The influence of glycerol level for freezing medium on the quality of ram semen

Research on the use of glycerol levels as a cryoprotectant in the semen freezing process of Palu ram has been carried out at the Reproduction Laboratory of the Faculty of Animal Husbandry and Fisheries, Tadulako University. The purpose of this study was to determine the best level of glycerol to maintain the quality of spermatozoa after thawing. This study used semen from 2.5 years old rams. The semen collection process was carried out two times a week. The treatments in this study were glycerol levels of 4%, 6% and 8% for P1, P2 and P3 respectively. The data was analyzed using analysis variance, the differences between treatments was analyzed by multiple range test Duncan. Based on the results of the study showed that the addition of glycerol at a level of 6% was able to maintain the best spermatozoa motility (45.42%) followed by a level of 9% (41.14 %) and a level of 3% (40.29). As a conclusion, that the level of glycerol significantly effected on motility, viability and abnormality.


Introduction
One of the efforts to maintain genetic material in gamete sources was through cryopreservation.The local sheep has genetic material that needs to be maintained because it was able to adapt to extreme environments.The problem that often occurs in the sperm clotting process is damage to the cell membrane caused by the occurrence of ice crystals and cold shock on frozen cells and intracellular changes due to water loss associated with the formation of ice crystals.Damage could be reduced by adding a cryoprotectant substance, cryoprotectant was a protective agent added to the diluent to protect spermatozoa from damage.Generally, the cryoprotectant used was glycerol, which functions as a protective agent.Factors that affect the quality of post-thawing spermatozoa include the lack of equilibration time so that glycerol absorption was not optimal, the using of glycerol at end process of the equilibration does not provide optimal protection against cold shock events and the process of ice crystal formation because the time interval to freezing was too short [1].The addition of glycerol as a cryoprotectant could help to improve semen quality.The role at glycerol in protected the plasma membrane, prevented physical and functional membrane cracking to sperm cells at process freezing of the semen due to the formation of ice crystals.With the process of preventing physical damage caused by ice crystals formation, the integrity of the sperm would increase [2].after the discovery of glycerol as a cryoprotectant agent in the process of freezing semen in most mammals, most of the processes of freezing almost all species till now was still a good cryoprotectant for cryopreservation of sperm.

Material and method
The experimental animal used was a 2-year-old local ram, the materials and tools used were ram semen, glycerol, Tris, egg yolk, penicillin and streptomycin, haemocytometer, microscope, incubator, 0.25 ml straw, and others.Semen of local rams was collected using an artificial vagina, the sperm collecting was carried out twice a week.Semen that has been collected was then observed macroscopically including volume, consistency, odor, color and pH, and and then microscopic examination was carried out including motility, abnormality and concentration.The semen that had been examined was then diluted with egg yolk-Tris extender with a composition of Tris Hydroxymethyl aminomethane 2.4 gr; citric acid 1.4 gr; fructose 0.9 gr; penicillin 100 IU and streptomycin 0.1 mg per ml diluent; distilled water 10 ml; 20 % egg yolk and 3%,6%,9% glycerol for P1, P2 and P3 respectively.Furthermore, semen was mixed into each treatment and diluted according to the research treatment using one and two step methods at room temperature and cold temperature (5°C).Semen that has received glycerolization treatment is prepared into straws.The equilibration period was carried out at 5°C for ± 4 hours.Straws were frozen using a styrofoam box, the straws were first steamed over liquid nitrogen for 8 minutes and then dipped into liquid nitrogen.Thawing was carried out using water at 39 o C for 30 seconds [3].Semen quality observed was motility, abnormality and intact plasma membrane of spermatozoa.All of the treatment was repeated seven times, data obtained was then analyzed by analysis of variance in Completely Randomized Design (CRD) and if there were differences between the treatments, a further test was carried out using Duncan's multiple range test (α = 0.05)

Fresh semen characteristics
The examination of the fresh semen which would be used as a standard for the feasibility of semen which will be processed further, besides that it was also used to calculate the need for diluent composition when it was diluted or frozen.Based on the above data, the sheep semen was feasible to proceed with the cryopreservation process.

Effect of glycerol on sperm motility
Motility was often used to assess the quality of frozen semen because this parameter is easy and fast to do [4].For the purposes of AI, the minimum motility allowed is 40%.The analysis of variance showed that the treatment of various glycerolization methods had significantly effect on the motility of post-thawing spermatozoa.The results of this study were in accordance with Yusuf et al ( 2006) that adding glycerol produce different post-thawing motility.These results suggest that in one-or two-stage mixing of diluents containing glycerol, there was an increase in osmotic pressure in the diluent, this increase can reach 800-1000 mosmol [5].According to Apriyanti (2012) changes in osmotic pressure could cause damage to spermatozoa [6].The results showed that the glycerolization method in the P2 treatment produced spermatozoa motility which tended to be higher.The addition of glycerol gradually gave spermatozoa the opportunity to adapt to the diluent so that a molecular balance between spermatozoa and diluent could be achieved.Glycerolization at 5°C could minimize the death of spermatozoa due to high metabolic activity and the negative effects of egg yolks at room temperature.One-step or two-stage glycerolization methods at room temperature and 5ºC produce motility above the normal motility limit [7].High concentration of glycerol increases the membrane osmotic pressure of extender and dehydrates of sperm, resulting in denaturation of protein and cracking of cell membrane structure and formation of sperm, known as "Osmotic Damage."The concentration of glycerol may affect to the morphology and function of sperm by adjusting the pressure of osmotic diluent, making it was reasonable to find an optimal concentration where the glycerol was introduces and protective the least membrane osmotic damage [8].

Effect of glycerol level on abnormality sperm
Observation of sperm abnormalities with differential staining using eosin fluid and observed under a microscope.Spermatozoa abnormalities resulting from this study were dominated by secondary abnormalities such as broken, broken or coiled tails.These secondary abnormalities could occur during ejaculation of semen, excessive heat, rapid cooling, contamination with water, urine or antiseptics, and so on.In addition, the process preparations could also affect the percentage of abnormalities.The results was found that the treatment of various glycerolization methods did not give significantly different results for abnormalities, but the P1 treatment had abnormalities that to be higher, this is thought to be caused by the negative effect of egg yolks at room temperature, because according to Feradis (2010) some elements of egg yolk could produce poison at room temperature, besides that the high activity of spermatozoa at room temperature would also accelerated the damage of spermatozoa [9].Abnormalities that occur were also thought to be the result of freezing treatments such as cold shock, changes in osmotic pressure and when making smear preparations.Spermatozoa that cold shock sometimes have their tails and body parts wrapped around the head and this causes abnormalities in spermatozoa According to Herdis (2005) that there is a change in osmotic pressure when given glycerol in solution and this can result in abnormalities in spermatozoa/ This change in osmotic pressure may cause abnormalities in spermatozoa [5].Furthermore, Herdiawan (2004) the occurrence of physical changes in the living media of spermatozoa, both changes pressure of osmotic, and the of intracellular ice crystals formation could cause changes in the structure of spermatozoa such as the tail of the spermatozoa bends or the head detaches from the tail [10].

Effect of glycerol level on intact plasma membrane
Spermatozoa An intact plasma membrane could be seen using a hypoosmotic.An intact of plasma membrane would be marked by the curvature of the spermatozoa tail, whereas spermatozoa with a damaged of plasma membrane do not see the curvature of the tail because the plasma membrane cannot retain a hypoosmotic solution.The results of the analysis of variance, showed that the different level of glycerol produced significantly different results.To find out the differences between treatments, The results of Duncan's multiple range test analysis in Table 5. Treatmen of Glycerol at 5º C gave results that were not significantly different from MPU spermatozoa of postthawing sheep.This was presumably in the one-stage and two-stage mixing of glycerol, an increase in osmotic pressure occurs in the diluent solution.According to Herdis (2005) this increase in osmotic pressure is high enough to cause damage to the membrane and can result in membrane damage [5].According to Apriyanti (2012) changes in osmotic pressure could cause rupture of spermatozoa plasma membrane [6].Good plasma membrane conditions would also have a good influence on motility because the plasma membrane contains many macromolecules that function in metabolic processes and protect spermatozoa cell organelles.The plasma membrane of spermatozoa was rich in unsaturated fats making it susceptible to lipid peroxidation [11].The result of lipid peroxidation was the lipid peroxides formation which would reaction with free radicals and stimulate autocatalytic reactions resulting in cracking to the spermatozoa plasma membrane [12].Room temperature could cause increased spermatozoa metabolic activity and produce free radicals as a result of cells metabolism.According to Yulnawati and Setiadi (2005) decreased quality of spermatozoa during storage due to toxicity from spermatozoa was dead or from substances contained in diluent which have undergone oxidation could cause high levels of free radicals which could damage the integrity of the plasma membrane [13].The glycerol was the most used cryoprotectant agent in process of cryopreservation semen.It was different concentrations of glycerol may result with varying effects on function of sperm and therefore fertilizing abilities.It has been indicated that the optimum concentration of glycerol was 7% (range of 6%-9%).In another research, showed that glycerol with different concentration of 4%, 6%, 7%, and 8% differed in motility and viability sperm [14].

Conclusion
-The use of glycerol at different levels did not significantly affect the motility and abnormal spermatozoa of post thawing local sheep, but had a significant effect on intact plasma membranes of spermatozoa.-Thr use Glycerol at 7% level produced the best Intact membrane of postthawing semen compared to treatments at 5% and 9% levels.

Table 1 .
Average characteristic value of fresh semen of Palu Sheep.

Table 5 .
Duncan test of intact membrane spermatozoa in various treatments.