Effect of an Organic Trace Mineral Premix on the Semen Quality , Testicular Morphology and Gene Expression Related to Testosterone Synthesis of Male Broiler Breeders

In order to investigate the effect of organic trace minerals premix (OTM) on the reproductive performance of breeder roosters, a total of 240 San Huang roosters (23 weeks of age) were randomly divided into two treatments with six replicates of 20 roosters each. The first group (n = 120) was fed a basal diet containing an inorganic trace minerals premix (ITM) and the other group (n = 120) was fed the basal diet in which ITM was replaced by OTM. The experiment period was 22 weeks. Semen from one randomly-selected rooster per replicate was collected two weeks after the beginning of the experiment and other 10 times every two weeks. Another rooster per replicate was randomly selected at 30, 35, and 45 weeks of age, and sacrificed. Results showed that OTM did not affect relative organ weights. There was a significant increase in semen parameters in OTM group (p<0.05), such as semen volume, semen density, and semen motility from 31 to 35 weeks. OTMfed roosters presented higher serum testosterone levels at 45 weeks of age, as well as higher testicular mRNA expression of the genes 3-beta dehydrogenase 2 (HSD3B2) and cytochrome P450 17A1 (CYP17A1) in the OTM-fed group at 45 weeks of age compared with those fed ITM (p<0.05). Considering the results of the present study, it was concluded that feeding organic instead of inorganic trace minerals to male broilers breeders improves semen quality, which may be attributed to their better testicular development and higher expression of enzymes related to testosterone synthesis.


INTRODUCTION
Trace minerals are daily required for poultry growth and production, and are important intermediates in physiological and biochemical processes (Leeson et al., 2005).Three main types of trace mineral supplements are added to animal feeds: inorganic metal elements, such as ferrous (Fe) sulfate, zinc (Zn) oxide, and cupric chloride; primary simple organic compounds, like Fe citric acid and Zn gluconate; and organic trace minerals (OTM), such as copper glycine (Cu), iron glycine, and Zn methionine, among others (Saeid et al., 2013).
Organic trace minerals are chelates, formed by ionic bond or covalent bonds between the ligand (amino acids, small peptides, proteins, organic acids, and polysaccharide derivatives) and the metallic element.Organic trace minerals present better rate of absorption and utilization, good chemical stability, higher biological potency, and delay the antagonism among different minerals (Spears, 1996) compared with inorganic and simple organic trace minerals.
Lipid peroxidation is lethal to the sperm cells.The antioxidant glutathione peroxidase (GSH-Px), containing selenium (Se) in its activity center, which can improve GSH-Px catalytic activity directly, plays a crucial role in protecting sperm from oxidative damage (Mohammad

Effect of an Organic Trace Mineral Premix on the Semen Quality, Testicular Morphology and Gene Expression Related to Testosterone Synthesis of Male Broiler Breeders
& Moslemi, 2011).Selenium deficiency may slow down the development of the testis and epididymis, decreasing sperm density in semen, and increasing the production of abnormal sperm cells (Ahsan et al., 2014;Graupner et al., 2015).
Zinc is closely related with the antibacterial property of the seminal fluid.In addition, Zn plays a role in the stabilization of the sperm membrane, preventing its degradation (Taniguchi et al., 2007).The early development of the prostate requires high Zn levels, and its deficiency delays the development of secondary sexual characteristics, and reduces testosterone serum levels (Xu et al., 2015;Kothari & Chaudhari, 2016).
Copper is required to maintain the stability of the luteotropic hormone (LH) and the follicle-stimulating hormone (FSH) in the serum.In addition, it enhances prostate capacity to secrete testosterone by improving the binding capacity of prostaglandin E2 (PGE2) to its receptor in the prostate (Rajeswari & Swaminathan 2014;Wang et al., 2014).PEG2 can regulate the synthesis of LH through copper, increasing LH serum levels, thereby inducing testosterone secretion to promote the differentiation and maturation of sperm cells (Sakumoto et al., 2014).
High concentration of manganese (Mn) chloride in diet can affect the development of testes, impairing spermatogenesis, interfering with sperm cell growth and development, and resulting in the production of abnormal sperm cells (Gu & Hecht 1996).In addition, serum Mn excess induces significant changes in testes histology and physiology, and cause seminiferous tubule degeneration (Gabrielsen & Tanrikut, 2016;McGough & Jardine, 2017).
Most previous studies evaluated the effects of single inorganic element on the male reproductive system, and there is limited research on the effects of the dietary addition of OTM on the reproductive performance of male broiler breeders.
In the present study, rooster of the San Huang Chinese chicken breed were fed with Zn, Mn, Cu, and Fe chelated with glycine, and Se yeast to determine their effect on semen quality, tissue accumulation, testicular morphology, serum testosterone level, and the gene expression of testosterone synthesis-related enzymes.

Birds, diets and experimental design
The experiments were carried out on a farm belonging to Jiangsu Lihua Animal Husbandry Stock Co, Ltd., Jiangsu, China.The San Huang roosters were housed in a commercial broiler breeder farm, and fed according to the Chicken Feed Regulations of Lihua.Roosters were used to semen collection every two days.
Birds were housed in individual cages and maintained under uniform management conditions (15 h light/day, 20-30 °C).Water was provided ad libitum.The following feed allowances were supplied, according to age: 129 g per bird from 23 to 26 weeks, 121 g per bird from 27 to 34 weeks, and 131 g per bird from 35 to 45 weeks of age.
Two hundred and forty San Huang roosters with 23 weeks of age were randomly divided into two treatments, with six replicates of 20 birds each.In the control treatment, the basal diet (Table 1) was supplemented with 100% inorganic trace minerals (ITM) at levels of 55, 60, 5.5, 70, and 0.15 mg of Zn, Mn, Cu, Fe, and Se per kg of diet, respectively, in the form of sulfate, except for Se, which was added as sodium selenite.The inorganic compounds contained 34.5% Zn, 31.8%Mn, 25% Cu, 30% Fe, and 0.45% Se, according to the products' specifications.In the second treatment, the basal diet did not contain ITM and was supplemented with 100% OTM, at the levels of 45, 49, 4.5, 58, and 0.13 mg of Zn, Mn, Cu, Fe, and Se per kg of diet, respectively, as methionine chelates,

Effect of an Organic Trace Mineral Premix on the Semen Quality, Testicular Morphology and Gene Expression Related to Testosterone Synthesis of Male Broiler Breeders
except for glycine-Fe and Se-yeast.The organic trace mineral product specifications are 16% Zn, 13% Mn, 15% Cu, 17% Fe, and 0.2% Se.The OTM were purchased from Novus International Trade Co, Ltd.
The basal diet was based on corn and soybean meal basal and formulated to contain adequate levels of all nutrients, as recommended by the National Research Council (NRC, 1994).The experimental period was 22 weeks.One rooster was randomly selected per replicate for semen collection.Semen was collected for the first time 2 weekss after the beginning of the treatments, and 10 times thereafter in 2-week intervals (at 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45 weeks of age).One rooster per replicate was randomly selected at three time points (30, 35, and 45 weeks of age) and sacrificed for blood and organ collection.

Sample collection
Blood samples were collected in plastic tubes and centrifuged at 1369.6 (×g) for 15 min at 4 °C.The serum was separated and stored at -20 °C until assayed for testosterone levels.Internal organs (heart, liver and spleen) and reproductive organs (testis and epididymis) were excised and immediately weighed.A subsample from each testis sample was fixed in 4% paraformaldehyde and processed for histological examination; the remaining testes were stored in liquid nitrogen for RT-PCR.

Determination of the five evaluated trace minerals concentrations in the feed, serum and testis
Diet and testis samples (0.5 g), as well as blood samples (0.5 mL) were digested with nitric acid (75%) and perchloric acid (25%) overnight at 25 ºC.After the solution became faint yellow, the samples were placed in a digestion System.After digestion, the solution was removed to 25-mL volumetric flask, then levels of the five supplemented trace minerals (Zn, Mn, Cu, Fe, and Zn) were measured by inductively coupled plasma optical emission spectrometry (ICP-OES, Perkin Elmer Optima 2100 DV, USA).The results were determined against the standard curve of each element.

Semen quality analysis
The semen was collected by dorso-abdominal massage (Burrows & Quinn, 1937).A semen sample was placed into a sterile stoppered graduated measuring glass, and its volume was immediately measured and recorded.After collection, the sample was incubated at 37 °C until analysis.A 10-μL sample was diluted (1:20) in hydrosaline solution (3%, wt/vol; NaCl) to determine sperm concentration using hemocytometer.Sperm motility was visually assessed in at least 10 microscopic fields, and the percentage of moving sperm relative to non-motile cells was calculated.Sperm morphology was assessed in 300 sperm cells per air-dried sample.

Testis histology
The testis samples fixed in formaldehyde were dehydrated in graded ethanol series, cleared in xylene, and embedded in paraffin wax.Sections (5-μm thickness) were then cut and were with hematoxylin and eosin (HE).The stained sections were mounted on slides and examined under light microscopy (400 ×).

Radioimmunoassay
The serum concentrations of testosterone were determined by the Nanjing General Hospital of the Nanjing Military Command using a 125 I-labeled radioligand double-antibody RIA kit.The commercial RIA kit was purchased from Beijing Beifang Institute of Biotechnology.The intra-and inter-assay coefficients of variation were less than 10% for testosterone.

Reverse transcription-polymerase chain reaction
Total RNA was extracted from the tissue using a RNeasy mini kit (Qiagen, China) according to the manufacturer's instructions.The concentration and purity of the isolated total RNA was determined spectrophotometrically at 260 and 280 nm with a Nanodrop 8000 (Thermo Fisher Scientific, Wilmington, USA).The total RNA (1 μg) was reverse transcribed to cDNA with an Omniscript Reverse Transcription kit (Takara, Japan) with Oligo-dT primers (Takara, Japan) according to the manufacturer's instructions.The target fragments were quantified by real-time PCR using a QuantiTectTMSYBR Green PCR Kit (F Hoffmann-La Roche Ltd., Switzerland) with 100 ng of the cDNA template.The gene expression data were normalized to β-actin expression.The specific primer sets, designed using Primer 5 Plus program, are described in Table 2.For the quantification of real-time PCR results, the threshold cycle Ct was determined for each reaction.Ct values for each gene of interest were normalized to the housekeeping gene; PCR amplification efficiencies were taken into account by amplifying various amounts of target cDNA for each reaction.Normalized values were used to calculate the degree of induction or inhibition expressed as a "fold difference" compared with normalized control values.

Effect of an Organic Trace Mineral Premix on the Semen Quality, Testicular Morphology and Gene Expression Related to Testosterone Synthesis of Male Broiler Breeders
Therefore, all of the data were statistically analyzed as "fold induction" between treated and control animals.

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM) and were analyzed by one-way analysis of variance (ANOVA).Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).Differences were considered statistically significant when p value was less than 0.05.

Semen quality
As shown in Table 3, semen volume increased in the OTM group both from 31 to 35 weeks and from 37 to 45 weeks, compared with the ITM group (p<0.01).Higher sperm density (p<0.001) and motility (p<0.01) were determined in the OTM group compared with the ITM group from 31 to 35 weeks.Lower abnormal sperm morphology was detected in the OTM group than in the ITM group from 25 to 45 weeks (p<0.05).

Body weight and relative organ weights
Body weight and relative organ weights at 3-time points were shown in Table 4. Body weight and relative organ weights were not significantly different between groups as determined from 30 to 45 weeks at 3-time points.

Trace minerals concentrations in the diet, serum and testis
The trace minerals levels were evaluated in the diet, and in the serum and testis of the birds fed OTM or ITM at 30, 35, and 45 weeks of age (Table 5).
The OTM diet presented lower Mn and Zn levels (p<0.001)than the ITM diet, but the levels of the other evaluated trace minerals (Cu, Fe, Se) were not different.Testicular Zn level at 45 weeks of age were higher (p<0.05) in the OTM group compared with the ITM group.In 30-week-old birds, serum Fe level was lower in the OTM group compared with the ITM group (p<0.01),but not at the other evaluated ages.Serum Mn levels in the OTM group were higher than in the ITM group at 45 weeks (p<0.05).

Testicular histology
Figure 1 shows the testicular histology of roosters evaluated at 30, 35, and 45 weeks of age.As shown in Fig. 1, the testicular histology was not different

Effect of an Organic Trace Mineral Premix on the Semen Quality, Testicular Morphology and Gene Expression Related to Testosterone Synthesis of Male Broiler Breeders
between ITM-and OTM-fed roosters at 30 and 35 weeks of age.The seminiferous epithelium was tighter in the OTM group than in the ITM group at 45 weeks of age, and the number of spermatogonia, primary spermatocytes and Leydig cells were higher in the OTM group compared with the ITM group.

Effect of an Organic Trace Mineral Premix on the Semen Quality, Testicular Morphology and Gene Expression Related to Testosterone Synthesis of Male Broiler Breeders
expression levels of the HSD3B2 and CYP17A1 genes were determined in the OTM-fed roosters compared with those fed ITM at 45 weeks of age (p<0.05) (Fig. 3A and 3B), but not at younger ages (30 and 35 weeks of age).In contrast with the ITM-fed birds, the mRNA expression level of the HSD17B4 gene in the OTMfed birds was higher at 30 (p<0.01) and 35 (p<0.05)weeks of age, but lower at 45 (p<0.05)weeks of age (Fig. 3C).The mRNA expression of the CYP19A1 gene was lower in the OTM-fed birds at 30 weeks of age (p<0.001), and higher at 35 and 45 weeks of age (p<0.01)than in those fed ITM (Fig 3D ).

DISCUSSION
This is the first report that focuses on the effects of the OTM on semen quality and testosterone synthesis in San Huang breeder roosters.The present study demonstrated that OTM had a positive impact on the growth of closed-packed spermatogonia and Leydig cells, and improved the semen quality of roosters from 31 to 35 weeks of age.At 45 weeks of age, roosters fed OTM presented significantly higher serum testosterone levels, as well as higher mRNA expression levels of the HSD3B2 and CYP17A1 genes in the testes.
Several markers are used to evaluate semen quality, such as semen volume, sperm density, sperm motility, and abnormal morphology.In the mammalian studies, trace minerals have been shown to be essential for improving semen quality.There is a positive correlation between OTM and high semen quality in broilers (Abdallah et al., 2009;Atig et al., 2013).The absence of Zn or Mn may suppress enzyme systems required for sperm motility and sperm count in rats (Yamaguchi et al., 2009;Babaei & Abshenas, 2013).The trace elements of Zn, Mn, and Se enhance spermatogenesis and improve semen quality in broiler breeders (Barber et al., 2005).It was reported that the supplementation of OTM, including Zn, Cu, Co, and Mn, improved the semen quality of bulls, even after freezing, compared with ITM (Rowe et al., 2013;Rowe et al., 2014).In the present study, the OTM-fed roosters presented better semen parameters, such as semen volume, density, motility, and number of normal sperm during the entire experimental period compared with those fed ITM, indicating that OTM improved semen quality.
Dietary organic mineral sources increase the intestinal absorption of trace elements as they do not form indigestible complexes with dietary antinutrient compounds (Świątkiewicz et al., 2014).Due to their high bioavailability, OTM supplementation of broiler diets is an efficient method to reduce trace mineral excretion in the environment (Bao et al., 2007).In the present study, the levels of the five trace minerals were higher in the ITM diet than in the OTM diet.However, both ITM-and OTM-fed roosters presented similar serum and testicular levels of these trace minerals, indicating that the organic forms had higher bioavailability and absorption rate than the inorganic forms.
A previous study reported that feeding high OTM levels increased pregnancy rate to artificial insemination of beef cows compared with ITM either at low or high levels (Stanton et al., 2000).Angus heifers fed OTM presented a higher number of fertilized oocytes than those fed ITM (Lamb et al., 2008).Feeding OTM benefitted sow reproductive performance compared with ITM (Peters & Mahan, 2008).In present study, the seminiferous epithelium was tighter in the OTM-fed roosters, which is consistent with mentioned previous studies showing that feeding OTM may improve reproductive performance.
Testosterone plays a vital role in the development of the testis, epididymis, an prostate, and on semen quality (Murashima et al., 2015).In the present study, dietary OTM increased serum testosterone levels.In order to investigate the potential mechanisms underlying the OTM-induced testosterone increase, the mRNA expression of enzymes related to the testosterone biosynthesis were measured.The HSD3B2, CYP17A1, hydroxysteroid 17-beta dehydrogenase 4 (HSD17B4) genes are known to encode enzymes involved in biosynthesis of testosterone (Taniguchi et al., 2007;Pierce et al., 2010;Rabbani et al., 2012).Cytochrome P450 19A1 (CYP19A1) converts androstenedione

Effect of an Organic Trace Mineral Premix on the Semen Quality, Testicular Morphology and Gene Expression Related to Testosterone Synthesis of Male Broiler Breeders
to estrone and testosterone to estradiol (Hosono et al., 2011).In the present study, the higher mRNA expression of the HSD3B2, CYP17A1 and HSD17B4 genes possibly indicates an increase in the activity of the corresponding enzymes, which accounts for the increase in the serum testosterone levels of the roosters fed OTM.Moreover, the testicular seminiferous epithelium may benefit from the elevated testosterone levels.

CONCLUSIONS
In summary, feeding male broiler breeders with organic trace minerals promoted better semen quality, as identified by its physical traits, compared with inorganic trace minerals.This result is possibly due to the increase in testosterone synthesis in OTM-fed roosters.The present experiment demonstrates that supplementing OTM to the basal diet is beneficial to the male broiler breeder reproductive system, and provides further evidence of the advantages of supplementing trace minerals in organic forms.

Figure 1 -
Figure 1 -Effects of dietary organic trace minerals premix (OTM) on spermatogonia and Leydig cells of the testes of male broiler breeders at 30 (A and B), 35 (C and D) and 45 (E and F) weeks of age.Sections were stained with hematoxylin and eosin (HE).Bar = 100 μm.Arrowhead shows spermatogonia and primary spermatocyte cells.Arrow shows Leydig cells (400 ×).

Figure 2
Figure2shows the serum testosterone levels of 30-, 35-, and 45-week-old roosters.Those fed OTM presented higher testosterone levels at 35 (p<0.05) and 45 (p<0.01)weeks of age than those fed ITM, but not at 30 weeks of age.

Figure 3
Figure 3 presents the mRNA expression levels of enzymes related to testosterone synthesis in the testes of 30-, 35-, and 45-week-old roosters.Higher mRNA ITM = inorganic trace minerals premix; OTM = organic trace minerals premix.The values shown are the mean ± SEM of 6 animals per group.The relative organ weights were calculated as the organ weights divided by BW (g/g BW).

Table 1 -
Composition of the basal diet.

Table 2 -
Primers for mRNA analyses.

Table 3 -
Effect of dietary organic trace minerals premix on semen quality.

Table 4 -
Effect of dietary organic trace minerals premix on body weight and the relative organ weight.