30-week-old hens as compared to 8-week-old hens [46]. Therefore, a different seminal concentration of Bacillus ssp. could be found in younger males as opposed to the present study, in which both (control and supplemented) groups were composed of aged roosters. Similarly, pH (P = 0.548) as well as gas concentrations of O2 (P = 0.159) and CO2 (P = 0.189) and electrolyte concentrations of Na+ (P = 0.849), K+ (P = 0.315), Ca2+ (P = 0.654), and Cl− (P = 0.928, Table 3) were not significantly affected by the dietary supplementation of B. subtilis. Avian semen pH ranges from 6.9 to 7.1, and seminal buffer activity plays an important role in maintaining sperm livability because pH changes can be detrimental to spermatozoa. In fact, temperature as well as concentrations of uric and lactic acid have been shown to affect semen pH [41]. Semen also contains several elements that surround sperm and ensure viability by controlling osmolality and participating in enzymatic activity [47]. Research suggests that the concentration of various semen components may be affected by different factors, such as location of semen in the male reproductive tract and temperature to which roosters are exposed [41, 47]. Table 3. Semen pH and ionic concentrations from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 1n = 42 (21 roosters per treatment). View Large Table 3. Semen pH and ionic concentrations from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 1n = 42 (21 roosters per treatment). View Large Additionally, other species of bacteria have been known to alter semen composition and pH and, ultimately, decrease semen quality. For example, in humans, the presence of U. urealyticum is associated with poor semen quality due to the utilization of microelements in the ejaculate by this bacterium [48]. Moreover, in avian species, the in vitro inoculation of semen with Lactobacillus and Bifidobacterium, commonly used as probiotics in animal feed, decreases sperm motility, probably due to the reduction in pH caused by the production of lactic acid [18]. However, in the current study, the results indicate that dietary addition of B. subtilis does not alter semen pH or composition, probably because B. subtilis is a natural inhabitant of the male reproductive tract and semen. Additionally, B. subtilis supplemented roosters in the current study showed similar feed intake (P = 0.636), body weight (P = 0.515), and body weight gain (P = 0.825, Table 4) as compared to untreated birds. Although improvements in feed conversion, body weight, and other meat production parameters have been observed in response to the addition of dietary Bacillus spp. [49, 50], there are studies that report no improvement in growth performance with supplementation. For example, in a commercial trial, the addition of Bacillus spp. in broiler diets did not significantly affect body weight, body weight gain, feed intake, or feed conversion ratio when compared to bacitracin methylene disalicylate and control treatments [51]. Furthermore, the previous studies on B. subtilis supplementation were focused mainly on broiler chicken performance. Therefore, because the current study tested this probiotic in mature male layer breeders that are no longer in the growth stage, a rapid body weight change was not expected. Thus, results obtained in this study might be different from the broiler research with B. subtilis. Table 4. Rooster feed intake and body weight condition from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 1n = 42 (21 roosters per treatment). View Large Table 4. Rooster feed intake and body weight condition from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 1n = 42 (21 roosters per treatment). View Large Additionally, supplementation of B. subtilis in the feed did not alter Bacillus spp. counts per mL semen (P = 0.199) or Bacillus spp. counts per billion sperm (P = 0.381, Table 5). Previous studies suggest that some directly fed microorganisms, including B. subtilis, must be continuously supplemented in the diet because they are partially excreted from the gastrointestinal tract through the cloaca [9]. Because the semen is in direct contact with the cloaca during ejaculation, the bacteria present in this region might be a source of contamination in both natural mating and artificially inseminated flocks [14, 52]. However, in our study, the presence of Bacillus also was observed in seminal samples of non-treated birds, likely because these bacteria naturally occur in the rooster's reproductive tract and semen. In fact, Bacillus spp. have been described in turkey semen, along with other bacteria, including Staphylococcus, Escherichia, and Enterococcus, at a concentration of approximately 9 log CFU/mL [15]. Additionally, Wilcox and Shorb [16] also revealed the presence of bacteria in rooster semen at a concentration of 6 log CFU/mL. Similarly, in the present study, the concentrations of Bacillus spp. in semen from control and treated roosters were found to be 6.9 and 6.6 log CFU/mL, respectively (Table 5). Table 5. Bacillus spp. concentration in semen from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 1n = 39 (20 roosters for control and 19 roosters for birds supplemented with B. subtilis). View Large Table 5. Bacillus spp. concentration in semen from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 1n = 39 (20 roosters for control and 19 roosters for birds supplemented with B. subtilis). View Large The presence of Bacillus spp. in semen has been identified in other species. For example, Ghoneim et al. (2014) [53] found that Bacillus is one of the most common bacteria present in dromedary semen. Bacillus spp. also were isolated from 22% of boar semen samples, at a concentration of 3.6 × 107 CFU/mL of semen, along with other aerobic bacteria [54]. However, a previous study suggests that the presence of Bacillus spp. in boar semen possibly originated from external sources, such as tubing and extending systems, rather than the animal [55]. In this current trial, the sources of contamination were avoided by using sterile microtubes and by autoclaving the diluent before the semen was collected and analyzed. Additionally, the presence of bacteria in the vas deferens has not been described in poultry species [14]. Therefore, it is possible that bacteria contamination occurs at the cloaca and not in the upper part of the reproductive tract [14]. In fact, the microbiota found in semen and the cloaca are of similar composition, suggesting that the microorganisms present in the cloaca contaminate semen through excretion [14]. Previous studies have shown that supplementation of B. subtilis in poultry diets modulates the intestinal microbiota by increasing the population of Lactobacilli and decreasing the population of pathogenic bacteria, such as E.coli [11] and Salmonella [12], which are commonly associated with foodborne diseases [56, 57]. Bacteria released from the intestinal tract through the cloaca can contaminate semen. Therefore, future studies should evaluate the impacts of dietary inclusion of B. subtilis on overall flock fertility and the relationship of this probiotic with horizontal and vertical transmission of pathogenic bacteria from the roosters to the hens and chicks, respectively, which could ultimately be transmitted to humans. CONCLUSIONS AND APPLICATIONS This study suggests that direct in vitro exposure to semen or supplementation in the diet with B. subtilis does not have any detrimental impact on rooster semen volume, pH, ion and gas composition, or sperm motility, concentration, or viability. Bacillus spp. naturally occur in rooster semen, and adding B. subtilis in the diet did not result in an increased presence of this bacterium in semen. Due to the ability of B. subtilis to modulate intestinal microbiota and decrease the population of harmful bacteria, future research should investigate the impact of this bacterium on bacterial pathogens in semen. The interaction of B. subtilis with harmful bacteria present in the ejaculate, which could be vertically and horizontally transmitted to the offspring, could impact the incidence of foodborne diseases. Primary Audience: Microbiologists, Physiologists, Nutritionists, Veterinarians, Researchers REFERENCES AND NOTES 1. Castanon J. I. R. 2007 . History of the use of antibiotic as growth promoters in European poultry feeds . Poult. Sci. 86 : 2466 – 2471 . Google Scholar CrossRef Search ADS PubMed 2. Huyghebaert G. , Ducatelle R. , Van Immerseel F. . 2011 . An update on alternatives to antimicrobial growth promoters for broilers . Vet. J. 187 : 182 – 188 . Google Scholar CrossRef Search ADS PubMed 3. Miles R. D. , Bootwalla S. M. . 1991 . Direct-fed microbials in animal production . Pages 117 – 132 in Direct-Fed Microbials in Animal Production. A Review , National Food Ingredient Association , West Des Monies, Iowa, IA . 4. FAO/WHO . 2001 . Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria . Pages 1 – 34 . In Food and nutrition paper . American Córdoba Park Hotel, Córdoba, Argentina . 5. Park Y. H. , Hamidon F. , Rajangan C. , Soh K. P. , Gan C. Y. , Lim T. S. , Liong M. T. . 2016 . Application of probiotics for the production of safe and high-quality poultry meat . Korean J. Food Sci. Anim. Resour. 36 : 567 – 576 Google Scholar CrossRef Search ADS PubMed 6. Gaggìa F. , Mattarelli P. , Biavati B. . 2010 . Probiotics and prebiotics in animal feeding for safe food production . Int. J. Food Microbiol. 141 : S15 – S28 . Google Scholar CrossRef Search ADS PubMed 7. Turnbull P. C. B. , Hutson R. A. , Ward M. J. , Jones M. N. , Quinn C. P. , Finnie N. J. , Melling J. . 1992 . Bacillus anthracis but not always anthrax . J. Appl. Bacteriol . 72 : 21 – 28 . Google Scholar PubMed 8. Turnbull P. , Kramer J. , Melling J. . 1990 . Bacillus In: Topley and Wilson Principles of Bacteriology . Pages 185 – 210 . In Virology and Immunity . 8th ed , Edward Arnold , London . 9. Souza V. L. D. 2012 . Desempenho e Utilização de Nutrientes Por Vacas Leiteiras Suplementadas Com Bacillus Subtilis . PhD Diss . Univ. Federal do Parana , Parana, Brazil . 10. Hoal N. T. , Baccigalupi L. , Huxham A. . 2000 . Characterization of Bacillus species used of oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders . Appl. Environ. Microbiol. 66 : 5241 – 5247 . Google Scholar CrossRef Search ADS PubMed 11. Wu B. Q. , Zhang T. , Guo L. Q. , Lin J. F. . 2011 . Effects of Bacillus subtilis KD1 on broiler intestinal flora . Poult. Sci. 90 : 2493 – 2499 . Google Scholar CrossRef Search ADS PubMed 12. Knap I. , Kehlet A. B. , Bennedsen M. , Mathis G. F. , Hofacre C. L. , Lumpkins B. S. , Lay E. . 2011 . Bacillus subtilis (DSM17299) significantly reduces Salmonella in broilers . Poult. Sci. 90 : 1690 – 1694 . Google Scholar CrossRef Search ADS PubMed 13. Sobczak A. , Kozłowski K. . 2015 . The effect of a probiotic preparation containing Bacillus subtilis ATCC PTA-6737 on egg production and physiological parameters of laying hens . Ann. Anim. Sci. 15 : 711 – 723 . Google Scholar CrossRef Search ADS 14. Smith A. U. 1949 . The control of bacterial growth in fowl semen . J. Agric. Sci. 39 : 194 – 200 . Google Scholar CrossRef Search ADS 15. Gale C. , Brown K. I. . 1961 . The identification of bacteria contaminating collected semen and the use of antibiotics in their control . Poult. Sci. 40 : 50 – 55 . Google Scholar CrossRef Search ADS 16. Wilcox F. H. , Shorb M. S. . 1958 . The effect of antibiotics on bacteria in semen and on the motility and fertility ability of chicken spermatozoa . Am. J. Vet. Res. 19 : 945 – 949 . Google Scholar PubMed 17. Vizzier-Thaxton Y. , Cox N. A. , Richardson L. J. , Buhr R. J. , McDaniel C. D. , Cosby D. E. , Wilson J. L. , Bourassa D. V. , Ard M. B. . 2006 . Apparent attachment of Campylobacter and Salmonella to broiler breeder rooster spermatozoa . Poult. Sci. 85 : 619 – 624 . Google Scholar CrossRef Search ADS PubMed 18. Haines M. D. , Parker H. M. , McDaniel C. D. , Kiess A. S. . 2013 . Impact of 6 different intestinal bacteria on broiler breeder sperm motility in vitro . Poult. Sci. 92 : 2174 – 2181 . Google Scholar CrossRef Search ADS PubMed 19. NRC . 1996 . Guide for the care and Use of Laboratory Animals . 8th. ed. Natl. Acad. Press , Washington, DC . 20. Burrows W. H. , Quinn J. P. . 1937 . The collection of spermatozoa from the domestic fowl and turkey . Poult. Sci. 16 : 19 – 24 . Google Scholar CrossRef Search ADS 21. King L. M. , Donoghue A. M. . 2000 . Adaptation of the sperm mobility test for the identification of turkey toms with low fertilizing potential . J. Appl. Poult. Res. 9 : 66 – 73 . Google Scholar CrossRef Search ADS 22. Bilgili S. F. , Renden J. A. . 1984 . Fluorometric determination of avian sperm viability and concentration . Poult. Sci. 63 : 2275 – 2277 . Google Scholar CrossRef Search ADS PubMed 23. Bacillus subtilis product: QST 713 ; Opti Bac , Huvepharma , Peachtree City, GA . 24. Nutrient Broth . Catalog no.234000 , Becton Dickinson , Sparks, MD . 25. VWR, Model 1535 , Cornelius, OR . 26. Model 3520 , Pittsburgh, PA . 27. Mannitol Egg Yolk Polymyxin Agar . Catalog no. 2281010 , Becton Dickinson , Sparks, MD . 28. Eppendorf minispin , Hamburg, Germany . 29. McDaniel C. D. , Hannah J. L. , Parker H. M. , Smith T. W. , Schultz C. D. , Zumwalt C. D. . 1998 . Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index . Poult. Sci. 77 : 888 – 893 . Google Scholar CrossRef Search ADS PubMed 30. VWR, West Chester, PA . 31. NRC . 1994 . Nutrient Requirements of Poultry . 9th rev. ed. Natl. Acad. Press , Washington, DC . 32. Thermo scientific QSP, San Diego, CA . 33. Parker H. M. , McDaniel C. D. . 2006 . The immediate impact of semen diluent and rate of dilution on the sperm quality index, ATP utilization, gas exchange, and ionic balance of broiler breeder sperm . Poult. Sci. 85 : 106 – 116 . Google Scholar CrossRef Search ADS PubMed 34. From each sample, 100 μL of semen were serially diluted in 900 μL of PBS and mixed using a vortex to provide a homogenous mixture. For each serial dilution, 100 μL were aspirated and spread plated on petri dishes containing MYP agar. All samples were plated within 2 to 5 h after semen collection. Two agar plates were incubated for each dilution at 37°C for 48 hours. After the plates were removed from the incubator, the numbers of CFU per plate were counted . 35. Days (n = 3) represented the blocks, and split plots were the 2 lengths of incubation (0 or 10 min). The measured variables were analyzed using the GLM statistical procedure of SAS. When global P ≤ 0.10, means were separated by Fisher's protected least significant difference with α = 0.05] . 36. Steel R. G. D. , Torrie J. H. . 1980 . Principles and Procedures of Statistics: A Biometrical Approach . 2nd. ed. McGraw-Hill, Inc. , New York, NY . 37. Parker H. M. , McDaniel C. D. . 2002 . Selection of young broiler breeders for semen quality improves hatchability in an industry field trial . J. Appl. Poult. Res. 11 : 250 – 259 . Google Scholar CrossRef Search ADS 38. Dávila S. G. , Campo J. L. , Gil M. G. , Castaño C. , Santiago-Moreno J. . 2015 . Effect of the presence of hens on roosters sperm variables . Poult. Sci. 94 : 1645 – 1649 . Google Scholar CrossRef Search ADS PubMed 39. Tabatabaei S. , Chaji M. , Mohammadabadi T. . 2010 . Correlation between age of rooster and semen quality in Iranian indigenous broiler breeder chickens . J. Anim. Vet. Adv. 9 : 195 – 198 . Google Scholar CrossRef Search ADS 40. Ljungh A. , Wadstrom T. . 2006 . Lactic acid bacteria as probiotics . Curr Issues Intest Microbiol . 7 : 73 – 90 . Google Scholar PubMed 41. Al-Aghbari A. M. 1992 . Demonstration of a link between seminal plasma proteins and male fertility in the domestic fowl (Gallus domesticus) . PhD dissertation. Oregon State Univ . 42. Mehta R. H. , Sridhar H. , Vijay Kumar B. R. , Anand Kumar T. C. . 2002 . High incidence of oligozoospermia and teratozoospermia in human semen infected with the aerobic bacterium Streptococcus faecalis . RBM Online . 5 : 17 – 21 . Google Scholar PubMed 43. Donoghue A. M. , Blore P. J. , Cole K. , Loskutoff N. M. , Donoghue D. J. . Detection of campylobacter or salmonella in turkey semen and the ability of poultry semen extenders to reduce their concentrations 1 2 . Poult. Sci. 83 : 1728 – 1733 . CrossRef Search ADS PubMed 44. Abaza I. M. , Shehata M. A. , Shoieb M. S. . 2006 . Evaluation of some natural feed additive in layer diets . Egypt. Poult. Sci. 26 : 891 – 909 . 45. Awad W. A. , Mann E. , Dzieciol M. , Hess C. , Schmitz-Esser S. , Wagner M. , Hess M. . 2016 . Age-related differences in the luminal and mucosa-associated gut microbiome of broiler chickens and shifts associated with Campylobacter jejuni infection . Front. Cell. Infect. Microbiol. 6 . 46. Cui Y. , Wang Q. , Liu S. , Sun R. , Zhou Y. , Li Y. . 2017 . Age-related variations in intestinal microflora of free-range and caged hens . Front. Microbiol. 8 : 1310 . Google Scholar CrossRef Search ADS PubMed 47. Barna J. , Boldizsar H. . 1996 . Motility and agglutination of fowl spermatozoa in media of different amino acid content and pH value in vitro . Acta Vet. Hung. 44 : 221 – 232 . Google Scholar PubMed 48. Fraczek M. , Kurpisz M. . 2007 . Inflammatory mediators exert toxic effects of oxidative stress in human spermatozoa . J. Androl. 28 : 325 – 333 . Google Scholar CrossRef Search ADS PubMed 49. Li Y. , Xu Q. , Huang Z. , Lv L. , Liu X. , Yin C. , Yan H. , Yuan J. . 2016 . Effect of Bacillus subtilis CGMCC 1.1086 on the growth performance and intestinal microbiota of broilers . J. Appli. Microbiol. 120 : 195 – 204 . Google Scholar CrossRef Search ADS 50. Opalinski M. , Maiorka A. , Dahlke F. , Cunha F. , Vargas F. S. C. , Cardozo E. . 2007 . On the use of a probiotic (Bacillus subtilis-strain DSM 17299) as growth promoter in broiler diets . Rev. Bras. Cienc. Avic. 9 : 99 – 103 . Google Scholar CrossRef Search ADS 51. Dersjant-Li Y. , Awati A. , Kromm C. , Evans C. . 2013 . A direct fed microbial containing a combination of three-strain Bacillus sp. can be used as an alternative to feed antibiotic growth promoters in broiler production . J. Appl. Anim. Nutri. 2 : 1 – 6 Google Scholar CrossRef Search ADS 52. Haines M. D. 2012 . Bacteria and their effects on fertility in the chicken . Master thesis. Mississippi State University. Mississippi . 53. Ghoneim I. M. , Waheed M. M. , Al-Hofofi A. N. , Fayez M. M. , Al-Eknah M. M. , Al-Busadah K. A. , Al-Humam N. A. . 2014 . Evaluation of the microbial quality of fresh ejaculates of camel (Camelus dromedarius) semen . Anim. Reprod. Sci. 149 : 218 – 223 . Google Scholar CrossRef Search ADS PubMed 54. Gączarzewicz D. , Udała J. , Piasecka M. , Błaszczyk B. , Stankiewicz T. . 2016 . Bacterial contamination of boar semen and its relationship to sperm quality preserved in commercial extender containing gentamicin sulfate . Pol. J. Vet. Sci. 19 : 451 – 459 . Google Scholar PubMed 55. Baracaldo M. , Ward J. . 2008 . Quality control of extended boar semen . In Pages 195 – 206 , Vol. 2 , 8th London Swine Conference Proceedings. Facing the new reality , London, Ontario, Canada . 56. Zhao C. , Ge B. , De Villena J. , Sudler R. , Yeh E. , Zhao S. , White D. G. , Wagner D. , Meng J. . 2001 . Prevalence of Campylobacter spp., Escherichia coli, and Salmonella serovars in retail chicken, turkey, pork, and beef from the Greater Washington, DC, area . Appl. Environ. Microbiol , 67 : 5431 – 5436 . Google Scholar CrossRef Search ADS PubMed 57. Antunes P. , Mourão J. , Campos J. , Peixe L. . 2016 . Salmonellosis: The role of poultry meat . Clin. Microbiol. Infect. 22 : 110 – 121 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Impact of in vitro inoculation and dietary supplementation with Bacillus subtilis on sperm quality of aged White Leghorn roosters

Loading next page...
 
/lp/ou_press/impact-of-in-vitro-inoculation-and-dietary-supplementation-with-qoQKefRVgx
Publisher
Applied Poultry Science, Inc.
Copyright
© 2018 Poultry Science Association Inc.
ISSN
1056-6171
eISSN
1537-0437
D.O.I.
10.3382/japr/pfx072
Publisher site
See Article on Publisher Site

Abstract

SUMMARY The probiotic Bacillus subtilis improves broiler performance; however, its effects on rooster reproduction are unknown. Therefore, 2 experiments (EXP) were conducted to evaluate impacts of B. subtilis on poultry semen quality. In EXP 1, B. subtilis was cultured for 48 h to a concentration of 108 CFU/mL. Semen from 72-week-old White Leghorn roosters was pooled and diluted 10-fold with the following treatments: 1) saline control, 2) sterile broth, 3) culture of B. subtilis, 4) supernatant from the culture, and 5) bacterial pellet from the culture. Semen pH and the sperm quality index (SQI) were obtained at 0 and 10 min post dilution to analyze effects of exposure length. The entire experiment was replicated 3 times. Semen pH and SQI were not affected by the B. subtilis pellet as compared to saline control. However, pH and SQI for every treatment containing broth were lower than the saline or pellet treatments. For EXP 2, 42 individually caged White Leghorn roosters, 74 wk old, were fed either 0 or 4.5 × 104 CFU of B. subtilis/g of feed. For each of 4 wk, individual semen samples were analyzed for pH, semen volume, sperm concentration, sperm viability, and the SQI. Additionally, semen concentrations of Na+, Ca2+, K+, Cl−, CO2, and O2 were measured. In the last week, ejaculates were serially diluted and plated to determine Bacillus spp. counts. The dietary addition of B. subtilis did not alter sperm quality characteristics, seminal ion concentrations, or Bacillus spp. counts. In conclusion, neither direct exposure of sperm nor dietary exposure of roosters to B. subtilis alters sperm quality, possibly because this bacterium is indigenous to the rooster's reproductive tract and semen. Therefore, feeding B. subtilis to roosters may not negatively affect fertility and may be an acceptable method to decrease pathogens, because B. subtilis alters intestinal microbiota. Future studies should investigate the effect of this probiotic on semen microbiota, especially regarding the presence of pathogenic bacteria that threaten public health. DESCRIPTION OF PROBLEM The use of antibiotics in broiler production has been widely practiced for decades to prevent diseases, improve broiler performance, and reduce mortality. In fact, apart from their therapeutic and prophylactic use, antibiotics have been supplemented into animal diets as antimicrobial growth promoters (AGP) for years [1]. The addition of antibiotics as AGP to livestock and poultry feed has been reported to improve animal performance by interacting with intestinal microbiota and by decreasing the population of pathogenic bacteria [1]. However, the risk associated with antimicrobial resistance has led to the use of alternatives to AGP, such as probiotics [2]. Probiotics are live microorganisms, including bacteria and fungi, that when adequately supplemented in the diet benefit host health [3, 4]. Supplementation with these feed additives helps to meet the consumer demand for antibiotic-free livestock and poultry products, decreases the risk to human health, and potentially alleviates the reduction in animal performance caused by the removal of antibiotics from animal feed [5]. Bacillus spp. are examples of microorganisms commonly exploited as probiotics for livestock and poultry [2, 6]. Bacillus spp. are Gram positive, aerobic or facultative anaerobic, and endospore-forming bacteria [7]. The genus encompasses a few pathogenic species and especially non-pathogenic bacteria, such as B. subtilis. This bacterium is commonly used as a dietary supplement to prevent gastrointestinal disorders and enhance growth performance [6, 8]. Because the population of B. subtilis gradually decreases after supplementation, its constant addition in the diet is required [9]. Unlike other non-pathogenic and Gram-positive bacteria, such as Lactobacillus and Bifidobacterium, B. subtilis can form spores (dormant life forms). In fact, these spores are predominantly provided in feed (rather than vegetative cells) due to their ability to resist heat, dehydration, and storage prior to consumption, as well as the low pH and bile salts found in the gastrointestinal tract [10]. Despite its complex and diverse effects, the modulation of intestinal microbiota by B. subtilis is an important mechanism of action to improve animal performance. For example, a previous study conducted on broiler chickens showed that the supplementation of cultured B. subtilis improved broiler intestinal microbiota by increasing the population of Lactobacilli and decreasing the population of E. coli as compared to the control group [11]. Furthermore, improvements in average daily gain and feed conversion rate were reported. Similar results were reported by Knap et al. [12] in broilers fed cultured B. subtilis, which showed a reduction of 58% and 3 log units in Salmonella-positive drag swabs and ceca counts, respectively, as opposed to the untreated group. Furthermore, a numerical improvement was reported in feed conversion rate and body weight gain at 42 days. In layer hens, the supplementation of a commercial probiotic containing B. subtilis was associated with improvements in egg quality by enhancing yolk color, albumen quality, shell strength, and shell thickness [13]. Although research concerning the effects of B. subtilis on poultry growth performance has been well documented, scarce information is available concerning the effect of this probiotic on rooster reproductive performance. However, B. subtilis is partially excreted through the cloaca, where the semen is also released during ejaculation. Therefore, it is possible that the microorganisms present in the cloaca can contaminate semen [14]. In fact, Bacillus spp. have been found in contaminated turkey semen, along with other bacteria species, such as Staphylococcus spp., coliforms, and Streptococcus spp. [15]. In addition, Wilcox and Shorb [16] also described the presence of different bacteria in rooster semen at a concentration of 2.2 × 106 CFU/mL. These findings suggest that semen contains several species of bacteria; however, their impacts on semen quality and fertility were not elucidated. Alternatively, research has demonstrated the direct effect of some species of bacteria on semen quality. For example, a previous in vitro study revealed that Salmonella and Campylobacter were apparently attached to different parts of spermatozoa when rooster semen was exposed to these bacteria [17]. In addition, the in vitro exposure of rooster semen to pathogenic bacteria (Salmonella, E. coli, Campylobacter, and Clostridium) decreased sperm motility, but the detrimental effect of bacteria on sperm motility was even more evident in the presence of Lactobacillus and Bifidobacterium, classified as non-pathogenic bacteria that are commonly used as probiotics [18]. However, research analyzing the effects of B. subtilis on semen quality is scarce. As a result, 2 experiments were conducted. The first objective was to evaluate if sperm motility was altered when rooster semen was directly exposed to B. subtilis or its metabolites, in vitro. The second objective was to determine the impact of dietary supplementation of B. subtilis on sperm quality as well as on semen pH, ionic composition, and Bacillus concentration. MATERIALS AND METHODS Experiment 1 Housing and care. In this experiment, semen from 30 White Leghorn roosters, 72 wk old, was obtained. Feed and water were provided ad libitum, and the birds received 16 h of light per day. The birds were fed a common basal diet (Table 1) for 4 wk before and also during the experimental period. Each rooster was caged in raised-wire cages and treated in accordance with the Guide for Care and Use of Laboratory Animals in Agricultural Research and Teaching [19]. Table 1. Experimental diet composition provided to 74- to 78-week-old White Leghorn roosters in Exp. 1 and 2. Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 1Sand was included to replace B. subtilis and maintain the inclusion level for remaining ingredients provided in the basal diet. 2The vitamin and mineral premix provided the following per kg diet: vitamin A, 7717 IU; vitamin D3, 2756 UI; vitamin E, 17 UI; vitamin B12, 0.01 mg; vitamin B6, 1.38 mg; niacin 28 mg; d- pantothenic acid, 6.6 mg; menadione, 0.83 mg; folic acid,0.69 mg; thiamine,1.1 mg; biotin 0.007 mg; choline, 386 mg; riboflavin, 6.61 mg; zinc;100 mg; iron, 50 mg; manganese, 100 mg; copper, 11.25 mg; iodine, 1.25 mg; selenium, 0.15 mg. View Large Table 1. Experimental diet composition provided to 74- to 78-week-old White Leghorn roosters in Exp. 1 and 2. Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 Diet formulation Ingredient name Percent inclusion Corn 60.973 SBM 14.958 Wheat midds 20.000 Poultry fat 0.500 Dicalcium phosphate 1.419 Sand or B. subtilis1 0.045 Limestone: Calcium carbonate 0.971 Salt(NaCl) 0.155 Sodium bicarbonate 0.358 L- Lysine HCL 0.232 DL- Methionine 0.071 Choline- Cl 0.069 Nutra blend Vit TM premix2 0.250 Calculated composition Crude protein, CP (%) 15.261 AME poultry (Kcal/Kg) 2825.690 Lys, digestible (%) 0.777 Met, digestible (%) 0.265 TSAA, digestible (%) 0.459 Thr, digestible (%) 0.452 Calcium (%) 0.750 Phosphorus, total (%) 0.694 Phosphorus, available (%) 0.376 Sodium (%) 0.180 1Sand was included to replace B. subtilis and maintain the inclusion level for remaining ingredients provided in the basal diet. 2The vitamin and mineral premix provided the following per kg diet: vitamin A, 7717 IU; vitamin D3, 2756 UI; vitamin E, 17 UI; vitamin B12, 0.01 mg; vitamin B6, 1.38 mg; niacin 28 mg; d- pantothenic acid, 6.6 mg; menadione, 0.83 mg; folic acid,0.69 mg; thiamine,1.1 mg; biotin 0.007 mg; choline, 386 mg; riboflavin, 6.61 mg; zinc;100 mg; iron, 50 mg; manganese, 100 mg; copper, 11.25 mg; iodine, 1.25 mg; selenium, 0.15 mg. View Large Semen collection and analysis prior to treatment. On each of 3 alternating d, ejaculates from 10 White Leghorn roosters (30 roosters total), 72 wk old, were collected by the abdominal massage method of Burrows and Quinn [20] and pooled into a sterile scintillation vile. Before the addition of treatment solutions, semen was examined to determine if sperm concentration and viability were within the normal range, using a photometer [21] and fluorometer [22], respectively. B. subtilis culture One wk prior to the experiment, 1 g of B. subtilis probiotic product [23] was cultured in 9 mL of sterile fresh nutrient broth [24]. To provide appropriate growth conditions, 1 mL of the culture was aseptically transferred to 9 mL of sterile fresh nutrient broth every 48 h. The culture was incubated under aerobic conditions at 37°C [25] and simultaneously kept in constant motion on an Orbit Junior Shaker [26]. Immediately before inoculation of semen samples, B. subtilis counts for the product were found to be 108 CFU/mL after 24 h of incubation on mannitol egg yolk polymyxin agar (MYP) [27]. Treatments. The pooled semen samples were exposed to the following 5 treatments: phosphate buffered saline (PBS) control, sterile nutrient broth, B. subtilis culture of 108 CFU/mL, supernatant from the B. subtilis culture, and pellet from the B. subtilis culture. To create the supernatant and bacterial pellet treatments, 1 mL of the B. subtilis culture was placed in a microcentrifuge tube and centrifuged for 5 min in a microcentrifuge [28] at 8400 rpm (4700 x g). After centrifugation, the supernatant was aspirated and used for the supernatant treatment. The pellet in the bottom of the microtube after centrifugation was reconstituted with PBS to the original volume and then added to the neat semen. For all treatments, semen was diluted 10-fold (50 μl of semen and 450 μl of treatment solution) and thoroughly mixed in a microcentrifuge tube before the tests were performed. Semen analysis after treatment. After the addition of treatments, diluted semen was analyzed for the sperm quality index (SQI), using a sperm quality analyzer (SQA) [29], and pH [18] was determined by pH indicator strips [30]. Two readings for SQI and pH were obtained for each treatment at both 0 and 10 min after exposure of semen to each treatment under aerobic conditions. The experiment was replicated 3 times, on alternate days. Experiment 2 Housing and care. A total of 42 White Leghorn roosters was used in this experiment. Feed and water were provided ad libitum, and the birds received 16 h of light per day. All the roosters were fed a basal diet (Table 1) for an adaptation period of 5 wk. Roosters were individually caged in raised-wire cages and treated in accordance with the Guide for Care and Use of Laboratory Animals in Agricultural Research and Teaching [19]. Experimental diets and procedures. The concentration of B. subtilis (QST 713) in the commercially available product used in this current study was previously evaluated in the first experiment and determined to be 108 CFU/g. One wk before the beginning of the study, 42 White Leghorn roosters were divided into 2 equal groups, with 21 males per group. For 4 wk, males were fed, ad libitum, the following experimental diets: a control conventional rooster basal diet with no inclusion of B. subtilis or a Bacillus diet with inclusion of 4.5 × 104 CFU of B. subtilis/g of feed (0.045% of Opti bac S- manufacturer recommendation). Both diets (Table 1) were formulated to meet or exceed the NRC recommendations [31]. Semen collection and analysis. Individual semen samples from 42 White Leghorn roosters, 74 wk old, were collected by abdominal massage [20] weekly, for 4 wk. Immediately after semen collection, semen analysis was performed. Semen volume was obtained with a graduated microcentrifuge tube [32]. The SQI, sperm concentration, and sperm viability also were obtained by using the same methods described in Experiment 1. Two readings were obtained for each parameter. Additionally, pH and semen concentrations of Na+, Ca2+, K+, Cl−, CO2, and O2 were measured using an ABL77 gas and electrolyte analyzer [33]. Live performance. Every week, unconsumed feed was weighed for each rooster to determine feed intake. Because all the roosters were over 70 wk old and no longer in the growth stage, body weight and body weight gain were individually obtained only every 2 wk, at 74, 76, and 78 wk of age. Seminal microbial analysis. During the last wk (wk 4) of semen collection and immediately after the semen parameters were estimated, semen samples were kept on ice for a maximum of 2 h and analyzed to determine Bacillus concentrations. The variables measured to determine the concentration of Bacillus in semen samples included log CFU of Bacillus per mL of semen and per billion sperm in the ejaculate [34]. Statistical analysis Data from Experiment 1 were analyzed using a randomized complete block design with a split plot in time [35]. In Experiment 2, data were analyzed using a split plot design, with individually caged roosters serving as the experimental units and dietary treatments split over wk of the study [36]. RESULTS AND DISCUSSION Experiment 1 Semen analysis is a useful tool to predict rooster reproductive performance, by determining the number of viable and motile sperm in the ejaculate that is capable of fertilizing the egg and ultimately producing offspring [37]. In this current study, neat semen analysis performed before addition of any treatments revealed that the semen samples contained 3.3 billion sperm/mL and 7.4% dead sperm, which were similar to values reported in previous studies [22, 38]. Due to semen being collected from old roosters, it was expected that these parameters could be slightly worse as compared to younger roosters [39]. When the different treatments were added to semen, the overall main effect revealed that all treatments containing broth (sterile broth, Bacillus culture, and supernatant from the culture) had similar SQI values that were all drastically lower than those of the saline control or bacterial pellet treatments (P = 0.0001; Figure 1A). However, a time by treatment interaction was found for the SQI (P = 0.0007; Figure 1B). The interaction was due to an increase over incubation in the SQI of the saline control and pellet of B. subtilis treatments. However, a reduction in the SQI was observed in all remaining treatments between 0 and 10 min of exposure of semen to the treatments. During both 0 and 10 min of incubation, no difference was detected between the saline control and pellet of B. subtilis. However, at each of these time periods, the SQI was reduced in all the remaining treatments (P < 0.0001). Figure 1. View largeDownload slide Sperm quality index (SQI) for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on SQI. Means with no common superscript are significantly different at P < 0.0001; SEM = 14.22; n = 6 per treatment (3 blocks * 2 incubation times). (B) SQI interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0001; SEM = 10.552; n = 3 blocks. Figure 1. View largeDownload slide Sperm quality index (SQI) for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on SQI. Means with no common superscript are significantly different at P < 0.0001; SEM = 14.22; n = 6 per treatment (3 blocks * 2 incubation times). (B) SQI interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0001; SEM = 10.552; n = 3 blocks. The SQI is a measure of general sperm movement that is influenced by how often and how many sperm move across a light path [29]. Because the same original pool of semen, with a constant sperm concentration, was utilized to create all in vitro treatments in the present study, the SQI could have been affected only by sperm motility changes among treatments. The lack of a detrimental effect on sperm motility when semen was exposed in vitro to the reconstituted bacterial pellet suggests that B. subtilis does not directly have a negative effect on sperm movement. Additionally, because the SQI of the supernatant was actually greater than that of the broth, it is unlikely that B. subtilis metabolites negatively affect sperm motility. The detrimental effect on sperm motility of the treatments containing broth was possibly due to components of nutrient broth that could inhibit sperm motility. Similarly, Haines and cohorts [18] described a decline in SQI when rooster semen was exposed in vitro to tryptic soy broth. Additionally, the authors also revealed a decline in pH as compared to the saline control when rooster semen was incubated with tryptic soy broth at both 0 and 10 minutes. This decline in pH possibly contributed to the reduction observed in the SQI. In fact, similar to the SQI, the main effect for in vitro treatments, in the current study, revealed that all treatments containing broth yielded lower pH (P = 0.0013; Figure 2A) values as compared to the saline control and pellet treatments. However, a time by treatment interaction also occurred due to a decrease in pH over incubation when semen was exposed to the saline control, but an increase in pH over incubation when semen was diluted in bacterial culture (P = 0.016; Figure 2B). At 0 min, semen exposed to the saline control exhibited the highest pH compared to the other treatments, whereas the bacterial pellet exhibited a higher pH than sterile broth, bacterial culture, or the supernatant. By 10 min of incubation, no significant difference in pH was found between the saline control and the bacterial pellet, whereas semen pH was lower in all the remaining treatments, with the broth diluent exhibiting the lowest pH. These data suggest that the nutrient broth used to culture B. subtilis is mostly responsible for not only the reduction in sperm motility, but also a reduction in pH, whereas the direct exposure of semen to B. subtilis cells only does not alter the SQI or semen pH. Figure 2. View largeDownload slide pH for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on pH. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.062; n = 6 per treatment (3 blocks * 2 incubation times). (B) pH interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.063; n = 3 blocks. Figure 2. View largeDownload slide pH for rooster semen exposed to B. subtilis and diluents in Exp. 1. (A) Main effect of treatment on pH. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.062; n = 6 per treatment (3 blocks * 2 incubation times). (B) pH interaction between treatment and time. Means with no common superscript are significantly different at P < 0.0013; SEM = 0.063; n = 3 blocks. However, the presence of other species of bacteria has been described to have a negative effect on sperm motility and semen pH. For example, a previous study revealed that sperm motility is reduced when rooster semen is directly exposed in vitro to Salmonella, E. coli, Campylobacter or Clostridium. However, in the same study, sperm motility was eliminated with exposure of rooster semen to Lactobacillus and Bifidobacterium, which, similar to B. subtilis, are Gram-positive bacteria commonly supplemented as probiotics in animal feed [18]. Furthermore, the direct exposure of rooster semen to all bacteria, except Salmonella, significantly lowered pH as compared to the saline control, and the greatest reduction in pH was again observed in semen exposed to Lactobacillus and Bifidobacterium as compared to the pathogenic bacteria. The reduction in pH upon exposure to Bifidobacterium and Lactobacillus was probably due to the production of lactic acid by these bacteria [40]. Because semen pH plays an important role in sperm function and movement, it is possible that this reduction in sperm motility was partially attributed to the reduction in pH [41]. In fact, in our study, the sterile broth treatment showed the lowest SQI and pH after 10 min of incubation, suggesting that the decrease in pH negatively affected sperm motility. Alternatively, the direct exposure to the pellet from the culture of B. subtilis did not alter sperm movement or pH after 10 min of incubation, as compared to the saline control. Because in the present study, the direct in vitro exposure of rooster semen to B. subtilis did not alter pH or motility, it is possible that B. subtilis do not use the damaging mechanisms described in other species of bacteria to reduce sperm function and semen quality. Similar to this current study, the presence of other Gram-positive bacteria, such as Micrococci and alpha-haemolytic Streptococci, in the ejaculate also did not alter human sperm movement or semen quality [42]. Perhaps B. subtilis does not have any detrimental effect on sperm quality, because Bacillus naturally occurs in the rooster reproductive tract and semen [15, 43]. Experiment 2 Throughout the study, no significant interactions were observed between dietary treatments and time (wk) for any parameter evaluated; therefore, only results for the main effect of diet will be discussed. Dietary supplementation of B. subtilis did not significantly alter SQI (P = 0.320), percentage dead sperm (P = 0.609), total sperm concentration (P = 0.929), live sperm concentration (P = 0.918), semen volume (P = 0.657), total sperm concentration per ejaculate (P = 0.727), or live sperm per ejaculate (P = 0.740; Table 2). These data suggest that the manufacturer recommended inclusion of B. subtilis (0.045% of Opti Bac S) does not alter rooster semen quality. Although the manufacturer claims that this probiotic contains 109 CFU of B. subtilis/g of product, in the current study the concentration of this bacterium was determined to be 108 CFU/g of product. Therefore, the concentration of B. subtilis added in the feed was about 4.5 × 104 CFU/g of feed. Because B. subtilis is provided in the feed as spores, which have been reported to survive harsh gastrointestinal tract conditions [9, 10], possibly the majority of B. subtilis included in the diet was still viable in the intestine. However, because in this current study the intestinal and fecal concentration of B. subtilis was not determined from control or roosters supplemented with B. subtilis, it is not possible to estimate the exact percentage of viable B. subtilis in the gut and feces that could have potentially contaminated rooster semen through the cloaca. Table 2. Semen quality parameters from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 1n = 42 (21 roosters per treatment). 2Sperm quality index. View Large Table 2. Semen quality parameters from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 Sperm concentration Ejaculated sperm Treatment SQI2 Dead sperm Total Live Volume Total Live billion sperm/ –%– billion sperm/mL mL ejaculate Control 453 8.1 2.7 2.4 0.44 1.18 1.08 B. subtilis 439 8.5 2.6 2.4 0.45 1.21 1.11 SEM 10.3 0.67 0.13 0.12 0.021 0.076 0.071 P-value 0.320 0.609 0.929 0.918 0.657 0.727 0.740 1n = 42 (21 roosters per treatment). 2Sperm quality index. View Large In contrast to this study, previous research suggests that the addition of B. subtilis and B. licheniformis in the rooster's diet improves semen volume, sperm concentration, and sperm motility, and decreases the percentage of abnormal and dead spermatozoa in comparison to a control group [44]. However, in that work, both B. subtilis and B. licheniformis were supplemented together in the rooster's diet. Hence, it is unknown if an individual bacteria species or the interaction between both bacteria species improved semen quality. Additionally, semen samples were collected only once from 43-week-old Al–Salam roosters (a local Egyptian strain), whereas in this present research, ejaculates were obtained weekly from 74- to 78-week-old White Leghorn roosters. In fact, in poultry species, intestinal microbiota concentration and composition have been found to be affected by age. For example, Awad et al. (2016) [45] found a more diverse microbiota in 28-day-old chickens as compared to younger birds, whereas in laying hens, a higher concentration of Firmicutes and Bacteroidetes as well as a more diverse bacterial community were found in 30-week-old hens as compared to 8-week-old hens [46]. Therefore, a different seminal concentration of Bacillus ssp. could be found in younger males as opposed to the present study, in which both (control and supplemented) groups were composed of aged roosters. Similarly, pH (P = 0.548) as well as gas concentrations of O2 (P = 0.159) and CO2 (P = 0.189) and electrolyte concentrations of Na+ (P = 0.849), K+ (P = 0.315), Ca2+ (P = 0.654), and Cl− (P = 0.928, Table 3) were not significantly affected by the dietary supplementation of B. subtilis. Avian semen pH ranges from 6.9 to 7.1, and seminal buffer activity plays an important role in maintaining sperm livability because pH changes can be detrimental to spermatozoa. In fact, temperature as well as concentrations of uric and lactic acid have been shown to affect semen pH [41]. Semen also contains several elements that surround sperm and ensure viability by controlling osmolality and participating in enzymatic activity [47]. Research suggests that the concentration of various semen components may be affected by different factors, such as location of semen in the male reproductive tract and temperature to which roosters are exposed [41, 47]. Table 3. Semen pH and ionic concentrations from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 1n = 42 (21 roosters per treatment). View Large Table 3. Semen pH and ionic concentrations from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 O2 K+ Treatment pH nmol/mL CO2 Na+ μmol/mL Ca2+ Cl− Control 6.98 1.4 104 132.3 9.3 1.48 78 B. subtilis 7.01 2.1 95 132.1 8.8 1.45 78 SEM 0.033 0.34 4.5 1.05 0.32 0.051 2.2 P-value 0.548 0.159 0.189 0.849 0.315 0.654 0.928 1n = 42 (21 roosters per treatment). View Large Additionally, other species of bacteria have been known to alter semen composition and pH and, ultimately, decrease semen quality. For example, in humans, the presence of U. urealyticum is associated with poor semen quality due to the utilization of microelements in the ejaculate by this bacterium [48]. Moreover, in avian species, the in vitro inoculation of semen with Lactobacillus and Bifidobacterium, commonly used as probiotics in animal feed, decreases sperm motility, probably due to the reduction in pH caused by the production of lactic acid [18]. However, in the current study, the results indicate that dietary addition of B. subtilis does not alter semen pH or composition, probably because B. subtilis is a natural inhabitant of the male reproductive tract and semen. Additionally, B. subtilis supplemented roosters in the current study showed similar feed intake (P = 0.636), body weight (P = 0.515), and body weight gain (P = 0.825, Table 4) as compared to untreated birds. Although improvements in feed conversion, body weight, and other meat production parameters have been observed in response to the addition of dietary Bacillus spp. [49, 50], there are studies that report no improvement in growth performance with supplementation. For example, in a commercial trial, the addition of Bacillus spp. in broiler diets did not significantly affect body weight, body weight gain, feed intake, or feed conversion ratio when compared to bacitracin methylene disalicylate and control treatments [51]. Furthermore, the previous studies on B. subtilis supplementation were focused mainly on broiler chicken performance. Therefore, because the current study tested this probiotic in mature male layer breeders that are no longer in the growth stage, a rapid body weight change was not expected. Thus, results obtained in this study might be different from the broiler research with B. subtilis. Table 4. Rooster feed intake and body weight condition from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 1n = 42 (21 roosters per treatment). View Large Table 4. Rooster feed intake and body weight condition from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 Treatment Feed intake Body weight Body weight change Kg Control 0.10 2.19 0.003 B. subtilis 0.10 2.15 0.009 SEM 0.004 0.041 0.019 P-value 0.636 0.515 0.825 1n = 42 (21 roosters per treatment). View Large Additionally, supplementation of B. subtilis in the feed did not alter Bacillus spp. counts per mL semen (P = 0.199) or Bacillus spp. counts per billion sperm (P = 0.381, Table 5). Previous studies suggest that some directly fed microorganisms, including B. subtilis, must be continuously supplemented in the diet because they are partially excreted from the gastrointestinal tract through the cloaca [9]. Because the semen is in direct contact with the cloaca during ejaculation, the bacteria present in this region might be a source of contamination in both natural mating and artificially inseminated flocks [14, 52]. However, in our study, the presence of Bacillus also was observed in seminal samples of non-treated birds, likely because these bacteria naturally occur in the rooster's reproductive tract and semen. In fact, Bacillus spp. have been described in turkey semen, along with other bacteria, including Staphylococcus, Escherichia, and Enterococcus, at a concentration of approximately 9 log CFU/mL [15]. Additionally, Wilcox and Shorb [16] also revealed the presence of bacteria in rooster semen at a concentration of 6 log CFU/mL. Similarly, in the present study, the concentrations of Bacillus spp. in semen from control and treated roosters were found to be 6.9 and 6.6 log CFU/mL, respectively (Table 5). Table 5. Bacillus spp. concentration in semen from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 1n = 39 (20 roosters for control and 19 roosters for birds supplemented with B. subtilis). View Large Table 5. Bacillus spp. concentration in semen from 74- to 78-week-old White Leghorn roosters1 in Exp. 2. Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 Bacillus spp. Treatment Log CFU/mL of semen Log CFU/billion sperm Control 6.9 2.8 B. subtilis 6.6 4.2 SEM 0.14 1.13 P-value 0.199 0.381 1n = 39 (20 roosters for control and 19 roosters for birds supplemented with B. subtilis). View Large The presence of Bacillus spp. in semen has been identified in other species. For example, Ghoneim et al. (2014) [53] found that Bacillus is one of the most common bacteria present in dromedary semen. Bacillus spp. also were isolated from 22% of boar semen samples, at a concentration of 3.6 × 107 CFU/mL of semen, along with other aerobic bacteria [54]. However, a previous study suggests that the presence of Bacillus spp. in boar semen possibly originated from external sources, such as tubing and extending systems, rather than the animal [55]. In this current trial, the sources of contamination were avoided by using sterile microtubes and by autoclaving the diluent before the semen was collected and analyzed. Additionally, the presence of bacteria in the vas deferens has not been described in poultry species [14]. Therefore, it is possible that bacteria contamination occurs at the cloaca and not in the upper part of the reproductive tract [14]. In fact, the microbiota found in semen and the cloaca are of similar composition, suggesting that the microorganisms present in the cloaca contaminate semen through excretion [14]. Previous studies have shown that supplementation of B. subtilis in poultry diets modulates the intestinal microbiota by increasing the population of Lactobacilli and decreasing the population of pathogenic bacteria, such as E.coli [11] and Salmonella [12], which are commonly associated with foodborne diseases [56, 57]. Bacteria released from the intestinal tract through the cloaca can contaminate semen. Therefore, future studies should evaluate the impacts of dietary inclusion of B. subtilis on overall flock fertility and the relationship of this probiotic with horizontal and vertical transmission of pathogenic bacteria from the roosters to the hens and chicks, respectively, which could ultimately be transmitted to humans. CONCLUSIONS AND APPLICATIONS This study suggests that direct in vitro exposure to semen or supplementation in the diet with B. subtilis does not have any detrimental impact on rooster semen volume, pH, ion and gas composition, or sperm motility, concentration, or viability. Bacillus spp. naturally occur in rooster semen, and adding B. subtilis in the diet did not result in an increased presence of this bacterium in semen. Due to the ability of B. subtilis to modulate intestinal microbiota and decrease the population of harmful bacteria, future research should investigate the impact of this bacterium on bacterial pathogens in semen. The interaction of B. subtilis with harmful bacteria present in the ejaculate, which could be vertically and horizontally transmitted to the offspring, could impact the incidence of foodborne diseases. Primary Audience: Microbiologists, Physiologists, Nutritionists, Veterinarians, Researchers REFERENCES AND NOTES 1. Castanon J. I. R. 2007 . History of the use of antibiotic as growth promoters in European poultry feeds . Poult. Sci. 86 : 2466 – 2471 . Google Scholar CrossRef Search ADS PubMed 2. Huyghebaert G. , Ducatelle R. , Van Immerseel F. . 2011 . An update on alternatives to antimicrobial growth promoters for broilers . Vet. J. 187 : 182 – 188 . Google Scholar CrossRef Search ADS PubMed 3. Miles R. D. , Bootwalla S. M. . 1991 . Direct-fed microbials in animal production . Pages 117 – 132 in Direct-Fed Microbials in Animal Production. A Review , National Food Ingredient Association , West Des Monies, Iowa, IA . 4. FAO/WHO . 2001 . Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria . Pages 1 – 34 . In Food and nutrition paper . American Córdoba Park Hotel, Córdoba, Argentina . 5. Park Y. H. , Hamidon F. , Rajangan C. , Soh K. P. , Gan C. Y. , Lim T. S. , Liong M. T. . 2016 . Application of probiotics for the production of safe and high-quality poultry meat . Korean J. Food Sci. Anim. Resour. 36 : 567 – 576 Google Scholar CrossRef Search ADS PubMed 6. Gaggìa F. , Mattarelli P. , Biavati B. . 2010 . Probiotics and prebiotics in animal feeding for safe food production . Int. J. Food Microbiol. 141 : S15 – S28 . Google Scholar CrossRef Search ADS PubMed 7. Turnbull P. C. B. , Hutson R. A. , Ward M. J. , Jones M. N. , Quinn C. P. , Finnie N. J. , Melling J. . 1992 . Bacillus anthracis but not always anthrax . J. Appl. Bacteriol . 72 : 21 – 28 . Google Scholar PubMed 8. Turnbull P. , Kramer J. , Melling J. . 1990 . Bacillus In: Topley and Wilson Principles of Bacteriology . Pages 185 – 210 . In Virology and Immunity . 8th ed , Edward Arnold , London . 9. Souza V. L. D. 2012 . Desempenho e Utilização de Nutrientes Por Vacas Leiteiras Suplementadas Com Bacillus Subtilis . PhD Diss . Univ. Federal do Parana , Parana, Brazil . 10. Hoal N. T. , Baccigalupi L. , Huxham A. . 2000 . Characterization of Bacillus species used of oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders . Appl. Environ. Microbiol. 66 : 5241 – 5247 . Google Scholar CrossRef Search ADS PubMed 11. Wu B. Q. , Zhang T. , Guo L. Q. , Lin J. F. . 2011 . Effects of Bacillus subtilis KD1 on broiler intestinal flora . Poult. Sci. 90 : 2493 – 2499 . Google Scholar CrossRef Search ADS PubMed 12. Knap I. , Kehlet A. B. , Bennedsen M. , Mathis G. F. , Hofacre C. L. , Lumpkins B. S. , Lay E. . 2011 . Bacillus subtilis (DSM17299) significantly reduces Salmonella in broilers . Poult. Sci. 90 : 1690 – 1694 . Google Scholar CrossRef Search ADS PubMed 13. Sobczak A. , Kozłowski K. . 2015 . The effect of a probiotic preparation containing Bacillus subtilis ATCC PTA-6737 on egg production and physiological parameters of laying hens . Ann. Anim. Sci. 15 : 711 – 723 . Google Scholar CrossRef Search ADS 14. Smith A. U. 1949 . The control of bacterial growth in fowl semen . J. Agric. Sci. 39 : 194 – 200 . Google Scholar CrossRef Search ADS 15. Gale C. , Brown K. I. . 1961 . The identification of bacteria contaminating collected semen and the use of antibiotics in their control . Poult. Sci. 40 : 50 – 55 . Google Scholar CrossRef Search ADS 16. Wilcox F. H. , Shorb M. S. . 1958 . The effect of antibiotics on bacteria in semen and on the motility and fertility ability of chicken spermatozoa . Am. J. Vet. Res. 19 : 945 – 949 . Google Scholar PubMed 17. Vizzier-Thaxton Y. , Cox N. A. , Richardson L. J. , Buhr R. J. , McDaniel C. D. , Cosby D. E. , Wilson J. L. , Bourassa D. V. , Ard M. B. . 2006 . Apparent attachment of Campylobacter and Salmonella to broiler breeder rooster spermatozoa . Poult. Sci. 85 : 619 – 624 . Google Scholar CrossRef Search ADS PubMed 18. Haines M. D. , Parker H. M. , McDaniel C. D. , Kiess A. S. . 2013 . Impact of 6 different intestinal bacteria on broiler breeder sperm motility in vitro . Poult. Sci. 92 : 2174 – 2181 . Google Scholar CrossRef Search ADS PubMed 19. NRC . 1996 . Guide for the care and Use of Laboratory Animals . 8th. ed. Natl. Acad. Press , Washington, DC . 20. Burrows W. H. , Quinn J. P. . 1937 . The collection of spermatozoa from the domestic fowl and turkey . Poult. Sci. 16 : 19 – 24 . Google Scholar CrossRef Search ADS 21. King L. M. , Donoghue A. M. . 2000 . Adaptation of the sperm mobility test for the identification of turkey toms with low fertilizing potential . J. Appl. Poult. Res. 9 : 66 – 73 . Google Scholar CrossRef Search ADS 22. Bilgili S. F. , Renden J. A. . 1984 . Fluorometric determination of avian sperm viability and concentration . Poult. Sci. 63 : 2275 – 2277 . Google Scholar CrossRef Search ADS PubMed 23. Bacillus subtilis product: QST 713 ; Opti Bac , Huvepharma , Peachtree City, GA . 24. Nutrient Broth . Catalog no.234000 , Becton Dickinson , Sparks, MD . 25. VWR, Model 1535 , Cornelius, OR . 26. Model 3520 , Pittsburgh, PA . 27. Mannitol Egg Yolk Polymyxin Agar . Catalog no. 2281010 , Becton Dickinson , Sparks, MD . 28. Eppendorf minispin , Hamburg, Germany . 29. McDaniel C. D. , Hannah J. L. , Parker H. M. , Smith T. W. , Schultz C. D. , Zumwalt C. D. . 1998 . Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index . Poult. Sci. 77 : 888 – 893 . Google Scholar CrossRef Search ADS PubMed 30. VWR, West Chester, PA . 31. NRC . 1994 . Nutrient Requirements of Poultry . 9th rev. ed. Natl. Acad. Press , Washington, DC . 32. Thermo scientific QSP, San Diego, CA . 33. Parker H. M. , McDaniel C. D. . 2006 . The immediate impact of semen diluent and rate of dilution on the sperm quality index, ATP utilization, gas exchange, and ionic balance of broiler breeder sperm . Poult. Sci. 85 : 106 – 116 . Google Scholar CrossRef Search ADS PubMed 34. From each sample, 100 μL of semen were serially diluted in 900 μL of PBS and mixed using a vortex to provide a homogenous mixture. For each serial dilution, 100 μL were aspirated and spread plated on petri dishes containing MYP agar. All samples were plated within 2 to 5 h after semen collection. Two agar plates were incubated for each dilution at 37°C for 48 hours. After the plates were removed from the incubator, the numbers of CFU per plate were counted . 35. Days (n = 3) represented the blocks, and split plots were the 2 lengths of incubation (0 or 10 min). The measured variables were analyzed using the GLM statistical procedure of SAS. When global P ≤ 0.10, means were separated by Fisher's protected least significant difference with α = 0.05] . 36. Steel R. G. D. , Torrie J. H. . 1980 . Principles and Procedures of Statistics: A Biometrical Approach . 2nd. ed. McGraw-Hill, Inc. , New York, NY . 37. Parker H. M. , McDaniel C. D. . 2002 . Selection of young broiler breeders for semen quality improves hatchability in an industry field trial . J. Appl. Poult. Res. 11 : 250 – 259 . Google Scholar CrossRef Search ADS 38. Dávila S. G. , Campo J. L. , Gil M. G. , Castaño C. , Santiago-Moreno J. . 2015 . Effect of the presence of hens on roosters sperm variables . Poult. Sci. 94 : 1645 – 1649 . Google Scholar CrossRef Search ADS PubMed 39. Tabatabaei S. , Chaji M. , Mohammadabadi T. . 2010 . Correlation between age of rooster and semen quality in Iranian indigenous broiler breeder chickens . J. Anim. Vet. Adv. 9 : 195 – 198 . Google Scholar CrossRef Search ADS 40. Ljungh A. , Wadstrom T. . 2006 . Lactic acid bacteria as probiotics . Curr Issues Intest Microbiol . 7 : 73 – 90 . Google Scholar PubMed 41. Al-Aghbari A. M. 1992 . Demonstration of a link between seminal plasma proteins and male fertility in the domestic fowl (Gallus domesticus) . PhD dissertation. Oregon State Univ . 42. Mehta R. H. , Sridhar H. , Vijay Kumar B. R. , Anand Kumar T. C. . 2002 . High incidence of oligozoospermia and teratozoospermia in human semen infected with the aerobic bacterium Streptococcus faecalis . RBM Online . 5 : 17 – 21 . Google Scholar PubMed 43. Donoghue A. M. , Blore P. J. , Cole K. , Loskutoff N. M. , Donoghue D. J. . Detection of campylobacter or salmonella in turkey semen and the ability of poultry semen extenders to reduce their concentrations 1 2 . Poult. Sci. 83 : 1728 – 1733 . CrossRef Search ADS PubMed 44. Abaza I. M. , Shehata M. A. , Shoieb M. S. . 2006 . Evaluation of some natural feed additive in layer diets . Egypt. Poult. Sci. 26 : 891 – 909 . 45. Awad W. A. , Mann E. , Dzieciol M. , Hess C. , Schmitz-Esser S. , Wagner M. , Hess M. . 2016 . Age-related differences in the luminal and mucosa-associated gut microbiome of broiler chickens and shifts associated with Campylobacter jejuni infection . Front. Cell. Infect. Microbiol. 6 . 46. Cui Y. , Wang Q. , Liu S. , Sun R. , Zhou Y. , Li Y. . 2017 . Age-related variations in intestinal microflora of free-range and caged hens . Front. Microbiol. 8 : 1310 . Google Scholar CrossRef Search ADS PubMed 47. Barna J. , Boldizsar H. . 1996 . Motility and agglutination of fowl spermatozoa in media of different amino acid content and pH value in vitro . Acta Vet. Hung. 44 : 221 – 232 . Google Scholar PubMed 48. Fraczek M. , Kurpisz M. . 2007 . Inflammatory mediators exert toxic effects of oxidative stress in human spermatozoa . J. Androl. 28 : 325 – 333 . Google Scholar CrossRef Search ADS PubMed 49. Li Y. , Xu Q. , Huang Z. , Lv L. , Liu X. , Yin C. , Yan H. , Yuan J. . 2016 . Effect of Bacillus subtilis CGMCC 1.1086 on the growth performance and intestinal microbiota of broilers . J. Appli. Microbiol. 120 : 195 – 204 . Google Scholar CrossRef Search ADS 50. Opalinski M. , Maiorka A. , Dahlke F. , Cunha F. , Vargas F. S. C. , Cardozo E. . 2007 . On the use of a probiotic (Bacillus subtilis-strain DSM 17299) as growth promoter in broiler diets . Rev. Bras. Cienc. Avic. 9 : 99 – 103 . Google Scholar CrossRef Search ADS 51. Dersjant-Li Y. , Awati A. , Kromm C. , Evans C. . 2013 . A direct fed microbial containing a combination of three-strain Bacillus sp. can be used as an alternative to feed antibiotic growth promoters in broiler production . J. Appl. Anim. Nutri. 2 : 1 – 6 Google Scholar CrossRef Search ADS 52. Haines M. D. 2012 . Bacteria and their effects on fertility in the chicken . Master thesis. Mississippi State University. Mississippi . 53. Ghoneim I. M. , Waheed M. M. , Al-Hofofi A. N. , Fayez M. M. , Al-Eknah M. M. , Al-Busadah K. A. , Al-Humam N. A. . 2014 . Evaluation of the microbial quality of fresh ejaculates of camel (Camelus dromedarius) semen . Anim. Reprod. Sci. 149 : 218 – 223 . Google Scholar CrossRef Search ADS PubMed 54. Gączarzewicz D. , Udała J. , Piasecka M. , Błaszczyk B. , Stankiewicz T. . 2016 . Bacterial contamination of boar semen and its relationship to sperm quality preserved in commercial extender containing gentamicin sulfate . Pol. J. Vet. Sci. 19 : 451 – 459 . Google Scholar PubMed 55. Baracaldo M. , Ward J. . 2008 . Quality control of extended boar semen . In Pages 195 – 206 , Vol. 2 , 8th London Swine Conference Proceedings. Facing the new reality , London, Ontario, Canada . 56. Zhao C. , Ge B. , De Villena J. , Sudler R. , Yeh E. , Zhao S. , White D. G. , Wagner D. , Meng J. . 2001 . Prevalence of Campylobacter spp., Escherichia coli, and Salmonella serovars in retail chicken, turkey, pork, and beef from the Greater Washington, DC, area . Appl. Environ. Microbiol , 67 : 5431 – 5436 . Google Scholar CrossRef Search ADS PubMed 57. Antunes P. , Mourão J. , Campos J. , Peixe L. . 2016 . Salmonellosis: The role of poultry meat . Clin. Microbiol. Infect. 22 : 110 – 121 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc.

Journal

Journal of Applied Poultry ResearchOxford University Press

Published: Jan 25, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off