Effect of dietary probiotics on the semen traits and antioxidative activity of male broiler breeders
Effect of dietary probiotics on the semen traits and antioxidative activity of male broiler breeders
OPEN Published: xx xx xxxx This study aimed to investigate the effect of probiotics on the intestinal morphology, intestinal microflora, oxidative activity (biological antioxidant potential), and semen quality of male broiler breeders. For this, 180 Cobb male broiler breeders (60 weeks of age) were randomly distributed into two groups. The control group was fed a basal diet, and the probiotics group was fed basal diet supplemented with probiotics for 6 weeks. Probiotics containing Bacillus amyloliquefaciens TOA5001 improved the above mentioned characteristics of the male broiler breeders. Thus, B. amyloliquefaciens TOA5001 might improve the reproductive performance of male broiler breeders.
period was 16 h?d?1. The birds in group 1 were fed a basal diet, and those in group 2 were fed basal diet
containing probiotics for 6 weeks. The probiotics (TOA Pharmaceutical Co. Ltd., Tokyo, Japan) containing Bacillus
amyloliquefaciens TOA5001 at 1 ? 108 colony-forming units?g?1 in rice bran was supplemented to the feed at 0.2%
(w/w). The basal diet consisted of commercially available antibiotic-free male broiler breeder feed (Kagoshima
Agricultural Economic Federation). The basal diet was used in a mashed form and formulated to meet the
nutritional requirements of 60?66-week-old male broiler breeders as per the guidelines of the Kagoshima Agricultural
Economic Federation. The composition of the basal diet and nutrient content are shown in Tabl1e?. The diet for
both the groups was provided at 125g feed per bird per day, and water was provided ad libitum.
Small intestinal morphology. After the semen, serum, and plasma samples were collected, all birds were
killed at 66 weeks of age to collect intestinal segments. The intestinal segment samples were collected from the
jejunum and ileum and, after their contents were flushed with physiological saline, the samples were submerged
in a fixative solution (0.1M collidine buffer, pH 7.3) containing 3% glutaraldehyde, 2% paraformaldehyde,
and 1.5% acrolein. They were then brought to the laboratory to determine the morphological changes. Three
cross-sections for each intestinal sample were prepared after staining with azure A and eosin by using standard
paraffin embedding procedures21. A total of 10 intact, well-oriented crypt-villus units were selected in triplicate
for each intestinal cross-section. The villus height, measured from the tip to the crypt junction, and crypt depth,
defined as the depth of the invagination between adjacent villi, were measured using Image-Pro Plus (Media
Cybernetics, Washington, USA) as described in detail by Touchette et a.l22. The villus height:crypt depth ratio
was also calculated.
pH of digestive tract contents. The pH of the different parts of the gastrointestinal tract was measured for
all birds as described previousl23y. Gut contents (10 g) were aseptically collected from the dissected jejunum and
ileum of each bird and placed in 90mL sterilised physiological saline (1:10 dilution; Terumo Corporation, Tokyo,
Japan), and the pH was determined using a pH meter (HORIBA Ltd., Kyoto, Japan).
Microbial enumeration. The microbial counts of jejunum and ileum were obtained as previously
described24. Approximately 1g of jejunal and ileal digesta was obtained from all birds and serially diluted 10-fold
(from 10?1 to 10?7) with sterile physiological saline solution (0.9% NaCl) and subsequently homogenised for
3 min by using an ultra-turrax. Dilutions were then plated onto selective agar medium for enumeration of the
target bacterial groups. Escherichia coliwere grown on MacConkey agar (Beijing Aoboxing Bio-tech Co., Ltd.,
Beijing, China). Lactobacilli were cultivated using de Man?Rogosa?Sharpe agar (Oxoid Ltd., Hampshire, UK).
Lactobacillus plates were incubated anaerobically, whereas E. coli plates were incubated aerobically at?3C7for
24 h. Bacteria were enumerated by visual counting of colonies by using the best replicate set from dilutions that
resulted in 30 to 300 colonies per plate. The microbial enumerations of jejunum and ileum were expressed as
base-10 logarithm colony-forming units per gram of jejunum and ileum digesta.
Serum ?-tocopherol concentration. For the analysis of serum ?-tocopherol concentrations, blood
samples of all birds were collected from the wing vein and allowed to clot fomr3in0. Blood clots were centrifuged
at 3,000 ? g for 15 min at 4 ?C; the top yellow serum layer was pipetted into two 1-mL conical tubes and held at
?80 ?C. The ?-tocopherol level in the serum was determined using high-performance liquid chromatograp2h5y.
Reactive oxygen metabolites and biological antioxidant potential. The reactive oxygen
metabolite-derived compound (d-ROM) test provides a measure of the whole oxidant capacity of plasma against
N,N-diethylparaphenylendiamine in acidic buffer. Such oxidant capacity is mainly attributed to hydroperoxides,
with the contribution of other minor oxidant factors. The biological antioxidant potential (BAP) test evaluates
the capacity of the plasma sample to reduce ferric ions to ferrous ions. BAP varies primarily as a function of the
titres of the major oxidative barriers in the plasma (vitamin C, vitamin E, uric acid, bilirubin, etc.). For the
analysis d-ROMs and BAP, blood samples of all birds were collected from the wing vein in EDTA-containing blood
collection tubes and centrifuged (1,000? g for 15 min). The plasma supernatants were stored at ?80 ?C until
assayed. The d-ROMs and BAP were determined using commercial kits (Diacron, Grosseto, Italy) and FRAS4 (H
& D, Parma, Italy), respectively26.
Semen traits. The semen from all birds from each group was collected using the massage method as per the
procedure of Burrows and Quinn27. The spermatozoa density or sperm count in the semen was estimated using
a colorimetric method28. The live and dead spermatozoa were differentiated by staining with eosin and nigrosine
by using the method described by Swanson and Bearden29.
Statistical analysis. Mann?Whitney U tests were performed using EZR software (Saitama Medical Center,
Jichi Medical University); EZR is a graphical user interface for R (The R Foundation for Statistical Computing,
version 2.13.0). The significance level was set at p< 0.05.
The villus height, crypt depth, and villus height:crypt depth ratio from each group are shown in Tab2l.eT? he crypt
depth of jejunum and ileum was not markedly different between groups 1 and 2. In contrast, the villus height and
villus height:crypt depth ratio of the jejunum and ileum were significantly higher in group 2 than in group 1. The
pH of the digestive tract contents from the jejunum and ileum was significantly lower in group 2 than in group
The viable counts of Lactobacilluswere significantly higher in group 2 than in group 1, whereas those of
E. coli were significantly lower in group 2 than in group 1 (p< 0.05; Table3?). The serum ?-tocopherol
concentration, BAP, and sperm density, and proportion of live sperms were significantly higher in group 2 than in group 1
(Tables4,5). However, the d-ROM levels were not markedly different between groups 1 and 2 (Tabl4e).?
The major microbes used as probiotics in poultry production include Lactobacillu, sSaccharomyces, Streptococcus,
Aspergillus spp., and Bacillus30; their success in providing beneficial effects to the host depends on their ability
to tolerate heat, osmotic stress, and oxygen stressors during processing and storage31. B. amyloliquefaciens are
spore-forming bacteria having resistance to high temperature and harsh storage conditions and are generally
regarded as safe for use as probiotics in poultry production32?35. Several studies have shown favourable results
with broilers by using B. amyloliquefaciens32?35. In the present study, B. amyloliquefaciens TOA5001 was mixed
with a carrier (rice bran) such that the addition of 2g?kg?1 of diet would yield 180colony-forming units per kg
diet. Probiotics are known to be efficacious in animals at the daily intake level of 71?0109 microorganisms36?38.
In this study, dietary supplementation with B. amyloliquefaciens TOA5001 did not show any adverse effects on
male broiler breeders. The structure and integrity of the intestinal epithelium are important factors contributing
to gut health and subsequent digestive capacity. Villus height is generally recognised as a good indicator of the
function and activation of intestinal vill39i. Better villus height and villus height to crypt depth ratio suggest an
improvement in nutrient digestibility and absorption capacity of the small intestin4e0. The present study showed
that B. amyloliquefaciens TOA5001 improved the gut structure and resulted in a greater absorption surface, as
indicated by the improved villus height and villus height to crypt depth ratio. The effects of probiotics on gut
structure and integrity have also been reported in the literature. Jayaraman et a.4l1. and Xinjian et al.32,
respectively, reported that the inclusion of B. subtilisand B. amyloliquefaciens in broiler diets led to better villus height
and villus height to crypt depth ratio. These two factors have been shown to be related to the epithelial cell
turnover42. Inflammatory responses induced by pathogens or their toxins might cause the rapid epithelial cell
turnover43. Thus, the suppression of pathogenic bacteria by B. amyloliquefaciens TOA5001 might have resulted in the
better villus height and villus height to crypt depth ratio.
In the gastrointestinal tract, numerous microorganisms co-exist and constitute a symbiotic ecosystem in
equilibrium44. Various studies have shown that probiotics can positively modulate the composition of the intestinal
microflora of chickens via the stimulation of potentially beneficial bacterial populations and/or the reduction of
potentially pathogenic bacteria45. In the present study, jejunum and ileum samples of birds fed a diet containing
B. amyloliquefaciens TOA5001 had lower pH, higherLactobacillus concentration, and reduced E. coli counts. The
B. amyloliquefaciens TOA5001-mediated reduction in intestinal pH might be favourable for the colonisation of
lactobacilli and the suppression of E. co4l6i.
Mitsuoka47 indicated that irregular intestinal microflora can cause malabsorption of vitamins. In mouse,
rotavirus infection was shown to cause acute diarrhoea and vitamin deficiency48. In the present study, serum
vitamin E (?-tocopherol) concentrations were significantly higher in group 2 than in group 1. Probiotics have been
shown to increase serum vitamin E concentration in cattle by improving the intestinal environmen4t9. Thus, the
probiotics containing B. amyloliquefaciens TOA5001 might improve the digestive health of male broiler breeders.
Reactive oxygen metabolites are produced as a by-product of oxidative metabolism or exposure to oxidants
in food or the environment. Oxidant exposure leads to the production of toxic reactive oxygen species such as
free radicals, which in turn modify biological macromolecules. Vitamin E is known as an excellent biological
chain-breaking antioxidant that protects cells and tissues from lipid peroxidation induced by free radica50l,5s1. It
also increased plasma BAP in sheep exposed to heat stres5s2. However, few studies have linked BAP and probiotic
supplements in chickens. In the present study, although d-ROM was not markedly different between groups 1
and 2, BAP was significantly higher in group 2 than in group 1. These data indicate that male broiler breeders in
groups 1 and 2 were under the same oxidative stress conditions, but probiotic supplements increased the
antioxidative activity of the male broiler breeders. B.amyloliquefaciens TOA5001 supplementation was suggested
to increase serum vitamin E concentrations and improve the antioxidative activity of male broiler breeders by
increasing antioxidant absorption in the intestine.
In the present study, the sperm density and proportion of live sperms were significantly higher in group 2 than
in group 1. The results of sperm density are in agreement with those of a previous study in which broiler breeders
were fed diets supplemented with yeast cultur5e3. The results of the proportion of live sperms was in agreement
with that of a previous study in which broiler breeders after zinc-induced moulting were fed diets supplemented
with probiotics54. The observed improvement in sperm concentration in the B. amyloliquefaciens TOA5001-fed
males might have been due to the enhanced availability of nutrients facilitated by more efficient nutrient
absorption by the gastrointestinal tract. Furthermore, several studies have indicated higher antioxidant activity in chic-k
ens fed probiotic-supplemented diet2s3,55, and B. amyloliquefaciens TOA5001 improved the antioxidative activity
of male broiler breeders in this study. Thus, the relationship between the improvement in the activity of
antioxidants such as glutathione peroxidase (GSH-Px) and superoxide dismutase in B. amyloliquefaciens TOA5001-fed
birds and spermatozoa production and maturation needs to be considered. High levels of GSH-Px are found
in the testes, and they act as powerful antioxidants in developing spermatids and spermatozo5a6. Spermatozoa
are adversely affected by high concentrations of peroxides in the testes, semen, and uterovaginal sperm host
glands57,58. In organs such as testes that have high metabolic rates, levels of antioxidants required to ensure the
survival of spermatozoa in aerobic environments are high. Thus, the high density of spermatozoa and high
proportion of live sperms recorded in B. amyloliquefaciens TOA5001-fed males in this study might be attributed to
the influence on the antioxidant activity.
Our results indicate that probiotics containing B. amyloliquefaciensTOA5001 improve intestinal morphology,
intestinal microflora, oxidative activity (biological antioxidant potential), and semen quality (sperm count and
liver sperm) of male broiler breeders. Therefore, their use would be advantageous to the poultry industry.
T.I. performed the experiments and drafted the manuscript. K.O. designed the study and supervised the project.
Competing Interests: The authors declare no competing interests.
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