The Motility and Ratio of X and Y Sperm Filial Ongole Cattle Using Different Sexed Semen Methods

Corresponding Author: Enike Dwi Kusumawati, Animal Husbandry Faculty, Universitas Kanjuruhan Malang, Jl. S. Supriadi No 48 Malang, Indonesia Email: enike@unikama.ac.id Abstract: Artificial Insemination (AI) using sexed semen produce sex of calf as expected. There are various methods of sexed semen has been found. The aim of this research is to determine the effect of sexed semen methods using egg white sedimentation and percoll density gradient centrifugation toward motility and ratio of X and Y sperm Filial Ongole Cattle. Semen was collected using Artificial Vagina (AV) from Filial Ongole Cattle, evaluated and then separated using egg white sedimentation and percoll density gradient centrifugation technique. Only fresh semen with a minimum of 70% individual motile sperm and 2+ mass motility used in this study. Andromed as a based extender was diluted using aquabidest with 1:4 ratio. The obtained data were analyzed with analysis of variance (ANOVA) and continued by Duncan test if there was significant or very significant different. The result showed that the sexing methods (non sexing sperm, upper and under fraction using percoll density gradient centrifugation, upper and under fraction using egg white sedimentation technique) had very significant effect (P<0.01) on motility (64.25±3.94%; 48.55±8.28%; 53±7.93%; 56.9±8.22%; 49.75±8.19% respectively) and significant effect (P<0.05) on the ratio of X and Y sperm. The ratio of X and Y using percoll density gradient centrifugation and egg white sedimentation methods for the upper fraction were 28.0±2.26%:72.0±2.26% and 77.5±1.26%:22.5±1.26%. The ratio of X and Y using percoll density gradient centrifugation and egg white sedimentation technique for the under fraction were 69.0±15.35%: 26.0±4.37% and 22.9±1.44%: 77.1±1.44%. The study concludes that the sexed with egg white sedimentation technique is better than percoll density gradient centrifugation on sperm motility and ratio of X and Y sperm.


Introduction
The Effort to increase the efficiency of using AI in cattle is how to obtain the effectiveness of sperm separation (sexed semen) technology to separate X and Y sperm chromosomes Saili et al., 2017). There are various methods of sexed semen has been found including the sedimentation method using albumin column and percoll density gradient centrifugation . Motility is an important indicator for characterization of sperm quality after processing. Sexing methods easily applied is Egg White Sedimentation (EWS) (Afiati, 2004;Saili et al., 2017) and Percoll Density Gradient Centrifugation method (PDGC). Percoll is a medium that can be made with various densities and does not penetrate cell membrane (Susilawati, 2014). Although sexed sperm is currently used, the high cost and the reduced pregnancy rates compared to conventional sperm (Carvalho et al., 2014). Therefore it is necessary to look for alternative other ingredients as a substitute of egg whites and to determine the effect of sexed semen methods using egg white sedimentation and percoll density gradient centrifugation toward motility and ratio of X and Y sperm Filial Ongole Cattle.

Materials and Methods
Semen was collected using AV from five Filial Ongole Cattle in Beef Cattle Research Station, Pasuruan, Indonesia. After collection, fresh semen was evaluated macroscopically (colour, pH, volume) and microscopically (concentration, mass motility, individual motility, sperm viability and sperm abnormality). Only fresh semen with a minimum of 70% individual motile sperm and 2+ mass motility used in this study. Semen collection was regularly conducted twice a week.
The parameters observed, percentage of X and Y sperm based on morphometry of sperm wide head, percentage of motility before and after separation of the sexed semen. The obtained data were analyzed with analysis of variance (ANOVA) and continued by Duncan test if there was significant or very significant different.
Motility and abnormality percentage of Filial Ongole Cattle sexed semen with different methods is shown in Table 1. X and Y sperm ratio of Filial Ongole Cattle sexed semen with different methods shown in Table 2.

Characteristic of Fresh Filial Ongole Cattle Semen
The semen used in this study was normal (Garner and Hafez, 2008;Ax et al., 2008;Pineda, 2003). The morphometry observation of the fresh semen is in accordance with the general state of the X and Y sperm ratio in the fresh semen is 50%:50% and after fertilization, 50% the embryo should be males and 50% should be females (Pineda, 2003). The cattle were used in this study has a standard percentage motility of 70% and no more than 70%, this corresponds to Ax et al. (2008) found a decent standard of sperm in the further process was 70%. If the percentage motility of sperms that have below 60% the results will not meet the standards in the process before freezing up in the freezing process. The viability was 95.12±0.98% which was still within in the normal range or more than 80% . The abnormalities 0.92±0.27% was less than 20% (Ax et al., 2008). Semen concentration values obtained in this study is 1.758±137.66×106/mL which shows that the value of the concentration of relatively normal because, according to Garner and Hafez (2008) sperm concentration of cattle is 800-2000×106/ml. Examination of the concentration needs to be done because the sperm concentration can be used to predict the fertility of cattle (Ax et al., 2008). The quality of fresh semen used in this study was the semen that has good quality, it was intended that the sperms were able to survive during the separation process.

Motility and Abnormality of Filial Ongole Cattle Sexed Semen
The result in Table 1 shows that the sperm motility was highly significant different (P<0.01) after sexing between sexing methods. The sperm motility of egg white sedimentation (Upper Fraction) sexed semen was better than other treatments and no different from non sexing method (control). The sperm motility of percoll density gradient centrifugation is lower than egg white sedimentation. This is because the centrifugation causes more damage the sperm membrane (Kusumawati et al., 2017). Furthermore, sperm motility will decline as an increase in the number of the broken membrane Sekosi et al., 2016;Kusumawati et al., 2016).   The sperm must have less than 25% major morphologic abnormalities (Pineda, 2003) or more than 80% of the sperm conform to normal morphology (Ax et al., 2008). Susilawati et al. (2014) found that centrifugation process might also damage the sperm membrane, thus causing a decrease in sperm motility. The centrifugation process provided free radicals namely Reactive Oxygen Species (ROS) that damaged the sperm membrane (Zanella et al., 2016). ROS are free radicals that play a crucial role in many sperm physiological processes such as capacitating, hyperactivation and spermoocytes fusion (Aitken et al., 2012;Ball, 2008;Bansal and Bilaspuri, 2011). The minimum standard post thawing motility is 25% (Pineda, 2003).

X and Y Sperm Ratio of Filial Ongole Cattle Sexed Semen
The result in Table 2 shows the significant effect (P<0,05) on the ratio of X and Y sperm sexed semen between sexing methods. The sexed semen with egg white sedimentation method is better than percoll density gradient centrifugation on the ratio of X and Y sperm. Under fraction have a high of X sperms, whereas the upper fraction have a high of Y sperms on percoll density gradient centrifugation method. The sexed semen with egg white sedimentation method produces a high of X sperms on upper fraction, whereas the under fraction have a high of Y sperms. From all of the research results, sexing sperm technology using egg white sedimentation technique is better than percoll density gradient centrifugation on the ratio of X and Y sperm.

Conclusion
The study concludes that the sexed with egg white sedimentation technique is better than percoll density gradient centrifugation on sperm motility and abnormality.

Acknowledgment
Beef Cattle Research Station which has allowed implementation of research.

Funding Information
Ministry of Finance through Indonesia's Endowment Fund for Education (LPDP) which has provided research funding (Contract Number?).