Raw Semen Characteristics of Three Different Indonesian Local Roosters

Indonesia has agreat variety of roosters, either indigenous type as well as exotic and cross breed. The purpose of this experiment was to study the characteristics of semen from three types of Indonesian local roosters such as Merawang, Kampung and crosses Sentul Kampung with Kedu (SK Kedu). A total of 15 roosters consist of Merawang roosters, Kampung, and SK Kedu roosters were 5 each. The semen was collected 3 times a week by dorso-abdominal and cloaca massage method. The parameters evaluation was macroscopic characteristics consist of volume, color, consistency, and pH. Microscopic evaluation of semen such as a mass movement, sperm motility, live sperm, sperm abnormality and sperm concentration. Results of this experiment showed that semen volume of Merawang (0.40±0.26 mL) was higher (p<0.05) compare to Kampung (0.24±0.12 mL) or SK Kedu (0.16±0.10 mL) but no difference on semen color, consistency and semen pH. There were no difference in the mass movement, sperm motility and live sperm as well as on sperm abnormality among three types of roosters. Sperm concentration of Merawang (4490 million mL-1) was significantly higher than Kampung (3245 million mL-1) and the SK Kedu roosters (3751 million mL-1). Its was conclude that Merawang roosters had good semen quality better than Kampung and SK Kedu roosters


Introduction
Increase productivity of local roosters is needed due to some types of Indonesia local roosters are an Indonesian native germplasm that need to be preserved. Local roosters play a role as meat and egg producer, contributed in animal food sufficiency up to 23% of meat and 40% of the eggs (Suprijatna, 2010). There is some indication that the demand of local roosters from year to year is increased. The tastes of meat local roosters more preferable, low fat and more organic meat product. This can be seen with establishing a new restaurant with a local roosters menu.
The advantages of local roosters are good adaptability to tropical environment, good mothering ability and resistance to certain diseases. However, there are several obstacles in the development of local roosters. The weaknesses of local roosters such as slow growth rate, egg production are low and small body size when compared to Broiler roosters (Dessie et al., 2011), having the nature of broodess, slow sexual maturity, low genetic quality, and relatively expensive price because of high demand which is not offset by increased production (Sulandari et al., 2007). The high demand for local roosters products may threaten the local roosters population if not balanced with conservation.
Problems often found in increasing the production of local roosters is the provision of quality breeds. In search of candidate breeds, rather than being based on the exterior appearance can also be done with the concept of breeding, so as to obtain quality breeds to increase livestock production and value-added or advantages of both types of crossbred roosters (Darwati, 2000). Breeding types of local roosters made to produce the new breeds roosters (proven breed) with a better genetic quality. The quality of local roosters needs to be improved through cross breeding with the aim of increasing the rate of its growth and improve reproductive efficiency, but while maintaining the original characteristics such as coat color of local roosters egg shape, color shell, meat flavor, and texture of the meat. Suprijatna (2010) stated that crosses local roosters can increase their productivity by not changing the characteristics of the product and phenotype.
Application of technology of Artificial Insemination (AI) is one of reproductive technology that can be used to improve the productivity of local roosters by improving the ability of local roosters that have superior production quality to fertilize several females. One of the factors that influence the success of AI is the quality of the semen. Hence the success of AI requires good quality semen. This study aim to compare the semen quality of 3 types Indonesian local roosters and to find out individual variation among breed roosters.

Materials and Methods
The present study conducted at breeding cages of Faculty of Animal Science and at the Laboratory of Reproduction Rehabili-tation Unit, Faculty of Veterinary Medicine, Bogor Agricultural University during February-May 2016.

Semen collection methods
Semen was collected from each roosters 3 times a week for 3 months by the dorsoabdominal and cloaca massage method. All roosters were trained for semen collection, and collected by a trained technician.

Semen evaluation
Immediately after collection, semen was evaluated macro-and Microscopically. Macroscopyc evaluation were conducted for semen volume (mL), color, consistency and pH. Microscopic evaluation was conducted on the percentage of motile sperm, live sperm, sperm abnormality and sperm concentration.

Macroscopic evaluation
The semen volume, color and consistency, were visually evaluated. The pH of semen sample of each roosters was measured using pH ranged from 6.4 to 8 (Merck special indicator paper).

Microscopic evaluation
Mass movement was examined by putting a drop of semen on a warm slide under a light microscope (Olympus CH 20) at 100 x magnification. The score assigned between 1+ (total sperm were motionless), 2+ (if the wave thick mass, but slow migratory or mass wave being but a quick move), and 3+ (wave motion varied rapidly, the eddies were present). Sperm motility was assessed by mixing 1 drop of semen with 4 drops of physiological saline and homogenized. One drop of the mixture was transferred to a clean, warm glass object and covered with a cover glass. The sperm motility was assessed subjectively from 5 fields, 0 (all not moving) to 100% (all progressive motile). The evaluation was conducted under a light microscope (400x magnification). Sperm motility estimations were performed from 5 different fields in each sample. Percentage of sperm motility was subjectively evaluated on a scale ranging from 0 to 100 %.
The percentage of live sperm was evaluated according to Ax et al. (2000). Briefly, a drop of well-mixed semen was mixed with a drop of eosin-nigrosin stain on a glass slide. Another glass slide was used to prepare the smear and dried at heating table (37 o C). 10 fields per slide (at least 200 sperm) were directly counted using light microscope (400x magnification). Sperm colored pink was considered nonviable and unstained (clear) cells were counted as a live (Arifiantini et al., 2013). Sperm abnormality, a smear was prepared from a mixture of diluted semen and eosin-nigrosin. The percentage was based on 200 sperm count (Arifiantini, 2012). Sperm concentration of the semen sample was counted by using a Neubauer Chamber. 2 µl semen was diluted with 998 µl formolsalin, in an 1.5 mL microtube. Total sperm number per ejaculate was obtained by multiplying the total volume with the sperm concentration.

Data analysis
The data were analyzed by using Analysis of Variance (ANOVA), followed by the Duncan's multiple range tests to determine significant differences in all the parameters among groups (Steel and Torrie, 1995)

Semen quality of Merawang, Kampung, and SK Kedu
Generally, poultry semen has a low semen volume and high sperm concentration. This present study macroscopically found no differences among colour, consistency and pH. All roosters demonstrated milky white in color with thick consistency and pH ranging from 6.94±0.25 to 6.97±0.31 (Table 1). pH range was still included in the normal range, a factor that could play a role is the technique of semen collection and stimulation of the accessory sex glands. The accessory sex gland fluid is generally alkaline (Bah et al., 2001;Peters et al., 2008;Tuncer et al., 2008).
The study showed that there was a significant difference (p<0.05) on semen volume. Semen volume of Merawang (0.40±0.19 mL) was higher than Kampung (0.24±0.15 mL) and SK Kedu (0.16±0.15 mL) (Tabel 1). In the present study, the semen volume was similar to the results reported for other roosters of different breeds Green-Legged Partridge, Black Minorca, White Crested Black Polish, and Italian Partridge (0.24-0.52 mL) in previous studies Siudzinska and Łukaszewicz (2008a). According to Malik et al. (2013) the variations among the breeds in semen volume were 0.33±0.16 mL, 0.29±0.18 mL, and 0.10±0.10 mL for red jungle fowl, domestic roosters, and bantam roosters, respectively. This defferences might be related to body weight. Merawang is 2.72±0.05 kg, Kampung is 2.57±0.05 kg and SK Kedu only 2.26±0.05 kg.
(2016) reported sperm concentration was 3650 × 10 6 mL -1 to 5890 ×10 6 mL -1 among genetic groups of roosters. Tarif et al. (2013) noticed that sperm concentration significantly varied among the line of roosters and varied from 9600×10 6 to 7500×10 6 per mL. The differences in sperm concentration between roosters are suspected to involve many factors such as intake of feed, and the body size could be attributed to their different genetic makeup, and body weight (Malik et al., 2013) also age and season (Elagib et al., 2012). Donoghue et al. (2000) observed the sperm concentration increace for heavy breeds.
Total number sperm per ejaculate were found significant differences (p<0.05) among three types local roosters. Total number sperm per ejaculate of Merawang (2066±318x10 6 cell per ejaculate) was higher than Kampung (789±255x10 6 cell per ejaculate) and SK Kedu (613±255 x 10 6 cell per ejaculate). The present study of Merawang roosters recorded the highest of total spermatozoa per ejaculate compare reported by Sonseeda et al. (2013) of Thai Indigenous roosters 1477 x 10 6 cell per ejaculate.

Individual semen quality of Merawang
This present study macroscopically found no differences between color and pH. All roosters demonstrated milky white in color and pH ranging from 6.90±0.10 to 7.0±0.05. Consistency ranged from thick until watery. A variation in the semen volume among individuals on Merawang roosters was recorded. It was found that there were significant differences (p<0.05) in semen volume. The greater amounts of ejaculated volume recorded for Merawang number 5 (M5) was 0.66±0.03 mL and the lowest in Merawang number (M3) only 0.17±0.03 mL (Fig. 1). Bah et al. (2001) reported semen volume of Sahel regional local breeding Roosters to be averaged 0.28 mL. Tuncer et al. (2006) reported semen volume of Denizli Roosters to be 0.70 mL. It also in the same range reported by Peters et al. (2008) 0.37-0.73 mL, for Nigerian indigenous breeds. Mosenene (2009) recorded semen volume was 0.3±0.10 to 0.4±0.10 mL in 4 South African roosters breeds. This present study microscopically found that there were significant differences (p<0.05) in the mass movement, motile sperm, live sperm, sperm abnormal and sperm concentration. Mass movement ranging from 2+ to 3+. From all individual, Merawang number 2 (M2) roosters had lowest motile sperm (71.67±2.92%) and live sperm percentages (80.90±2.80%). However, for M1 motile sperm and live sperm did not differ with roosters M3, M4 and M5. The greatest percentages of motile sperm and live sperm (83.67±1.30% and 91.13±1.20%) were found in the individual roosters M4 and M5, respectively (Fig. 2). Tuncer et al., (2006) determined the sperm motility for Denizli roosterserels and found it to be on average 72.3±0.08%. Researchers have also found the sperm motility for the White Leghorn breed to be 83.2±0.60%, and 77.6±0.20% of the New Hampshire (Tuncer et al., 2008). The variation in sperm motility among genetic groups could be due to the genetic potential of individual line. Also previously, results of Tuncer et al. (2006), Peters et al. (2008), and Tarif et al. (2013), showed that there were differences in strain with respect to motility. Earlier study conducted by Hermiz et al, (2016) reported that the crossed roosters percentage of live sperms were 93.08%. Tarif et al. (2013) stated that the proportion of live sperms significantly varied from 82.20% to 87.30% among the line of roosters. Nevertheless, the high percentage of live sperms in the present study were good enough for routine AI in poultry.

Individual semen quality of Kampung
This present study macroscopically found no differences between color and pH. All roosters demonstrated milky white in color and pH ranging from 6.91±0.05 to 7.0±0.05. Consistency ranged from thick until watery. A variation in the semen volume among individuals on Kampung roosters was recorded. It was found that there were significant differences (p<0.05) in semen volume. The greater amounts of ejaculated volume recorded for K1 (0.29±0.03 mL) and K5 (0.28±0.03 mL) and lowest in K3 (0.17±0.03 mL) (Fig. 4). In this study microscopically, found that there were no differences in motile sperm, live sperm, and sperm abnormality, while sperm concentrations were significantly different (p<0.05) among individuals Kampung roosters. Mass movement ranging from 2+ to 3+. All roosters showed motile sperm, live sperm, and sperm abnormality ranging from 81.33±1.30% to 84.00 ±1.30%; 90.59±1.26% to 92.46±1.26%; and 1.60±0.91% to 4.88±0.91%, respectively Figure 5 showed sperm concentrations and total number sperm per ejaculate of individual of Kampung. Statistical analysis showed that sperm concentration and total number sperm per ejaculate were significantly different (p<0.05). As can be seen, Kampung number 2 (K2) roosters recorded the highest of extreme sperm concentration (5913±445 x 10 6 mL -1 ) and total number sperm per ejaculate (1299±147 x 10 6 ) and the lowest sperm concentration and total number sperm per ejaculate in Kampung number 3 (K3) were 1885±445 x 10 6 mL -1 and 300±147 x 10 6 , respectively. The sperm concentration in the present study was lower than Mphaphathi et al. (2016) reported an average sperm concentration of 6800 × 10 6 mL -1 in Venda roosters.

Conclusions
The study of raw semen characteristics of three local roosters concluded that Merawang roosters had good semen quality better than Kampung and SK Kedu roosters. Semen volume of Merawang was higher compare to Kampung or SK Kedu but no difference on semen color, consistency and semen pH. Sperm concentration of Merawang (4490 million mL -1 ) was significantly higher than Kampung (3245 million mL -1 ) and the SK Kedu roosters (3751 million mL -1 ).