Dietary Supplementation with Sesame Seeds to Improve Semen Quality of Ho Cocks

High levels of polyunsaturated fatty acids in chicken spermatozoa make them susceptible to lipoperoxidation and reduce their fertility. This study was conducted to assess the effect of sesame seed suplementation in the diet on the semen quality of Ho cocks. Eighteen 13-14 month-old cocks were randomly divided into three groups and were assigned to one of the following treatments: 0% SS (control), 5% SS, or 7% sesame seeds per kg of diet for ten consecutive weeks after a two-week adaptation period. Semen characteristics were evaluated once a week. In the 7% sesame seed treatment group, seminal traits including semen ejaculate volume (1.02mL), sperm concentration (3.68 x 10 sperm), and abnormal spermatozoa (10.51% were improved (P <0.05) compared to the control group (0.82mL, 2.81 x 10 sperm, and 11.04% for semen ejaculate volume, sperm concentration, and abnormal spermatozoa, respectively). Supplementation with sesame seeds did not significantly affect sperm motility, mass movement, or semen pH. Our results demonstrate that sesame seed supplementation at 7% successfully improved the ejaculate volume, sperm concentration, and normal spermatozoa percentage of Ho cocks.


Introduction
In Vietnam, the national poultry flock is dominated by local breeds. Among indigenous chicken breeds in Vietnam, the Ho chicken breed is famous for its massive body weight (3.78kg per cock and 2.64kg per hen) (Duy et al., 2015). However, this heavy body weight leads to their poor natural mating ability and hence, a low rate of fertile eggs (72.81%) (Duy et al., 2015). In addition, semen production and quality of Ho cocks are considered as poor. Hue et al. (2015) reported that the semen qualities of Ho cocks in terms of average ejaculate volume, motility, and sperm concentration were 0.63mL, 57.6%, and 950.6 million sperms mL -1 , and feeding strategies to improve the fertility of Ho chicken flocks have been increasing in recent years.
To the best of our knowledge, the effect of sesame seeds on the semen quality of Ho chickens has never been investigated. Therefore, the objective of this study was to assess the impact of sesame seed dietary supplementation on the semen quality parameters of Ho cocks.

Materials and Methods
This study was implemented at the chicken farm of the Faculty of Animal Science, Vietnam National University of Agriculture.

Experimental design, animals, and housing
A total of 18 sexually mature and clinically healthy Ho chicken breeder males, which were 13-14 months old and had an average body weight of 3.73 ± 0.47kg per animal, were assigned to three treatments, comprised of diets containing different ratios of sesame seeds, namely 0%, 5%, and 7%, taking into account body weight. There were six replicates per treatment. The length of the adaptation period was 14 days, followed by an experimental period of 70 days. The cocks were housed individually in 1 x 1 x 1m cages with solid concrete floors and rice hull bedding. The photoperiod was 16h of light per day. All the necessary vaccinations and medications were administered accordingly.

Experimental diets and feeding
Three different diets were formulated for this experiment ( Table 1). The dietary treatments were corn-soybean-rice bran based diets supplemented with 0%, 5%, and 7% sesame seeds (denoted as control, 5% SS, and 7% SS, respectively). Sesame seeds were purchased from a local farmer in Thua Thien Hue province. Proximate analysis of the sesame seeds showed that they contained 5.96% humidity, 4121 Kcal kg -1 , 21.63% crude protein, 45.60% crude lipid, 15.21% crude fiber, and 5.44% ash. The sesame seeds were incorporated into the diet by replacing equivalent amounts of soybean seeds. Minor adjustments were made in the ingredients to keep the diets isonitrogenous and isocaloric. The feed was served twice per day and water was provided ad-libitum.

Semen collection and characterization
Semen was collected from the cockerels once a week using the dorso-abdominal massage, a modified technique as described by Lake (1962). Semen was collected into 1.5mL Eppendorf tubes. After ejaculate collection, semen samples were stored at 4-10°C and examined for the following traits: (1) Ejaculate volume (mL) from individual cocks was measured visually with the use of a prepared clean graduated collection tube; (2) Semen pH was determined by using pH test strips; and (3) Concentration of spermatozoa was measured by implementing the direct cell count method. A Neubauer chamber, which is used for counting blood cells, was used to measure the concentration of spermatozoa. Five µL of semen was mixed with 495µL of saline solution (3% NaCl). Ten µL of the diluted semen was then placed on one end of the hemocytometer using a micropipette and also on the other end to settle. The loaded hemocytometer was then placed on a microscope at a magnification of 40x. The heads of spermatozoa that fell within the subdivided smaller squares at the four edges and center of the hemocytometer were counted. The average sperm count per individual was calculated from two repeated measurements. The concentration of sperm per volume was estimated using the formula: C = N x 5 x 10 6 , where: C is the concentration of spermatozoa per volume (sperm count mL -1 ), and N is the number of spermatozoa counted.
To assess mass motility of sperm (%), a drop of semen was placed on a microscope slide using a micropipette, and then covered with a glass coverslip to spread the semen in order to have a uniform thickness and to prevent drying. Thereafter, it was placed on a microscope with a magnification of 40x for examination. The motility determination of the semen sample was expressed as the percentage of sperm cells showing forward motion, under their own power (Ax et al., 2000). Sperm motility was also scored to assess mass movement, aspects of wave motion. The score was assigned between 0 (total sperm are motionless) and 5 (wave motion varied rapidly, eddies are present) (Sonseeda et al., 2013).
The percentage of abnormal sperm was determined by using a 2% eosin stain. A drop of fresh semen was mixed with a drop of eosin stain on a glass slide followed by making a thin smear of it. The spermatozoa were examined under a digital camera (Optilab, Miconos, Indonesia) connected to a conventional microscope (Ceti, Belgium) and a computer at 40x magnification. At least 200 live spermatozoa were counted to determine the percentage of abnormal sperm according to Abu et al. (2013) and Bah et al. (2001). The dead spermatozoa were stained with eosin and appeared pink in color, while the live spermatozoa were not stained with eosin and appeared without any color. Spermatozoa with spindle-shaped heads and visible tails were considered as normal, while spermatozoa with structural defects, e.g. a simple bend at the midpiece, coiled head, broken tail, loose head, or tail coiled below the head of the spermatozoa, were considered abnormal (Figure 1).

Statistical analysis
Data were analyzed using Microsoft Excel (2016) and Minitab software version 16.1.0 (2010 Minitab Inc.). The overall means of the semen quality characteristics were expressed as the means and standard error (SE) of the means. One-way analysis of variance (ANOVA) was used to compare means with the Tukey test. Pvalues of <0.05 were considered statistically significant.

Semen quality characteristics during the adaptation period
The semen quality characteristics of the Ho cocks in the three treatment groups were similar during the adaptation period ( Table 2).

Semen ejaculate volume
The semen ejaculate volume showed similar values (0.64-0.73mL) in the control, 5% SS, and 7% SS groups during the adaptation period. These values are proportional to the results of Hue et al. (2015) and Xuan et al. (2017) who reported that the average ejaculate volumes of Ho cocks were 0.63 and 0.70mL, respectively. Almahdi et al. (2014) and Masindi & Mphaphathi (2016) recorded that the average ejaculate volume of Venda cockerels (a local chicken breed from South Africa with a low  reproductive potential) and Arabic cockerels (an indigenous chicken from Europe) were both 0.3mL. Differences in the live body weights of the mature cocks, which were around 3.7kg in the Ho breed, 2.3kg in the Venda breed, and 1.65kg in the Arabic breed, may account for the substantial variation in ejaculate volume.

Sperm concentration
Sperm concentration in the control, 5% SS, and 7% SS groups were identical (1.81-2.19 x 10 9 sperm mL -1 ) during the adaptation period. This result was equivalent to the sperm concentration of 2.07 x 10 9 sperm mL -1 in Ho cocks published by Xuan et al. (2017) but higher than that of 0.95 x 10 9 sperm mL -1 in the same breed reported by Hue et al. (2015). The relative influence of various reproductive glands, management, and the extent to which the genetic potential is exploited may be responsible for this differentiation (Gee et al., 2004). Indeed, many studies have published higher sperm counts in other native chicken breeds. Tuncer et al. (2008) and Obidi et al. (2008) reported the mean sperm concentrations of 2.4 x 10 9 sperm mL -1 in Gerze cocks and 3.6 x 10 9 sperm mL -1 in Shikabrown cocks, respectively. Siudzinska & Lukaszewick (2008) stated sperm counts of 4.2 x 10 9 sperm mL -1 in the Black Minorcas breed and 4.7 x 10 9 sperm mL -1 in White Crested Black Polish cocks. Sonseeda et al. (2013) reported the average sperm count of 4.3 x 10 9 sperm mL -1 in Thai indigenous chickens.

Sperm motility
The motility of spermatozoa of the Ho cocks in the control, 5% SS, and 7% SS groups were similar (67.50-70%) ( Table 2). These values are in agreement with Peters et al. (2008) who reported that sperm motility in fresh semen of seven chicken breeds ranged from 60 to 90%. However, these results were lower than the average value of sperm motility (80.64%) in Ho cocks as published by Xuan et al. (2017) and that of sperm motility (90%) in the same breed as reported by Doan et al. (2016). According to Sonseeda et al. (2013), sperm motility of Thai indigenous chickens was 88.2%. Akhlaghi et al. (2014a) reported that a sperm motility of 80% was observed in Arabic chickens whilst this value in the Lingnam, Bangkok, and Kedu chicken breeds was 84%.

Mass movement
The average mass movement scores of the Ho spermatozoa in the control, 5% SS, and 7% SS groups were 2.86-3.08. These values were lower than the results of Hue et al. (2015) and Xuan et al. (2017) who reported the average mass movement scores of Ho cocks were 3.3 and 3.8, respectively. Moreover, the values of the mass movement scores in Ho spermatozoa were lower than those in other native chickens. Nataamijaya et al. (2003) recorded the movement of spermatozoa in Arabian chicken had a score of 4.02 (as cited in Almahdi et al. (2014)). According to Sonseeda et al. (2013), the average mass movement score of Thai indigenous chicken spermatozoa was 4.3. Donoghue & Wishart (2000) stated that chicken semen pH often ranges from 6.0-8.0. Semen with a pH below 6.0 generally has decreased sperm motility, lactic acid production, and oxygen uptake, while a high semen pH (>8.0) increases the metabolic rate during in vitro semen storage (Donoghue & Wishart, 2000). The correlation between semen pH, sperm motility, and the metabolic rate was also reported by Masindi & Mphaphathi (2016). The semen pH values of the Ho cocks in the control, 5% SS, and 7% SS groups were 7.52-7.68 ( Table 2) which are in accordance with the findings of the mentioned studies. The results in the current study are higher than the semen pH values of 7.44 and 7.20 in Ho cocks which were reported by Xuan et al. (2017) and Hue et al. (2015), respectively. Moreover, Almahdi et al. (2014) recorded lower average values of semen pH in four local chicken breeds including Lingnam (from China), Bangkok (from Thailand), Kedu (from Indonesia), and Arabic chickens (from Europe) which were 6.92, 6.98, 6.98, and 7.04, respectively.

Semen quality characteristics during the experimental period
To our knowledge, no previous studies have examined the effect of SS on the sperm quality of chickens. In general, our results proposed that sesame seeds had a positive influence on the semen quality characteristics of Ho cocks, significantly shown in the three parameters of semen volume, sperm concentration, and abnormal spermatozoa ( Table 3). Semen ejaculate volume and sperm concentration were greater in the 7% SS group compared with the control group (P <0.05), whereas the percentage of abnormal spermatozoa was lower (P <0.05). The control group showed the lowest semen volume and sperm concentration but the highest abnormal spermatozoa percentage. Other traits involving sperm motility, mass movement, and semen pH were not affected by the dietary treatments.
The effects of the treatments on specific traits during the ten weeks of the experimental period are presented in Figures 2-7. Semen volume was improved in the group fed 7% SS compared to the control and 5% SS groups, however, the differences were not significant across the ten weeks (Figure 2). Sperm concentration showed increasing values in the three groups throughout the study period, and the 7% SS group presented slightly higher values than the other groups, but the values were not considerably different (Figure 3). When comparing sperm concentration within each group, no variation for a considered group was observed during the period of semen analysis. Sperm motility showed no substantial differences between the three groups throughout the study period (Figure 4). Mass movement and semen pH showed the same patterns as sperm motility with no significant improvement in the treatment groups compared to the control group from weeks 1-10 (Figures 5 and 6, respectively).
Concerning the abnormal spermatozoa trait, significant improvement in the group fed 7% SS was observed compared to the control (Figure 7).
Chicken spermatozoa are rich in PUFAs which make them vulnerable to oxidative stress and lipid peroxidation (Surai et al., 1998;Eid et al., 2006) and therefore, reduce their motility and fertility (Sanocka & Kurpisz, 2004;Khan, 2011). To lessen chicken spermatozoa quality loss, different antioxidants have been investigated. Lycopene, a carotenoid existing in vegetables and fruits, showed a positive effect on semen volume and sperm concentration of broiler breeder males when supplemented in drinking water (Mangiagalli et al., 2010). Likewise, dietary ginger powder improved sperm forward motility and live sperm percentage, and decreased abnormal sperm in aged breeder cocks (Akhlaghi et al., 2014a). It was also previously reported that sperm concentration and sperm membrane integrity were significantly enhanced in aging Ross 308 breeder cocks fed dried apple pomace (Akhlaghi et al., 2014b). Moreover, positive effects were established on semen concentration, sperm forward motility and viability, semen volume,   Week 1-2 Week 3-4 Week 5-6 Week 7-8 Week 9-10 Semen volume (mL)

Control
5% SS 7% SS Figure 4. Sperm motility (%) in the studied groups (control: no supplementation, 5% SS: receiving 5% of sesame seeds per cock, and 7% SS: receiving 7% of sesame seeds per cock) during the 10 weeks of semen analysis. Values are expressed as means ± SE.
Values are expressed as means ± SE. and sperm plasma membrane functionality when feeding rosemary leaf powder to breeder cocks (Borghei-Rad et al., 2017).
In the current study, the improvements observed are possibly associated with the high levels of minerals, vitamins, and antioxidant lignans (phytoestrogens) in sesame products (Shittu & Bankole, 2007). Amini et al. (2013) reported that sesame seed intake (30% of total diet) improved testicular parameters (number of epithelium cells and percentage volume of epithelial, lumen, and interstitial of these tubules, P <0.0001), increased LH concentration (P <0.03), increased fertility, and increased sperm production in male Wistar rats. According to Dimitrous (2006), sesame seeds and sesame lignans could also work to enhance the activity of vitamin E to potentially protect low-density lipoproteins against oxidative damage. In addition, dietary vitamin E and organic selenium have been shown to have a synergistic effect in reducing lipid peroxidation and enhancing the antioxidative status of chicken semen, therefore, improving the spermatozoa count and number of live spermatozoa (Ebeid, 2012).
In our study, no significant differences were shown regarding sperm motility, mass movement, and semen pH between the group receiving 5% SS and the group receiving 7% SS per cock. These results propose that the effects of sesame seeds are articulated throughout spermatogenesis by providing antioxidant substances.

Conclusions
Semen quality characteristics were improved in Ho cocks by supplementing their diet with sesame seeds. Semen volume and sperm concentration were increased and abnormal spermatozoa were reduced in Ho chickens receiving 7% of sesame seeds in their diets. Therefore, dietary supplementation with sesame seeds is recommended in terms of malesavings to enhance the reproductive and economic efficiencies. Week 1-2 Week 3-4 Week 5-6 Week 7-8 Week 9-10 Abnormal spermatozoa (%)